Debra O. Prosser

Evaluation of Bioinformatic Programmes for the Analysis of Variants within Splice Site Consensus Regions

The increasing diagnostic use of gene sequencing has led to an expanding dataset of novel variants that lie within consensus splice junctions. The challenge for diagnostic laboratories is the evaluation of these variants in order to determine if they affect splicing or are merely benign. A common evaluation strategy is to use in silico analysis, and it is here that a number of programmes are available online; however, currently, there are no consensus guidelines on the selection of programmes or protocols to interpret the prediction results. Using a collection of 222 pathogenic mutations and 50 benign polymorphisms, we evaluated the sensitivity and specificity of four in silico programmes in predicting the effect of each variant on splicing. The programmes comprised Human Splice Finder (HSF), Max Entropy Scan (MES), NNSplice, and ASSP. The MES and ASSP programmes gave the highest performance based on Receiver Operator Curve analysis, with an optimal cut-off of score reduction of 10%. The study also showed that the sensitivity of prediction is affected by the level of conservation of individual positions, with in silico predictions for variants at positions −4 and +7 within consensus splice sites being largely uninformative.

Novel mutation in the TMEM127 gene associated with phaeochromocytoma

Phaeochromocytomas and paragangliomas are rare neuroendocrine tumours that arise from the adrenal glands or paraganglia (paragangliomas) within the abdomen, thorax and neck. Although it was originally suggested that approximately 10% of these tumours were inherited, it is now recognised that up to approximately 30% of these tumours are associated with a germline mutation in one of the phaeochromocytoma/paraganglioma susceptibility genes. Of the 12 currently known genes predisposing to these tumours, the TMEM127 gene is one of the more recently identified and appears to be present in approximately 2% of apparently sporadic phaeochromocytomas. We report a 33‐year‐old man who presented with an apparently sporadic adrenal phaeochromocytoma and was identified as carrying a novel TMEM127 germline mutation, p.Gln139X. Patients harbouring a germline TMEM127 mutation most commonly present with an apparently sporadic solitary adrenal phaeochromocytoma. Testing patients who present with a phaeochromocytoma or paraganglioma for an underlying germline mutation needs to be considered in all patients due to implications for family members, but a strategy based on clinical and immunohistochemical findings would be prudent to limit costs.

Array Comparative Genomic Hybridization Identifies a Heterozygous Deletion of the Entire KCNJ2 Gene as a Cause of Sudden Cardiac Death

Background—Large gene rearrangements, not detectable by standard molecular genetic sequencing techniques, are present in a minority of patients with long QT syndrome. We aimed to screen for large rearrangements in genes responsible for long QT syndrome as part of the molecular autopsy of a 36-year-old woman who died suddenly and had a negative autopsy. A retrospective analysis of an ECG identified a long QT interval, but sequencing of known LQT genes was uninformative. Methods and Results—Array comparative genomic hybridization was used to screen for deletions and duplications in 101 genes implicated in cardiac disorders and sudden death using a postmortem blood sample. A 542 kb deletion encompassing the entire KCNJ2 gene was identified in the decedent. The mother had electrocardiographic U-wave changes consistent with Andersen–Tawil syndrome and exaggerated by exercise but none of the characteristic noncardiac features. Fluorescence in situ hybridization confirmed the deletion in the decedent and established its presence in the mother. Conclusions—A novel application of array comparative genomic hybridization and fluorescence in situ hybridization has identified that long QT syndrome and sudden cardiac death may occur as a result of a deletion of an entire gene. The case also supports recent research suggesting that noncardiac features of Andersen–Tawil syndrome occur only with missense or minor gene rearrangements in the KCNJ2 gene, resulting in a dominant negative effect on Kir2.x channels.

Citrullinemia type I: molecular screening of the ASS1 gene by exonic sequencing and targeted mutation analysis

We developed a mutation-screening protocol for the ASS1 gene in order to guide clinical management of neonates with elevated citrulline detected during routine newborn screening. An exon-based amplification and sequencing method was designed and successfully applied to patients to identify disease-associated mutations. The sequencing-based method was applied to three patients with mild or asymptomatic clinical courses. Identification of a homozygous mutation in these patients, c.787G>A (p.Val263Met), led to the development of a tetra-primer ARMS-PCR method that successfully detected the mutation in DNA extracted from blood or from Guthrie card spots.

A Novel Glycine Decarboxylase Gene Mutation in an Indian Family With Nonketotic Hyperglycinemia

Nonketotic hyperglycinemia is an inborn error of glycine metabolism. It manifests mostly as an acute encephalopathy in the neonatal period, although later, atypical presentations have also been reported. Mutations in 3 different genes have been implicated in nonketotic hyperglycinemia. Here we report a novel mutation, c.2296G>T (p.Gly766Cys), in exon 19 of the glycine decarboxylase (GLDC) gene (Refseq accession number NM_000170.2) in a consanguineous Indian couple with a history of 4 neonatal deaths.

