Article Cytogenetic investigations in couples with repeated miscarriages and malformed children: report of a novel insertion

Transcription

1 RBMOnline - Vol 14. No Reproductive BioMedicine Online; on web 22 January 2007 Article Cytogenetic investigations in couples with repeated miscarriages and malformed children: report of a novel insertion Priya Iyer, Leena Wani, Smruti Joshi, Jyothi Lakshmi, Rupa Dalvi, Deepak Chavan, Bibhu Ranjan Das, Swarna Mandava 1 Cytogenetic Division, SRL Ranbaxy Ltd, Plot 124, 15th Street, MIDC, Andheri (E), India Correspondence: Swarna@srlranbaxy.co.in Dr Swarna Mandava is head of the Cytogenetic Division at SRL Ranbaxy Limited. She obtained her PhD in Genetics from Osmania University. SRL Ranbaxy is a leading clinical diagnostic laboratory in India and the biggest in South East Asia, spread over all major cities in India. It is the first laboratory of its kind in India having received the accreditation from NABL and The College of American Pathologist (CAP) in India. The R & D facility is equipped with highly qualified and experienced professionals with focus on research and development of advanced new diagnostic tests. SRL Ranbaxy Ltd is widely acknowledged for its commitment to quality and customer service through its corporate mission statement care for you, care for accuracy. Abstract Recurrent miscarriage and the birth of a malformed child continue to be challenging problems for the patient and clinician. Often, cytogenetic studies have an important role in the evaluation of couples with a poor obstetric history. The present study deals with analysis of chromosome abnormalities in 2150 couples from India, and is the largest group ever reported in the literature. The observed incidence of chromosomal rearrangements in these couples was 3.5%. Apart from reciprocal, Robertsonian, inversions, a unique case of chromosome insertion was identified, which is perhaps only the second report in literature. Minor chromosome variants such as inv(9),inv(y),9qh+, D/G variants were observed in 108 subjects. Cytogenetic studies should be performed for all couples with repeated miscarriages and bad obstetric history, and in cases of detected chromosomal aberration, the patient should be counselled individually according to the type of anomaly. Keywords: balanced translocation, insertion, inversion, poor obstetric history, recurrent miscarriage Introduction 314 Globally, the annual number of parental karyotypes with repeated miscarriages and poor obstetric history is increasing gradually. Chromosomal disorders and mutant genes could disturb gamete formation, thereby impairing normal embryonic development. In the general population, the frequency of balanced translocations has been estimated as 0.17% (Jacobs, 1977), while in a group of patients with altered reproductive fitness, the percentage of chromosomal anomalies was always higher. Approximately 15 20% of clinically recognizable pregnancies end in spontaneous miscarriages (Dejmek et al., 1992). The incidence of chromosomal abnormalities in those miscarriages is as high as 60%. A clinically significant proportion of spontaneous miscarriages is caused by a balanced chromosomal aberration in one of the parents, which results from the production of gametes and embryos with unbalanced chromosome sets. The clinical consequences of such abnormal gametes include sterility, repeated miscarriages, and giving birth to malformed children. Couples with a history of multiple miscarriages and malformed children are at risk of carrying a balanced translocation, as these carriers might produce unbalanced gametes that will be associated with fetal abnormalities and mental retardation. The genetic aetiology for poor reproductive failure might be an unbalanced chromosome rearrangement, which could be the result of one