Screening of rural children in West Bengal for Fragile-X syndrome

Samikshan Dutta, Manali Das, Aneek Das Bhowmik, Swagata Sinha, Anindita Chattopadhyay & Kanchan Mukhopadhyay

Manovikas Biomedical Research & Diagnostic Centre, Kolkata, India

Received October 17, 2007

Background & objectives: Screening for Fragile X syndrome (FRAXA), the most common genetic cause for mental retardation (MR), has mostly been carried out among MR patients. The present study was conducted to find out prevalence of FRAXA amongst children residing in the rural areas of West Bengal.

Methods: Demographic details including age, sex, nutritional status as well as birth, medical, and developmental histories, were collected amongst rural children (n=38,803) of West Bengal, India, over three years (2004-2007). Based on the records of scholastic backwardness, 179 children were short-listed and examined by a team of experts comprising of child psychiatrist, clinical psychologist, paediatrician and special educator. Blood samples were collected and molecular and cytogenetic studies were performed for identification of CGG repeats and determination of FMR1 gene promoter methylation.

Results: Of the selected 179 children, six were diagnosed as Down syndrome, one as cerebral palsy and 140 as non-syndromic MR. These 140 children with MR were grouped as mild (56), moderate (60), and severely (4) retarded based on IQ; children <5 yr were grouped as developmental delay (20). FRAXA was not detected in any of these children (frequency being 0% with 0-.02% confidence interval). Prevalence of MR was found to be low (about 4/1000 children). Down syndrome also had a lower frequency (0.15/1000 children).

Interpretation & conclusion: The data obtained in the present study indicated that familial disorders like FRAXA were less frequent in the studied population.

Key words Environment - Fragile-X syndrome - genetic factors - malnutrition - mental retardation

Fragile X syndrome (FRAXA) (MIM 309550) is the most common cause of monogenic X-linked mental retardation (XLMR) with a worldwide prevalence of nearly 1 in 4000 males and 1 in 8000 females1. Primary features of FRAXA are mild to severe mental retardation2 (MR), macro-orchidism3 associated with connective tissue disorder such as hyperextensible finger joints, double jointed thumbs, etc. In most cases, a fragile

site is detected at Xq27.3, which harbours the FMR1 gene4. Positional cloning of FMR1 gene has revealed expansion of a tri-nucleotide CGG repeat (>200-220 repeats; i.e., full mutation) at the 5'UTR of exon 1 in association with extensive methylation of the promoter5, hindering expression of fragile X mental retardation protein (FMRP). Routine clinical analysis has shown that in normal individuals CGG repeat length varies

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from 6-50 repeats. Repeats with >50 to 200 CGG units are responsible for increased transcriptional activity6 of FMR1 and individuals carrying these fall under pre-mutation category, since pre-mutation repeats are susceptible to further expansion. The exact cause of repeat expansion is not known, but is assumed to occur during DNA replication of proliferating cells7. Syndromic manifestation of FRAXA is observed only in cases with hypermethylated CpG island8. However, individuals with a rare mutation having un-methylated >200 CGG repeats shows normal intelligence9. Studies have shown that triple repeat instability depends on the increasing repeats in pre-mutation zone and it only increases if maternal transmission is considered10. Therefore familial screening, with special emphasis to the maternal side, is necessary for identification of the disorder.

Studies carried out on Indian FRAXA patients show a frequency of ~7 per cent among mentally retarded males attending different types of outpatients11-15. The present study was conducted to explore the occurrence of fragile X phenotype especially FRAXA amongst rural children of West Bengal to assist in early developmental intervention.

Material & Methods

Population screening: A three step screening programme was implemented for identification of FRAXA cases among the rural population of Sonarpur block, South 24 Parganas, in the State of West Bengal, India. The site was selected because of its close proximity to the organization and location of a branch office of Manovikas Kendra Rehabilitation and Research Institute for the Handicapped (MRIH) in that locality. Initially, all 11 Gram Panchayats in the Sonarpur block with an estimated population of 1,45,000, were targeted. Of these, 7 Gram Panchayats (population size ~ 80,250) actively participated in the programme and were screened for FRAXA. The remaining four Gram Panchayats were excluded from the study. Actual assessment was carried out on more than 38000 children in 7 Panchayats, with a fraction of 0.76 of estimated children. Elimination of four Gram panchayats may not have resulted in any bias because the study was designed to estimate the frequency of MR/FRAXA in the population actually screened which was done with a confidence interval of less than 0.4 (estimated sample size needed was 33767 with a 99% confidence at 0.4 confidence interval)16 .

