The month of July: an early experience with pandemic influenza A (H1N1) in adults with cystic fibrosis

<p>Abstract</p> <p>Background</p> <p>Pandemic Influenza A (H1N1) 2009 is a novel viral infection that emerged in March 2009. This is the first report addressing the clinical course of patients with cystic fibrosis (CF) and H1N1 infection.</p> <p>Methods</p> <p>All patients with an influenza-like illness (ILI) attending our adult centre during July 2009 were identified. Baseline respiratory function, nutritional status, approach to management and short-term clinical course were recorded.</p> <p>Results</p> <p>Most patients experienced a mild course and were able to be managed with antiviral agents as an outpatient. Robust infection control policies were implemented to limit transmission of H1N1 infection within our CF centre. Patients with severe lung disease, poor baseline nutritional reserve and presenting with more than 48 hours of ILI experienced a more severe course. Prompt antiviral therapy within the first 48 hours of illness may have been important in improving outcomes.</p> <p>Conclusions</p> <p>This observational study demonstrates that most adults with CF with H1N1 infection had mild clinical courses and recovered rapidly.</p

Effect of BRCA2 sequence variants predicted to disrupt exonic splice enhancers on BRCA2 transcripts

Background: Genetic screening of breast cancer patients and their families have identified a number of variants of unknown clinical significance in the breast cancer susceptibility genes, BRCA1 and BRCA2. Evaluation of such unclassified variants may be assisted by web-based bioinformatic prediction tools, although accurate prediction of aberrant splicing by unclassified variants affecting exonic splice enhancers (ESEs) remains a challenge

BRCA1 and BRCA2 5′ noncoding region variants identified in breast cancer patients alter promoter activity and protein binding

© 2018 The Authors. Human Mutation published by Wiley Periodicals, Inc. The widespread use of next generation sequencing for clinical testing is detecting an escalating number of variants in noncoding regions of the genome. The clinical significance of the majority of these variants is currently unknown, which presents a significant clinical challenge. We have screened over 6,000 early-onset and/or familial breast cancer (BC) cases collected by the ENIGMA consortium for sequence variants in the 5′ noncoding regions of BC susceptibility genes BRCA1 and BRCA2, and identified 141 rare variants with global minor allele frequency \u3c 0.01, 76 of which have not been reported previously. Bioinformatic analysis identified a set of 21 variants most likely to impact transcriptional regulation, and luciferase reporter assays detected altered promoter activity for four of these variants. Electrophoretic mobility shift assays demonstrated that three of these altered the binding of proteins to the respective BRCA1 or BRCA2 promoter regions, including NFYA binding to BRCA1:c.-287C\u3eT and PAX5 binding to BRCA2:c.-296C\u3eT. Clinical classification of variants affecting promoter activity, using existing prediction models, found no evidence to suggest that these variants confer a high risk of disease. Further studies are required to determine if such variation may be associated with a moderate or low risk of BC

Detection of splicing aberrations caused by BRCA1 and BRCA2 sequence variants encoding missense substitutions: implications for prediction of pathogenicity

Missense substitutions in high-risk cancer susceptibility genes create clinical uncertainty in the genetic counseling process. Multifactorial likelihood classification approaches and in vitro assays are useful for the classification of exonic sequence variants in BRCA1 and BRCA2, but these currently rely on the assumption that changes in protein function are the major biological mechanism of pathogenicity. This study investigates the potentially pathogenic role of aberrant splicing for exonic variants predicted to encode missense substitutions using patient-derived RNA. No splicing aberrations were identified for BRCA1c.5054C&gt;T and BRCA2c.7336A&gt;G, c.8839G&gt;A, and c.9154C&gt;T. However, RT-PCR analysis identified a major splicing aberration for BRCA1c.4868C&gt;G(p.Ala1623Gly), a variant encoding a missense substitution considered likely to be neutral. Splicing aberrations were also observed for BRCA2c.7988A&gt;T(p.Glu2663Val) and c.8168A&gt;G(p.Asp2723Gly), but both variant and wildtype alleles were shown to be present in full-length mRNA transcripts, suggesting that variant protein may be translated. BRCA2 protein function assays indicated that BRCA2p.Glu2663Val, p.Asp2723Gly and p.Arg3052Trp missense proteins have abrogated function consistent with pathogenicity. Multifactorial likelihood analysis provided evidence for pathogenicity for BRCA1 c.5054C&gt;T(p.Thr1685Ile) and BRCA2c.7988A&gt;T(p.Glu2663Val), c.8168A&gt;G(p.Asp2723Gly) and c.9154C&gt;T(p.Arg3052Trp), supporting experimentally derived evidence. These findings highlight the need for improved bioinformatic prediction of splicing aberrations and to refine multifactorial likelihood models used to assess clinical significanc

