The present study was conducted on 50 patients with mental retardation and clinical features suggestive of fragile X syndrome

The present study was conducted on 50 patients with mental retardation and clinical features suggestive of fragile X syndrome. The patients were referred to the genetic’s clinic, Abo El-Reech hospital Kasr El Ainy Medical school. They were 50 males. Their ages ranged from 3 to 21 years. 50 healthy age matched volunteers were also conducted as a control group. Their age ranged from 3 to 17 years. Informed consents were obtained from adult patients and parents of studied children. The study design was approved by the Scientific Research Committee of the Clinical Pathology Department, Faculty of Medicine, Cairo University.
All patients were subjected to full history taking including family history. Thorough clinical examination with carful assessment of clinical and neurological features using a 15-item checklist (Guruju et al., 2009) that includes physical (big ears, joint hyperextensibility, Simian crease, wide forehead, macro-orchidism and elongated face) and neurological features (mental retardation (MR), family history of MR, poor eye contact, hand biting, hyperactivity, perservative speech, tactile defensiveness, hand flapping and short attention span).
Cytogenetic Studies: Peripheral blood samples were collected for chromosomal analysis using GTG-banding technique (Verma and Babu, 1995). Metaphases with good banding quality were karyotyped using image analysis system (Applied Imaging USA). Individual chromosomes were identified and arranged according to the International System for human cytogenetices and nomenclature ISCN (2016).
Genotyping Method for the detection of expanded alleles of the FMR1gene by methylation sensitive PCR: Three mL blood were withdrawn aseptically from every patient and control then collected in sterile ethylenediaminetetra-acetic acid (EDTA) vacutainer tube. DNA was extracted from the whole blood using DNA extraction kit (GeneJET™ Genomic DNA Purification Kit, Fermentas LIFE SCIENCE, catalogue number: #K0721, Vilnius, Lithuania). Based upon the method described by (Chaudhary et al., 2014), extracted DNA was used in the bisulfite reactions. PCR amplification of the methylated CpG island located upstream of the repeats (The primers were designed for the modified antisense strand and are specific for PCR amplification of the methylated C residues present in affected individuals and on the inactive X chromosome in normal females). Forward Primer (5′-AAC GAC GAA CCG ACG ACG-3′) and reverse Primer (5′-CGT CGT CGC GTT GTC GTAC-3′).
All reactions were performed in a total volume of 25 ?l. After an initial denaturation step (5 min at 94°C), the samples were subjected to 32 cycles of 93°C for 30 seconds, 65°C for 30 seconds , and 72°C for 30 s, with a final extension of 10 min at 72°C.
PCR amplification of fragments containing the unmethylated CGG repeats (The primers were designed for the modified antisense strand after bisulfite treatment of DNA for amplifying the unmethylated sequence in normal males). Forward primer (5′-CAA CCT CAA TCA AAC ACT CAA CTC CA-3′) and reverse primer (5′-GGG AGT TTG TTT TTG AGA GGT GGG -3′) (Chaudhary et al., 2014).
All reactions were performed in a total volume of 25 ?l. After an initial denaturation step (5 min at 94°C), the samples were subjected to 32 cycles of 93°C for 60 seconds, 58°C for 60 seconds , and 74°C for 120 s, with a final extension of 10 min at 74°C. PCR products were analyzed on a 2% agarose gel.
Normal males showed no bands using methylated primers and a band at 280 bp representing the normal range of 19 to 40 CGG repeats by unmethylated primers. FXS-positive males showed band at 80 bp using methylated primers and nothing by unmethylated primers. Premutation carrier males showed no bands using methylated primers and band at 400bp by unmethylated primers.