At first, door-to-door survey for MR was conducted by a group of rural community workers,

trained and employed by MRIH during 2004-2007 and details regarding age, sex, family income, nutritional status of the family, educational and medical status of family members, family history, maternal delivery history, birth details of children and present status of the affected individuals, if there is any were collected using a structured questionnaire.

At the second stage, data collected were analyzed by the mental health professionals to identify candidates for further screening. Inclusion criteria for selection at this stage were based on record of scholastic backwardness, developmental delay, clinical manifestation, cognitive disability, birth defect and family history of MR.

Recruitment of MR subjects: In the third and the final stage, a team of child psychiatrist, paediatrician, clinical psychologist and special educator examined the short-listed candidates. Mental health status was assessed following the Diagnostic and Statistical Manual of Mental Disorders – IV17. IQ of children above 5 yr was measured by Wechsler Intelligence Scale for Children18 and cases were classified as mild, moderate or severe on the basis of IQ. For children below 5 yr, developmental quotient (DQ) was measured as a ratio of the developmental age to the chronological age using the Developmental Screening Test19 and those with DQ <85 were separately grouped under developmental delay (DD) category. The study protocol was approved by the Human Ethical Committee of MRIH.

Molecular analysis: Peripheral blood samples were collected from the cubital vein after obtaining informed written consent from the parents/guardians of the proband. Genetic testing for FRAXA was carried out for all the family members of MR cases recruited.

Detection of fragile X mental retardation protein (FMRP): Peripheral blood smear on glass slides were collected and stored at -20°C (<2 wk). Prior to staining, smear was fixed with 3 per cent paraformaldehyde (Sigma, USA) in phosphate buffer (pH 7.3), permeabilized with methanol (SRL, India) and immunostaining of peripheral blood lymphocytes20,21 was carried out. Incubation with 1st antibody (goat FMRP-specific polyclonal antibody, Santa-Cruz Biotechnology, USA) was carried out for 2 h. A 2nd

antibody (rabbit anti-goat-FITC conjugate, Santa-Cruz Biotechnology, USA) was added for 60 min at room temperature. Cells were viewed under Zeiss Axioskop 2 plus fluorescence microscope (Germany). For each slide, nearly 1000 cells were scored, and per cent of

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leukocytes exhibiting immunofluorescent staining (i.e., expressing FMRP) was calculated (Fig. 1).

Identification of CGG repeats: Genomic DNA was isolated by standard high salt precipitation method22 from peripheral blood collected in EDTA and used for PCR amplification. Primer sequences used to amplify the CGG repeat region were procured from Sigma, USA. PCR amplification was carried out using Perkin Elmer thermal cycler (Gene Amp #2400, USA), in a final reaction volume of 20 µl containing 75 ng of genomic DNA, 20 pmoles of each primer, 1.0 U Taq polymerase (Bangalore Genei, India), 200 μM dNTP mix (Bangalore Genei, India), 1X magic amplification solution (Bangalore Genei, India) 10 mM Tris buffer (NEB, USA) with 50 mM KCl and 2.0 mM MgSO4. After an initial denaturation at 95°C for 5 min, amplification was performed for 35 cycle of denaturation at 94°C for 1 min, annealing at 68°C for 40 sec and extension at 74°C for 40 sec; cyclic reaction was followed by a final extension at 74°C for 5 min. PCR amplicons were analyzed by 12 per cent polyacrylamide gel electrophoresis and repeat sizes were determined using DNA marker (Fig. 2).

Determination of FMR1 gene promoter methylation: Analysis of methylation status of the FMR1 promoter and an internal control Xist gene (X-inactive specific transcript) was carried out following the method of Weinhausel and Hass23. Genomic DNA (approximately 5 μg) was primarily deaminated using EZ DNA methylation kit (Zymo Research, USA) and was used for methylation analysis. PCR was carried out in a final reaction volume of 20 µl containing 75 ng of deaminated DNA, 5 pmoles of each primer, 1.0 U

Taq polymerase, (Bangalore Genei, India) 200 μM dNTP mix, 1X Taq buffer B and 1.5 mM MgCl2. This PCR condition simultaneously amplified sequences of FMR1 and Xist gene23. After an initial denaturation at 95°C for 5 min, amplification was performed for 35 cycle of denaturation at 95°C for 30 sec, annealing at 60°C for 20 sec and extension at 72°C for 40 sec; cyclic reaction was followed by a final extension at 72°C for 7 min. Fragment sizes were analyzed by 2.5 per cent agarose gel electrophoresis (Fig. 3).