parent being a carrier for a balanced chromosome rearrangement (Gadow et al., 1991) Several studies have been carried out globally to determine the chromosomal abnormalities in couples with reproductive failure for the last 3 decades (Tharapel et al., 1985; Campana a et al., 1986; Muneera et al., 2000; Dubey et al., 2005; Nagham et al., 2005; Rao et al., 2005). So far as is known, this study reports the cytogenetic investigations in the largest number of couples with repeated miscarriage from India that has ever been reported in the literature Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB3 8DB, UK

2 Materials and methods Cytogenetic analysis was carried out in 4300 persons (2150 couples) with clinical diagnosis of repeated miscarriages or bad obstetric history referred from all parts of the country by different gynaecologists. Patients included women with two or more miscarriages up to 20 weeks of gestational age, and couples with poor obstetric history such as history of neonatal deaths, live-born malformed child, normal child and miscarriages. Cytogenetic analysis was performed from peripheral blood lymphocyte cultures using the modified standard protocol (Moorhead et al., 1960). About 0.5 ml of sample was inoculated in 6 ml of Roswell Park Memorial Institute (RPMI) medium supplemented with 20% fetal bovine serum and 0.2 µl phytohaemagglutinin (PHA). The culture was incubated for 72 h in a 5% CO 2 incubator; the cells were harvested using hypotonic solution treatment followed by chilled fixative treatment to fix the cells. Fixed cells were dropped on slides and stained for GTG banding (Seabright, 1971).The banded metaphase chromosomes were examined at resolution 550 band level. At least 20 metaphase plates were analysed from each sample and three or four well spread plates were photographed and karyotyped using Ikaros Metasystem Software (Gmbh, Germany). In abnormal cases, at least 30 metaphases were evaluated. Results A total of 2150 couples were analysed for cytogenetic study, out of which 75 cases were found to have major chromosomal aberrations, while 108 cases revealed chromosomal variants. The distribution pattern of various chromosome abnormalities is given in Figure 1. The details of various major chromosomal cytogenetic observations are presented in Table 1. Among the 75 major chromosomal aberrations detected, 44 (58.7%) cases were found to be in women and 31(41.3%) were in men. Several major aberrations detected were reciprocal translocations (n = 42, 56.0%), Robertsonian translocations (n = 18, 24.0%), inversions (n = 5, 6.7%), deletions (n = 5, 6.7%), numerical (n = 4, 5.3%) and insertion (n = 1, 1.3%) (Table 1). Five subjects showed deletions; one was deletion Xq and the other four were mosaic with deletion of the chromosome 9q region. Numerical abnormalities were found in four subjects. Of these, two showed an extra marker chromosome, one had mosaicism with extra marker chromosome and one woman had 47,XXX karyotype. Case history of a man with a chromosome insertion This young couple, in a phenotypically normal, nonconsanguineous marriage, were referred to the gynaecologist with a history of miscarriage and three neonatal deaths due to congenital malformations. There was no family history of congenital malformations or recurrent miscarriages on either side. The husband showed a unique type of chromosome insertion with the karyotype 46,XY,ins(1;13)(q22;q31q3 3) (Figure 2) in all the cells analysed, while his wife had a normal karyotype. The observed chromosome insertion in this investigation is a unique abnormality and is the second report published so far (Rao et al., 2005). Apart from these, minor chromosomal variants were found in 108 (2.5%) of subjects, which included 1qh, 9qh, 16qh, inversion of chromosome 9 and Y (Table 2). In this study, three