Predicting the pathogenic potential of BRCA1 and BRCA2 gene variants identified in clinical genetic testing

Missense variants are very commonly detected when screening for mutations in the BRCA1 and BRCA2 genes. Pathogenic mutations in the BRCA1 and BRCA2 genes lead to an increased risk of developing breast, ovarian, prostate and/or pancreatic cancer. This study aimed to assess the predictive capability of in silico programmes and mutation databases in assisting diagnostic laboratories to determine the pathogenicity of sequence-detectable mutations. Between July 2011 and April 2013, an analysis was undertaken of 13 missense BRCA gene variants that had been detected in patients referred to the Genetic Health Services New Zealand [Northern Hub] for BRCA gene analysis. The analysis involved the use of 13 in silico protein prediction programmes, two in silico transcript analysis programmes and the examination of three BRCA gene databases. In most of the variants, the analysis showed different in silico interpretations. This illustrates the interpretation challenges faced by diagnostic laboratories. Unfortunately, when using online mutation databases and carrying out in silico analyses, there is significant discordance in the classification of some missense variants in the BRCA genes. This discordance leads to complexities in interpreting and reporting these variants in a clinical context. The authors have developed a simple procedure for analysing variants; however, those of unknown significance largely remain unknown. As a consequence, the clinical value of some reports may be negligible

Diagnostic screening workflow for mutations in the BRCA1 and BRCA2 genes

Screening for mutations in large genes is challenging in a molecular diagnostic environment. Sanger-based DNA sequencing methods are largely used; however, massively parallel sequencing [MPS] can accommodate increasing test demands and financial constraints. This study aimed to establish a simple workflow to amplify and screen all coding regions of the BRCA1 and BRCA2 [BRCA1/2] genes by Sanger-based sequencing as well as to assess a MPS approach encompassing multiplex polymerase chain reaction [PCR] and pyrosequencing. This study was conducted between July 2011 and April 2013. A total of 20 patients were included in the study who had been referred to Genetic Health Services New Zealand [Northern Hub] for BRCA1/2 mutation screening. Patients were randomly divided into a MPS evaluation and validation cohort [n = 10 patients each]. Primers were designed to amplify all coding exons of BRCA1/2 [28 and 42 primer pairs, respectively]. Primers overlying known variants were avoided to circumvent allelic drop-out. The MPS approach necessitated utilisation of a complementary fragment analysis assay to eliminate apparent false-positives at homopolymeric regions. Variants were filtered on the basis of their frequency and sequence depth. Sanger-based sequencing of PCR amplified coding regions was successfully achieved. Sensitivity and specificity of the combined MPS/homopolymer protocol was determined to be 100% and 99.5%, respectively. In comparison to traditional Sangerbased sequencing, the MPS workflow led to a reduction in both cost and analysis time for BRCA1/2 screening. MPS analysis achieved high analytical sensitivity and specificity, but required complementary fragment analysis combined with Sanger-based sequencing confirmation in some instances

Implications of a Chr7q21.11 microdeletion and the role of the PCLO gene in developmental delay

We report here a 4-year-old boy with global developmental delay who was referred for karyotyping and fragile X studies. A small interstitial deletion on chromosome 7 at band 7q21 was detected in all cells examined. Subsequent molecular karyotype analysis gave the more detailed result of a 6.3 Mb heterozygous deletion involving the interstitial chromosome region 7q21.11. In this relatively gene-poor region, the presynaptic cytomatrix protein, Piccolo [PCLO] gene appears to be the most likely candidate for copy number loss leading to a clinical phenotype. G-banded chromosome analysis of the parents showed this deletion was inherited from the father. Molecular karyotype analysis of the father's genome confirmed that it was the same deletion as that seen in the son; however, the father did not share the severity of his son's phenotype. This cytogenetically-visible deletion may represent another example of a chromosomal rearrangement conferring a variable phenotype on different family members

Clinical outcomes and counseling issues regarding partial trisomy of terminal Xp in a child with developmental delay

Female carriers of balanced translocations involving an X chromosome and an autosome present genetic councelling challenges. This is in view of the number of possible meiotic outcomes, but also due to the impact of X chromosome-localised genes that are no longer subject to gene silencing through the X chromosome inactivation centre. We present a case where delineation of the extent of X chromosome-localised genes on the derivative autosome using molecular karyotyping offers critical information in the context of genetic councelling

Array-based identification of copy number changes in a diagnostic setting. Simultaneous gene-focused and low resolution whole human genome analysis

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. 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 widelyspaced 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. 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. 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

Molecular analysis of a case of thanatophoric dysplasia reveals two de novo FGFR3 missense mutations located in cis

Thanatophoric dysplasia [TD] is the most common form of lethal skeletal dysplasia. It is primarily an autosomal dominant disorder and is characterised by macrocephaly, a narrow thorax, short ribs, brachydactyly, and hypotonia. In addition to these core phenotypic features, TD type I involves micromelia with bowed femurs, while TD type II is characterised by micromelia with straight femurs and a moderate to severe clover-leaf deformity of the skull. Mutations in the FGFR3 gene are responsible for all cases of TD reported to date. The objective of the study here was to delineate further the mutational spectrum responsible for TD. Conventional polymerase chain reaction [PCR], allele-specific PCR, and sequence analysis were used to identify FGFR3 gene mutations in a fetus with a lethal skeletal dysplasia consistent with TD, which was detected during a routine antenatal ultrasound examination. In this report we describe the identification of two de novo missense mutations in cis in the FGFR3 gene [p.Asn540Lys and p.Val555Met] in a fetus displaying phenotypic features consistent with TD. This is the second description of a case of TD occurring as a result of double missense FGFR3 gene mutations, suggesting that the spectrum of mutations involved in the pathogenesis of TD may be broader than previously recognised