Hum Mutat [journal]
- Comprehensive Mutation Analysis of PMS2 in a Large Cohort of Probands Suspected of Lynch Syndrome or Constitutional Mismatch Repair Deficiency (CMMRD) Syndrome. [JOURNAL ARTICLE]
- Hum Mutat 2016 Jul 20.
Monoallelic PMS2 germline mutations cause 5-15% of Lynch syndrome, a midlife cancer predisposition, whereas biallelic PMS2 mutations cause approximately 60% of constitutional MMR deficiency (CMMRD), a rare childhood cancer syndrome. Recently improved DNA and RNA-based strategies are applied to overcome problematic PMS2 mutation analysis due to the presence of pseudogenes and frequent gene conversion events. Here, we determined PMS2 mutation detection yield and mutation spectrum in a nationwide cohort of 396 probands. Furthermore, we studied concordance between tumor IHC/ MSI (immunohistochemistry/ microsatellite-instability) profile and mutation carrier state. Overall, we found 52 different pathogenic PMS2 variants explaining 121 Lynch syndrome and nine CMMRD patients. In vitro MMR assays suggested pathogenicity for three missense variants. Ninety-one PMS2 mutation carriers (70%) showed isolated loss of PMS2 in their tumors, for 31 (24%) no or inconclusive IHC was available, and eight carriers (6%) showed discordant IHC (presence of PMS2 or loss of both MLH1 and PMS2). Ten cases with isolated PMS2 loss (10%; 10/97) harbored MLH1 mutations. We confirmed that recently improved mutation analysis provides a high yield of PMS2 mutations in patients with isolated loss of PMS2 expression. Application of universal tumor pre-screening methods will however miss some PMS2 germline mutation carriers. This article is protected by copyright. All rights reserved.
- Discovery and Functional Annotation of PRSS1 Promoter Variants in Chronic Pancreatitis. [JOURNAL ARTICLE]
- Hum Mutat 2016 Jul 18.
Recently, our resequencing of the promoter region of PRSS1 in French Caucasian individuals led to the identification of a functional variant (c.-204C>A) that is in perfect linkage disequilibrium with the 'chronic pancreatitis (CP)-protective' PRSS1 c.-408C>T variant. Here, we extended the resequencing to 626 French Caucasians (242 idiopathic CP patients and 384 controls). We discovered three additional variants (c.-184G>A, c.-173C>T and c.-147C>T), each being found only once in either patients or controls. We analyzed these three variants, together with a known PRSS1 promoter variant (c.-30_-28delTCC) long considered to be causative for CP, by luciferase promoter reporter assay in AR42J cells treated with dexamethasone. This analysis revealed that c.-30_-28delTCC resulted in reduced rather than increased PRSS1 gene expression, suggesting that it is not a CP risk factor as originally claimed. We provide evidence that c.-147C>T probably confers protection against CP by reducing the affinity of an ATF4 transcription factor binding site. This article is protected by copyright. All rights reserved.
- 4th Generation of NGS Technologies: Promise and Consequences. [REVIEW, JOURNAL ARTICLE]
- Hum Mutat 2016 Jul 13.
In this review, we discuss the emergence of 4th generation sequencing technologies that preserve the spatial coordinates of RNA and DNA sequences with up to subcellular resolution thus enabling back mapping of sequencing reads to the original histological context. This information is used for example in two current large-scale projects that aim to unravel the function of the brain. Also in cancer research, 4(th) generation sequencing has the potential to revolutionize the field. Cancer Research UK has named "Mapping the molecular and cellular tumor microenvironment in order to define new targets for therapy and prognosis" one of the grand challenges in tumor biology. We discuss the advantages of sequencing nucleic acids directly in fixed cells over traditional NGS methods, the limitations and challenges that these new methods have to face to become broadly applicable, and the impact that the information generated by the combination of in situ sequencing and NGS methods will have in research and diagnostics. This article is protected by copyright. All rights reserved.
- Deep Intronic Sequence Variants in COL2A1 Affect the Alternative Splicing Efficiency of Exon 2, and May Confer a Risk for Rhegmatogenous Retinal Detachment. [JOURNAL ARTICLE]
- Hum Mutat 2016 Jul 13.
COL2A1 mutations causing haploinsufficiency of type II collagen, cause type 1 Stickler syndrome which has a high risk of retinal detachment and failure of the vitreous to develop normally. Exon 2 of COL2A1 is alternatively spliced, expressed in the eye but not in mature cartilage and encodes a region that binds growth factors TGFβ1 and BMP-2. We investigated how both an apparently de novo variant and a polymorphism in intron 2 altered the efficiency of COL2A1 exon 2 splicing and how the latter may act as a predisposing risk factor for the occurrence of posterior vitreous detachment (PVD) associated rhegmatogenous retinal detachment (RRD) in the general population. Using amplification of illegitimate transcripts and allele specific minigenes expressed in cultured cells, we demonstrate variability in exon 2 inclusion not only between different control individuals, but also between different COL2A1 alleles. We identify trans-acting factors that bind to allele specific RNA sequences, and investigate the effect of knockdown and over expression of these factors on exon 2 splicing efficiency. Finally, using a specific cohort of patients with PVD associated RRD and a control population we demonstrate a significant difference in the frequency of the COL2A1 intronic variant rs1635532 between the two groups. This article is protected by copyright. All rights reserved.
- Regulatory Single Nucleotide Variant Predictor (RSVP) Increases Predictive Performance of Functional Regulatory Variants. [JOURNAL ARTICLE]
- Hum Mutat 2016 Jul 13.
