GENETIC AND CYTOGENTIC STUDIES ON PRIMARY AND SECONDARY AMENORRHEA IN TAMIL NADU POPULATION

Transcription

1 GENETIC AND CYTOGENTIC STUDIES ON PRIMARY AND SECONDARY AMENORRHEA IN TAMIL NADU POPULATION Thesis submitted in Partial fulfillment for the award of Degree of Doctor of Philosophy in BIOTECHNOLOGY Submitted by K. CHITRASANKARI M.Sc., M.Phil., Under the Guidence Dr. C. K. HINDUMATHY, M.Sc., Ph.D., Dean, Department of Bio-Science, VMKV Engineering College Salem, Tamil Nadu, India VINAYAKA MISSIONS UNIVERSITY SALEM, TAMILNADU, INDIA November 2014

2 VINAYAKA MISSIONS UNIVERSITY DECLARATION I, K. CHITRASANKARI declare that the thesis entitled GENETIC AND CYTOGENETIC STUDIES ON PRIMARY AND SECONDARY AMENORRHEA IN TAMIL NADU POPULATION submitted by me for the Degree of Doctor of Philosophy in Biotechnology is the record of work carried out by me during the period from FEBRUARY 2011 to NOVEMBER 2014 under the guidance of Dr. C. K. HINDUMATHY, M.Sc., Ph.D., (Dean), Head of the Department of Bio-Science, VMKV Engineering college Salem, India and has not formed the basis for the award of any degree, diploma, associate-ship, fellowship, titles in this or any other University or other similar institutions of higher learning. Place: Salem Signature of the Candidate Date:

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4 ACKNOWLEDGEMENT I express my sincere and humble gratitude to GOD ALMIGHTY for the grace and abundant blessings and peace of mind to complete my course successfully. I offer my profound thanks to head Sister. Suseela, M.A TPM, Ambatur, Chennai encouraged to join this study. First and foremost I express my thanks from depth of my heart to my guide, Dr.C.K.HINDUMATHY, M.Sc., Ph.D., P.D.F Dean, Department of Bio Science, VMKV Engineering College, Salem for her incessant help, steadfast encouragement, meticulous guidance in planning and execution of my research work. It is my privilege to work under her supervision and I am ever grateful to her for inspiring me with her wise council and guidance. I express my sincere thanks to Late Dr. A. SHANMUGASUNDARAM, Former Chancellor, and Dr. S. GANESAN, Chancellor Vinayaka Missions University, Salem for giving me an opportunity to do my Ph.D. I deem it as great privilege to convey my sincere thanks to Dr. K. RAJENDRAN, Dean-Research, Vinayaka Missions University, Salem for his constant encouragement and suggestions for the successful completion of my work. I specially thank Dr. A. NAGAPAN, Principal, VMKV Engineering College, Salem for his immense encouragement and valuable suggestions during the period of study. I owe my sincere thanks to Dr. S. Samundi Selvamani, M.D., D.G.O, D.A., FICOG (Lon) Dean Department of Obest & Gynec, Kilpauk Medical College Chennai, for her help in the identification of Genetic infertility cases to carry out the study. I express my deep gratitude to Mr. Anand Assistant Professor, VMKV Salem for his help throughout this study. My uncle Mr G. D. Damodharan (Rtd Asst

5 Director fisheries) and Mrs. Sundari Damodharan (BSNL) for their everlasting love and affection, guidance towards me through out this study. I place on record my sincere thanks to Dr. S. Divya Sivaraman MBBS., DNB(DRM)., DRCOG (Gynec), Director, Srushti IVF and Genetics Medical Research Pvt. Ltd, Ramapuram, Chennai - 91 for her guidance and keen interest in the study. Finally, on a personal note, I wish to owe my respected and very special thanks to my husband Mr. L. Philip Ajit Kumar B.Sc., B.Tech., PGDBA, DBF for his unending love and without whose co-operation and encouragement my research would not have been successful. K.CHITRASANKARI

6 CONTENTS Sl No TITLES Page No 1 LIST OF TABLES 2 LIST OF FIGURES 3 LIST OF PLATES 4 INTRODUCTION 5 OBJECTIVE OF THE STUDY 6 REVIEW OF LITERATURE 7 NEED OF THE STUDY 8 MATERIALS AND METHODS 9 RESULTS 10 DISCUSSION 11 SUMMARY 12 CONCLUSION 13 REFERENCES i-ii ii iii-vi i

7 Table No I II III IV V VI LIST OF TABLES Title of Tables The frequency of chromosomal aberrations associated with PA & SA (collected from reports having less than cases Recent cytogenetic studies of chromosomal abnormalities of PA Classification of primary amenorrhea patients of their syndromic nature and karyotypes Frequency of ovarian dysgenesis in various categories of PA & SA Frequency of consanguineous marriages in various categories of PA Frequency of familial incidence in various categories of PA Page VII Mean parental age in various categories of PA 66 VIII Analysis of Birth- Order in PA 67 IX Incidence of associated disorders among of PA & SA 67 X Incidence of congenital anomalies among sibs of PA 68 XI XII XIII XIV XV XVI XVII Reproductive history of parents in various categories of PA & SA Antenatal medical history of proband s mothers in various categories of PA Types and frequency of chromosomal abnormalities in 221 cases of PA and SA Recent cytogenetic studies of chromosomal abnormalities of SA Types and frequencies of clinical features in various categories of PA having the karyotypes of 45,X0; 45,X0/46,XX; (5 groups) and 46,XX Types and frequencies of clinical features in various categories of PA&SA having the karyotyotype of 46,XiXq, 45,X/46,XiXq, 46,XXp- (partial deletion) and 46,XXq- and 46,XXq- (partial deletion) Clinical features associated with individual cases of PA having the karyotype of 46,X(Xp-) complete deletion; 46,X(Xq-) complete deletion 45,X/46,X(rX); 45,X/47,XXX; 45/46,,XX/47,XXX and 46,XX/47,XXX i

8 XVIII Variation in the clinical featuers of XY females 75 XIX XX XXI XXII XXIII XXIV XXV XXVI Clinical features associated with 45,X/46,XY and 46,XX/46,XY Karyotypes Radiosensitivity of patients with XX, XO, XO/XX karyotypes and normal XX females Student s t values for the comparison of means for the data given in Table XXVIII Radiosensitivity of chromosomes of the primary amenorrheas: Relative frequency of translocations and brake + Deletions Student s t values for the comparison ofmeans or the data given In Table XXIX Radio sensitivity of individual chromosomes in syndrome associated with primary amehorrhea A comparative analysis of the radiosensitivity of individual chromosome in Syndrome associated with primary amenorrhea Radiosensitivity of chromosomes in primary amenorrhea relative to the chromosome of normal individual FIG.1 GENE MAPPING CHROMOSOME FIG.2 GENE MAPPING CHROMOSOME X FIG.3-5 FIG.6-10 Densitometric profiles of the normal and deleted X chromosome in a patient of primary amenorrhea A Normal X chromosome B Deleted X with break point Xp22 Densitometric profiles of the normal and deleted X chromosome in a patient of primary amenorrhea A Normal X chromosome B Deleted 7 with break point 7q ii

9 LIST OF PLATES Plate No 1 & 1a TITLE OF PLATES Clinical features of the true Turner syndrome A, B, C Clinical features of mosaic Turner (XO/XX, 50:50 ) A - Short Stature B - Poor breast development C - Infantile external genitalia D - Short 4 th metacorpel E - Infantile external genitalia 2a Karyotype of normal female (G-banded chromosomes) 97 2b Karyotype of true Turner, 45,XO (Patient in plate No.1) a 5b 6 A, B - Clinical features of mosaic Turner (XO/XX,50:50) A - Short Stature B - Poorly developed secondary sexual features D, E - Clinical features of mosaic Turner (XO/XX, 21:79) D - Average height with well developed secondary sex characters E - Well developed secondary sex characters A, B, C1 - Clinical features of primary amenorrhea with 45,X/46,X,r(X) (72:28 ) A - Short Stature B - Poor breast development and absence of axillary hair, Low set ear D, E - Clinical features of a patient with 45,X/47,XXX D - Short Stature E - Absence of axillary hair and normal external genitalia Karyotype of one of the cell lines 46,X (rx) (Ring chromosome) in plate No.4 (A-C) Karyotype of one of the cell lines (47,XXX) in plate No.4 (D-F) A,B - Clinical features of a primary amenorrhea patient with 46,Xi(Xq) A - Short Stature B - Absence of axillary hair, poor breast development, infantile external genitalia with sparse pubic hair D, E - Clinical features of a patient with 45,X/46,Xi(Xq) (30;47) Page No iii

10 D - Short Stature, shield chest E - Absence of axillary hair and infantile external genitalia 7 Karyotype of 46,Xi(Xq) (patient in plate No.6 - A,B,C) 99 8 A, B, C - Clinical features of a primary amenorrhea patient with 46, X(Xp-) (bp Xp22) A - Short Stature B - Absence of axillary hair, infantile external genitalia D, E - Clinical features of a primary amenorrhea patient with 46, X(Xq-) (bp Xq28 ) D - Average height E - Normally developed axillary hair and breast and well developed external genitalia 9 Karyotype of 46,X(Xp-) bp Xp22 (patient in plate No.8 A-C) Karyotype of 46,X(Xq-) bp Xq22 (patient in plate No.8 D-F) Clinical features of a primary amenorrhea patient with 46,X(Xp-) complete p arm deletion (bp Xp11) A - Short Stature B - Sparse axillary hair and infantile external genitalia D - Presence of small Barr bodyt E - Ultrasonogrram scan showing absence of left ovary F - Presence of right ovary Karyotype of 46,X(Xp-) complete short arm deletion (patient in plate No.11) Clinical features of a primary amenorrhea patient with 46,X(Xq-) complete long arm deletion with break point at Xq11 A - Short Stature, with poorly developed secondary sexual charecters B - Absence of axillary hair, C - Infantile external genitalia, D - presence of small Barr body, E, F - Absence of both left and right ovary Karyotype of 46,X(Xq-) complete long arm deletion (patient in plate No.13) B,C - Clinical features of patient with pseudo vaginal perineoscrotal hypospadias syndrome, B- Eunuchoid body habitus, absence breast development presence of axillary hair C - Enlarged clitoris like a phallus and testes situated in the labioscrotal folds iv

11 Karyotype of primary amenorrhea patient with 46,XY (patient in plate No. 15-D-F) Clinical features of mosaic Turner 45,X/46,XX with multiple chromosome aberration (Note the typical Turner features) A - Short Stature, cubitus valgus, knock knees, poorly developed secondary sex cheracters B - Absence of axillary hair C - Low set ears, short webbed neck, infantile external genitalia, and pigmented nevi Multiple chromosome aberration of patient in plate No. 17 A - 6q break, B, D - 2q gap, C - 46,XX 20, E - Metaphase plate showing 7,14 translocation Clinical features of a primary amenorrhea patient with 46,XX gonadal dysgenesis A - Average height with poorly developed secondary sex characters B - Absence of axillary hair, saddle shaped neck and infantile external genitalia Multiple chromosome aberration of a primary amenorrhea (patient in plate no.19) A - 2q break, B - a gap at 5p break, C - 11q arm break D, E, F- break at Xq22 Clinical features of two sisters with 45,X/46,XX/47,XXX (30:50:20) A - Full view of the patient and her sister B,C - Absence of breast development in patient (B) and her sister (both showed infantile external genitalia and flexion contracture of little finger) Effect of folic acid free medium in patients with 46,XX GD chromosome plates. Radiation induced chromosomal break points in 7q arm A, B, D - break at 7q31 locus C, E, F, G, H, I - break at 7q22 locus 24 Radiation induced g - banded metaphase spreads showing various chromosomal rearrangements involved at 7q22 locus. 25 Radiation induced chromosomal break points of X chromosome A,B,- break at Xq22 locus v

12 26 C, D, E, F, G - Deletion at Xp22 locus Radiation induced g - banded metaphase spreads showing various chromosomal rearrangements involved at Xq22 locus A - X,2 translocation B - X,3 translocation C,F - X,5 translocation D - X, 7 translocation E - X,9 translocation G - Xp; 3q translocation 99 TITTLE OF FIGURE FIG.1 GENE MAPPING CHROMOSOME FIG.2 GENE MAPPING CHROMOSOME X 86 FIG.3-5 FIG.6-10 Densitometric profiles of the normal and deleted X chromosome in a patient of primary amenorrhea A Normal X chromosome B Deleted X with break point Xp22 Densitometric profiles of the normal and deleted X chromosome No.7 seen in 46,XX gonadal dysgenesis A Normal No. 7 B Deleted 7 with the break point 7q Abbreviations : PA SA PRIMARY AMENORRHEA SECONDARY AMENORRHEA Pphs TFS GD PSEUDO VAGINAL PERINEOSCROTAL HYPOSPADIAS TESTIGULAR FEMINIZATION SYNDROME GONADAL DYSGENESIS vi