families were found where malformed children were born with abnormal karyotype, as one of the parents was a carrier of balanced translocation. Case report 1 A couple was referred for cytogenetic analysis with a history of four miscarriages and one male child with multiple congenital malformations. Cytogenetic investigation of the wife showed normal female karyotype, while the husband showed 46,XY,t(6;12)(q27;p12) and the malformed child karyotype was 46,XY,der(6),t(6;12)pat (Figure 3). Figure 1. Overall distribution of various abnormalities in couples. * Indicates minor chromosome variants. 315

3 Table 1. Major chromosomal abnormalities observed in couples. Men Women Balanced translocations Balanced translocations Age (years) Karyotype Age (years) Karyotype 46 46,XY,t(2;12)(q31;q24) 23 46,XX,t(1;21)(42.2;q22.1) 26 46,XY,t(5;13)(q34;q33) 25 46,XX,t(7;12)(q24;q22) 32 46,XY,t(5;9)(q23.3;p24) 26 46,XX,t(8;13)(24.1;q22) 32 46,XY,t(7;21)(q32;q22.2) 41 46,XX,t(7;18)(q32;q12.3) 39 46,XY(94%)/46,XY,t(5;6)(q14;q27) 22 46,XX,t(4;13)(q34;q22) 38 46,XY,(2;21)(p22;q23) 25 46,XX,t(4;8)(q24;q24.2) 36 46,XY,t(12;17)(q24.3;q23) 29 46,XX,t(5;18)(q31.3;q22) 27 46,XY,t(1;2)(p36.1;p11.2) 33 46,XX,t(11;12)(q25;q13) 28 46,XY,t(12;13)(q23;q21) 25 46,XX,t(8;9)(p23;p13) 33 46,XY,t(6;11)(q23;q24) 29 46,XX,t(19;21)(q13.1;q12) 29 46,XY,t(3;6)(q28;q21) 29 46,XX,(2;21)(p22;q23) 33 46,XY,t(4;!4)(q34;q23) 27 46,XX,t(1;15)(q42.2;q13) 32 46,XY,t(6;12)(q27;p12) 30 46,XX,t(3;8)(p25;p22) 40 46,XY,t(2;8)(p24;q22.1) 26 46,XX,t(10;12),inv(9) 28 46,XY,t(13;15)(q31;q23) ,XX,t(11;17)(q12;p11) Robertsonian translocations 35 46,XX,t(10;11)(q22;q14) 42 45,XY,t(13;21)(q11;q11) 25 46,XX,t(1;2)(q44;q22) 23 45,XY,t(14;21)(q11;q11) 28 46,XX,t(15;18)(q13;p11.3) 26 45,XY,t(13;14)(q11;q11) 27 46,XX,t(6;7)(p21.2;p21) 26 45,XY,t(13;14)(q11;q11) 28 46,XX,t(4;12)(q34;q21) 34 45,XY,t(13;14)(q11;q11) 25 46,XX,t(9;18)(p23;q21) 30 45,XY,t(13;14)(q11;q11),15ps ,XX,t(7;18)(p11.1;p11.2) 30 45,XY,t(14;15)(q11;q11) 26 46,XX,t(1;11)(q41;q22) 29 45,XY,t(13;15)(q11;q11) 31 46,XX,t(6;10)(q24;q14) Numerical 30 46,XX,t(11;22)(q25;q13) 32 47,XY,+mar 24 46,XX,t(5;6)(q23;q26) 32 47,XY,+mar 24 46,XX,t(11;15)(q12;q26.4) Inversions Robertsonian translocations 30 46,XY,inv(4) 31 45,XX,t(13;14)(q11;q11) 42 46,XY,inv(2) 24 45,XX,t(13;14)(q11;q11) 28 46,XY,inv(5) 33 45,XXt(14;21)(q11;q11) Deletions 26 45,XX,t(13;14)(q11;q11) 30 46,XY/46,XY,del(9q) 25 45,XX,t(14;21)(q11;q11) 34 46,XY/46,XY,del(9q) 26 45,XX,t(13;14)(q11;q11) Insertion 24 45,XX,t(13;14)(q11;q11) 27 46,XY,ins(1;13)(q22;q31q33) 32 45,XX,t(14;21)(q11;q11) 34 45,XX,t(15;15)(q11;q11) 21 45,XX,t(13;14)(q11;q11) Numerical 28 47,XXX 28 46,XX/47,XX,+mar Inversions 27 46,XX,inv(3) 34 46,XX,inv(18) Deletions 36 46,XX/46,XX,del(9q) 28 46,X,del(Xq) 27 46,XX(72%)/46,XX,del(9q)(28%) 316

4 Figure 2. Karyotype showing novel chromosome insertion. Table 2. Frequency of minor chromosome variants in couples. Sex No. of 1qh+,16qh inv(9) Yqh+/Yqh- D/G inv(y) Total couples +,9qh+ group Men Women Case report 2 A couple was referred with a poor obstetric history of two miscarriages and two malformed children. The husband showed normal male karyotype and his wife showed 46,XX, t(1;15)(q42.2;q13) pattern. The two malformed children showed the karyotype 45,XY, t(1;15),-15 pattern (Figure 4). Case report 3 A couple who were first cousins (consanguineous marriage) were referred for cytogenetic analysis with a history of repeated miscarriages. Interestingly, both showed the same type of balanced translocation, i.e. 46,XY,t(2;21)p22;q23) and 46,XX,t(2;21)(p22;q23) (Figure 5). 317

5 Figure 3. Pedigree and karyotype showing balanced translocation 46,XY,t(6;12) in father and proband with 46,XY,der(6),t(6;12)pat pattern. 318 Figure 4. Pedigree chart showing t(1;15) in mother and her two sons with 45,XY,t(1;15),-15.