Two Novel GLDC Mutations in a Neonate with Nonketotic Hyperglycinemia

Nonketotic hyperglycinemia, also known as glycine encephalopathy (OMIM #605899), is an autosomal recessive disorder of glycine metabolism resulting from a defect in the glycine cleavage system. We report two novel mutations of the glycine decarboxylase (GLDC) gene observed in a compound heterozygous state in a neonate of mixed Maori and Caucasian parentage: c.395C>T p.(Ser132Leu) in exon 3, and c.256-?_334+?del p.(Ser86Valfs*119), resulting in an out-of-frame deletion of exon 2. Additionally, we describe our experience of implementing the ketogenic diet, alongside standard pharmacological therapy, and highlight its potential therapeutic benefit in severe nonketotic hyperglycinemia, particularly in seizure management.

Gene Dosage Analysis in a Clinical Environment: Gene-Targeted Microarrays as the Platform-of-Choice

The role of gene deletion and duplication in the aetiology of disease has become increasingly evident over the last decade. In addition to the classical deletion/duplication disorders diagnosed using molecular techniques, such as Duchenne Muscular Dystrophy and Charcot-Marie-Tooth Neuropathy Type 1A, the significance of partial or whole gene deletions in the pathogenesis of a large number single-gene disorders is becoming more apparent. A variety of dosage analysis methods are available to the diagnostic laboratory but the widespread application of many of these techniques is limited by the expense of the kits/reagents and restrictive targeting to a particular gene or portion of a gene. These limitations are particularly important in the context of a small diagnostic laboratory with modest sample throughput. We have developed a gene-targeted, custom-designed comparative genomic hybridisation (CGH) array that allows twelve clinical samples to be interrogated simultaneously for exonic deletions/duplications within any gene (or panel of genes) on the array. We report here on the use of the array in the analysis of a series of clinical samples processed by our laboratory over a twelve-month period. The array has proven itself to be robust, flexible and highly suited to the diagnostic environment.

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

Massively Parallel Sequencing of Genes Implicated in Heritable Cardiac Disorders: A Strategy for a Small Diagnostic Laboratory

Sudden cardiac death (SCD) in people before the age of 35 years is a devastating event for any family. The causes of SCD in the young can be broadly divided into two groups: heritable cardiac disorders that affect the heart structure (cardiomyopathies) and primary electrical disorders (cardiac ion channelopathies). Genetic testing is vital as those suffering from cardiac ion channelopathies have structurally normal hearts, and those with cardiomyopathies may only show subtle abnormalities in the heart and these signs may not be detected during an autopsy. Post-mortem genetic testing of SCD victims is important to identify the underlying genetic cause. This is important as family cascade screening may be undertaken to identify those who may be at risk and provide vital information about risk stratification and clinical management. The development of massively parallel sequencing (MPS) has made it possible for the simultaneous screening of multiple patients for hundreds of genes. In light of this, we opted to develop an MPS approach for SCD analysis that would allow us to screen for mutations in genes implicated in cardiomyopathies and cardiac ion channelopathies. The rationale behind this panel was to limit it to genes carrying the greatest mutation load. If no likely pathogenic gene variant were found then testing could cascade to whole exome/genome sequencing as a gene-discovery exercise. The overarching aim was to design and validate a custom-cardiac panel that satisfies the diagnostic requirements of LabPLUS (Auckland City Hospital, Auckland, NZ) and the guidelines provided by the Royal College of Pathologists of Australasia and the Association for Clinical Genetic Science.

A workflow for the detection and classification of variants in the BRCA1 and BRCA2 genes

PLK1 promotes cell cycle and is involved in recovery from DNA damage. PLK1 overexpression is present in CRC, and it is associated with poor patient prognosis. The upstream proteins are affecting the expression of PLK1. However, the contribution of the intrinsic factors to the causes of deregulation remains uncertain. This study investigates mutation and CpG island methylation of PLK1 in CRC and their associations with PLK1 overexpression. PLK1 gene mutations in HCT116, SW48, Colo320DM and T84 cells were assessed by Sanger sequencing. The methylation statuses were analysed by mass spectrometry. Subsequently the methylation study was expanded to a cohort CRC patients using pyrosequencing. The in vitro studies show only single deletions were found in the silencer region upstream of promoter region of PLK1 gene in SW48 and a subpopulation of HCT116 cells. Furthermore, no specific trend of methylation status was observed in the majority of the CG sites of the irradiated and control cells. In the patient cohort, low methylation was detected in CRC tissues independent of the expression of PLK1 and normal mucosa tissues. In conjunction with the findings on the literature, overexpression of PLK1 appears to be due to the effects of upstream proteins rather than intrinsic factors.