13 1. INTRODUCTION Primary Amenorrhea Amenorrhea or lack of menstrual cycles as the term denotes, is one of the prime causes for female infertility and can be either primary or secondary in nature. According to Palmer and Reichmann, (1976) one in every 674 females born with an X chromosome abnormality. The incidence however, is considered to be much higher (63.5%) in cases where ovarian failure is associated with primary amenorrhea (PA) (McDonough et al., 1977). World Health Organization (WHO 1992,pp 1-111) which accounted for 81 percent of female infertility, among which ovulatory disorders alone 25%. Primary amenorrhea can be clinically suspected when patient has lymphedema of the hands and feet, excessive skin at the nape of the neck, cardiac anomaly in newborns, short stature, and delayed puberty in girls (Turner, 1938). Puberty begins with gonadal maturation and increasing production of sex steroids and delayed if there are no secondary sexual characteristics by 13 years of age (Bratberg et al., 2006). PA is a common feature among subjects with Turner syndrome, XO - and XY-gonadal dysgenesis and testicular feminization syndrome (Acheimann et al., 2008). Primary and secondary amenorrhea is one of the major disorders of females and has a complex etiology. The main causes attributed to PA is genetic disorders but these genetic abnormalities due to single gene, multiple genes or part or whole of a chromosomal mutation (Peris, 1

14 1968; Rajangam and Nanjappa, 2007). Clinical features of Turner syndrome are due to complete or partial absence of an X chromosome. Genes which are essential for normal ovarian development located on both arms of the X-chromosome (Fitch et al., 1982). An abnormality in the number or structure of the chromosome results in a disturbance in the normal process of translation of genetic sex and ends with phenotypic sex (Jost, 1959, 1960 and 1972). For example, some patients, ovotestis is present in the form of both testicle and ovary (Sales et al., 2000 and Páll-Gergely, 2008). Intersex cases are having congenital abnormality in their gonads associated with GD (Chase et al., 2004). The main causes of PA are pituitary/hypothalamic disorders (27.8%); gonadal/ovarion disorders (50.4%); outflow tract (uterinevaginal) abnormalities (21.8%) Schorge et al., The incidence 92.84% had PA and 7% had SA (Lacramioara et al., 2011). It has been reported that the percentage of chromosomal abnormalities varies from 15.9% to 63.3% in patients with primary amenorrhea (Joseph and Thomas, 1982; Wong and Lam, 2005). Secondary Amenorrhea Females with secondary amenorrhea (SA) there are one or more bleeding episodes followed by a minimum of three months of amonorrhea Doody and Carr, 1990; Schorge et al., 2008 and it ranged 2

15 from 6 months to 7 years (Akbar Safai et al., 2012). Secondary amenorrhea is more often due to cortical or hypothalamic disturbances, polycystic ovary, resistant ovary syndrome, and premature ovarian failure all are due to hormonal cause (Lauritzen, 1983; Wong and Lam, 2005). Genetic cause of chromosomal abnormalities are divided into numerical, structural, and mosaicism which are single gene disorder or multifactorial. The numerical or structural chromosomal abnormalities have been reported at levels ranging from various parts of the world 3.8% to 44%; 4.42% (Jyothy et al., 2002). Several cytogenetic studies were carried out to understand in primary amenorrhea but most patients were selected for karyotyping on the basis of certain severe clinical features and not all of them were screened for karyotypic analysis. Hence the frequency of chromosomal abnormalities associated with primary amenorrhea has to be considered with certain reservation. Hence it is proposed to study several patients in Tamil Nadu population with varying X-chromosome anomalies to make a correlation between the karyotype of PA & SA. Until about two decades ago, most of the patients carrying an abnormality of the X chromosome could be described only with reference to the presence or absence of Barr body (Wilkins et al., 1954). Later studies employing non-banded, conventionally stained chromosome preparations have made it possible to distinguish various numerical and gross morphological differences in Turner patients. It was 3

16 in 1959 that Ford et al confirmed the presence of 45,X karyotype in a 14 year old girl with ovarian dysgenesis who was chromatin negative. With the advent of numerous banding procedures, it is now possible, not only to identify the various structural aberrations like isochromosomes, deletions and translocations involving either the X chromosome or autosomes but also to precisely define the regions involved in the breaks and exchanges. Although chromosomal aberrations were frequently reported in many cases of primary amenorrhea but not obvious (Lindsten, 1963). It can be assumed that certain chromosomal are probably instrumental in the etiology of this category of PA. Minute errors or rearrangements of chromosomal materials are, of course, far more difficult to demonstrate than gross chromosomal change. A normal chromosomal complement in a given patient with certain clinical defects does not always mean the chromosome changes have not occurred. A superficially normal chromosomal complement does not always imply a normal genome. In a number of congenital diseases in which no obvious chromosome abnormality has been detected by the application of routine cytogenetic techniques, it is most likely that specific genetic regions might have undergone certain modification. We must, therefore, grant the possibility that a variety of chromosome aberrations, not detectable by present day technique may be masked by a superficially normal karyotype. 4

17 So, We plan to study with gonadal dysgenesis having the chromosomal constitution of XX, XO and XO/XX to find out whether there exists an increased incidence of radiosensitivity in gonadal dysgenesis syndrome. Hence an attempts made to use sophisticated technique to analyse chromosome abnormalities. The application of newly developed leukocyte culture techniques for the high resolution banding of human chromosome and the combined application of computer assisted densitometric chromosome analysis systems on Q banded and G banded chromosome and semi automated densitometric system on Q banded chromosomes have not only the numerical chromosome anomalies but also minute structural anomalies like deletions and translocations possible (Caspersson et al., 1970; Yunis et al., 1971; Ladda et al., 1974). In 1983, Sutherland by applying his newly developed techniques showed a fragile site on the terminal region of the long arm of one of the X chromosomes in fragile X patients. It seems possible to bring out the hidden chromosomal anomalies in a genetically defective syndrome having a seemingly normal karyotype by the application of appropriate cytogenetic technique. Hence in our present study, we selected ionizing radiation technique as our tool to bring out the chromosomal weak spots, if any, hidden on any of the chromosomes of amenorrhea having a normal karyotype. 5

18 Radiation was selected as a tool because it was established by several workers that the chromosomal breaks induced by radiation are non-randomly distributed on the chromosomes of the normal individuals. A significantly increased number of breaks on the long arm of the G group chromosomes were observed (Ishihara et al., 1970). Caspersson et al, 1972 reported different sensitivity of different parts of one and the same chromosome. Holmberg and Jonasson observed a preferential location of breaks on the bands of weak fluorescence; Lee and Kamra, (1981), showed in their study of in vitro chromosomal radiosensitivity in human chromosomes of normal individuals, and showed that non-random involvement of chromosome 1, 7 and 12, and a significantly large number of breaks in the light band regions and also in the terminal segments. Hence, radiation to the chromosome may bring about the weak spots on them. Individuals with Turner syndrome are prone to gonadal (Bonadkar and Peisner, 1980) and extragonadal (Sandberg, 1980) tumours suggesting an inherent tendency for chromosome damage and a defective repair mechanism (Countryman et al., 1977). Streak gonads are ovarian tissues but look like stroma are elongated and whitish in color. In Swyer syndrome 20% to 30% of these gonadoblastoma were observed (Karimian et al., 2010). Investigations were carried out to examine the radiosensitivity of these patients by exposing their lymphocytes to x - rays -لا and then analyzing the dividing cells for 6

19 cytogenetic damage Sasaki et al., (1970); Lee (1981); Heras and Coco, (1986) and Kiuru et al., 2000 examined the frequency of chromosome aberrations among patients with different chromosomal make-up and also sex chromosomal. There exists disgreement amongst the investigators on the effect of radiation on the lymphocytes of patients with abnormal karyotypes. In this context, it was thought desirable to carry out a cytogenetic study on the irradiated lymphocytes of Turner subjects. An attempt is being made to correlate the clinical features with cytogenetics since there is an increasing demand in clinical medicine for the establishment of the cytogenetic characters of patients with sexual disorders. Tortora et al., 1977 have not only permitted a more precise description of structural abnormalities of human chromosomes but also made the detection of minute deletions and complicated translocations, possible to detect finer chromosomal changes associated with genetic disorder. Hence in our present study we selected ionizing radiation technique as our tool to bring out the chromosomal weak spot, if any, hidden on any of the chromosomes of gonadal dysgenesis (Muhlenstedt et al., 1979; Alex et al., 2012). 7

20 2. OBJECTIVE OF THE STUDY 1. To detect the incidence and the clinical nature of PA and SA associated syndrome. 2. To apply various classic and modern cytogenetic techniques for a thorough chromosome analysis and correlation study. 3. To apply radiation technology to bring out the inherent minute chromosome anomalies in so called normal karyotype. 4. To study the chromosomal sensitivity of XX, XO and XX/XO syndromes with special reference to gonadal dysgenesis. 8

21 3. REVIEW OF THE LITERATURE The volume of literature dealing with all those categories of PA is vast and hence it is proposed to restrict our review to the areas of our present study only hypergonadotropic hypogonadism, the main theme of our thesis. In that class also we will deal with only those aspects that were taken up for our studies and those that are related to those aspects. Those aspects are based on epidemiological studies, on PA and SA of females, which are Frequency of ovarian dysgenesis, Consanguinity, Familial incidence, Parental age, Birth order, Associated disorders, Incidence of congenital anomalies among probands sibs, Maternal reproductive history, and Maternal antenatal medical history. Primary Amenorrhea Associated with Sex Chromosome Anomalies with Number and Structure In patients with number and size abnormalities of Barr body ( 1Mm), suggesting X chromosomal numerical and structural abnormalities (Gorduza et al., 2003; Lacramiora et al., 2011). Primary amenorrhea associated with sexual infantilism is caused by the majority of patients by sex chromosome anomalies it involve numerical variations and structural rearrangements. Aneuploidy results in XO or XXX conditions observed include isochromosomes, 9

22 partial deletions, X-autosome and X-X translocaions and rings, in addition to other types of anomalies. Hook, (1983) has estimated 25% have chromosome abnormalities (16% - 45, X; 3% - 45,X/46,XX; 1% - 45, X/46, XX; 4% - X structural abnormalities; 1% other). X mosaicism (25%-36%); X- Structural (8%); and 46,XY female (16%). Rajangam et al., 2007 and Vijalakshmi et al., 2010 observed 26% structural abnormalities; whereas 11.64% observed by Rajesekhar et al., Secondary amenorrhea the genetic in patients with increased gonodatropin level Brovik, (1984) has estimated 25% have chromosomal abnormalities. The ratio of numerical to structural was 22:9 observed by Jyothy, (2002); 9.9% Wong and Lam,(2005) and 5.3% Akbar Safai et al., Incidence of Ovarian Dysgenesis (Court Brown et al.,1964) estimated the frequency of chromatin negative female infants to be 0.37/1000 live female birth, a considerably lower frequency than is found for either, 47, XXY or 47, XXX. Turner syndrome occurs at a rate of 0.4 per 1000 live born females. The most common is 45.X constitution and the prevalence of 45, X is 1/1000 (Guidozzi et al., 1994). Other constitution found in which a second X chromosome is present but is structurally abnormal, and of these an isochromosome of the long arm of the X is the most common. In one 10

23 study, the relative proportions of 45, X; 45, X/46, XX; 45, X/46, XXi and 45, X/46,XX karyotypes among Turner syndrome patients were 66, 16, 9 and 9% respectively (Ferguson-Smith, 1965). The incidence of 45, X is said to be much higher in girls with short structure (Suri et al., 1995). Whereas spontaneously aborted conceptuses it is 4%. There fore a low incidence of 45, X individuals at birth is due to the high prenatal death of XO zygotes (Vijalakshmi et al., 2010). Frequency of Total Chromosomal Aberrations in PA and SA Several investigators have examined the frequency of abnormal karyotypes associated with PA and SA (Jacobs et al., 1961; Philip et al., 1965; Sarto et al., 1974; Van Niekerk et al., 1978; Rita sarkar, (1978); Ko, (1982); Opitz et al., 1983; Mulye et al., 1983; Anglani et al., 1984; Chuang et al., 1985; Ghalib et al., 1988; Ten et al., 1990; Park and Kang, 1999). Before this pioneering work evidences have mounted in literature to show that a number of sex chromosomal abnormalities in women are associated with PA. Wide ranges ( %) of frequency of chromosomal anomalies are associated with PA and SA. These reported variations in the frequency of total chromosome anomalies may be due to the variation in the methods of ascertainment of patients, selected for karyotyping. 11

24 Studies On X Chromosomal Abnormalities Mixed gonadal dysgenesis of 27 year old female showed X0/Xq - mosaicism chromosomal complement (Takayasu et al.,1970). A 22 year old woman with primary amenorrhea with three cell lines identified in peripheral blood lymphocytes 45, X (30%); 46, XX (60%) and 46, X tandom dic (Y) (10%) was reported by Herva et al., Grass et al., 1981 reported a 16 yr. old female patient with primary amenorrhea, with the region of q22 through q24 of the abnormal X is inserted into region p11 of the same chromosome. Robertoson et al., 1982 reported a 17 yr. old patient with PA had an isodicentric X chromosome 46, Xi dic(x) (pter - qter::qter - pter) of only one functional centromere. Joseph and Thomas, (1982) reported a patient with primary amenorrhea who had 46, X inv. (Y). Petit et al., 1982 reported a 16 yr. old girl with a 45, X/46, X idic(x) (pter - 21:: q21 - pter) karyotype. Zuffardi et al., 1983 reported a patient with multiple chromosomal aberrations and abnormalities involving 7 and 4 similar to those described in patients with Ataxia telangiectasia. Gall et al., 1984 reported a female patient with a chromosome of one X and a marker chromosome 21 with deleted short arm. Fryns, (1988) 45,X/46, del(x) p ter---- p21.3)/46,x,idic(x) (qter---- p21.3::p qter) was fertile. Radha Krishna et al., 1991 reported 5 year boy with infantile uterus and unilateral gonadal dysgenesis with both testes and fallopian tube on the right side the chromosomal compliment showed both 45,X/46,XX inverted Y (p11.2q11.23) cell line 12

