1.   
Alternative
methodologies

5A. Triplet repeat
disorders Huntington’s
disease(HD)

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 HD is caused
by an expansion of 36 or more CAG trinucleotide repeats in exon 1 of the HTT genei. Triplet
repeat expansions are not easily detected using NGSii.

The CAG triplet repeat sizes can usually be determined
by a PCR assay. This involves amplification using primers flanking the CAG
repeat region, followed by capillary electrophoresis (CE) which is run out on a
polyacrylamide gel alongside standard repeat size markers, and the bands are
revealed by silver staining or a fluorescence assay, allowing qualitative
detection of triplet repeat expansions.

If a single band is
detected, additional investigations including a  PCR amplification of the adjacent CCG regioniii and/or
 Southern blot can be carried out to investigate
the possibility of a  PCR amplification
failure of a large expanded alleleiv.

To investigate the possibility of a larger
pathogenic repeat not amplified using flanking primersv, triplet
repeat primed PCR (TP PCR) is used. A TP PCR involves a locus-specific primer
flanking the repeat alongside paired primers amplifying from multiple sites
within the repeat vi. TP PCR
gives a characteristic ladder on the fluorescence trace when there is a large
pathogenic repeatvii. This method
can determine presence of a CAG expansion, but does not inform of the number of
repeats in the expansion. This method is occasionally useful in finding large CAG repeats associated with juvenile-onset HD.

5B. Epigenetic changes

Epigenetic
changes are not easily detected by WES or WGS. Loss of methylation at the
paternal imprinting centre 1 (IC1) on chromosome 11p15.5, is the cause of  ~35% to 50% of cases of Russel Silver syndrome
(RSS).

 

DNA methylation is
an 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 cytosine
ring resulting in 5-methylcytosine (5-mC)), consequently altering  gene expression,

 

Methylation
sensitive Multiplex ligation-dependent probe amplification (MS-MLPA) studies of IC1 will detect  methylation abnormalities at this locus, as
well as uniparental disomy of chromosome 11, and/or deletions and duplications in the locus. viii.
MS-MLPA involves. DNA denaturation and hybridisation
of MLPA probes, followed by ligation and digestion, then a PCR, and  separation of amplification products by
capillary electrophoresis.  If the sample
DNA is methylated, the DNA-probe hybrids are protected against HhaI digestion
and the ligated probes will generate a peak.

i
GeneReviews Huntington Disease Warby et al https://www.ncbi.nlm.nih.gov/books/NBK1305/
Cited 17.12.2017

ii Singleton A.B. Exome sequencing: a transformative
technology. Lancet Neurology. 2011;10(10):942–946.

iii
Andrew SE et al. . A CCG repeat polymorphism adjacent to the CAG repeat in the
Huntington disease gene: implications for diagnostic accuracy and predictive
testing. Hum Mol Genet. 1994; 3(1):65–7..

iv
Guida M, Fenwick RG, Papp AC, Snyder PJ, Sedra M, Prior TW. Southern transfer
protocol for confirmation of Huntington disease. Clin Chem. 1996;
42(10):1711–2.

v Ciotti P
et al.  Triplet repeat primed PCR
(TP PCR) in molecular diagnostic testing for Friedreich ataxia. J Mol Diagn. 2004 Nov;6(4):285-9.

vi Triplet
Primed Repeat PCR (TP-PCR) in molecular diagnostic testing for trinucleotide
repeat disorders (PDF Download Available).
Available from: https://www.researchgate.net/publication/262684078_Triplet_Primed_Repeat_PCR_TP-PCR_in_molecular_diagnostic_testing_for_trinucleotide_repeat_disorders
. Cited 16.12. 2017

 

viii Weksberg R, Shuman C, Beckwith JB. Beckwith-Wiedemann
syndrome. Eur J Hum Genet.2010;18:8–14.