Cytogenetic Diagnosis of Fragile X Syndrome: Study of 305 Suspected Cases in Saudi Arabia

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1 Cytogenetic Diagnosis of Fragile X Syndrome: Study of 305 Suspected Cases in Saudi Arabia M. Anwar Iqbal, PhD, FACMG; Nadia Sakati, MD; Michael Nester, PhD; Pinar Ozand, MD, PhD From the Departments of Pathology, Section of Cytogenetics/Molecular Genetics, Pediatrics, Sections of Pediatric Genetics/Endocrinology and Metabolic Diseases, and Neurosciences, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia. Address reprint requests and correspondence to Dr. Iqbal: Section of Cytogenetics/Molecular Genetics, Department of Pathology and Laboratory Medicine, MBC-10, King Faisal Specialist Hospital and Research Centre, P.O. Box 3354, Riyadh 11211, Saudi Arabia. iqbal@kfshrc.edu.sa. Accepted for publication 27 June Received 4 July MA Iqbal, N Sakati, M Nester, P Ozand, Cytogenetic Diagnosis of Fragile X Syndrome: Study of 305 Suspected Cases in Saudi Arabia. 2000; 20(3-4): Background: Fragile X syndrome is the most common cause of inherited mental retardation. Patients with fragile X syndrome show variable mental disability, typical long and narrow facial appearance with large ears and prominent fontanelle and frequent macro-orchidism. It is generally associated with a fragile site at Xq 27.3, which can be observed in the metaphase chromosome following selective culture conditions. At the molecular level, the fragile X syndrome is associated with an amplification of CGG repeat sequence of the FMR1 gene. The prevalence estimates are reported as one per 1500 males and one per 2500 females. Estimated prevalence rates of fragile X syndrome in different ethnic groups range from per 1000 in males and per 1000 in females. In this study, we have determined the frequency of fragile X-positive cases in 305 preselected patients. Materials and Methods: Three hundred and five Saudi patients with mental retardation/developmental delay/clinical suspicion of fragile X syndrome were screened for fragile X chromosome by cytogenetic methods. The majority of patients (95.59%) screened were under the age of 20 years. Results: Two hundred and ninety-nine patients (98.03%) were in the category of mild to moderate mental retardation. Twenty-four males (7.86%) and two females (0.65%) were found to express fragile X site at q27.3. The frequency of fragile X-positive cells in males ranged between 7% and 58% (mean 26±13.11), while in the females it was between 14% and 21% (mean 12.5±35), respectively. Conclusion: The frequency of fragile X positive cases found in this study is similar to other reports of fragile X syndrome in preselected patients. Ann Saudi Med 2000;20(3-4): KEY WORDS: Fragile X syndrome, mental retardation. The fragile X syndrome is the most common cause of inherited mental retardation. The syndrome is mainly characterized by a variable degree of mental retardation, typical long and narrow facial appearance with large ears, prominent fontanelle and large testes. 1,2 The syndrome is inherited as an X-linked dominant trait with reduced penetrance: 80% in males and 30% in females. 3 The penetrance of the disease was shown to increase in successive generations in a fragile X family. 4,5 This peculiar mechanism of variable penetrance in the fragile X syndrome is frequently termed the Sherman paradox. 2 The syndrome is also associated with a fragile site at Xq27.3, designated as FRA X A, which can be observed in the metaphase chromosome following selective culture conditions. In 1991, the gene responsible for fragile X syndrome was identified, and was designated as fragile X mental retardation 1 (FMR1). 6-8 At the molecular level, the FMR1 gene has a size of 38 kb with 17 exons 8 and an untranslated polymorphic DNA sequence made up of CGG repeats within the first exon. 9 The FMR1 gene contains between 6 and 54 CGG repeats in normal individuals. 10 Amplification of CGG repeat sequence in affected fragile X syndrome patients was shown to be greater than 200 repeats to lengths exceeding The lengths of the CGG repeat in the phenotypically normal transmitting male and obligate female carriers range from CGG repeats, and are generally known as premutation alleles. Two of the fragile sites distal to the FRA X A in the Xq27.3-Xq28 regions have been cloned. These are termed FRA X E 11 and FRA X F, 12 both of which can be detected by routine cytogenetic methods, although differentiating between all three fragile sites is extremely difficult. FRA X E positive individuals were shown to be associated with a milder form of mental retardation compared to fragile X syndrome. Both FRA X E and FRA X F were shown by molecular techniques to be due to expansion of GCC trinucleotide repeats in the vicinity of CpG islands in the Xq28 region