25 (98:2). Asha et al., 1997 reported a girl and her karyotype was considered to be 46, X,del(Xq13 q22)inv(x)(q23-q27). Xiao Zhao et al., 2008 she observed three novel abnormal karyotype 46,X,t(X,1)(Q22;p34); t(11;14)(q23;q32) and Xp11.2 and Xq22 associated with primary amenorrhea. Margaret et al., (2010) reported a patient with 45,X/47,X,i(X)(q10)/46,X,i(X)(q10) isochromosome Xq in mosaic Turner. Vijayalakshmi et al., 2010 reported a rare chromosome compliment of a patient with 45,X (80%)/46,X,inv(Xq)(q21-22)(20%). Kalavathy et al., 2010 reported a female with a structural abnormality 45,X/46,X,idic(X)(q24). Vijaya Laxmi et al., 2012 observed a 14 year old girl showing 46,XX, 15ps+,i(Xq) with GD. Rajasekhar et al., 2013 he observed a rare case of 45/46,XX,t(12:14). Bhavani et al., 2014 observed in two patients 45,X/46,X,i(X)(q10). Thus several X chromosome variants in pure lines and mosaic lines were found to be associated with primary amenorrhea. Among the X chromosome variants 45, X is most frequently associated with primary amenorrhea. Fewer 45, X individuals will be detected if primary amenorrhea is the presenting complaint that if the short stature or various somatic anomalies are the presenting complaints. Phenotype - Karyotype Correlations The phenotype-karyotype correlation in patients with an X-chromosome rearrangement is very complex problem deserving 13

26 special attention because of the inactivation mechanism of that chromosome. Nevertheless attempts have been made to locate genetic determinants influencing stature, ovarian development and some somatic features of sex chromosome monosomy from studies of structural and numerical anomalies of the X-chromosome. Further the report on deletions of the X chromosome with different break points is of great interest for several open questions such as: 1) occurrence of specific symptoms due to the loss of definite segments; 2) prognosis of infertility; 3) inactivation behaviour; and 4) structural rearrangements during evolution. Phenotypic Features Associated with 45, X Turner Syndrome Most patients with X chromosome homogenous,45,x presented clinical features of Turner syndrome, have oedema at birth or show physical evidence of in utero lymphoedema, streak gonads seen hall marks of the condition (Sybert, 2001). The phenotype of this syndrome as a prototype of the disorder is regularly characterized by short stature and a variable number of clinical anomalies such as pigmented nevi, a short-webbed neck, a high arched palate, epicanthal folds, short fourth 14

27 metacarpals, cubitus valgus, infantile external and internal genitalia, coarctation of aorta and certain other skeletal anomalies. Some Turners affected with 17-60% cardiac anomaly (German, 1971; Sybert and McCauley, 2004). Kaffe, (1983) menstruation and fertility occur very rarely in 45,X patients. In the 45,X condition the embryonic ovaries appear to develop normally for about the first 3 months of fetal life. Subsequently it disappears and at birth there may be few, or perhaps no germ cells left. At puberty the ovarian dysgenesis resulting from this karyotype almost inevitably leads to primary amenorrhea (Jacobs et al., 1961; Rao et al., 1997; Chen et al., 2009). However, the phenotypic expression varies as evidence by observations that (a) an occasional 45, X individuals has oocytes, and (b) differences exist with respect to stature and the number of somatic anomalies present in a given 45, X individuals. Complete Turner syndrome is due to monosomy of loci in the short arm of the X chromosome. Streak gonads are almost invariably present in 45, X patients; however, 3% occasionally menstruate and 5% show some breast development. The rare cases of apparently normal secondary sexual characters in the presence of 45, X karyotypes are probably related to a hidden mosaic pattern (Ferguson-Smith, 1965). 15

28 Phenotypic Features Associated with 45, X/46, XX Karyotype Like all mosaics, the eventual phenotype depends very much on the relative proportions of cells of the two cell lines, either in the body as a whole, or in individual tissues. Accordingly, any phenotype may be found in a spectrum between normal female and full blown Turner phenotype (Rajesekhar et al., 2013). Simpson, 1975 one might expect fewer anomalies in 45, X/46, XX individuals than in 45, X individuals. Twelve percent of 45,X/46,XX, individuals menstruate, compared to only 3% of 45,X individuals. Eighteen percent undergo breast development, compared to 5% of 45, X individuals. The mean adult height is greater in mosaic patients than in non-mosaic patients, and better prognosis (Simpson, 1975; Koeberl, 1995). Thus, the 45, X/46, XX mosaics cannot be considered as a single entity because it showed a great variation in the clinical features ranging from mild forms to severe forms. Rita Sarkar and Marimuthu, (1983) observed 32 cases of 45, X and 50 of 45, X/46, XX Turner mosaics having different ratios of those two mosaic cell lines, but the severity of the syndrome in Turner mosaics was high with the relative increase in the abnormal cell line population. 16

29 The monosomy mosaicism, and the variable clinical features of patients was explicated by theories of Fraccare, (1977) and ovarian failure were studied by Ogata (2001) and Wieacker, (2009). Phenotypic Features Associated with 46, X, r(x) and 45, X/46,X,r(X) Karyotype Patients with a ring X chromosome mosaicism have the clinical signs of the Turner syndrome, 45, X, due to the partial deletion of short arm of an X chromosome. A mosaicism involving the ring X chromosome associated with gonadal dysgenesis. There is wide variation in the frequency with which the abnormalities associated with Turner syndrome are found in these patients, 100% short stature and some are fertile. Because of their mosaicism the ring X patients might be expected to have phenotype intermediate between that of the 45, X patients and the mosaic 45, X/46,XX (Berkovitiz et al., 1983). As with instances of deletions and translocations, phenotypic variations among ring X cases reflect the size of the deleted portions on the short and long arm (Alter, 1969; Rajasekhar et al., 2013). Phenotypic Features Associated with 46 Xi (Xq) Karyotype Patients have streak gonads, short stature and Turner stigmata 15% (Hook and Warburton, 1983); 7-17% (Sybert et al., 2004; Catoviv, 2005). Almost all individuals have primary amenorrhea (Therman, 1991). However, some 46,Xi(Xq) individuals menstruate Stafford et al., 17

30 1973. The mean height of this is similar to that of 45, X individuals. Exactly like TS this patients have partially developed nipples, mental retardation, low posterior hair line, webbed neck, and hypoplastic nails, and rate is 12% (Etem Akbas et al., 2012). Phenotypic Features Associated with 45,X/46.Xi(Xq) Karyotype A considerable variability of phenotypical manifestation occurs due to the existence of XO cells in some patients (Davidenkova et al., 1978). They tried to correlate the clinical pictures of 11 patients with 46 Xi(Xq) and 24 patients with 45,X/46,Xi(Xq) showed no difference. Similarly no difference in the phenotype of patients with different proportion of XO cells and cells with abnormal X chromosome in mosaicism was detected. In previous studies this karyotype observed was 8% (Lacramiara et al., 2011). Phenotypic Features Associated with 45,X/47, XXX Karyotype Spontaneous menarche is much more likely in subjects with mosaicism for an XXX cell line. This type of chromosomal constitution is less common than 45, X/46,XX. It is uncertain whether 45,X/47,XXX individuals have fewer anomalies than 45, X individuals because of rarity of these cases. It has been estimated that in 45,X/47,XXX individuals, non-disjunction probably occurred in the zygotes. Facial abnormality of non specific craniofacial dysmorphism, (downs - slanting palpebral fissures, epicanthus) poor development of secondary sexual 18

31 characters with normal intiligence were observed (Lacramiora et al., 2011). Mental retardation seen (7/39, 18%), tubercular meningitis and pachygyria of the frontal lobe and cerebellar atrophy (Terao, 1996). Phenotypic Features Associated with 45,X/46,XY Karyotype This is also an another variants of Turner syndrome found in phenotypic females with gonadal dysgenesis caused and exhibits a wide variety of phenotypes ranging from normal males, ambiguous, to infertile females (German, 1971). Simpson, (1976) grouped 45,X/46, XY into three categories, namely, individuals with (a) unambiguous female external genitalia, (b) ambiguous external genitalia, and (c) almost normal male external genitalia. The testis determining genes are lost in female karyotype of 45,X/46,XY (Oster et al., 1989). The XY gonadal constitution in females was found in 18-20% of the patients with PA (Anupam et al., 2004). In such mosaic cases, the imbalance caused by the sex chromosome abnormalities appears to disturb certain stages of embryonal development the normal differentiation of the gonadal element leading to the degeneration or maldevelopment of the gonad (Rey and Grinspon, 2011). Müllerian duct develops in females and degenerates in males. The reproductive system of females having two embryological segments. The one is called urogenital sinus and the other one is called Müllerian ducts (Yasmin, 2011). Both male and female Müllerian ducts are present in the embryo. The fallopian tubes are formed only in the females. These tubes are main reproductive 19

32 organ. Males these tubes are absent, instead of this they develop the adjoining duct as a main reproductive organ. The sex based differences in the contributions of the Müllerian ducts to reproductive organs is based on the presence, and degree of presence, of anti Mullerian hormone (Hashimoto, 2011; Ball et al., 2011). a) Unambiguous female external genitalia About 5% of patients with gonadal dysgenesis are with unambiguous external genitalia and have 45,X cells and cells containing a Y chromosome. These individuals may have the Turner stigmata and thus be clinically indistinguishable from 45,X individuals. b) Ambiguous genitalia In 1964, Sohval reported several individuals who had one streak gonad and one dysenteric testis and the term mixed gonadal dysgenesis was suggested. The clitoral enlargement or ambiguous external genitalia occurs but these female external genitalia have Mullerian derivatives. Occasionally the uterus is rudimentary or a fallopian tube fails to develop on the side on which a testis is present. On the side containing streak gonad a fallopian tube is invariably present. This formerly called intersex condition (Lambert, 2010). 20

33 c) Almost Normal Male Genitalia Occasionally 45, X/46, XY mosaicism is detected in individuals with almost normal male external genitalia. These males having normal penis and bilateral testis but no spermatogenesis and no uterus was present which is a deviation from the group of 45, X/46,XY individuals described above. It would be interesting to know whether that these normal male external genitalia developed tumours as often as individuals with ambiguous or female external genitalia. Thus the Y-gonadal dysgenesis which was first described by Sohval, (1964); Oster et al., 1989, consisting of a 45, XO line and a 46,XY male line was 46,XY male line with Y were present as the determinant for testis-differentiation, occasionally the Y is structurally abnormal. The Y in significant percentage of the patient with PA was 3.3% % Safaei et al., Phenotypic Features Associated with XY Females Female with XY sex chromosome constitution indistinguishable from that of a normal male has been variously discussed in the literature such as true agonadisum, pseudohermaphroditism with multiple congenital anomalies. XY females with absence of gonads, vestigial pelvic organs agonadism, mixed gonadal dysgenesis and gonadal dysgenesis with androgenic manifestations. These XY females with gonadal dysgenesis have streak gonads, but look like normal females, 21

34 absence of secondary sexual characters and menstruation. They do not have the somatic stigmata of Turner Syndrome. A high incidence of germ cell tumors in streak gonads patients with the XY karyotype and in Swyer syndrome. A familial occurrence of XY females with pure gonadal dysgenesis also occurs (Coutin et al., 1996; Aneulekha et al., 2013). (a) Testicular Feminization Syndrome This syndrome is characterized by female breast, completely female external genitalia, but without a uterus maldeveloped male gonad seen. In 60% of the testes in the inguinal region 19% in labia majora and 21% in abdomen were studied gonadal disgenesis cases (Frauser, 1963; Lacromioara et al.,2010). The vagina ends blindly, short in length or absent. Sparse pubic and axillary hair. The patients with this syndrome are phenotypically females but genotypically males. Statural growth and body proportions are normal with long arms, and legs, hands and large feet. It is a well defined form of male pseudohermaphroditism. This syndrome may affect sisters, aunts and relatives of the maternal line. Hauser, (1963) has commented that the condition is either transmitted by a sex-linked recessive gene, but more probably dominant (Hamerton, 1969) although an autosomal gene cannot be rule out in all cases. By using specific probe for FISH study SRY/CEPX, which 22

35 revealed complete absence of SRY gene in the patient cell line (Lim, 1998). b) Pseudovaginal Perineoscrotal Hypospadias Hypospadias is a birth congenital defect. Individuals a puberty they undergo virilisation, phallic enlargement, increased facial hair, muscular hypertrophy voice deepening and no breast development. The opening of the urethra on the under side, affects 4 in 1000 and hereditary Kraft et al., 2010; Elder, (2007). The external genitalia consist of phallus that resembles a clitoris more than a penis, a perinea or face that resembles a vaginea.ppsh patients under the category of non-familial perineal hypospadias. PPSH is an autosomal recessive trait (Simpson et al., 1971; Opitz et al., 1972). c) Pure Gonadal Dysgenesis Pure gonadal dysgenesis (PGD) has been used to distinguish a group of patients from gonadal dysgenesis related to Turner syndrome. In the latter a disdinct chromosomal aberrations is present either into two types according to the Sex chromosome complement XX (Swyer, 1955) and XY. Through each occurs rarely, XX or XY gonadal dysgenesis tend to affect several members of a family (autosomal) more frequently than does gonadal dysgenesis associated with 45, X or X-rearrangements. Epibulbar dermoid (eye disorder) a new type of syndrome with XX gonadal dysgenesis (Quayle, 1991). 23