6 Figure 5. Pedigree and karyotype showing t(2;21)(p23;q22) in a couple who had a consanguineous marriage. Discussion Chromosome rearrangements in carrier parents are one of the causes of reproductive failure. In this study, it was observed that 44 women and 31 men showed chromosomal abnormalities with female to male ratio of 1.42:1. A higher female to male ratio has been observed in most studies (Chandley et al., 1975; Braekeleer and Dao, 1990; Muneera et al., 2000; Dubey et al., 2005). This female predominance could explain why chromosomal abnormalities that are compatible with fertility in women might be associated with sterility in men. The incidence of chromosomal abnormalities varied in different studies from different parts of the world, from none (Rowley et al., 1963) to as high as 25% (Nagham et al., 2005). This variation might be due to size of the sample and criteria used for the sample. In the present study, the incidence of chromosomal abnormalities among the couples was 3.5%, which is comparatively low when compared with other studies. One cumulative study has reported 2.86% abnormalities in couples with recurrent miscarriages, which is less than the present study (Kalpana et al., 2004). Comparison of structural chromosomal abnormalities in the present study with others reported worldwide is given in Table 3. From South India, Rao et al. (2005) reported 11.25% chromosomal abnormalities in 160 couples, which includes minor chromosome variants such as inversion (9). The expected rate of future infants with chromosomal aberration is especially high in couples with a mixed history of miscarriages, intrauterine fetal death and malformation (Hamamah et al., 1997). Detailed studies of miscarriage material from known translocation carriers will be helpful in the investigation of meiotic malsegregation and interchromosomal effects to provide genetic counselling for couples with a balanced translocation. Interestingly, a unique interchromosomal abnormality of insertion in a husband with karyotype 46,XY,ins(1;13) was found. Rao et al. (2005), from the South Indian population, reported for the first time this novel pattern in a woman with 46,XX,ins(12;6). The mechanism for this chromosome breakage is unknown. Intrachromosomal insertion with risk of miscarriages have been reported (Allderdice et al., 1983), whereas interchromosomal insertions are rarely reported. Further molecular characterization might help in understanding the cause of fetal loss due to insertion In the present study, two women, each with inversion (3) and inversion (18) and in two men with inversion (2) and (4) were observed. Dubey et al. (2005) also observed inversion (4) in their study. Rao et al. (2005) reported 8.1% inversions, including inversion (9), in their study. The finding of a balanced translocation or an inversion is a strong indication for prenatal diagnosis in subsequent pregnancies, translocations and inversions predisposing to high levels of chromosome 319

7 Table 3. Comparison of present structural chromosomal abnormalities with other studies worldwide a. Country No. of Reciprocal Robertsonian Inversions Other Total no. couples (%) b Spain (18.8) Netherlands (13.4) Italy (9.7) Netherlands (9.6) Belgium (8.3) Switzerland (7.3) Saudi Arabia (6.7) France (5.1) Japan (4.5) France (2.8) Egypt (25) China (11.5) Turkey (4.9) North India (4.2) Present study (3.5) a Boue et al. (1985); Chandley (1990); Butler and Hamil (1995); Jiang et al. (2001); Duczan et al. (2003); Dubey et al. (2005); Nagham et al. (2005). b % calculated from the total no. of couples studied. 320 imbalances in the zygote, and increasing the likelihood of spontaneous miscarriages (Campana et al., 1986). A mosaic deletion of chromosome 9 and mosaic deletion of Xq was observed. Jung et al. (2002) found mosaic deletion of Xq in their study. Numerical chromosomal abnormalities were less frequent among couples with recurrent miscarriages. There were two cases with an extra marker chromosome, one woman with seven miscarriages having a mosaicism with marker chromosome, and one woman with 47,XXX karyotype. Wolstenholme et al. (1983) also reported a mosaic carrier of marker chromosome in their study of 78 couples for familial translocation carriers. In addition to the structural abnormalities, 108 subjects (5.1%) were found with chromosomal variants, Inv(9), Inv (Y), 9qh+, 21p+, 1qh+, 16qh+ etc. However, this