1. Alternativemethodologies5A. Triplet repeatdisorders Huntington’sdisease(HD) HD is causedby an expansion of 36 or more CAG trinucleotide repeats in exon 1 of the HTT genei. Tripletrepeat expansions are not easily detected using NGSii. The CAG triplet repeat sizes can usually be determinedby a PCR assay.
This involves amplification using primers flanking the CAGrepeat region, followed by capillary electrophoresis (CE) which is run out on apolyacrylamide gel alongside standard repeat size markers, and the bands arerevealed by silver staining or a fluorescence assay, allowing qualitativedetection of triplet repeat expansions. If a single band isdetected, additional investigations including a PCR amplification of the adjacent CCG regioniii and/or Southern blot can be carried out to investigatethe possibility of a PCR amplificationfailure of a large expanded alleleiv.To investigate the possibility of a largerpathogenic repeat not amplified using flanking primersv, tripletrepeat primed PCR (TP PCR) is used. A TP PCR involves a locus-specific primerflanking the repeat alongside paired primers amplifying from multiple siteswithin the repeat vi. TP PCRgives a characteristic ladder on the fluorescence trace when there is a largepathogenic repeatvii. This methodcan determine presence of a CAG expansion, but does not inform of the number ofrepeats in the expansion.
This method is occasionally useful in finding large CAG repeats associated with juvenile-onset HD.5B. Epigenetic changesEpigeneticchanges are not easily detected by WES or WGS. Loss of methylation at thepaternal imprinting centre 1 (IC1) on chromosome 11p15.5, is the cause of ~35% to 50% of cases of Russel Silver syndrome(RSS). DNA methylation isan epigenetic alteration involving the addition of a methyl (CH3) group to DNA (such as the covalent addition of a methyl group at the 5-carbon of cytosinering resulting in 5-methylcytosine (5-mC)), consequently altering gene expression, Methylationsensitive Multiplex ligation-dependent probe amplification (MS-MLPA) studies of IC1 will detect methylation abnormalities at this locus, aswell as uniparental disomy of chromosome 11, and/or deletions and duplications in the locus. viii.
MS-MLPA involves. DNA denaturation and hybridisationof MLPA probes, followed by ligation and digestion, then a PCR, and separation of amplification products bycapillary electrophoresis. If the sampleDNA is methylated, the DNA-probe hybrids are protected against HhaI digestionand the ligated probes will generate a peak.iGeneReviews Huntington Disease Warby et al https://www.ncbi.nlm.nih.gov/books/NBK1305/Cited 17.
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