36 a) XX-Gonadal Dysgenesis The XX type is characterized by primary amenorrhea, female phenotype, normal or eunuchoidal proportions underdevelopoment of the breast and secondary sexual characteristics, hypoplastic external genitalia and streak gonads. Most individuals with X gonadal dysgenesis are normal in stature; about 15% are less than 150 cm tall. Testicular feminization syndrome and the Turner syndrome present some clinical features in common with XX gonadal dysgenesis. Simpson et al., 1971; Boczkowsi, (1970); Maximilian et al., 1970; Hamet et al., 1973 reported a family with four 46, XX siblings affected by the pure gonadal dysgenesis syndrome. The three sisters and the proband herself present the same clinical history, primary amenorrhea, absence of breast development, scanty pubic and axillay hair and no functional ovaries present due to low estrogen and high FSH-LH. The 46,XX females who have normal ovarian function and secondary sexual characteristics but show some physical stigmata, such as webbing of the neck, resembling those of Turner syndrome may represent undetected mosaicism, of 45, X cell line. 46, XY Gonadal Dysgenesis These Individuals have sexual infantilism, amenorrhea and streak gonads (Achermann et al., 2008). Almost all individuals have been normal in stature and undetected, 45, X cells may have been 24

37 present in individuals with short stature (De Grouchy and Jobi, 1965). Somatic anomalies absent but common somatic anomalies are cubitus valllgus and pigmented nevi, although neither occurs in more than one fourth of the individuals with gonadal dysgenesis. Sometimes the clitoris is enlarged. The gonads are differ from those of patients with monosomy but X-which forms gonadoblastomas. The explanation for the increased predisposition to tumors in these patients is unknown. However, at least two possibilities might be offered. First, the mutant gene might cause aneupolidy. The aneuploidy might normally be limited to sex chromosomes of germ cells but it sometimes could involve other chromosomes of germ cells and thus lead to a neoplastic alone, that is adenoblastoma in the dysgenetic gonads Sybert, (2004); Grarholt (2000). Phenotypic Features Associated With Xp-Karyotype The phenotype associated with Xp- is difficult to determine because of the frequency of associated mosaicism, with deletion of this short arm (Xp-) usually results in GD, short stature ( Kalousek et al., 1979; Simpson,1979). Women with Xp deletions have given birth Fraccaro et al., 1977; Nurmi et al., 1981 and woman of extremely short stature (146 cm) who had inherited partial Xp deletion with the break point at p22 was presented by Bartsch-Sandhoff et al., (1976). 25

38 Petrinelli et al.,1978 described break point localizedin X(p22) d with growth retardation, absence of labia and vaigna, hypoplastic uterus, small left gonad and absence of right gonad, but no Turner stigmata. The distal part of the Xp has no influence on an individuals phenotype (Fraccaro, 1975; Hoo, 1975), however there are also evidence indicating that genes controlling the growth of an individual are precisely in that segment, but loss of a larger segment, which includes the X(p21) band, results short stature and ovarian insufficiency, as well as the well known somatic characteristics of Turner syndrome (Giraud et al.,1974; Ogata, 2001; Wieacker, 2009). Goldman et al., 1982 suggested that there must be an accessory factors necessary for the maintenance of oocytes situated on the short arm of the X chromosome. Since women heterozygous for deficiency of the (p21 - pter) region of the X chromosome are fertile (Fraccaro et al., 1977). These factors are thought to be between X(p21) and the centromere. Interstitial Deletion The structural X chromosome aberration with interstitial and presumptive terminal deletion of short arm found in a case of Turner syndrome associated with a 45, X/46, X del X (p22) (p22 :: p11-1qter) was reported (Silesen et al., 1976). 26

39 Herva et al., 1979 they reported that short stature was a phenotypic symptoms or signs. He suggested that a gene or genes controlling stature is located in band X(p21) immediately adjacent to this band. He further suggested since the absence of this region does not cause streak gonads it does not contain genes controlling the formation of the ovaries. The same observation was found in her mother and in one of her 4 sisters, all phenotypically normal. The report appeared to be the first example of a heritable chromosome deletion compatible with a normal phenotype and reproduction. Pericentric Inversion A tall patient with eunuchoid appearance (181 cm) was reported by Nikolis and Stolevic (1978) and he observed that pericentric inversion of the X chromosome: 46, X inv (X) (p22q24), from G and R banding was responsible. The deficiency of Xq produce gonadal dysgenesis but neither short stature nor other somatic features like Turner stigmata (Ferguson- Smith, 1965; Sybert, 1984; Vijayalakshmi et al., 2010). Most patients with a simple X long arm deletion have streak gonads but menstruation and breast development occur than in 45, X (Simpson, 1975). Jacobs,(1969) suggested that short IV metacarpal and pigmented nevi might occur in individuals with deletions of the long arm. 27

40 About half the 46, X del (Xq) patients are taller than 152 cm but short stature have an undetected mosaicism. Whereas normal stature having a deletion of almost all of Xq (Baughman et al., 1968) as well as individuals with short stature having only a smaller deletions of Xq (Hecht et al., 1970), showing that the determinant of stature is not associated with the long arm of X. Therman and Patau, (1974) concluded that distal deletion of Xq amounting to about 38% causes gonadal dysgenesis only, whereas deletion of more than 60% of Xq gives a number of somatic symptoms of Turner syndrome (our case come under the category only). According to Hoo s hypothesis on X chromosome evolution (1975), the Xq distal part represents the original segment responsible for gametogenesis, which must be present on both x chromosomes for the development of functional ovaries. Bronkovic et al., 1979 reported a 19 yr. old female with the break point 46, X del (X) (q21). The patient presented with normal phenotype and secondary amenorrhea, infantile external and internal genitalia. It has recently become apparent that the X chromosome contains factors necessary for normal fertility on the long arm. These factors have been localized by Forabosco et al., 1979 to an area between Xq and Xq This area must remain intact if fertility is to be maintained, and it also appeared to be functional even when separated 28

41 from the rest of the X chromosome in the form of balanced X-autosome translocations Therman and Susman (1990). Goldman et al., 1982 stated that a region around X(q21) is sufficiently close to the centromere on the long arm is involved in the maintenance of ova. Wyss et al., 1982 on the other hand proposed a localization of genes determining gonadal development on the distal part of Xq(Xq26-q28). Kaiser et al., 1984 reported clinical and cytogenetic data from a 20 year old female with del X(q24 - ter). She was noted by all normal clinical features except small uterus. In their conclusion they stated that the loss of tiny terminal segment distal to q26 is possible without affecting body growth and with only minor gonadal dysfunction which allows fertility (Sybert, 2001). Radiosensitivity of the chromosomal syndrome Aurias et al.,1980 have reported from their study of Ataxia telengiectasia patients and 6 of their relatives. Thus it is clear that hot spots for the breakage of the chromosome do occur on specific chromosome, in clinically normal people, patients with normal karyotypes and patients with chromosomal instability syndrome. 29

42 Radiation induced chromosomal break points in normal human chromosome Holmberg and Jonnasson, (1973) have shown that X-ray induced breakage points wee non-randomly distributed according to the mitotic length of the chromosome and break points non-randomly (Seabrights, 1973; Dubos et al., 1978; Lee and Kamra, 1981; Kucerova et al., 1976). From these studies it is clear that there exists a strong agreement among the workers (1) that almost all the break points are located on the negative band region with respect to G band (2) there exists a disagreement with respect to the correlation between the length of the chromosomes and frequency of break points, showing a non random distribution of break points on the chromosomes. It has been known that although certain type of chromosomal aberrations both numerical and structural occur in somatic cells as well as in germinal cells at a low incidence in nature, those aberrations are more frequent upon exposure to ionizing radiation (Aurias et al.,1980). Such induced aberrations are of prime importance on account of their genetic hazards and their significance has been recognized to certain extent, as a subject of both biological and medical interest. With special reference to their association with certain congenital disorders and their possible role in the causation of neoplasia. 30

43 The association between chromosomal congenital anomalies and neoplasia is well documented from the increased incidence of leukemia in Down syndrome, retinoblastoma in the 13q- syndrome, and gonadoblastoma, seminoma or dysgerinoma in patients with the karyotype of 45,X/ 46,XY mosaicism, Wilm s tumor in 11q13 deletion syndrome, breast tumor in Klinefelter syndrome (Miller 1963 and 1966). Further, it has been shown that chromosomel instability syndrome such as Bloom syndrome, xeroderma pigmentosa, Ataxia talenjiectasia and Fanconi anemia leading to retinoblastoma. Several hereditary disorders including Immuno deficiency syndrome, Nijmegen breakage syndrome were also associated with an elevated risk of cancer (Taalman et al., 1983; Alex George et al., 2012). Thus, It has been shown that several chromosomal syndromes are predisposed to the development of cancer. Further, the chromosomes of that syndrome have been shown to have greater radiosensitivity than the syndrome having normal karyotypes. Peripheral blood culture obtained from 49 individuals with various types of chromosomal anomalies (21 trisomy and their variants, 18 trisomy and their variants D trisomy and their variants, B p deletion, 45,X and its variants) and 34 subjects from normal karyotypes were irradiated and the frequency and the nature of chromosomal aberrations were estimated (Sasaki,1970). 31

44 They concluded that the chromosomal radiosensitivity is significantly higher in cells which are trisomic for the whole or a part of a chromosome than in cells with a normal karyotype and that the radiosensitivity of the cells with balanced type of anomalies and the monosomic cells is found to be the same level of sensitivity as that of the normal karyotype (Caspersson, 1970). In general chromosomes of primary amenorrhea with gonadal dysgenesis are more sensitive to the radiation induced genetic damages. It is interesting to note that more number of chromosomes (8, 21, X, 20 and 22) showed a hypersensitivity to radiation in gonadal dysgenesis group than in any other amenorrhea group. Further, chromosome 7 and X showed a siginificantly high number of breaks at 7q22 and at Xq22 respectively. Out of a total number of 167 breaks on No.7 and 178 breaks on X, 67 breaks were on 7q22 and 59 where on Xq22. The mechanism associated with X: 18 tanslocation describe a pattern of inheritance, where break points and translocations of the Xq22.3; 18q 23 regions have resulted in variable fertility (Attila Szvetko et al., 2012). 32

45 4. NEED OF THE STUDY Treatment of amenorrhea depends on the cause. Primary amenorrhea often requires no treatment, but it s always important to discover the cause of the problem in any case. Not all conditions can be treated, but any underlying condition that is treatable should be treated. Hormonal imbalance is the problem, progesterone for one to two weeks every month or two may correct the problem. a) Hormone replacement therapy may also be effective b) Polycystic ovary syndrome (secondary amenorrhea), birth control pills are often prescribed. c) A pituitary tumor is treated with bromocriptine, a drug that reduces certain hormone (prolactin) secretions. d) Clomiphene can be administered for follicle formation. e) In very rare cases surgery may be needed for women with ovarian or uterine cysts, or with anatomical deformities etc. If the amenorrhea is caused by one of the following conditions, it is unlikely that the amenorrhea can be corrected by any method. Congenital abnormalities of the genital system, Gonadal dysgenesis,turner s syndrome (XO), Testicular feminization Syndrome, True hermaphroditism, cystic fibrosis, craniopharyngioma, Prader-Willi syndrome. If the amenorrhea cannot be corrected, it is sometimes possible to create a pseudomenstruation with medications to help the young women feel like her friends or family. 33

46 5. MATERIALS AND METHODS The study consists of 221 women with primary amenorrhea and secondary amenorrhea cases that were referred for cytogenetic investigations from Chennai city hospitals, most of the samples were collected from the genetic out-patient unit at the Government hospital at kasthurba Gandhi Hospital Chennai and from various parts of Tamil Nadu. Out of those 221 cases, 128 cases were confirmed of having ovarian dysgenesis and the rest having normal ovaries. Of the 221 patients, 201 were subjects with primary amenorrhea, 20 cases with secondary amenorrhea. Detailed clinical examinations of each patient were done carefully, and recorded with the help of physicians. Clinical pictures were routinely taken. Ultrasonogram pictures and X-ray photographs were taken wherever necessary. Secondary sexual charecteristics and hormonal conditions were recorded serum luteinizing hormone (LH) and follicular stimulating hormone (FSH) concentration data were analysed from hospital record. Epidemiological study of informations regarding parental age, parental consanguinity, familial incidence of the anomaly, congenital anomalies among the sibs of probands, reproductive capabilities of both male and female siblings, maternal teratogenicity, maternal reproductive 34

47 history and the various associated disorders of probands were recorded for genetic and pedigree analysis. All the above mentioned informations and chromosome constitution of the probands were coded and fed into a computer (IBM PC compatible). Various tables were prepared from the coded data and standard statistical techniques were used. Cytogenetic Methods Sex Chromatin Study Sex chromatin analysis on buccal smears stained with thionin using the method of Klinger and Ludwig (1957), was carried out for all the patients and the frequency of Barr body was recorded. Lymphocyte Culture Method Chromosome preparations from the lymphocyte cultures for karyotypic analysis were made according to the modified method of Hungerford (1965). Preparation of Culture Medium The culture medium used was McCoy s 5 a medium (GIBCO). A stock (10X) solution of the medium was prepared and filtered through Seitz filter. From this stock soultuion, 30 ml was added to 255 ml sterile distilled water.15 ml of sterile sodium bicarbonate was added to adjust 35

