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Comparative Study
. 2012 Feb;16(2):113-22.
doi: 10.1089/gtmb.2011.0102. Epub 2011 Oct 24.

Fragile X CGG repeat variation in Tamil Nadu, South India: a comparison of radioactive and methylation-specific polymerase chain reaction in CGG repeat sizing

Nagarathinam Indhumathi  1 , Deepika Singh, Samuel S Chong, B K Thelma, Ramesh Arabandi, C R Srikumari Srisailpathy
Affiliations
Comparative Study

Fragile X CGG repeat variation in Tamil Nadu, South India: a comparison of radioactive and methylation-specific polymerase chain reaction in CGG repeat sizing

Nagarathinam Indhumathi et al. Genet Test Mol Biomarkers. 2012 Feb.

Abstract

Fragile X syndrome is the most frequent hereditary cause of mental retardation after Down syndrome. Expansion of CGG repeats in the 5' UTR of the fragile X mental retardation gene 1 (FMR1) causes gene inactivation in most of the cases. The FMR1 gene is classified into normal 5-44; gray zone 45-54; premutation 55 to <200; and full mutation ≥ 00 repeats. Precise sizing of FMR1 alleles is important to understand their variation, predisposition, and for genetic counseling. Meta-analysis reveals prevalence of premutation carriers as 1 in 259. No such reports are available in India. About 705 women from Tamil Nadu, South India, were screened for the FMR1 allelic variation by using radioactive polymerase chain reaction-polyacrylamide gel electrophoresis (PAGE) analysis. The women who were homozygous by radioactive polymerase chain reaction (rPCR) were reanalyzed by methylation-specific polymerase chain reaction (Ms-PCR) and GeneScan analysis. The techniques were validated and compared to arrive at a correction factor. Among 122 genotypes, 35 repeat variants ranging in size from 16 to 57 were observed. The most common repeat is 30 followed by 29. One in 353 women carried the premutation. No full mutations were observed. Screening populations with low frequency of premutations may not be applicable. Ms-PCR is more suitable for routine screening and clinical testing compared with rPCR-PAGE analysis.

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Figures

FIG. 1.
FIG. 1.
An autoradiogram of FMR1 PCR products. Lanes 1, 4, 5, 9, 10, 13, 14, 16, and 19: normal homozygous woman; lanes 2, 6, 7, 8, 11, 12, and 17: normal heterozygous woman; Lanes 15 and 18: woman with normal and full mutation allele; lane 20: full mutation male; lane 3: woman with normal and premutation FMR1 allele; Lane 21: normal homozygous woman with 29 repeats (reference standard). FMR1, fragile X mental retardation gene 1; PCR, polymerase chain reaction.
FIG. 2.
FIG. 2.
GeneScan profiles of three fluorescent methylation-specific (Ms)-PCR samples used in validation-I. The Met-Triplet primed, met, and nonmet-PCR products appear as black, blue (#), and green (*) peaks, respectively. FMR1 alleles are sized by using Rox-labeled internal size calibrator. The CGG repeat size calculated is indicated in the middle of the peak. The amplicon size is indicated at the bottom of the peak. Color images available online at www.liebertonline.com/gtmb
FIG. 3.
FIG. 3.
Representative GeneScan profiles of four fluorescent Ms-PCR samples used in validation-II. The mTP, met, and nonmet-PCR products appear as black, blue (#), and green (*) peaks, respectively. FMR1 alleles are sized by using Rox-labeled internal size calibrator. The CGG repeat size calculated is indicated in the middle of the peak. The amplicon size is indicated at the bottom of the peak. Color images available online at www.liebertonline.com/gtmb
FIG. 4.
FIG. 4.
Chemiluminescence analysis of 23 samples used in validation-II. Lane 1: normal homozygous woman; lanes 2, 3, 4, 5, 6, 7, 8, 10, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, and 23: normal heterozygous woman; lanes 9 and 11: heterozygous woman with normal and premutation; lane 12: heterozygous woman with normal and full mutation FMR1 allele. Two unmarked lanes are pBR322 digested with MspI, a size marker.
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