Fragile X Syndrome

Fragile X Syndrome (FXS) is a genetic disease inherited through the X chromosome, which was described for the first time in 1943 by Martin and Bell. It is regarded the most common inherited cause of intellectual disability after Down syndrome. FXS is permenantly inherited with many members in the family tree, either affected or a carrier (Ciaccio et al., 2017).
It is inherited as an X linked dominant trait with a fragile site at Xq27.3 locus named fragile X mental retardation gene (FMR 1) (Niu et al., 2017). The FMR1 gene consists of 17 exons spaning 38 kb. The polymorphic CGG (cytosine- guanine- guanine) trinucleotide repeat is located in its 5′ untranslated region of exon 1 within the 4.4 kb of FMR1 transcript. The number of the trinucleotide repeats ranges between 5 and 44 repeats in normal individuals, 45 to 54 triplets in intermediate expansion (gray zone), and 55 to 200 repeats in premutation carriers and beyond 200 repeats in full mutation carriers (Ciaccio et al., 2017).
Fragile X Syndrome results from an expansion mutation of a CGG repeats in the first exon of the FMR1 gene leading to transcriptional gene silencing and absence or remarkable reduction of its product, fragile X mental retardation protein. This protein is important for proper neuronal morphology, cognitive development and synaptic plasticity and its absence leads to changing levels of Intellectual disability (ID). Boys are more seriously affected than girls because FMR1 gene is located on the X chromosome and the presence of an unaffected second X chromosome in girls (Kidd et al., 2014).
In addition to ID, there are notable phenotypic characteristics of FXS that may be present (Rajaratnam et al., 2017). Phenotypic manifestations common to several clinical checklists include large forehead, large ears, elongated face, simian crease, high arched palate, large testicles, hyperextensibility of joints, tactile defensiveness, hyperactivity, poor eye contact, short attention span, hand biting, hand flapping , perseverative speech and a positive family history of mental retardation. To increase the effectiveness of the screening programs, preselection of subjects based on clinical features is required (Guruju et al., 2009).
Rapid screening of large numbers of individuals suspected to have FXS could be facilitated by methods based on the polymerase chain reaction (PCR). It accurately detects the number of CGG repeats (Panagopoulos et al., 1999). PCR techniques for amplification of CGG repeats are, however, inefficient and unreliable because of their 100% C+G composition. Thus, most of the described PCR protocols require subsequent Southern blot analysis and autoradiography (Kanwal et al., 2015).
Methylation sensitive PCR is found to be a quick and relatively inexpensive technique useful in the diagnosis of fragile X syndrome. The primary principle of that method is that it depends on the ability of bisulphite to deaminate C residues in a single strand DNA. A characteristic of the bisulphite treated DNA is that after modification, the sense and antisense strand are no longer complimentary. Thus, the modified strands can be amplified separately by designing primer pairs specific for each of them. The C residues of all CpG dinucleotides flanking the CGG repeats as well as those of the CGG repeats are methylated in affected males and in the inactive X chromosome in females (Karunasagar et al., 2005). The same C residues are however, unmethylated in healthy males, normal transmitting males and in the active X chromosome in females. The disadvantage of this methylation PCR is that it cannot reliably diagnose affected females with fragile X syndrome due to the fact that the inactive X chromosome is already methylated. During screening, all positive female samples will have to be subjected to Southern blot analysis for confirmation of diagnosis (Karunasagar et al., 2005).
The aim of the present work was to determine the expected alleles for FMR 1 gene by methylation sensitive PCR based method. Clinical correlation to molecular characterization was also evaluated.

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