48 ph to The antibiotics added was streptopenicillin. This was stored at 20 c. Culture Technique About 2.0 ml of venous blood was drawn into a sterile heparinized syringe and inoculations were made into vials containing 5.0 ml of culture medium (McCoy s 5a), 1.0 ml of human AB serum and 0.2ml of phytohaemoagglutinin (a crude extract from red kidney bean prepared in our laboratory. Cultures were incubated for a period of 69 ½ -71 hrs at 37 C and were shaken periodically, and CO² was released from the vials once in a day. After incubation at 37 C for 69 ½ - 71 hrs, the cultures were arrested by adding 0.001% colchicine (Colchicine volume adjusted to get a final concentration 0.1 μg/ml culture medium). The cultures were further incubated for 30 min at 37 C and at the end of the incubation period they were centrifuged at 800 rpm for 5 to 6 min. After removing the supernatant, 8.0 ml of pre-warmed (37 C) M potassium chloride solution (hypotonic) was added to the cell button and thoroughly mixed. It was incubated for 6 minutes at 37 C and followed by 5 to 6 minutes of centrifugation (800 rpm). After removal of supernatant, the cells were fixed in freshly prepared fixative (3 parts methanol: 1 part glacial acetic acid). 2 to 3 changes of the fixative were given at 1 to 2 hours interval, to get a colorless suspension of the fixed 36

49 cells. After the final wash the cell button was suspended in a minimum quantity of fixative (0.5 to 0.7ml). A drop of the suspension was placed on a previously cleaned cold slide and gently blown to obtain spread with nonoverlapping chromosomes. It was immediately dried over a slide warmer kept at 40 C. If the cell density was poor or too dense on the slide, then the concentration of the suspended cells was suitably adjusted by addition or by removal of the fixative. Staining and Banding Techniques Slides were stained routinely in Giemsa. Giemsa banding was carried out adopting the method of Marimuthu et al The air dried fresh slides (2 5 days old) were treated with percent trypsin in Ca ˉ and Mgˉ free saline solution for 4 10 sec. washed in distilled water and stained in giemsa stain for 3 minutes (Fisher Scientific Co.) (4 parts of stock Giemsa stain solution, 4 parts of 10 per cent Na2 HPO4 and 42 parts distilled water, ph ). The slides were washed in tap water for two minutes and air dried. Scoring and Microphotography A minimum of 50 technically good metaphase plates of each patient was analysed to detect the nature of the karyotype of the patient. If a patient showed mosaicism, a minimum of 75 cells were scored to estimate the ratio of each cell line. 37

50 Photomicrographs of selected metaphase plates were taken using Leica camera and DK5 ORWO high contrast document copying film. Karyotypes were made following the international system for human cytogenetic nomenclature (1978). Phenotypic Karyotypic Correlations Grouping of Mosaics for the Analysis of Phenotypic Variation (1) 45, XO; (2) 46,XX/45,XO; (3) 45, X/47,XXX; (4) 45,X/46,XX/47,XXX; (5) 46,XX/47,XXX; (6) 46,XXpˉ (partial and complete); (7) 46,XXqˉ (partial and complete); (8) 46,XiXq; (9) 45,X/46,XiXq; (10) 45,X/46,X,r(X); (11) XY females (TFS; XY GD; PPHS); (12) 45, X/46,Y; (13) 46, XX/46,XY and (14) 46, XX gonadal dyagenesis. Selection of Phenotypic Features 23 common characteristic features of Turner syndrome were selected and the number of features present in each patient was estimated. Check List 1. Height in cm 2. Epicanthal fold 3. High arched palate 4. Low set ear 5. Low posterior hair line 6. Short neck 7. Webbed neck 8. Shield chest 9. Pigmented nevi 10. Cubitus valgus 11. Short IV metacarpal 12. Heart abnormality 13. Hearing abnormality 14. Mental 38

51 retardation 15. Visual abnormality 16. Vertebral abnormality 17. Axillary hair 18. Pubic hair 19. Breast development 20. Vagina 21. Clitoris 22. Uterus 23. Ovaries. Radiosensitivity of Chromosomes of Primary Amenorrhea: The patients studied were classified into 3 groups consisting of 12 patients with 45, X complement (Turner syndrome), 10 patients with 45, XO/46, XX karyotype (Turner mosaics), 26 patients with 46,XX karyotype. A control group of 10 age matched healthy female individuals with proven fertility and with normal karyotype were also investigated simultaneously. Method of Irradiation About 1.0 ml of pheripheral blood samples were obtained from each patient and the control individuals. 0.5 ml of blood sample was inoculated into each of 2 culture vials containing 5.0 ml of growth medium (McCoy s 5a), 1.0 ml of human AB serum and 0.2 ml of phytohaemoaggultinin. Two vials per subject were irradiated at a dose of 300 rads [Caesa Gammatron machine (Siemens) available at Cancer Institute, Adyar, Madras] at a focus distance of 20 cm and a dose rate of R/min. Cultures were incubated for 69 ½ - 70 hrs at 37 C. Metaphase chromosome preparations were obtained and banded. 39

52 Scoring In all patients and controls, 50 metaphases were scored by direct microscopic analysis. Various structural abnormalities such as dicentrics, multicentrics, rings, translocations, breaks and deletions were recorded. The chromosomes involved and the location of the break points in each of these aberrations was also simultaneously noted. The total number of aberrations per cell was obtained by adding the number of chromosome deletions and twice the number of two hit aberrations (which includes rings and translocations) and dividing the sum by the number of cells examined. Method of estimation of radiosensitivity of individual chromosomes in syndrome associated with primary amenorrhea, Radiosensitivity of the individual chromosomes was computed as follows a/xn x 10³ a = number of chromosomal aberrations in one particular chromosome. n = total number of chromosomes studied = 2 x no. of metaphases/individuals x no. of individuals studied. X = the relative length of the chromosomes. (Birth defects, original articles service, 1972). 40

53 6. RESULTS Incidence and Clinical Nature of PA and SA and then Associated Syndrome All the 221 cases of primary and secondary amenorrhea were investigated for epidemiological features which included a variety of parameters, namely, (1) Frequency of ovarian dysgenesis (Tab IV), (2) Consanguinity (Tab V), (3) Familial incidence (Tab VI), (4) Parental age (Tab VII), (5) Birth order (Tab VIII), (6) Associated disorders (Tab IX), (7) Incidence of congenital anomalies among probands sibs (Tab X), (8) Maternal reproductive history (Tab XI), and (9) Maternal antenatal medical history (Tab XII). All these cases were karyotyped and were classified into 7 syndromic categories (Table III) and every parameter was analysed on the basis of these karyotypes. Frequency of Ovarian Dysgenesis Out of the 221 cases investigated, 128 cases (58%) were found to have ovarian dysgenesis. The data presented in the Table IV shows the frequency of ovarian dysgenesis in various categories of primary amenorrhea. The frequency of ovarian dysgenesis is significantly 41

54 higher (x 2 1df = ; p << 0.001) in categories II to VII (chromosomally abnormal group) than in category I (chromosomally normal group). Cytogenetic Analysis X-Chromatin Studies From Buccal Smears X - Chromatin studies from buccal smears of 32 cases bearing a 46, XX Karyotype revealed a range of 29 to 40 percent positive X- chromation. 25 individuals with a mosaic pattern of 45, X/46, XX, revealed a range of 4 to 18 per cent (I Group - 4-6%), (II Group - 6-9%), (III Group %), (IV Group %), (V Group %). The frequency of sex chromatin was 18% in a patients who exhibited 46, XX/47, XXX karyotype. At least 9 cells in this case were with two Barr bodies. One case with 45, X/46, XX/47, XXX karyotype were 7 per cent positive for single Barr body and negative for two Barr bodies. The sex chromatin is negative in a case with 45, X/47, XXX. All the 10 patients with 46, XY and 2 patients with 45, X/46, XY and all the 45, X0 patients were negative for sex chromatin. The X-chromatin masses were smaller than controls in 2 cases, one with 46, X(Xp - ) (complete deletion (Plate 11, D) and another with 46, X (Xq - ) (complete deletion) (Plate 13, D). The X-chromatin in all the XiXq patients revealed a range of percent positive sex chromatin and was larger than controls. Three 42

55 patients with 45, X/46, Xi (xq) showed a range of 7-11 per cent large X- chromatin body. Karyotypic analysis The results of karyotypic analysis carried out on 221 cases of primary and secondary amenorrhea are summarized in Table XIII. Sex chromosomes abnormalities were found in 92 cases (42%). These patients were grouped into 7 categories, having 16 types of karyotypes. Category I This group consists of 129 patients (58%) with a normal karyotype (46, XX). It was of great interest to observe multiple chromosomal aberrations in one patient with 46, XX karyotype in 12% of the metaphase plates. These aberrations were found to be localised to the following sites 2q33 (one cell), 5p15 (one cell), 11q23 (one cell) and Xq22 (3 cells) (Plate 20). Category II 20 Cases (9,17%) included in this category were found to have 45, X0 karyotype (Plate 2b). Category III 31 Patients (11.73%) with 45, X/46, XX karyotype and one each with the karyotype of 45, X/47, XXX (Plate 5b), 45, X/46, XX/47, XXX 43

56 and 46, XX/47, XXX are included in this category. How ever certain non-random chromosomal aberrations were noted in one of these patients which included a break at 6q27 (2 cells), a gap at 2q33 (2 cells) and monosomy of chromosome (3 cells) no. 20. Further, a 7, 14 translocation was observed in a single cell. The overall frequency of chromosomal aberrations was found to be 16% (Plate 18). Category IV 13 patients (5.98%) this includes 2 cases with 46, Xi (Xq), 3 with 46, X del X (p22) (partial short arm deletion) (Plate 9), 3 with 46, X del X (q 28) (Partial long arm deletion) (Plate 10), 3 with 46 del X (q- ) (complete long arm deletion) (Plate 14) and two with 46, X del X (P 11.1) (complete short arm deletion) (Plate 13). Category V This category 10 cases (4.58%) consists of 6 cases with 45, X/46, Xi (Xq) (Plate 7) and four cases with 45, X/46, X(rX) karyotype (Plate 5a). Category VI This category includes 13 (5.95%) cases of primary amenorrhea with a male 46, XY pattern (Plate 16). Of which 6 cases are of Testicular feminization syndrome (TFS), 4 cases of XY pure gonadal 44

57 dysgenesis and 3 cases of pseudovaginal perineoscrotal hypospadias syndrome (PPSH). Category VII This includes 6 (2.71%) patients of which 3 cases of 45,X/46,XY and 3 cases of 46,XX/46,XY are classified under this category. Phenotypic Karyotypic Correlations The phenotypic features of all the 221 cases of PA & SA were analysed and classified on the basis of their karyotypes and presented in Tables XV, XVI, XVII, XVIII and XIX. A check list for Turner syndrome features including all characters described in Materials and Methods was also presented. The data from the selected works were collected and presented in three tables (Tab I), PA (Tab II) and SA (Tab XIV). Phenotypic Features Associated with 46,XX Gonadal Dysgenesis Most individuals with 46, XX gonadal dysgenesis were normal in stature. Somatic features of the Turner stigmata were present in a few XX gonadal dysgenesis patients, but even in those patients there are fewer anomalies than usually present in 45, X (Table XV). Phenotypic Features Associated with 45,X0 and 45, X0/46, XX Karyotypes The frequency of phenotypic features observed in pure Turner syndrome and in all the 5 groups of Turner mosaics were presented in 45

58 Table XV. The patients with 45, X carried the clinical features common to those of Turner syndrome (Plate 1). The most constant features of pure Turner syndrome are shortness of stature and gonadal dysgenesis. The average hight of 45, X patients in the present investigation was cm. Rudimentary ovaries were presented in 53% and almost absent in 47% of the cases. Lack of breast development and absence of pubic hair was observed in 100% of cases. Uterus was rudimentary in 47% while absent in 53%. Mental retardation is not ordinarily considered to be a feature of the Turner syndrome but it was noted in about 5% of our cases. Twenty five cases of Turner mosaics manifested a phenotypic variations ranging from characteristic features of typical Turner syndrome to those of normal female phenotypes. Correlations were made with the percentage of affected cell lines (Table XV) (Plate 3). The frequency of somatic and as well as secondary sexual features occur less often in this group than 45, X group and increased with increase of abnormal cell population. Clinical Features Associated with 46, Xi (Xq) and 45, X/46,Xi (Xq) The phenotypic features of 2 cases of 46, Xi (Xq) and two groups each consisting 3 cases of 45, X/46, Xi(Xq) patients based on the percentage of affected cell lines were analysed and presented in Table XVI. It shows that the phenotype associated with individuals having the 46

59 karyotype of Xi(Xq) were indistinguishable from the mosaics (45, X/46, Xi(Xq)) (Plate 6). 45, X/46, Xi(Xq) patients invariably had streak-gonads, short stature and other Turner stigmata. Clinical Features Associated with 46, X del X(p22) There were 3 patients with this karyotype revealing short stature (average height cm), short neck and cubitus valgus and scanty pubic hair in common. However, in one patient the axillary hair was scanty and vagina was blunt (Plate 9) and in another patient the clitories was narrow. In both the cases, ovaries and uterus were normal (Table XVII). Clinical Features Associated with 46, X del X(q22) Three patients with this karyotype revealed no signs of abnormal clinical features except cubitus valgus, scanty pubic hair, poor breast development, absence of uterus and ovaries in one patient (Plate 10). The other 2 patients manifested only rudimentary uterus and ovaries. Case Reports In the present study, two patients, one with 46,XX and another with 45,X/46,XX chromosomal constitution showed multiple chromosomal aberrations. The detailed case histories of those two patients are given below. 47

