Classifying MLH1 and MSH2 variants using bioinformatic prediction,splicing assays,segregation, and tumor characteristics

Reliable methods for predicting functional consequences of variants in disease genes would be beneficial in the clinical setting. This study was undertaken to predict, and confirm in vitro, splicing aberrations associated with mismatch repair (MMR) variants identified in familial colon cancer patients. Six programs were used to predict the effect of 13 MLH1 and 6 MSH2 gene variants on pre‐mRNA splicing. mRNA from cycloheximide‐treated lymphoblastoid cell lines of variant carriers was screened for splicing aberrations. Tumors of variant carriers were tested for microsatellite instability and MMR protein expression. Variant segregation in families was assessed using Bayes factor causality analysis. Amino acid alterations were examined for evolutionary conservation and physicochemical properties. Splicing aberrations were detected for 10 variants, including a frameshift as a minor cDNA product, and altered ratio of known alternate splice products. Loss of splice sites was well predicted by splice‐site prediction programs SpliceSiteFinder (90%) and NNSPLICE (90%), but consequence of splice site loss was less accurately predicted. No aberrations correlated with ESE predictions for the nine exonic variants studied. Seven of eight missense variants had normal splicing (88%), but only one was a substitution considered neutral from evolutionary/physicochemical analysis. Combined with information from tumor and segregation analysis, and literature review, 16 of 19 variants were considered clinically relevant. Bioinformatic tools for prediction of splicing aberrations need improvement before use without supporting studies to assess variant pathogenicity. Classification of mismatch repair gene variants is assisted by a comprehensive approach that includes in vitro, tumor pathology, clinical, and evolutionary conservation data. Hum Mutat 0, 1–14, 2009. © 2009 Wiley‐Liss, Inc.     

Consortium for osteogenesis imperfecta mutations in the helical domain of type I collagen: regions rich in lethal mutations align with collagen binding sites for integrins and proteoglycans

Osteogenesis imperfecta (OI) is a generalized disorder of connective tissue characterized by fragile bones and easy susceptibility to fracture. Most cases of OI are caused by mutations in type I collagen. We have identified and assembled structural mutations in type I collagen genes (COL1A1 and COL1A2, encoding the proalpha1(I) and proalpha2(I) chains, respectively) that result in OI. Quantitative defects causing type I OI were not included. Of these 832 independent mutations, 682 result in substitution for glycine residues in the triple helical domain of the encoded protein and 150 alter splice sites. Distinct genotype-phenotype relationships emerge for each chain. One-third of the mutations that result in glycine substitutions in alpha1(I) are lethal, especially when the substituting residues are charged or have a branched side chain. Substitutions in the first 200 residues are nonlethal and have variable outcome thereafter, unrelated to folding or helix stability domains. Two exclusively lethal regions (helix positions 691-823 and 910-964) align with major ligand binding regions (MLBRs), suggesting crucial interactions of collagen monomers or fibrils with integrins, matrix metalloproteinases (MMPs), fibronectin, and cartilage oligomeric matrix protein (COMP). Mutations in COL1A2 are predominantly nonlethal (80%). Lethal substitutions are located in eight regularly spaced clusters along the chain, supporting a regional model. The lethal regions align with proteoglycan binding sites along the fibril, suggesting a role in fibril-matrix interactions. Recurrences at the same site in alpha2(I) are generally concordant for outcome, unlike alpha1(I). Splice site mutations comprise 20% of helical mutations identified in OI patients, and may lead to exon skipping, intron inclusion, or the activation of cryptic splice sites. Splice site mutations in COL1A1 are rarely lethal; they often lead to frameshifts and the mild type I phenotype. In alpha2(I), lethal exon skipping events are located in the carboxyl half of the chain. Our data on genotype-phenotype relationships indicate that the two collagen chains play very different roles in matrix integrity and that phenotype depends on intracellular and extracellular events.     

Splicing and multifactorial analysis of intronic BRCA1 and BRCA2 sequence variants identifies clinically significant splicing aberrations up to 12 nucleotides from the intron/exon boundary

Clinical management of breast cancer families is complicated by identification of BRCA1 and BRCA2 sequence alterations of unknown significance. Molecular assays evaluating the effect of intronic variants on native splicing can help determine their clinical relevance. Twenty-six intronic BRCA1/2 variants ranging from the consensus dinucleotides in the splice acceptor or donor to 53 nucleotides into the intron were identified in multiple-case families. The effect of the variants on splicing was assessed using HSF matrices, MaxEntScan and NNsplice, followed by analysis of mRNA from lymphoblastoid cell lines. A total of 12 variants were associated with splicing aberrations predicted to result in production of truncated proteins, including a variant located 12 nucleotides into the intron. The posterior probability of pathogenicity was estimated using a multifactorial likelihood approach, and provided a pathogenic or likely pathogenic classification for seven of the 12 spliceogenic variants. The apparent disparity between experimental evidence and the multifactorial predictions is likely due to several factors, including a paucity of likelihood information and a nonspecific prior probability applied for intronic variants outside the consensus dinucleotides. Development of prior probabilities of pathogenicity incorporating bioinformatic prediction of splicing aberrations should improve identification of functionally relevant variants and enhance multifactorial likelihood analysis of intronic variants.     

