Elaine Doherty

Simple Repeat-Primed PCR Analysis of the Myotonic Dystrophy Type 1 Gene in a Clinical Diagnostics Environment

Myotonic dystrophy type 1 is an autosomal dominant neuromuscular disorder that is caused by the expansion of a CTG trinucleotide repeat in the DMPK gene. The confirmation of a clinical diagnosis of DM-1 usually involves PCR amplification of the CTG repeat-containing region and subsequent sizing of the amplification products in order to deduce the number of CTG repeats. In the case of repeat hyperexpansions, Southern blotting is also used; however, the latter has largely been superseded by triplet repeat-primed PCR (TP-PCR), which does not yield a CTG repeat number but nevertheless provides a means of stratifying patients regarding their disease severity. We report here a combination of forward and reverse TP-PCR primers that allows for the simple and effective scoring of both the size of smaller alleles and the presence or absence of expanded repeat sequences. In addition, the CTG repeat-containing TP-PCR forward primer can target both the DM-1 and Huntington disease genes, thereby streamlining the work flow for confirmation of clinical diagnoses in a diagnostic laboratory.

Array-based Identification of Copy Number Changes in a Diagnostic Setting: Simultaneous gene-focused and low resolution whole human genome analysis

OBJECTIVES The aim of this study was to develop and validate a comparative genomic hybridisation (CGH) array that would allow simultaneous targeted analysis of a panel of disease genes and low resolution whole genome analysis. METHODS A bespoke Roche NimbleGen 12x135K CGH array (Roche NimbleGen Inc., Madison, Wisconsin, USA) was designed to interrogate the coding regions of 66 genes of interest, with additional widely-spaced backbone probes providing coverage across the whole genome. We analysed genomic deoxyribonucleic acid (DNA) from 20 patients with a range of previously characterised copy number changes and from 8 patients who had not previously undergone any form of dosage analysis. RESULTS The custom-designed Roche NimbleGen CGH array was able to detect known copy number changes in all 20 patients. A molecular diagnosis was also made for one of the additional 4 patients with a clinical diagnosis that had not been confirmed by sequence analysis, and carrier testing for familial copy number variants was successfully completed for the remaining four patients. CONCLUSION The custom-designed CGH array described here is ideally suited for use in a small diagnostic laboratory. The method is robust, accurate, and cost-effective, and offers an ideal alternative to more conventional targeted assays such as multiplex ligation-dependent probe amplification.

Postmortem DNA: QC Considerations for Sequence and Dosage Analysis of Genes Implicated in Long QT Syndrome

Long QT syndrome is a rare disorder of cardiac ion channels, characterised by a prolonged QT interval and T-wave abnormalities on electrocardiogram (ECG) and the occurrence of the ventricular tachycardia torsade de pointes. Sodium, potassium or calcium channels present in heart muscle may be affected, altering the regulation of electrical current in the cells [1-3]. Individuals with this condition will be predisposed to cardiac events such as arrhythmias and polymorphic ventricular tachycardia, which may lead, if untreated, to sudden cardiac death [2,3]. Thirteen genes are associated with the condition, and hundreds of mutations have been identified [3-5]. Currently, more than 95% of the pathogenic mutations listed in disease databases (Gene Connection For the Heart, http://www.fsm.it/cardmoc/; online Hu‐ man Gene Mutation Database, www.hgmd.cf.ac.uk/) are sequence variants (including point mutations and small insertions or deletions), but the importance of whole or multi-exon de‐ letions and duplications has more recently been recognised [6] and it is now recommended to use both sequence and dosage techniques in order to provide comprehensive analysis [3].

Primer Design to Sequence Analysis - a Pipeline for a Molecular Genetic Diagnostic Laboratory

Confirmation of a clinical diagnosis of a heritable disorder usually involves the analysis of the coding exons of a gene, plus surrounding intronic sequences. Variants that are identified using this strategy can be classed as either disease-causing or non-pathogenic polymorphisms. The variants comprise point mutations, micro insertions and deletions (indels), and copy number changes. The detection of small variants (such as substitutions and indels) can be identified using a number of techniques including SSCP (Orita et al., 1989), DHPLC (Yu et al., 2006) and highresolution melt-curve analysis (Garritano et al., 2009; Montgomery et al., 2010; NgyyenDumont et al., 2009). These techniques can de difficult to optimise and can result in both false negative and false positive results. The accepted gold standard of mutation detection, however, is dideoxy-based sequencing. This is the most direct approach and can be the most sensitive of all techniques. However, it is also the most expensive option and can be the most time-consuming due to bottle-necks arising from designing appropriate primers, the analysis of subsequent sequence data, and the interrogation of data to filter out sequence variants that are not disease-causing. Within our laboratory, we experienced difficulties in three principal areas: standardising the primer design; objectively evaluating sequence data that can be performed in an automated manner thereby addressing the bottle-neck of trace sequence evaluation; and responding to the increasing need for in-depth analysis of sequence variants to inform referring physicians as to the clinical significance of the data. To resolve the above issues, we developed a system that addressed each of these areas. The first involved designing primers that are assessed for unique amplification of targeted genomic regions using standard conditions. The aim here was not to tailor PCR conditions to a primer pair, but to force all primer designs to satisfy only one condition. The ancillary design feature of this module was to tail all primers such that all amplicons could be sequenced bi-directionally using only two primers (the “tail” sequences), thereby avoiding sequencing using amplicon-specific primers. The second area involved the wholesale adoption of Variant ReporterTM software (Applied Biosystems Ltd) for the automated analysis of all sequence data. This decision led to a significant initial investment in time to establish appropriate reference sequence projects for each of the genes analysed by our

Molecular characterisation of a der(Y)t(Xp;Yp) with Xp functional disomy and sex reversal

Sex reversal due to duplication of the Xp21 dosage-sensitive sex reversal locus results in XY females with gonadal dysgenesis. Pure Xp disomy (without a concurrent loss of genetic material) can occur by translocation or interstitial duplication. The case reported here is the rare form with a t(Xp;Yp). The combination of conventional clinical cytogenetic techniques, microsatellite analysis and high-density microarrays identified the X-chromosome breakpoint as centromeric of the NR0B1 gene and its control elements. Cytogenetics and array technology complemented each other in characterizing the translocation event and the extent of the dosage-sensitive sex reversal critical region on the derivative Y-chromosome. The implications of this analysis also lie in genetic counseling that highlight the likely de novo nature of a paternal meiotic event.