In silico methods for detecting functionally relevant genetic variants are important for identifying genetic markers of human inherited disease. Much research has focused on protein-coding variants since coding-regions have well-defined physicochemical and functional properties. However, many bioinformatics tools are not applicable to variants outside coding-regions. Here, we increase the classification performance of our Regulatory Single Nucleotide Variant Predictor (RSVP) for variants that cause regulatory abnormalities from an AUC of 0.90 to 0.97 by incorporating genomic regions identified by the ENCODE project into RSVP. RSVP is comparable to a recently published tool, Genome-Wide Annotation of Variants (GWAVA); both RSVP and GWAVA perform better on regulatory variants than a traditional variant predictor, Combined Annotation Dependent Depletion (CADD). However, our method outperforms GWAVA on variants located at similar distances to the transcription start site as the positive set (AUC: 0.96) compared to GWAVA (AUC: 0.71). Much of this disparity is due to RSVP's incorporation of features pertaining to the nearest gene (expression, GO terms, etc), which are not included in GWAVA. Our findings hold out the promise of a framework for the assessment of all functional regulatory variants, providing a means to predict which rare or de novo variants are of pathogenic significance. This article is protected by copyright. All rights reserved.
- The Complementarity Between Protein-Specific and General Pathogenicity Predictors for Amino Acid Substitutions. [JOURNAL ARTICLE]
- Hum Mutat 2016 Jul 10.
The usage of Next-Generation Sequencing (NGS) with biomedical/clinical purposes has fuelled the demand for tools that assess the functional impact of sequence variants. For single amino acid variants, general methods (GM), based on biophysics/evolutionary principles and trained by pooling variants from many proteins, are already available. Until now, their accuracy range (∼80%) has limited their usage in clinical applications. In parallel, a series of studies indicate that protein-specific predictors (PSP), using only information from the protein of interest, could frequently surpass the performance of GM. However, two reasons suggest that this may not always be the case: the existence of a performance threshold affecting both GM and PSP, and the effect of training data scarcity. Here, we characterize the relationship between the two approaches deriving 82 PSP and comparing them with several GM (PolyPhen-2, SIFT, PON-P2, MutationTaster2, CADD). We find a complementary relationship between PSP and GM, with no approach always outperforming the other. However, the relationship varies between two limiting situations, e.g. PSP are frequently outperformed by PON-P2, the best general method; however, the opposite happens when we compare PSP and SIFT. Finally, we explore how the observed complementarity could lead to increased success rates in pathogenicity prediction. This article is protected by copyright. All rights reserved.
- Silent Tyrosinemia Type I Without Elevated Tyrosine or Succinylacetone Associated With Liver Cirrhosis and Hepatocellular Carcinoma. [JOURNAL ARTICLE]
- Hum Mutat 2016 Jul 10.
Tyrosinemia type I (TYRSN1, TYR I) is caused by fumarylacetoacetate hydrolase (FAH) deficiency and affects approximately 1 in 100,000 individuals worldwide. Pathogenic variants in FAH cause TYRSN1, which induces cirrhosis and can progress to hepatocellular carcinoma (HCC). TYRSN1 is characterized by the production of a pathognomonic metabolite, succinylacetone (SUAC) and is included in the Recommended Uniform Screening Panel for newborns. Treatment intervention is effective if initiated within the first month of life. Here we describe a family with three affected children who developed HCC secondary to idiopathic hepatosplenomegaly and cirrhosis during infancy. Whole exome sequencing revealed a novel homozygous missense variant in FAH (Chr15(GRCh38):g.80162305A>G; NM_000137.2:c.424A > G; NP_000128.1:p.R142G). This novel variant involves the catalytic pocket of the enzyme, but does not result in increased SUAC or tyrosine, making the diagnosis of TYRSN1 problematic. Testing this novel variant using a rapid, in vivo somatic mouse model showed that this variant could not rescue FAH deficiency. In this case of atypical TYRSN1, we show how reliance on SUAC as a primary diagnostic test can be misleading in some patients with this disease. Augmentation of current screening for TYRSN1 with targeted sequencing of FAH is warranted in cases suggestive of the disorder. This article is protected by copyright. All rights reserved.
- microRNA Genetic Variation, from Population Analysis to Functional Implications of Three Allele Variants Associated with Cancer. [JOURNAL ARTICLE]
- Hum Mutat 2016 Jul 10.
Nucleotide variants in microRNA regions have been associated with disease, nevertheless still few studies have addressed the allele-dependent effect of these changes. We studied microRNA genetic variation in human populations and found that while low frequency variants accumulate indistinctly in microRNA regions, the mature and seed regions tent to be depleted of high frequency variants, probably as a result of purifying selection. Comparison of pairwise population fixation indexes among regions showed that the seed had higher population fixation indexes than the other regions suggesting the existence of local adaptation in the seed region. We further performed functional studies of three microRNA variants associated with cancer (rs2910164:C > G in MIR146A, rs11614913:C > T in MIR196A2 and rs3746444:A > G in both, MIR499A and MIR499B). We found differences in the expression between alleles and in the regulation of several genes involved in cancer, such as TP53, KIT, CDH1, CLH and TERT, which may result in changes in regulatory networks related to tumorigenesis. Furthermore, luciferase-based assays showed that MIR499A could be regulating the cadherin CDH1 and the cell adhesion molecule CLH1 in an allele-dependent fashion. A better understanding of the effect of microRNA variants associated with disease could be key in our way to a more personalized medicine. This article is protected by copyright. All rights reserved.
- Skewed X-inactivation and Females with Intellectual Disability. [Journal Article]
- Hum Mutat 2016 Aug; 37(8):717.