Cytogenetic analysis: Peripheral blood of probands, collected in heparin, was used to carry out lymphocyte culture in folate deficient as well as folate enriched RPMI 1640 media (Sigma, USA)24 for detection of Xq27.3 deletion and other chromosomal abnormalities25 respectively. Plasma or whole blood (0.5 ml), added to 4.5 ml of medium (Gibco BRL, USA) supplemented with 10 per cent foetal bovine serum (Gibco BRL, USA) and

Fig. 1. Peripheral blood leukocyte stained with anti-FMRP-goat Ab and anti-goat rabbit IgG-FITC conjugate.

Fig 2. Pictorial presentation of CGG repeat units in the FMRI gene promoter region. Lane 1: contains 21, 30 and 107 repeats; Lane 2: contains 21, 28, 86 and 91 repeats; Lane 3: 30 repeat; Lane 4 : fX174 HaeIII digest DNA marker.

Fig. 3. Analysis of methylation status of the FMR1 and Xist gene promoter regions by methylation specific polymerase chain reaction. Lanes 1 & 2 normal female with unmethylated (PU) and methylated (PM) promoter of FMR1 and Xist (XU, XM) genes; Lanes 3 & 4 normal male with PU and XM.

716 INDIAN J MED RES, DECEMBER 2009

100 ng phytohaemagglutinin (PHA-M, Gibco BRL, USA), was incubated for 69 h at 37°C in presence of 5 per cent CO2 (Incubator, Heraeus, Germany). At the 69th h, 0.3 µg of colcemid (N-deacetyl-N-methylcolchicine; Gibco BRL, USA) was added to arrest cell division. Cultures were harvested after 45 min of colcemid treatment, subjected to centrifugation, treated with 0.075 M KCl solution (Merck, India) and incubated at 37°C for 25min. It was centrifuged at 1000 g for 10 min, followed by fixation of cell pellet in Carnoy’s fixative (methanol: acetic acid: 3:1) (SRL, India). Two to three subsequent washes in fixative were given to remove remaining cell debris.

GTG banding analysis26 was carried out with fixed cells. Cells on glass slides were digested with 0.5 per cent trypsin (Difco, USA) and stained in 1 per cent Giemsa (BDH, India). Slides were air dried and viewed under oil-immersion lens of Zeiss Axioskop 2 plus microscope for evaluation. At least 50 well-spread metaphase plates were counted and dividing cells in late prophase/early metaphase with about 450 band resolutions were karyotyped using the karyoimager software (Carl Zeiss, Germany).

Results

A total of 38,803 children and other members from 19,810 families were screened. Depending on the selection criteria, 179 children were short-listed for the final stage screening. Among these children, 147 were diagnosed as MR and others were found to have normal IQ but with various problems leading to scholastic backwardness (29 cases with bilateral hearing defect/

speech problem, 2 with affective disorder or conduct disorder, and 1 with attention deficit).

Of the 147 children with MR, 140 were identified as non-syndromic MR, and were further classified into developmental delay (n=20; DQ <85), mild (n=56; MiMR, IQ 66.03±9.85), moderate (n=60; MoMR, IQ 45.93±4.03) and severe MR (n=4; SMR, IQ 29.75±1.71) categories. Remaining seven cases were identified as Down syndrome (DS, n=6) and cerebral palsy (n=1). Overall frequency of MR children in the studied population was approximately 4/1000 (147/38803); male: female ratio for MR was approximately 1.3 (age average 10.66 ± 4.84 yr).

Dysmorphic facial features were observed in <10 per cent cases (Table I). Speech problem was in about 36 per cent cases while hearing problem was observed in only a few (about 6%).