60 A 46, XX Gonadal Dysgenesis Patient with Multiple Chromosomal Aberrations Case No. 132 The patient was 18 years old female. She was born to consanguineously married parents. Height 149 cm. She had increased carrying angle, saddle shaped nose, hypertelorism, low set ears, shield chest, absence of axillary and pubic hair and external genitalia was infantile (Plate 19). Scanning with ultrasonograph showed rudimentary ovaries and uterus Cytogenetics Chromosomal analysis revealed 46, XX pattern. However, 12% of the metaphase plates showed multiple chromosomal aberrations localised to the following sites 2q33 (in one cell), 5p15 (in one cell), 11q23 (in one cell) and Xq22 (in 3 cells) (Plate 20). A case of 45,X/46,XX Patients with Multiple Chromosomal Aberrations Case No. 142 A 18 yr. old girl came with the complaint of not attaining menarche. She is the third born of non-consanguineously married parents. First 2 siblings were dead in neonatal period. She has no history of prolonged fever, respiratory infection or any other chronic 48

61 illness. Her speech and other motor development were delayed in appearance. Phenotypic Features Height 143 cm, span 138, head circumferance 50 cm, mentally retarded with Iq of 34. She had hypotelorism with malformed pinna of the left ear, Sabacious cyst on the left side of the cheek, was appeared. Head was small ; neck was short with slight webbing. She had low set ear low posterior hair line cubitus valgus, flat feet and knock knees. Pigmented nevi was present in the left side of the body (Plate 17) examination of cardiovascular system revealed ejection systolic murmur in the pulmonary region suggestive of pulmonary stenosis. Axillary hair and pubic hair was poorly developed. Breast development absent, She had infantile external genitalia with enlarged clitoris. Ultrasonography revealed absence of uterus, left normal and right rudimentary ovaries. Cytogenetics Chromosome analyses of blood lymphocytes revealed 45, X/46, XX mosicism in the ratio of 56 : 44. The culture grown in folic acid MEM medium have shown two cells with 6q27 fragile site, two cells with a break at 2q33, one cell with 7, 14 translocation and monosomy of chromosome No.20 in 3 cells (Plate 18). 49

62 Case report This study further recorded 6 individual cases of primary amenorrhea each having the following rare karyotypes 46, X del X (p 11.1), 46, X del X (q11), 45, X (46, r(x), 45, X/47, XXX, 45, X/46, XX/47, XXX and 46, XX /47, XXX. The phenotypic features of all these 6 cases were shown in Table XXIV and the detailed case reports are given below. Case Report 134: 46, X del X(p11.1) A 22 yr. old girl of remarkably short stature, referred for cytogenetic investigation with the complaint of primary amenorrhea. She first born to non-consanguineous parents 2 younger brothers, 2 mentally retarded from mothers side and one congenital heart disease. At the time of birth she was blue for a short period. No history of any other chronic illness. She got spotting twice after administration of the hormones. On physical examination she was alert and answered question intelligently. Clinical Features Height 136 cm, weight 39.5 kg, upper segment 64 cm, arm span 151 cm, Her neck was short but no web. There was adduction deformity of terminal plangr of both middle fingers. Llow set ears, low posterior hair line and cubitus valgus were present. Breast development was in grade III. Axillary and pubic hair was scanty. The external genitalia were 50

63 infantile ultrasonography revealed normal sized uterus. But there was no structure morphogically resemling the ovaries were seen in the left adenexa (Plate 11, E) But the ovary was normally present in the right adnexa (Plate 11, F). Cytogenetics The chromosome analysis of peripheral blood lymphocytes revealed a deletion of the complete short arm 46, X Del X (Xp11.1-q28 :) (Plate 12). There was no indication for a chromosomal mosaic in more than 50 metaphases analysed. Case No. 154 : 46, X del X (q11) A 19 yr. old girl with the lack of secondary sexual characters, referred for cytogenetic investigation because of PA. Her growth velocity has been slow since birth. She is the second born of consanguineous parents of a family of 6 children. Her elder sister got married and has a daughter. Clinical Features Height 139 cm, weight 30 kg. She had high arched palate, cubitus valgus, and her breast development was in stage III. Axillary and pubic hair absent, Infantile external and internal genitalia, ovaries absent and hypoplastic uterus (Plate 13). 51

64 Cytogenetics Chromosome analysis of blood lymphocytes revealed a deletion of the complete long arm 46, X del X (Xq11-Xp22) (Plate 14). Case No. 105: 45, X /46, X(rx) The patient aged 19 years she was the second child of 34 year old father and 30 years old mother. She had an elder sister who attained menarche at the age of 13 years. There was no similar defect in the family. She had delayed milestones. The patient had low set ear, low posterior hair line, short neck and cubitus valgus. Secondary Sex Characters Breast development was poor. She had no axmillary and pubic hair, both labia majora and labia minora were underdeveloped, clitoris was small (Plate 4, A-C1), ultrasonic scan showed the rudimentary ovaries and uterus. Cytogenetics Chromosome analysis revealed an abnormal pattern of chromosomal complement of 45, X/46, X r(x) (Plate 5a) with ratio of 72:28 cell lines. Case No. 46: 45, X/47, XXX The girl was 17 year old, retarded growth non-consanguineously married parents. She was the 1st child of 48 year old father and 36 52

65 years old mother. Prenatal history was full term, normal, and post natal history was also normal. The family history revealed that there was no similar illness in the family. The patient was short a (136 cm), webbed neck, low set ear, low posterior hair line, cubitus valgus and had sparse axillary hair, uterus and ovaries are normal. Cytogenetics Chromosome analysis revealed 45, X/47, XXX mosaicism with ratio of 60:40 cell lines (Plate 5b). Case No. 60: 45, X/46, XX/47, XXX The patient aged 24 years was referred for not having attained menarche. She was the second child of non-consanguineous healthy parents. She had one younger sister, aged 19 years who had also not attained menarche and had a elder brother aged 32 who had an abnormal clinical features characteristics of Klinefelter syndrome. Both the patient, and her sister had abnormal clinical features such as, high arched palate, cubitus valgus, absence of axillary and pubic hair, flexion contracture of the little fingures, wide 1st interdigital clefts, short IV toe (Brachyphalangy) under developed labia majora and minora, and small clitoris, in common. Breast was poorly developed in patient and was totally absent in her sister (Plate 21). The patient had 53

66 rudimentary uterus and ovaries. Both the patient and her sister was slightly mentally retarded. Cytogenetics Chromosome pattern was 45, X/46, XX/47, XXX with a ratio of 30:50:20 in patient and 36:24:40 in her sister. The karyotype of the patients elder brother was 46, XX/47, XXY. Case No.62 : 46, XX/47, XXX The patient was 16 yr. old and she presented herself with the complaint of primary amenorrhea and mental retardation. She was the second child of 45 year old father and 37 year old mother. She had two younger sisters. The first sister aged 20 years attained menarche. The second sister was 12 years old and did not attain menarche. She had normal height and was moderately built with well developed secondary sex characters. Breast well developed. Axillary and puic hair were present. External genitalia were normal, ultra scan showed normal uterus and ovaries. Cytogenetics mosaicism. The karyotyping exhibited 75:25 ratio of 46, XX/47, XXX 54

67 The clinical features of XY females There were 13 cases of XY females, five of them were Testicular feminization syndrome, 4 of them were XY gonadal dysgenesis and 3 were pseudovaginal perineoscrotal hypospadis syndrome. The phenotypic features of all the cases of XY females were analysed and presented in Table XVIII. There were 6 cases with Testicular feminization syndrome were appeared to be normal by body habitus. The average height was 159 cm. The absence of axillary and pubic hair was noted in all 6 cases. External genitalia were those of normal female type. Vagina ended as a blind pouch, Mullerian duct derivatives were not noticed. The gonads (testes) were situated in the inguinal region in 4 cases and in one case in the abdominal region. Uterus was absent in all the 6 cases. Four patients with XY gonadal dysgenesis were tall with more or less eunuchoid body habitus, with an average height of cm. The breast were small and had scanty axillary and pubic hair. External genitalia was female type with normal vagina. Clitoris was infantile. Uterus and gonads were absent in all the three cases. There were two cases of pseudovaginal perineoscrotal hypospadis syndrome (PPSH) aged 20 years and 23 years with 160 cm and 159 cm in height respectively. The two cases studied clinically were characterized by eunuchoid body habitus, no breast development, 55

68 enlared clitoris like a phallus, labio-scrotal folding with testes, blind vagina with perineal hypospadias. Clinical features associated with 45, X/46, XY karyotype Two cases with XO/XY mosaicism manifested as phenotypic females with shortness of stature (average height 138 cm). The first case consisted of 26 per cent of 45, X cell lines and 74 per cent of XY cell line and the second case consited of 58% of 45, X cell line and 42% of XY cell line. The second was severely affected than the first case (Table XIX). Clinical features associated with 46, XX/46, XY karyotype: The clinical data on two cases with 46, XX/46, XY karyotype were shown in Table XXVI. The first case consisted of 74% of XX cells and, 26% of XY cells the second case consisted of 82% of XX cells and 18% of XY cells. The two patients with this karyotype had no extragenital anomalies, except for blind vagina and infantile clitoris. Uterus was rudimentary in the first case while normally present in the second case. The right ovary was streak and the left ovary was absent in both the cases. The results of radiosensitivity studies of the genomes of the syndromes associated with primary amenorrhea Radiosensitivity of the chromosomes of primary amenorrheas of various categories relative to the normal individuals, viz., gonadal 56

69 dysgenesis (46,XX), Turner (45, XO) and Turner mosaics (45, XO / 46, XX) was assessed by exposing the leukocyte in vitro to the gamma radiation. The leukocyte culture was established for each individual and exposed to 300 rads of radiation -لا at O hr. The cultures were maintained for 69 ½ hours. For each individual chromosomal aberrations were analysed in 50 well spread metaphases. In case of Turner mosaics the data was recorded separately for both 46, XX and 45, XO cell lines. Information was also recorded for the individual chromosomes. The chromosomal aberrations scored include translocations, dicentrics, tricentrics, multicentrics and rings and also deletions and breaks. Table XX shows the frequency of chromosomal aberrations in various categories of primary amenorrhea and the control individuals. Student s t values for the comparison for two sample means are given in Table no XXI. Among primary amenorrheas, radiosensitivity of the chromosomes is greater in patients of gonadal dysgenesis (0.95 ± 0.08) with the normal karytype (0.58 ± ) and than in true Turners (0.71 ± 0.08) and mosaic Turners (0.69 ± 0.071). Chromosomes of true Turners and mosaic Turners are equally radiosensitive. Although the mean number of aberrations/cell in 45, XO cells of mosaic Turners (0.96 ± 0.52) is greater than that found in 45, XO cells of true Turners (0.71 ± 57

70 0.08), their difference do not attain statistical difference due to greater variability shown by the 45, XO cells of mosaic Turners. In spite of this fact the radiosensitivity of the chromosomes in 45, XO cells of mosaic Turners is greater than could be found in normal individuals. In contrast, the chromosomes of 46, XX cells in mosaic Turners are as sensitive to the radiation induced genetic damage as those of normal control individuals. Thus, it could be seen that the radiosensitivity of the chromosomes in the two cell lines (46, XX and 45, XO) of mosaic Turners are comparable to those of normal individuals (incase of 46, XX cells) and true Turners (incase of 45, XO cells). Another important observation is that the chromosomes of gonadal dysgenesis patients who have normal karyotype are more radiosensitive than not only of the normal control individuals but also of any other categories of the primary amenorrheas (XO, and XX/XO mosaics). It is also to be observed that XO cells in general are more radiosensitive than the XX cells in general, are more radiosensitive than the XX cells irrespective of the fact they being of mosaic Turners or normal individuals. Table no XXII shows the frequency of translocations and chorosome breaks and deletions in various groups of primary amenorrhea. Table no XXIII shows the student s t values for the data given in Table no XXII. In general translocations, breaks and deletions are equally frequent in all categories of primary amenorrhea and control individuals. The frequency of translocations is highest in primary 58

71 amenorrhea of gonadal dysgenesis (46,XX) (number of translocations /cell: ) and lowest in mosaic turers (0.31 ± 0.07). The frequency of translocation is similar in both mosaic turners and control individuals. When the individual cell lines in mosaics turners are considered separately the frequency is higher in X0 cell line but less in XX cell line. The frequency of chromosomal breaks and deletions is highest in cases of primary amenorrhea of gondadal dysgenesis (46,XX) followed by mosaic Turners and Turners. The chromosomes of X0 cell line in mosaic Turners is highly sensitive and comparable to the chromosome of 46,XX gonadal dysgenesis. A Chromosome was computed as follows: Of metaphases / individuals x no. of individuals studied X = the relative length of the chromosomes The relative radiosensitivity of the individual chromosomes were expressed as the ratios between the patients and that of the control. The values so computed are shown in table XXV. When the ratios for any specific chromosome are 1.5 or greater are considered as sensitive chromosomes for the radiation. Table XXVI shows the distribution of ratios for the individual chromosomes in each of the category of primary amenorrheas. Irrespective of the category of primary amenorrhea, chromosomes 20, 59