Type I Procollagen C‐Propeptide Defects: Study of Genotype–Phenotype Correlation and Predictive Role of Crystal Structure

The type I procollagen carboxyterminal(C‐)propeptides are crucial in directing correct assembly of the procollagen heterotrimers. Defects in these domains have anecdotally been reported in patients with Osteogenesis Imperfecta (OI) and few genotype–phenotype correlations have been described. To gain insight in the functional consequences of C‐propeptide defects, we performed a systematic review of clinical, molecular, and biochemical findings in all patients in whom we identified a type I procollagen C‐propeptide defect, and compared this with literature data. We report 30 unique type I procollagen C‐propeptide variants, 24 of which are novel. The outcome of COL1A1 nonsense and frameshift variants depends on the location of the premature termination codon. Those located prior to 50–55 nucleotides upstream of the most 3’ exon–exon junction lead to nonsense‐mediated mRNA decay (NMD) and cause mild OI. Those located beyond this boundary escape NMD, generally lead to production of stable, overmodified procollagen chains, which may partly be retained intracellularly, and are usually associated with severe‐to‐lethal OI. Proα1(I)‐C‐propeptide defects that permit chain association result in more severe phenotypes than those inhibiting chain association. We demonstrate that the crystal structure of the proα1(III)‐C‐propeptide is a reliable tool to predict phenotypic severity for most COL1A1‐C‐propeptide missense variants, whereas for COL1A2‐C‐propeptide variants, the phenotypic outcome is milder than predicted.     

Prediction of Mutant mRNA Splice Isoforms by Information Theory‐Based Exon Definition

Mutations that affect mRNA splicing often produce multiple mRNA isoforms, resulting in complex molecular phenotypes. Definition of an exon and its inclusion in mature mRNA relies on joint recognition of both acceptor and donor splice sites. This study predicts cryptic and exon‐skipping isoforms in mRNA produced by splicing mutations from the combined information contents (R_i, which measures binding‐site strength, in bits) and distribution of the splice sites defining these exons. The total information content of an exon (R_i,total) is the sum of the R_i values of its acceptor and donor splice sites, adjusted for the self‐information of the distance separating these sites, that is, the gap surprisal. Differences between total information contents of an exon (ΔR_i,total) are predictive of the relative abundance of these exons in distinct processed mRNAs. Constraints on splice site and exon selection are used to eliminate nonconforming and poorly expressed isoforms. Molecular phenotypes are computed by the Automated Splice Site and Exon Definition Analysis ( http://splice.uwo.ca ) server. Predictions of splicing mutations were highly concordant (85.2%; n = 61) with published expression data. In silico exon definition analysis will contribute to streamlining assessment of abnormal and normal splice isoforms resulting from mutations.     

Mutations in the COL1A2 gene of type I collagen that result in nonlethal forms of osteogenesis imperfecta

Although virtually all mutations that result in osteogenesis imperfecta (OI) affect the genes that encode the chains of type I procollagen, the effects of mutations in the COL1A2 gene have received less attention than those in the COL1A1 gene. We have characterized mutations in 4 families that give rise to different OI phenotypes. In three families substitutions of glycine residues by cysteine in the triple helical domain (a single example at position 259 and 2 families in which substitution of glycine at 646 by cysteine) have been identified, and in the fourth a G for A transition at position + 4 in intron 33 led to use of an alternative splice site and inclusion of 6 amino acids (val-gly-arg-ile-leu-phe) between residues 585 and 586 of the normal triple helix. The relation between position of substitution of glycine by cysteine in the COL1A2 gene does not follow the pattern developed in the COL1A1 gene. To determine how COL1A2 mutations produce OI phenotypes, we have produced a full-length mouse cDNA into which we plan to place mutations and examine their effects in stably transfected osteogenic cells and in transgenic animals.     