Analysis of leukocyte FMRP showed 46.58±12.49 per cent positive cells in MR cases (Fig. 1; Table II). CGG repeat analysis revealed presence of 25 diffferent alleles with repeat numbers varying between 13-107 (Fig.2, Table II). Methylation analysis of FMR1 promoter and Xist genes revealed normal methylation pattern for both the genes (Fig. 3; Table II). Data obtained revealed 0 per cent frequency of FRAXA in the studied population with 0-0.02 per cent confidence interval.

Discussion

During this rural screening programme for FRAXA frequency of MR was found to be low as (4/1000 i.e.,

Table I. Details of children with MR (n=147) No. of children screened

No. of

affected

Malnutrition Co-morbid features Dysmorphic featuresConduct/

behavioral disorder

Speech problem

Hearing problem

Elongatedface

Hypertelorism Ear anomalies

Microcephaly Macrocephaly

38,803 147 38(25.85)

19(12.95)

53(36.05)

9(6.12)

7(4.76)

10(6.80)

14(9.52)

10(6.80)

1(0.68)

Most of the MR probands exhibited more than one clinical featureValues in parentheses are percentages

Table II. Evaluation of MR cases for diagnosis of FRAXANo. of sample % of FMRP positive

leukocytes (range)No. of CGG repeats

(range)Promoter methylation status Xq27.3 deletionMale Female

PM PU PM PU179 46.58 + 12.5 (30-70) 25 (13-107) -ve +ve +ve +ve None

PM, fmr1 promoter methylated; PU, fmr1 promoter unmethylated; female have two X chromosomes, one active (unmethylated) and the other inactive (methylated). On the other hand, male have single X chromosome (unmethylated)

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about 0.4%). An unusually low frequency of SMR amongst rural children was also noticed (~ 0.1 per 1000) in contrast to the other developing countries with poor socio-economic status, where prevalence rate of SMR was found to be much higher27 (> 5 per 1000 children). In our study, frequency of DS among live births was also very low (~0.15 per 1000 individuals) as compared to the global frequency (1.2/1000)28 and other studies29,30 from India. Another observation was lack of any gross chromosomal abnormality in the 147 MR cases studied apart from six DS cases. Disorders like FRAXA, Huntington, bipolar disorder, attention deficit hyperactivity disorder, etc., which are known to have a genetic basis, were apparently absent. Whether this is due to rarity of these disorders in this rural population or is due to some other unknown factor is yet to be worked out.

A Kolkata-based study (n=158; 60 controls and 98 MR) reported 21 distinct normal CGG repeats (8-44) and among MR individuals, 7 per cent cases with higher repeats were confirmed as FRAXA14. Studies from north India (predominantly Caucasoid population) revealed variations in the frequency of FRAXA amongst male MR cases. Studies on Delhi-based MR patients revealed about 8 per cent FRAXA13,31 where as that from Lucknow showed 2.5 per cent prevalence32. Another investigation on hospital-based samples revealed about 10 per cent FRAXA in Indian population of mixed origin29. From the southern part of India, CGG repeat analysis of the FMR1 gene showed the 28 and 31 repeat alleles to be most predominant11. In contrast, in our study, 25 alleles were observed with a variation in repeat sizes within 13-107; the 30 repeat allele was the most frequent (26.43%) followed by the 26 repeat (11.43%) and 28 repeat (10%) alleles. Frequency of FMRP positive leukocytes was more than 25 and methylation pattern was normal. Two individuals, who have CGG repeats in the premutation zone (ID 424 with 21/28/86/97 and ID 438 with 21/30/107 CGG repeats), also exhibited normal methylation pattern of the promoter and absence of any prominent clinical feature. All these findings point toward the absence of FRAXA in the studied population.

In conclusion, our findings indicate that the distribution pattern of CGG repeat is extremely heterogeneous even within the same ethnic group. The studied population had a low frequency of MR as compared to other parts of India and none had FRAXA. It would be interesting to study more people in the rural areas to validate our observation.

Acknowledgment Authors acknowledge the Department of Biotechnology, New Delhi, for financial support. The first author (SD) thanks the Indian Council of Medical Research (ICMR), New Delhi, for providing senior research fellowship. Authors also thank the rural people and Gram Panchayat Pradhans and the rural community workers for active participation during the project.

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Reprint requests: Dr Kanchan Mukhopadhyay, Manovikas Biomedical Research & Diagnostic Centre, 482, Madudah Plot I-24, Sec.-J, E.M. Bypass, Kolkata 700 107, India e-mail: kanchanmvk@yahoo.com

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