observation was not in accordance with either Duzcan et al. (2003) or Madon et al. (2005), who observed 7.5 and 7.6% chromosome variants, which is high, or with Dubey et al. (2005) who found 2.8% cases from 742 couples with minor chromosome variants, which is low. Chatzimeletiou et al. (2006) has found one very small and one normal sized signal with sequential fluorescence in-situ hybridization indicating polymorphism in the heterochromatin region of chromosome 16 with centromere-specific probes which was inherited from the mother and grandfather. Pooled published data of the general population from several worldwide series showed the frequency of 9qh+, Yqh+ and D/G group variants as 2.44, 2.85 and 3.96% respectively. The prevalence of chromosomal abnormalities is much higher than that in the general population when chromosomal variants are also considered as chromosomal abnormalities, as mentioned in some reports (Fryns et al., 1998). For all these years, the precise role of heterochromatin in the human genome remained a mystery, as its frequent polymorphisms did not appear to have any functional and phenotypic effect (Mattei and Luciani, 2003). These variants are presently considered normal and are often transmitted from one of the parents, though they could arise de novo. At the same time, recent research by cell biologists suggests that heterochromatin may have more important cellular roles than previously thought and these should be re-evaluated and recorded for future use, as all polymorphic variants may not be normal. These normal polymorphic variants may be found to play a significant role in different clinical conditions including fertility with the research develops (Madon et al., 2005) In the present study, four families were observed in which the couples were phenotypically normal, with one partner having balanced reciprocal translocation, who produced children who inherited the derivative chromosome from the parent causing malformations. Few cases were reported with unbalanced translocation in a child which inherited from the parent who had a balanced translocation (Jun Mo et al., 2003; Prabhakara et al., 2004). It has been reported previously that balanced reciprocal translocations are present in about 0.1% of the population (Hook and Hamerton, 1977). Of these, about twothirds of translocations have been inherited (Jacobs, 1974). A possible casual association between congenital malformations and inherited balanced translocation has been suggested by Fryns et al. (1986). In inherited reciprocal translocation cases, the breakpoint could inactivate genes that could subsequently unmask a recessive allele inherited from the other parent, which suggests the importance of parental chromosome studies in cases of familial structural abnormalities and frequent miscarriages. It is necessary to detect these cases as early as

8 possible for adequate genetic counselling, and to allow parents to make a decision regarding subsequent pregnancies. Chromosomal analysis should be considered as an integral part of the evaluation of patients who have had multiple miscarriages and malformed children because of the clear relationship between a chromosomal abnormality in a parent and subsequent miscarriages and a malformed child. The detection of couples with chromosomal anomalies can undoubtedly help to prevent the birth of malformed infants. Acknowledgement The authors are grateful to the management of SRL Ranbaxy Ltd for providing the necessary infrastructure facilities. References Allderdice P, Eales B, Onyett H et al Duplication 9q34 syndrome. American Journal of Human Genetics 35, Boue A, Boue J, Groppe A 1985 Cytogenetics in pregnancy wastage. In: Harris H, Hirschhorn K (eds) Advances in Human Genetics. Plenum Press, New York, 14, pp Braekeleer M, Dao T 1990 Cytogenetic studies in couples experiencing repeated pregnancy losses. Human Reproduction 5, Butler M, Hamill T 1995 Blood specimens from patients referred for cytogenetic analysis: Vanderbilt University Experience. Southern Medical Journal 88, Campana M, Serra A, Neri G 1986 Role of chromosome aberrations in recurrent abortion; a study of 269 balanced translocations. American Journal of Medical Genetics 24, Chandley AC 1990 Infertility and recurrent abortions. In: Emery A, Rimoin D (eds) Principles and Practice of Medical Genetics. Churchill Livingstone, London, pp Chandley A, Edmond P, Christie S et