72 21 and 22 are more radiosensitive as compared to those of the control. X chromosome is radiosensitive in both the gonadal dysgenesis with normal karyotype and mosaic Turners but not in Turners of 45, X0 karyotype. With reference to the other chromosomes considerable variation was observed. In primary amenorrhea of gonadal dysgeniesis (46,XX) chromosomes, 7, 8 are highly sensitive; chromosomes 6, 9, 10, 12 are moderately sensitive. In cases of primary amenorrhea of Turners (45,X0) chromosomes 7, 8, 12, 14 are moderately sensitive. In case of mosaic Turners all other chromosome are as sensitive as was found in control individuals. When the individual cell lines of the mosaic Turners are considered separately chromosomes of the XX cell line are more sensitive than the chromosomes of the X0 cell line. In case of XX cell line chromosomes 20 is highly sensitive and 21, 22 and X chromosomes are moderately sensitive, whereas in X0 cell line chromosome No. 22 is highly sensitive and chromosomes 2, 3, 4 and 12 are moderately sensitive. In general chromosomes of primary amenorrheas with gonadal dysgenesis are more sensitive to the radiation induced genetic damages. It is also interesting to note that the chromosomes of smaller length, viz., 20, 21 and 22 are more prone to radiation damage, than those of longer chromosomes. X chromosomes invariably was found to 60

73 be radiosensitive in all the categories of primary amenorrhea with the exception of true Turners. It is interesting to note that more number of chromosomes (8, 21, X, 20 and 22) showed a hypersensitivity to radiation in gonadal dysgenesis group than in any other amenorrhea group. Further, chromosome 7 and X showed a siginificantly high number of breaks at 7q22 and at 3 xq22 respectively. Out of a total number of 167 breaks on No.7 and 178 breaks on X, 67 breaks were on 7q22 and 59 where on Xq22. However, the chromosomes in Turner mosaics, and X0 cell line Turner mosaic showed an increased number of breaks, yet those breaks were distributed at random on the chromosome. The densitometric profiles of the deleted or translocated chromosome were obtained using Leitz MPV2 microscope densitometer. The profiles of the normal homologus were also traced in similar manner. The exact break point on chromosome involved in any deletions or trnslocations were then determined on a comparitive basis. The densitometric profiles of abnormal chromosomes involved in the various structural anomalies (Both spontaneous and radiation induced break points) observed are presented along with those of the homologous chromosomes in fig X XVIII. From those chromosome profiles the spontaneous break points 7q22 and Xq22 are clearly demonstrated. 61

74 Table I The frequency of chromosomal aberrations associated with PA & SA (Less 50) S.No Authors and years Total No.of patients No. of patients chromosome abnormalities Percentage of cases with chromosome abnormalities 1. De la Chapelle Reitalu Jagiello Rigo (group) WenTsuochaing Lakshmi Pardo Gupta Sulewski Payne Opitz Radhakrishnan Castillo Lin and Yu Temocin Devi and Benn Vijaya Laxmi Anulekha

75 Table II Recent Cytogenetic studies on Chromosomal Abnormalities in PA Karyotype Results Lacramiora et al Rajasekar et al Wong et al Kong et al Viyajalakshmi et al Present Study Kalvathi et al Ramirez et al Safaei et al Frequency of cases 54.56% ( % (146) 24.5% (58) 58.8% (10) 27.8% (39) 41.6% (92) 25.82% (220) 36.7% (96) 20% (44) X Chromosome (Homogeneu/mosaics) Aneuploidies 82.15% 22.59% 50% (29) 20% (2) 74% (29) 22.17% (49) 45.45% (100) 89.58% (92) 52.27% (23) X Chromosome Unbalanced Structural abnormalities 8.17% (22) 11.64% (17) 12.06% (7) 50% (5) 8.69% (3) 9.04% (20) 27.27% (60) 4.16% (4) 15.90% (7) Maker Chromosome , XY 5.20% (14) 27.27% (12) 1.72% (1) 8.4% (20) % (3) 17.9% (7) 8.59% (19) 23.63% (52) 7.85% (3) Other anomalies 1.36% % % (12) 63

76 Table III Classification of Primary Amenorrhea Patients on the Basis of their Syndromic Nature and Karyotypes Categories of PA Description of Various Categories No. of Cases Karyotypes I Pheno typically Normal females ,XX II Pure Turner 20 45,X III Mosaic Turners and variants 31 45,X0/46,XX; 46,XX/47,XXX; 45,X/47,XXX; 45,X/46, XX/ 47, XXX IV Pure structural abnormalities of the X chromosome partial and complete 12 46,X(Xiq); 46, X del (Xp) 46,X del (Xq partial and complete V Mosaic structural abnormalities of the X chromosome 10 45, X/46, X (X1q); 45, X/46, Xr (X) VI Pure XY females 13 VII Mosaic XY females 6 46,XY Gonadal dysgenesies; 46,XY TEF 46,XYPPHS 45, X/46, XY; 46, XX/46,XY 64

77 Table IV Frequency of ovarian dysgenesis in various categories of PA & SA Categories of PA Category I verses II VII No. of cases No. of cases with Gonadal dysgenesis % of cases with ovarian dysgenesis I II III IV V VI VII II VII All Categories Table V Frequency of Consangunous Marriages in Various Categories of PA Categories of PA N Consangunous n Marriages % I II III IV V VI VII II VII All Categories General Population

78 Table VI Frequency of Familial Incidence in Various Categories of PA Categories of PA Total Cases Familial Cases No % Relatives affected I a 7.75 II Sibs 5 Father 1 Mother sib -1 First cousin 1 Fathers Mother s Sister 1 III 31 2b 6.45 Sib 1 Father sib 1 IV V VI 13 2c Father s Mother s Brothers daut-1 VII 6 1d Sib a. 1,2,3,4,56,7,8,9,10,11 b. 15,16 c.17,19 d. all these are pedigrees Categories of PA Table VII Mean Parental Age in Various Categories of PA Maternal Age Paternal Age No. of Cases Mean SD Mean SD I II III IV V VI VII II-VII All Categories Category I verses Category II VII; Maternal Age: t = 2.21, p<0.05; Paternal Age: t = 3.04, p<

79 Table VIII Analysis of Birth Order in PA Categories of PA N No of affected 6A E(6A) V(6A) 6A-E(6A) 2SE Mean Birth Order I II III IV V VI VII II-VII A = Observed of birth ranks; E(6A) = Expected sum of Birth ranks 6; V(6A) = Variance of Birth ranks; SE = Standard error; n = Number of cases. Table IX Incidence of Associated Disorders among patients of PA & SA Categories of PA & SA No of cases Nervous disorders psyochic Utrrine TB Hypothyroidism Obesity Tuber culosis Bronchopneumonia Diabetes Total I II III IV V VI VII

80 Table X Incidence of congenital anomalies among sibs of PA CateGories of PA No. of cases No. of sibs excluding proband Cleft palate Mental retar dation Blind Deaf mute Congeital eart diseas Total congenital anomalies % of sibs affected I II III IV V VI VII II VII Categories of all Table XI Reproductive History of Parents in Various Categories of PA & SA Categories of PA&SA Total Pregancies Total Live Birth Sex Ratio Among Live Births M/F Total Dead Sibs Spontaneous Obortions (SA) Still Births (SB) Fetal Death (SA+SB) I II III IV V VI VII II VII Total No. PA + SA

81 Table XII Antenatal Medical History of Proband s Mothers in Various Categories of PA Categories of PA No of Cases Frequency of other who are affected and taken drugs N % I II III Diseases for which drugs have been taken General health Urinary Infection Antiemetics Abortion Antiematics Abortion Allergic General health Antiemetics Abortion IV Abortion 1 V Urinary insfection 1 General 1 VI health Antiemetics 1 Abortion 1 VII Abortion II VII Total No. cases PA + SA General health Urinary Infection Antiemetics Abortion General health Urinary Infection Antiemetics Abortion psychotherapy Allergic Gastrointestinal Problem Hypothyroidism psychotherapy Allergic Asthmetic Allergic Asthmetic Gastrointestinal Hypothyroidism

82 Table XIII Types and Frequency of Chromosomal abnormalities in 221 Cases of PA & SA S.No Karyotypes No. of Cases % of Cases with Chromosome abnormalities 1 46, XX , XO , XO/46,XX ,XX/47,XXX ,XO/47,XXX ,XO/46,XX/47,XXX , X(Xiq) , XO/46, X (Xiq) , X del (Xp) Partial , X Del (Xp) Complete , X del (Xq) Partial , X Del (Xq) Complete , XO/46, X(rX) , XY Gonadal dysgenesis , XY TFS , XY PPHS , X O/46, XY , XX/46, XY

83 Table XIV Chromosomal abnormalities datetced in cases with secondary amenorrhea in different studies of ethnic populations Karyotype Results Casttiloe et al (1992) Lacrami ara et al (2011) Wong (2205) Kalvathi et al (2010) Rajangam et al (2007) Daevi et al (1999) Lin et al (1996) Opitz et al (1983) Present Study Akbar et al (2012) Jyothy (2002) Vannierkerk et al (1978) No.of Cases ,XX Karyotype 32 (68%) 31 (81.57%) 281 (90.1%) 118 (92.91%) 215 (87.75%) 26 (86.7%) 10 (55.6%) 10 (66.7%) 16 (80%) 89 (94.6%) 324 (95.5%) 99 (96.1%) Abnormal Karyotype 15 (32%) 7 (18.42%) 31 (9.9%) 9 (7.08%) 40 (16.32%) 4 (13.3%) 8 (44.4%) 5 (33.3%) 4 (20%) 5 (5.3%) 15 (4.4%) 4 (3.8%) 45,X (1.6%) (0.79%) (16.6%) (1.06%) - - Monosomy mosaic (18.42%) (3.5%) (6.77%) (8.32%) (13.3%) (11.1%) (2.6%) (5.0%) , del (Xq) (1.9%) (4.23%) (3.3%) (1.06%) , X,i (Xq) (0.3%) (2.85%) (10.0%) t (X:A) (0.6%) (6.6%) (5.5%) ,XXX (1%) (0.79%) (7.5%) (11.1%) ,X/46, XX/47,XXX (1%) (4.65%) (5.0%) (5.0%)

84 Tables XV Types and Frequencies of Clinical features in Various Categories of PA having the Karyotype of 45,X0; 45,X0/46,XX; (5 Groups) and 46, XX. 45,X/46, 45,X/46, 45,X/46, 45,X/46, 45,X/46, 46, Abnormal Cell 45,X XX XX XX XX XX XX (45,X) No. of Cases Height in Cm Epicanthal fold Higharchedpalate Low set ear Low posterior line Short neck Webbed chest Shield chest Pigmented nevi Cubitus Valgus Short IV Heart abnormality Hearing ab Mental retardation Visual Vertebral Axillary Hair 18.Public hair 19.Breast Development 20.Vagina 21.Clitoris 22.Uterus 23.Ovaries S A S A P A B A N E R A R C

85 Table XVI Types and Frequencies of Clinical Features in Various Categories of PA & SA having the Karyotyotype of 46, XiXq, 45,X/46, XiXq, 46,XXp (Partial deletion) and 46,XXq- and 46,XXq (partial deletion) No of Cases 46,XiXq 100,2 45,X/46, XiXq ,X/46, XiXq , XXp- Partial 2 XXq- Partial 3 1.Height Epicanthal fold Higharchedpalate Low set ear Low poste Short neck Webbed chest Shield chest Pigmented nevi Cubitus Valgus Short IV Metacarpal Heart abnormality Hearing abnormal Mental retardation Visual Vertebral Axillary Hair 18.Public hair 19.Breast Development 20.Vagina 21.Clitoris 22.Uterus 23.Ovaries S A S A P A B A N E R A R C A Scanty, Absent, Poor, Blunt, Narrow, Enlarged, Rudimentary, Cystic 73

86 Table XVII Clinical Features Associated with Individual cases of PA having Karyotype of 46,X(Xp-) Complete deletion; 46,X(Xq-) Complete deletion; 45,X/46,X(rX); 45,X/47,XXX; 45/46, XX/47, XXX and 46,XX/47,XXX. No of Cases 46,X(Xp- Complete deletion 46, X(Xq- Complete deletion 45,X/46,X(rX); (72:28) 45,X/47,XXX (60:40) 45/46, XX/47, XXX (30:50:20) 46,XX/47, XXX(75:25) 1.Height Epicanthal fold Higharchedpalate Low set ear Low poste Short neck Webbed chest Shield chest Pigmented nevi Cubitus Valgus Short IV Metacarpal Heart abnormality Hearing abnormal Mental retardation Visual Vertebral Axillary Hair S A Public hair S Breast Development 20.Vagina 21.Clitoris 22.Uterus 23.Ovaries A P A B A N E R A R C A

87 Table XVIII Variation in the clinical Featuers of XY females No Clinical Features 1 Body Habitus Testicular Feminization Syndrome Normal or Eunuchoid Pseudovaginal Perineoscrotal Hypospadias Eunuchoid XX Gonadal Agenesis Eunuchoid 2 Breast Well developed Absent Absent 3 Family History a) Clitoris Female Enlarged like phallus Female b) Labia Infantile Labio Scrotal folds with testis (bifid scrotum) Infantile c) Vagina Blind Pouch Absent, Blind Pouch Absent 5 Urethra Normal Perineal hypospadias 6 Mullerian duct differentiation Absent 7 Wolfian duct None Absent Normal Wolfian Duct derivatives Urogenital sinus Absent Rudimentary fallopian tubes, No Uterus None Axillary and Absent / 8 Absent Present public hair Scant Absent/Male 9 Escutcheon Absent Male at puberty type 10 Gonads Testes Testes Absent Sex 11 Negative Negative Negative Chromation 12 Chromosomes 46, XY 46, XY 46, XY 75