2 The laboratory diagnosis of fragile X syndrome can be achieved easily and reliably by molecular techniques such as Southern blotting or polymerase chain reaction (PCR) methodology. In addition, fragile X chromosome screening by special cytogenetic techniques can also diagnose the syndrome in a majority of affected males (99%) and in 90%-95% of affected females. 13 Recently, a comparison of the results of direct DNA analysis for the CGG repeat were shown to be in complete agreement with the cytogenetic diagnosis of fragile X syndrome in a study of 434 mentally retarded Japanese patients. 14 Table 1. Age distribution of 305 mentally retarded patients screened for fragile X chromosome (males=259; females=46). Age No. of males % No. of females % > Table 2. Summary of clinical features observed in 259 mentally retarded male patients screened for fragile X chromosome. Clinical feature Number of cases Percentage Autism Seizures Hyperactivity Poor eye contact Perseverant speech Large ears Large testis Consanguinity (first cousin) Family history of MR Degree of MR Moderate (IQ >50) Severe (IQ <50) Table 3. Summary of clinical features observed in 46 mentally retarded female patients screened for fragile X chromosome. Clinical feature Number of cases Percentage Autism Seizures Hyperactivity Poor eye contact Perseverant speech Large ears Consanguinity (first cousin) Family history of MR Degree of MR Moderate (IQ >50) Severe (IQ <50) Based on fragile X screening by cytogenetic methods, prevalence rates of fragile X syndrome in different ethnic groups have been reported. The prevalence rate estimates range from one per 1500 males to one per 2500 females. 15 In the present study, the frequency of fragile X-positive cases was determined in 305 Saudi patients from cytogenetic data taken between the years 1989 and These patients were selected on the basis of clinical criteria compatible with mental retardation/developmental delay or fragile X syndrome phenotype.

3 Materials and Methods A total of 305 patients were referred from the Pediatric Department of the King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia. The hospital serves as a tertiary care facility for the Kingdom of Saudi Arabia. Patients in this study were from various regions of the Kingdom. The main criteria for fragile X chromosome analysis in these patients was mental retardation, development delay or clinical suspicion of fragile X syndrome. Patients were also screened for biochemical defects, and those with a positive biochemical test result were excluded from the study. Systematic neurological, psychometric and genetic evaluations were conducted on each patient and various clinical features were recorded. The degree of mental retardation was categorized into two groups based on cognitive testing: a mild/moderate group with test scores showing IQ greater than 50, and a moderate/severe group with IQ less than 50. This somewhat arbitrary division of our sample into two groups was done to best categorize our patients within the limits of our test instrumentation. Children were evaluated primarily using the Leiter International Performance Test, 16 an instrument with minimal cultural and linguistic bias, used extensively over the past 10 years at our institution. 17 The tests were administered and scored by Saudi technicians under supervision of a qualified neuropsychologist. For cytogenetic analysis, peripheral blood samples were set up according to standard laboratory protocol for fragile X analysis. A combination of two culture systems was used: 1) media 199 with 5% fetal bovine serum, antibiotics, and 300 μg/ml thymidine; and 2) media 1640 with 5% fetal bovine serum, 0.25 μg/ml 5-fluoro- 2'deoxyuridine (FudR) and 300 μg/ml thymidine. All cultures were incubated at 37 C for 72 hours and harvested after the addition of two drops of colcemid (10 μg/ml) with a 21 g needle for 2 hours. Afterwards, the cultures were treated with 0.4% potassium chloride hypotonic solution and fixed in 3:1 methanol acetic acid mixtures. Slides were Giemsa-banded and 50 to 100 metaphases were scored for the fragile X site at Xq27.3. A fragile X-positive lymphoblastoid cell line or scoring of common fragile sites was utilized to determine the efficacy of the culture conditions for fragile X expression. A routine chromosome analysis was also performed on all cases, and those with other chromosomal abnormalities were excluded from the study. Results Fragile X analysis results were obtained in all the cases. There were 259 male (84.91%) and 46 female (15.00%) patients. The majority of patients (95.59%) were under the age of 20 years (Table 1). The various clinical features for males and females, respectively, are shown in Tables 2 and 3. Overall hyperactivity was observed in 117 cases (38.36%), seizures in 59 cases (21.85%), autism in 29 cases (10.74%), consanguinity was found in 32 patients (12.13%), and a family history of mental retardation was recorded in 81 cases (26.55%). In the mild/moderate group, 299 patients (98.03%) were found and only six patients were in the moderate-severe group. Out of 305 patients, 24 male (7.86%) and 2 female (0.65%) patients were found to express the fragile X site at q27.3 (Table 4). The frequency of fragile X-positive cells in males ranged between 7% and 58% (mean 26±13.11), while in the females it was between 14% to 21% (mean 12.5±3.5), respectively. Table 4 summarizes the clinical features as percentages in patients with positive test results for fragile X. The most common clinical features observed in fragile X-positive cases were hyperactivity (92.3%), mild/moderate mental retardation (100%), family history of mental retardation (46.15%), large ears (30%) and large testes (15.38%). Autism was not recorded in any of the fragile X-positive patients. Similarly, none of our fragile X positive cases exhibited severe mental retardation. Discussion The present study of fragile X screening in 305 patients with mental retardation or developmental delay is the first survey from Saudi Arabia. A number of genetic epidemiological studies from various geographical areas have previously been reported based on cytogenetic screening or DNA testing Prevalences of /1000 in males and /1000 in females were reported based on cytogenetic screening However, de Vries recently reported a prevalence of 0.6% by combining DNA analysis studies from Europe, Australia and the United States. 24 The frequency of new fragile X cases may. be significantly increased if patients are preselected based on abnormal physical features In selected populations of mentally retarded patients, an overall prevalence rate of 4.8% of fragile X was reported by Proops et al. 30 Based on fragile X screening of the mentally impaired from three different sources community placement, day care facilities and special education schools Jacobs et al. reported an overall frequency of 1.9% fragile X males and 0.3% fragile X females. 31 Different estimates of fragile X-positive cases among patients with severe and mild mental retardation are also available. In our study, all the fragile X- positive cases were from the moderate (IQ>50). This is consistent with the recent report in which it was found that