al Cytogenetics and infertility in man. I. Karyotype and seminal analysis results of a five-year study survey of men attending a subfertility clinic. Annals of Human Genetics 39, Chatzimeletiou K, Taylor J, Marks K et al Paternal inheritance of a 16qh- polymorphism in a patient with repeated IVF failure: a case report. Reproductive BioMedicine Online 13, Dejmek J, Vojtassak J, Malova J 1992 Cytogenetic analysis of 1508 spontaneous abortions originating from South Slovakia. European Journal of Obstetrics, Gynaecology and Reproductive Biology 46, Dubey S, Chowdhury M, Prahalad B et al Cytogenetic causes for recurrent spontaneous abortions an experience of 742 couples (1484 cases). Indian Journal of Human Genetics 11, Duzcan M, Ozan G, Bagci H 2003 Cytogenetic studies in patients with reproductive failure. Acta Obstetricia et Gynecologica Scandinavica 82, Fryns J, Van Buggenhout G 1998 Structural chromosome rearrangements in couples with recurrent fetal wastage. European Journal of Obstetrics, Gynaecology and Reproductive Biology 81, Fryns J, Kleczkowska A, Kubien E et al Excess of mental retardation and/or congenital malformations in reciprocal translocation in man. Human Genetics 72, 1 8. Gadow E, Lippold S, Otano l et al Chromosome rearrangements among couples with pregnancy losses and other adverse reproductive outcomes. American Journal of Medical Genetics 41, Hamamah S, Fingnon A, Lansac J 1997 The effect of male factors in repeated spontaneous abortion: lesson from in vitro fertilization and intracytoplasmic sperm injection. Human Reproduction Update 3, Hook EB, Hamerton JL 1977 The frequency of chromosome abnormalities detected in consecutive newborn studies differences between studies results by sex and by severity of phenotypic involvement. In: Hook EB, Porter LH (eds) Population Cytogenetics. Academic Press, New York, pp Jacobs PA 1977 Structural rearrangement of the chromosomes of man. In: Hook EB, Porter IH (eds) Population Cytogenetics Studies in Humans. Academic Press, New York, pp Jacobs PA 1974 Correlation between euploid structural rearrangements and mental subnormality in humans. Nature 249, Jiang J, Manfen F, Wang D 2001 Cytogenetic analysis in 61 couples with spontaneous abortions. Chinese Medical Journal 114, Jung A, Donke N, Tolkendrof E 2002 Cytogenetic study of peripheral lymphocyte culture in couples with habitual abortion. Gynecology 113, Kalpana V, Satyanarayana M, Sunil P, Giri C 2004 Chromosomal aberrations in recurrent aborters. Bionature 24, Madon PF, Arundhati S, Firuzza R 2005 Polymorphic variants on chromosomes probably play a significant role in infertility. Reproductive BioMedicine Online 11, Mattei M, Luciani J 2003 Heterochromatin from chromosome to protein. In: Atlas of Genetics and Cytogenetics in Oncology and Haematology. Infobiogen, France, p. 7. See atlasgeneticsoncology.org/ [accessed 16 January 2007]. Moorhead P, Nowell P, Mellman W et al Chromosome preparations of leukocytes cultured from human peripheral blood. Experimental Cell Research 20, Muneera A, Lulu A, Zainab A et al Cytogenetic study in cases with recurrent abortion in Saudi Arabia. Annals of Saudi Medicine 20, Nagham A, El Ameer, Nagwa A et al Cytogenetic and fluorescence in situ hybridisation study in couples with repeated spontaneous abortion. The Egyptian Medical Journal of the National Research Centre 930, Prabhakara K, Angalena R, Radha Devi 2004 Familial(9;11)(p22;p15. 5) Pat translocation and XX sex reversal in a phenotypic boy with cryptoorchidism and delayed development. Genetic Counselling 15, Rao L, Murthy K, Babu A et al Chromosome inversions and a novel chromosome insertion associated with recurrent miscarriages in South India. Archives of Gynecology and Obstetrics 272, Rowley P, Marshall R, Ellis J 1963 Genetic and cytological study of repeated spontaneous abortion. Annals of Human Genetics 27, Seabright M 1971 A rapid banding technique for human chromosomes. Lancet 2, Tharapel A, Tharapel S, Bannerman R 1985 Recurrent pregnancy losses and parental chromosome abnormalities: a review. British Journal of Obstetrics and Gynecology 92, Wolstenholme J, Faed M, Robertson J et al Chromosome abnormality in couples with histories of multiple abortions. The outcome of pregnancies subsequent to ascertainment and a study of familial translocation carrier. Human Genetics 63, Received 24 October 2006; refereed 13 November 2006; accepted 11 December