Naturally occurring BRCA2 alternative mRNA splicing events in clinically relevant samples

Background BRCA1 and BRCA2 are the two principal tumour suppressor genes associated with inherited high risk of breast and ovarian cancer. Genetic testing of BRCA1/2 will often reveal one or more sequence variants of uncertain clinical significance, some of which may affect normal splicing patterns and thereby disrupt gene function. mRNA analyses are therefore among the tests used to interpret the clinical significance of some genetic variants. However, these could be confounded by the appearance of naturally occurring alternative transcripts unrelated to germline sequence variation or defects in gene function. To understand which novel splicing events are associated with splicing mutations and which are part of the normal BRCA2 splicing repertoire, a study was undertaken by members of the Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium to characterise the spectrum of naturally occurring BRCA2 mRNA alternate-splicing events. Methods mRNA was prepared from several blood and breast tissue-derived cells and cell lines by contributing ENIGMA laboratories. cDNA representing BRCA2 alternate splice sites was amplified and visualised using capillary or agarose gel electrophoresis, followed by sequencing. Results We demonstrate the existence of 24 different BRCA2 mRNA alternate-splicing events in lymphoblastoid cell lines and both breast cancer and non-cancerous breast cell lines. Conclusions These naturally occurring alternate-splicing events contribute to the array of cDNA fragments that may be seen in assays for mutation-associated splicing defects. Caution must be observed in assigning alternate-splicing events to potential splicing mutations

Consequences of germline variation disrupting the constitutional translational initiation codon start sites of MLH1 and BRCA2: Use of potential alternative start sites and implications for predicting variant pathogenicity

Variants that disrupt the translation initiation sequences in cancer predisposition genes are generally assumed to be deleterious. However, few studies have validated these assumptions with functional and clinical data. Two cancer syndrome gene variants likely to affect native translation initiation were identified by clinical genetic testing: MLH1:c.1A>G p.(Met1?) and BRCA2:c.67+3A>G. In vitro GFP-reporter assays were conducted to assess the consequences of translation initiation disruption on alternative downstream initiation codon usage. Analysis of MLH1:c.1A>G p.(Met1?) showed that translation was mostly initiated at an in-frame position 103 nucleotides downstream, but also at two ATG sequences downstream. The protein product encoded by the in-frame transcript initiating from position c.103 showed loss of in vitro mismatch repair activity comparable to known pathogenic mutations. BRCA2:c.67+3A>G was shown by mRNA analysis to result in an aberrantly spliced transcript deleting exon 2 and the consensus ATG site. In the absence of exon 2, translation initiated mostly at an out-of-frame ATG 323 nucleotides downstream, and to a lesser extent at an in-frame ATG 370 nucleotides downstream. Initiation from any of the downstream alternative sites tested in both genes would lead to loss of protein function, but further clinical data is required to confirm if these variants are associated with a high cancer risk. Importantly, our results highlight the need for caution in interpreting the functional and clinical consequences of variation that leads to disruption of the initiation codon, since translation may not necessarily occur from the first downstream alternative start site, or from a single alternative start site. (c) 2013 Wiley Periodicals, Inc

RT-PCR results for <i>BRCA1</i> c.4484G>C(p.Arg1495Thr) and <i>BRCA2:</i>c.7828G>A (p.Val2610Met).

<p><i>M</i> - 100bp DNA marker (New England Biolabs). A) <i>BRCA2</i>:c.7828G>A (p.Val2610Met). Lane 1: RT-PCR products from variant carrier derived cycloheximide treated LCL. Lane 2–7: Cycloheximide treated LCLs from unaffected female controls. There is no evidence for a predicted loss of 149bp from exon 17 as a result of a <i>de novo</i> donor site. The Δexon 18 (540bp) and Δexon 17/18 (369bp) are detected in the variant carrier and all but one control samples. B) <i>BRCA1</i> c.4484G>C(p.Arg1495Thr). Lane 1: RT-PCR products from whole blood derived RNA from the variant carrier showing the Δexon 14 and Δexon 14/15 splicing aberration. Lane 2: RT-PCR carried out on whole blood derived RNA from an unaffected female control (collection and extraction methods as per the variant carrier). Lane 3–7: Cycloheximide treated LCLs from unaffected female controls.</p

BRCA1 RING-domain variants with reported loss of function on the basis of <i>in-vitro</i> functional assays and/or (likely) clinically significant from multifactorial likelihood analysis.

*<p>p.Met18Thr, p.Cys61Gly and p.Cys64Gly are also shown to have abrogated function using mouse embryonic stem cell assays <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086836#pone.0086836-Bouwman1" target="_blank">[44]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086836#pone.0086836-Chang1" target="_blank">[49]</a>. (No other variants listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086836#pone-0086836-t003" target="_blank">Table 3</a> were assayed using this method).</p