4 the degree of mental retardation in most prepubertal fragile X males is moderate, and in adults it is moderate to severe A correlation between macro-orchidism and fragile X syndrome has also been reported, however, macroorchidism is generally difficult to identify early in life and is frequently absent in younger patients. 1-2 However, different frequencies of fragile X-positive cases were reported among patients with macro-orchidism as one of the clinical features; this figure ranges from 11% to 80%. 15 In our study, since the majority of patients (83.43%) were of prepubertal age (5-15 years), machro-orchidism was found in only six cases. Among these, four patients were positive for fragile X chromosome (66%). This is similar to published reports in which 70% of fragile X males were shown to have macro-orchidism. Table 4. Frequency of typical clinical features in 26 fragile X positive patients. Clinical feature Number with feature Percentage Autism 0 0 Seizures Hyperactivity Poor eye contact Perseverant speech Large ears 8 30 Large testis Consanguinity (first cousin) Family history of MR Degree of MR Moderate (IQ >50) Severe (IQ <50) 0 0 Table 5. Mean number of cells expressing fragile X site in male and female patients. Number of patients with fragile X Percentage Mean no. of cells with fragile X Male ±13.1 Female ±3.5 Studies also suggest a relationship between autism and the fragile X syndrome, however, the relationship between these two characteristics is still questionable More recently, Kluck et al. 37 tested 141 autistic patients for the amplification of the CGG repeat at the fragile X syndrome locus by Southern blot analysis. They did not find any autistic patients with fragile X syndrome. In our study, autism was found in 10.74% of the patients, and none of these were found to be fragile X-positive. Fragile X in patients with a family history of mental retardation (MR) has also been reported. Carpenter et al. 38 studied 36 patients with a family history of MR and found 13.9% to be fragile X-positive cases, whereas Froster- Iskenius et al. 39 studied more than 200 patients with a family history of MR and found only 3.6% to be fragile X- positive. The authors in the present study found 81 patients with a family history of MR; among these, 12 patients (14.8%) were found to be fragile X-positive, which is similar to the report by Carpenter et al. 38 Seizures have been observed in 20% of fragile X males and 5% of females. 2,40-41 Interestingly, in our study population, approximately 4% of fragile X patients were affected with seizures. In summary, our result of 8.52% of fragile X-positive cases among 305 mentally retarded Saudi patients is similar to reports in other parts of the world. The findings presented in this study are mainly derived from data at a time when fragile X diagnosis was done by cytogenetic analysis. However, DNA testing for fragile X, which is more sensitive and cost effective, is currently being used at our center for diagnostic purposes. Nevertheless, cytogenetic analysis should be done initially on all cases referred for fragile X testing to detect cases which show other chromosome abnormalities besides FRA X A or FRA X E. Acknowledgements The authors thank the technical staff of the Cytogenetics Laboratory for their effort in this study. The authors also thank Rita Johnston for her secretarial assistance.

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