Introduction Sandeepa Chauhan, Madhumita Roy Chowdhury, Neerja Gupta, Sheffali Gulati*, BK Thelma #, Anjali Dabral #, Madhulika Kabra Genetic Unit, Department of Pediatrics, *Department of Pediatrics, AIIMS, New Delhi # Department of Genetics, University of Delhi South Campus, New Delhi Fragile X syndrome is the most common inherited mental impairment, affecting roughly 1 in 4,000 males and 1 in 7,000 females, arising from expansion of CGG repeats in the FMR1 gene. Our laboratory strategy was to do initial screening by PCR method and to test PCR positive cases and family members including at-risk females by Southern blotting. Recently, we have started using AmplideX FMR1 PCR kit to validate our results. Eight patients clinically diagnosed with Fragile X Syndrome were screened by PCR. Southern blotting was done to confirm the result and to detect the carrier status of 25 females in these eight families. AmplideX FMR1 PCR Kit was used to detect the accurate size of alleles up to 200 CGG. Out of eight males, all were found to be affected by all three techniques used. Out of 25 females, full mutation (FM) was found in 3, premutation (PM) in 13 and 9 females were normal Fragile X arises from expansion of CGG repeats in the FMR1 gene. Generally, in normal individuals there are 28 to 32 repeats. Expansions between 55 to 200 (PM) are associated with autism spectrum disorders, reproductive disorders in females (FXPOI), and cognitive/motor disorders in both older males and females (FXTAS). An expansion of repeats to >200 (FM) results in inactivation of the gene through methylation of CpG islands. Applying AmplideX FMR1 PCR Kit to detect the actual number of CGG repeats in the FMR1 gene to identify the carrier status of females and validating the results with Southern blot. Eight patients clinically diagnosed with Fragile X Syndrome were initially screened by PCR. Southern blotting was done to confirm the result and to detect the carrier status of 25 females in these eight families. AmplideX FMR1 PCR Kit was further used to detect the accurate size of alleles up to 200 CGG, identification of FM alleles >200 CGG and a characteristic product peak profile that resolves zygosity in females samples. FMR1 Gene PCR amplification In PCR simultaneous amplification of FRAXA and FRAXE triplet repeats is carried out using 100 ng of DNA in 25 µL reaction Results 5.2Kb Southern Blot PM N – Normal FM – Full mutation PM – Pre mutation Southern blotting was done by using PstI enzyme for Premutation and EcoRI and EagI for Full mutation N N FM N FM FM N N AmplideX ™ FMR1 PCR Kit Protocol The test protocol involves three key sets of procedures: PCR master mix setup and thermal cycling Capillary electrophoresis Fragment sizing analysis Conversion peak size to CGG repeat length After capillary electrophoresis, the size of the target amplicon is derived from comparison to a co-injected size standard, e.g. ROX 1000 Size Ladder The AmplideX FMR1 PCR Kit incorporates two correction factors for conversion of size in base pairs to the number of CGG repeats for each allele The size of each peak may be converted to repeat length by the equation Peak i - size in base pairs of a given product peak, c 0 - size correction factor, m 0 - mobility correction factor for each CGG repeat Size and mobility correction factors for standard instrument configurations Configurationc0c0 m0m0 3130, 36 cm Data interpretation Normal male with no AGG interruption Female with two normal X AGG interruption in both X in 2 locations 29 CGG Female 29/29 CGG homozygous Male FM mosaic with all peak populations >200 Premutation78 CGG 125 CGG 108 CGG Premutation Female CGG Female heterozygous Normal/FM with 2 AGG interruption FM >200 AGG interruption Two X, with one normal X and one pre-mutation mosaic X. 2 AGG interruptions on 1 X Total families screened :8 Total no. of females: 25 PCR Positive Confirmed by southern blotting Confirmed by AmplideX FMR1 PCR Kit Confirmed by southern blotting Confirmed by AmplideX FMR1 PCR Kit Full Mutation:3 Premutation: 13 Normal: 9 S noPCRSouthern BlottingAmplideX FMR1 1Unable to detect pre mutation Unable to detect full mutation in females Can not interpret accurate CGG repeats, Can not detect interrupting AGG sequences accurately detects full mutations having 200 CGG repeats to 1300 Can detect interrupting AGG sequences resolves female zygosity 2No radioisotopesUsage of radioisotopes, which are hazardous No radioisotopes 3Easy to performLabor intensiveEasy to perform 4RapidTime consumingLess time consuming than Southern blotting Comparison of PCR, Southern blotting and AmplideX FMR1 Conclusion The FMR1 CGG Primer is specific for CGG repeats and will not hybridize to AGG sequences commonly found in FMR1 alleles. Signal intensity dips in the CGG RP PCR profile correspond to the presence of interspersed AGG. These AGG “interruptions” are thought to confer DNA stability and to reduce the risk of expansion in the next generation intermediate and PM alleles because AGG “interruptions” are thought to confer DNA stability and to reduce the risk of expansion in the next generation Therefore, the risk of CGG repeat expansion for mothers with AGG “interruptions” may be lower than mothers with the same number of repeats but without at least one AGG. Hence helpful in genetic counselling in the families with Fragile X Abstract Objective Material and Methods 2 pairs of primers, 20 pmole of FXD and FXE for amplification of the FRAXA CGG repeat and 35 pmole of 598 and 603 for amplification of FRAXE CCG repeat are used. In a normal individual two distinct bands are be visible, the upper band is of FRAXE and a lower band is of FRAXA. Individual showing absence of any of these bands indicates fragile X positive case. FRAXE positive case is extremely rare thus this band acts as a control band. Total no.of males:8