88 Table XIX Clinical Features Associated with Individual cases of PA having Karyotype of 46,X(Xp-) Complete deletion; 46,X(Xq-) Complete deletion; 45,X/46,X(rX); 45,X/47,XXX; 45/46, XX/47, XXX and 46,XX/47,XXX. % of Cell Lines Clinical Features 45, X/46, XY 26:74 45, X/46, XY 58:42 46, XX/46, XY 74:26 46, XX/46, XY 82:18 Height Epicanthal fold absent absent absent absent Higharchedpalate absent absent absent absent Low set ear absent absent absent absent Low posterior line absent absent absent absent Short neck absent absent absent absent Webbed chest absent absent absent Absent Shield chest absent absent absent Absent Pigmented nevi absent absent absent Absent Gonads tests Streak l-absent, r-stre l-absent, r-stre Cubitus Valgus absent present absent Absent Short IV Metacarpal absent absent absent Absent Axillary hair absent absent present Present Pubic hair absent absent present Present Breast Vagina poor absent blind Poor blind Blind blind Blind Clitoris large Large infantile Infantile Uterus absent absent absent Absent 76

89 Table XX Radiosensitivityof Patients with XX, XO, XO/XX Karyotypes and Normal XX Females Normal (XX) (n=10) Primary Amenorrheas Gonadal Dysgenesis (XX) (n=26) Turner (X0) (n=12) XX cell line Turner mosaics (n=10) X0 Cell line Total (XX/X0) (n = 500) (26) 20 (24) (12) 13 (38) (23) 18 (27) (29) 24(21) (28) 21 (22) (33) 24 (17) (35) 25(15) (18) 14(32) (18) 12(32) (38) 21(12) X = X = X = X = SD = SD = SD = SD = _ X = SD = _ X = SD = Chromosomal aberrations includes translocations (dicentrics, Multicentrics, rings) Deletions and breaks n Number of individuals studied. 77

90 Figure given in paranthesis, refers to the number of cels with 46, XX or 45, X0 chromosomal constitution. For every individual 50 well spread metaphases were studied. 1 = Control Females (46,XX): 2 = Gonadal dysgenesis (46,XX): 3 = Turner (45,X0); 4 = XX cell line in Turner Mosaics; 5 = X0 cell line in Turner mosaics;. 6 = Turner mosaics (46,XX/45,X0) Table XXI Student s t values for the comparison of means for the data Comparison between T DF P 1 x *** 1 x *** 1 x NS 1 x * 1 x ** 2 x *** 2 x *** 2 x NS 2 x *** 3 x NS 3 x NS 3 x NS 4 x NS 78

91 1 = 46, XX normal; 2 = 46, XX Gonadal dysgenesis; 3 = 45, XO Turner; 4 = 46,XX cell line of Turner mosaic; 5 = 45, XO cell line of Turner mosaic; 6 = 46, XX/45, XO Turner mosaics. ***p < 0.01; **p < 0.01; *p < NS Not significant. Table XXII Radiosensitivity of Chromosomes of the Primary Amenorrheas Relative Frequency of Translocations, Break & Deletions. S.No Category N x s Breaks x Deletions S 1 46, XX Normal , XX GD , XO Turner 12 4 XX cell line 10 5 XO cell line , XO/XX Mosaics includes dicentrics, tricentrics, multicentrics and rings. x = Weighted average, no. of aberrations/cell; S = Standard deviations and N = Number of individuals studied. 79

92 Note: For each individual 50 cells are counted. In case of XO and XX cell lines of mosaics. The number of cells actually counted is used in the denominator. Table XXIII Student s t values for the comparison of means for the data given In Table XXIX Comparison between T Df P Translocations 1 x 2 1 x 3 1 x 6 4 x Breaks 1 x 2 1 x 3 1 x 6 4 x = 46, XX normal; 2 = 46,XX Gonadal dysgenesis; 3 = 45, XO Turners; 4 = 46, XX cell line of Turner mosaics; 5 = 45, XO cell line of Turner mosaics; 6 = 46, XX/45, XO Turner mosaics. 80

93 Table XXIV Radio sensitivity of individual chromosomes in syndrome associated with primary amehorrhea Chromosomes Nos Relative Length of the Chromsomes 1 Normal 46,XX Observed in 500 Cells (n = 10) 2 46,XX GD Observed in 1300 Cells (n=26) ,X0 Observed in 600 Cells 4 46,XX Cell line from Turner Mosaic Observed in 260 Cells 5 45,X0 Cell line from Turner mosaic observed in 240 Cells (n=10) 6 45, X/46,XX Observed in 500 Cells (n =10) X * N = Number of Patients studied.

94 Table XXV A comparative analysis of the radiosensitivity of individual chromosomen in Syndrome associated with primary amenorrhea Chr. Nos Control 46.XX (GD) X (Turner) XX Call Line (Mosaic Turner) X0 Cell Line (Mosaic Turner) 45,X/46, XX (1.0) 8.29 (1.1) 7.06 (0.9) 9.87 (1.3) 8.41 (1.0) (1.1) 8.10 (1.3) 6.47 (1.1) 9.61 (1.6) 7.98 (1.3) (1.2) 8.54 (1.3) 5.35 (0.8) (1.7) 7.91(1.2) (1.4) 6.48 (1.3) 4.27 (0.8) 8.60 (1.7) 6.35(1.2) (1.3) 6.72(1.1) 5.06 (0.8) 6.85 (1.1) 5.92(1.0) (1.9) 5.51 (1.2) 4.56 (0.8) 5.65 (1.2) 5.08(1.1) ) 4.51 (1.5) 2.87 (1.0) 4.28(1.4) 3.54(1.2) (3.1) 3.55(1.5) 1.95(0.9) 2.11 (0.9) 2.03(0.9) (1.6) 6.08 (1.3) 3.61 (0.8) 5.21 (1.1) 4.38(1.0) (1.5) 4.18 (1.1) 2.09 (0.5) 2.72 (0.7) 2.40(0.6) (1.3) 3.98 (1.1) 2.09 (0.6) 1.81 (0.5) 1.95 (0.5) (1.5) 4.47 (1.4) 3.30(1.1) 4.92 (1.6) 4.08(1.3) (1.3) 3.16 (1.2) 1.22 (0.4) 1.32 (0.5) 1.27 (0.5) (1.3) 6.55 (1.5) 2.70 (0.6) 4.68 (1.1) 3.65 (0.8) (1.2) 4.34 (1.1) 2.22 (0.5) 2.41 (0.6) 2.31 (0.6) (0.9) 3.72 (0.9) 1.72 (0.4) 2.48 (0.6) 2.08 (0.5) (1.1) 4.44 (1.1) 2.56 (0.6) 2.78 (0.6) 2.67 (0.6) (1.0) 2.28 (0.9) 1.31 (0.5) (0.2) (1.3) 1.87 (1.0) 1.44 (0.7) (0.4) (6.9) 1.30 (3.3) 1.50(3.8) (2.0) (3.6) 1.75 (1.6) 2.02 (1.9) 1.09(1.0) 1.58 (1.5) (4.2) 0.82 (1.6) 0.94 (1.9) 3.06 (6.2) 1.96 (4.0) (3.6) 4.56 (1.2) 6.76 (1.8) (3.2) 9.30 (2.3) Figures given in brackets refer to the ratios: of the patient or control 82

95 Table XXVI Radio sensitivity of chromosomes in primary amenorrhea relative to the chromosome of normal individual Primary Amenorrhea Relative damage : Patient / Control Above Gonadal Dysgenesis (46,XX) (2) 1,2,3,4,5, 11,13,14,15, 16,17,18,19 6,9,10,12 8,21,X 7,20,22 Turner Syndrome (3) 1,2,3,4,5, 6,9,10,11, 13,15,16,17, 18,19,X 7,8,12,14,2 1, ,XX Cell line From Turner Mosaic (4) 1,2,3,4,5, 6,7,8,9,10, 11,12,13,14, 15,16,17, 18,19 21,22,X 20 45,X0 Cell line From Turner Mosaic ( 5) 1,5,6,7,8,9,10, 11,13,14,15,16,17,18,19,20,21 2,3,4,12 X 22 Turner Mosaics 46,XX/45,X0 (6) 1,2,3,4,5,6,7,8,9, 10, 11,12,13,14,15,1 6, 17,18, ,X 22 83

96 GENE MAPPING CHROMOSOME 7 List of Abbreviations: Figure I 1. pter p14 : GCTG Gamma-glutamyl cyclotransferase 2. p15 : TRCG T cell receptor ( rearranging) gamma- polypeptide 3. p14 cent : BLVR Biliverin reductase 4. pter p14 : ERBB Avian erythroblastic leukemia viral ( v-erb-b) oncogene homolog 5. p13 p11 : EGF Epidermal growth factor rceptor 6. p11.2 : FRA7A Fragile site, rare folic acid type 7. p11 q11 : ASNS Asparagine synthetase 8. cent q22 : GUSB Glucuronidase beta 9. q21.3 q22.1 : 014 Osteogenesis imperfects type IV 10. q21.3 q22.1 : COLIA2 Collagen, type I, alpha 2 NJ3, NJ1, HPC1 genomic q22-qter : NM Neutrophil migration 12. q22-qter : TRY 1 Tr H2A ypsin q32: TCRB T cell receptor (rearranging) beta polypeptide B q32 q36 : H1 H1 histone 15. q32 q36 : H2A H2A histone 16. pter p14 : H2B H2B histone 17. q32 - q36 : H3F1 H3 histone family 84

97 FIGURE I GENE MAPPING CHOROMOSOME 85

98 Figure II GENE MAPPING CHROMOSOME X List of Abbreviations: 1. pter p22.32 : CDPX Chondrodysplasia punctata 2. pter p22.32 : STS Steroid sulfatase (microsomal) 3. pter p22.3 : XG Xg blood group 4. pter p22 : OA Ocular albinism 5. pter - p22 : AIC Aicardi syndrome 6. p22.1 p21.3 : POLA DNA polymerase alpha 7. p21.2 p21.3 : AHC Adrenal hypoplasia (congenital) 8. p21.2 p21.3 : GK Glycerol kinase 9. p 21 : BMD Muscular dystrophy Becker type 10. p21 : DMD Muscular dystrophy Duchenne 11. p21 : OTC Ornithine carbamoyl transferase 12. pter p21 : RS Retinoschisis 13. pter p21 : XK Kell blood group precursor 14. pter p21 : CGD Chronic granulomatous disease 15. p11 : IP Incontinentis pigmenti 16. p21 cent : NDP Norrie disease (pseudoglimoa) 17. p21 cent : RP2 Retinitis pigmentosa p11 q11 : DHTR Dihydrotestoterone receptor p11 q11 : MNK Menkes syndrome 19.q13 : PGDY Faciogenital dysplasia 20.q21 q22 : GLA Galactosidase, alpha 21.q26 q27.3 : F9 Coagulation factor (HEMB) Hemophilia B 86

99 FIGURE - II GENEMAPPING CHROMOSOME X 87

100 FIGURE III A. Normal B. Deleted X with break point Xp22 88

101 FIGURE IV A Normal B. Deleted X with break point Xp11 89

102 FIGURE V A. Normal B. Deleted X with break pointxp28 90

103 FIGURE VI A. Normal No.7 B. Deleted 7 with the break point 7q22 91

104 FIGURE VII A. Normal No.7 B. Deleted 7 with the break point 7q22 92

105 FIGURE VIII A. Normal No. 7 B. Deleted 7 with the break point 7q22 93

106 FIGURE IX A. Normal No. 7 B. 7,10 and translocations t(q22 : q22) 94

107 FIGURE X A. Normal No.7 B.. 7, 12 and translocation t(q22: p12) 95

108 Plate No.1 True Turner syndrome Plate No, 3 Mosaic Turner Plate No. 4 PA with,x/46,x,r(x) Plate No.6 PA with 46,Xi(Xq) Plate No.8 PA with 46,X(Xp-)( bp Xp22) Plate No. 11 PA with 6,X(Xp-) 96

109 Plate No 2a Karyotype of normal female (G-banded chromosomes) Plate No 2b Karyotype of true Turner, 45,XO (Patient in plate No.1) 97

110 Plate No 5a Karyotype of one of the cell lines (47,XXX) in plate No.4 (D F) Plate No 5b Karyotype of one of the cell lines (47,XXX) in plate No.4 (D F) 98

111 Plate No 7 Karyotype of 46,Xi(Xq) Plate No 26 Radiation induced G banded metaphase spreads showing various chromosomal rearrangements involved at Xq22 locus Plate No ,X(Xp-) bp Xp22 (patient in plate No. 8 A C) Plate No.10 46,X(Xq-) bp Xq22 (patient in plate No. 8 D F) 99

112 Plate No.12 46,X(Xp-) complete short arm deletion 46,X(Xq-) (patient in plate No.11) Plate No. 13 Clinical features of PA with complete long arm deletion with break point at Xq11 Plate No 14 46,X(Xq-) Plate No 16 PA with 46,XY complete long arm deletion patient in plate No 13. (patient in plate No. 15) 100

113 Plate No 17 Mosaic Turner 45,X/46,XX with multiple chromosome aberration Plate No 18 Multiple chromosome aberration 17 Plate No 19 PA with 46,XX gonadal dysgenesis Plate No 15 pseudo vaginal perineoscrotal Plate No 21 Two sisters with 45,X/46,XX/47, hypospadias syndrome XXX (30:50:20) 101

114 Plate No 20 Multiple chromosome aberration of a primary amenorrhea (patient in plate no.19) Plate No 22 Effect of folic acid free medium in patients with 46,XX gonadal dysgenesis Plate No 23 Radiation induced chromosomal break points in 7q arm 102

115 Plate No 24 Radiation induced g banded metaphase spreads showing various Chromosomal rearrangements involved at 7q22 locus Plate No 25 Radiation induced chromosomal break points of X chromosome 103