Contribution of bioinformatics predictions and functional splicing assays to the interpretation of unclassified variants of the BRCA genes

A large fraction of sequence variants of unknown significance (VUS) of the breast and ovarian cancer susceptibility genes BRCA1 and BRCA2 may induce splicing defects. We analyzed 53 VUSs of BRCA1 or BRCA2, detected in consecutive molecular screenings, by using five splicing prediction programs, and we classified them into two groups according to the strength of the predictions. In parallel, we tested them by using functional splicing assays. A total of 10 VUSs were predicted by two or more programs to induce a significant reduction of splice site strength or activation of cryptic splice sites or generation of new splice sites. Minigene-based splicing assays confirmed four of these predictions. Five additional VUSs, all at internal exon positions, were not predicted to induce alterations of splice sites, but revealed variable levels of exon skipping, most likely induced by the modification of exonic splicing regulatory elements. We provide new data in favor of the pathogenic nature of the variants BRCA1 c.212+3A>G and BRCA1 c.5194−12G>A, which induced aberrant out-of-frame mRNA forms. Moreover, the novel variant BRCA2 c.7977−7C>G induced in frame inclusion of 6 nt from the 3′ end of intron 17. The novel variants BRCA2 c.520C>T and BRCA2 c.7992T>A induced incomplete skipping of exons 7 and 18, respectively. This work highlights the contribution of splicing minigene assays to the assessment of pathogenicity, not only when patient RNA is not available, but also as a tool to improve the accuracy of bioinformatics predictions.     

Variant effect on splicing regulatory elements,branchpoint usage,and pseudoexonization: Strategies to enhance bioinformatic prediction using hereditary cancer genes as exemplars

It is possible to estimate the prior probability of pathogenicity for germline disease gene variants based on bioinformatic prediction of variant effect/s. However, routinely used approaches have likely led to the underestimation and underreporting of variants located outside donor and acceptor splice site motifs that affect messenger RNA (mRNA) processing. This review presents information about hereditary cancer gene germline variants, outside native splice sites, with experimentally validated splicing effects. We list 95 exonic variants that impact splicing regulatory elements (SREs) in BRCA1, BRCA2, MLH1, MSH2, MSH6, and PMS2. We utilized a pre‐existing large‐scale BRCA1 functional data set to map functional SREs, and assess the relative performance of different tools to predict effects of 283 variants on such elements. We also describe rare examples of intronic variants that impact branchpoint (BP) sites and create pseudoexons. We discuss the challenges in predicting variant effect on BP site usage and pseudoexonization, and suggest strategies to improve the bioinformatic prioritization of such variants for experimental validation. Importantly, our review and analysis highlights the value of considering impact of variants outside donor and acceptor motifs on mRNA splicing and disease causation.     

Functional Analysis of a Large set of BRCA2 exon 7 Variants Highlights the Predictive Value of Hexamer Scores in Detecting Alterations of Exonic Splicing Regulatory Elements

Exonic variants can alter pre‐mRNA splicing either by changing splice sites or by modifying splicing regulatory elements. Often these effects are difficult to predict and are only detected by performing RNA analyses. Here, we analyzed, in a minigene assay, 26 variants identified in the exon 7 of BRCA2, a cancer predisposition gene. Our results revealed eight new exon skipping mutations in this exon: one directly altering the 5′ splice site and seven affecting potential regulatory elements. This brings the number of splicing regulatory mutations detected in BRCA2 exon 7 to a total of 11, a remarkably high number considering the total number of variants reported in this exon (n = 36), all tested in our minigene assay. We then exploited this large set of splicing data to test the predictive value of splicing regulator hexamers’ scores recently established by Ke et al. ( 2011 ). Comparisons of hexamer‐based predictions with our experimental data revealed high sensitivity in detecting variants that increased exon skipping, an important feature for prescreening variants before RNA analysis. In conclusion, hexamer scores represent a promising tool for predicting the biological consequences of exonic variants and may have important applications for the interpretation of variants detected by high‐throughput sequencing.     

Different somatic and germline HPRT1 mutations promote use of a common,cryptic intron 1 splice site

Aberrant hypoxanthine phosphoribosyltransferase (HUGO‐approved gene symbol HPRT1; MIM# 308000) mRNA splicing, promoted by splice site mutation or loss, is a common mechanism for loss of the purine salvage enzyme HPRT1 from human cells. We report here two in vivo somatic HPRT1 mutations in human kidney tubular epithelial cells that disrupt HPRT1 intron 1 splicing and lead to the inclusion of intron 1 sequence. We propose an explanation for the use of a common, cryptic intron 1 splice donor site by these two mutations, and by 14 additional human HPRT1 mutations that lead to aberrant splicing with the incorporation of intron 1 sequence into mRNA. Hum Mutat 13:504–505, 1999. © 1999 Wiley‐Liss, Inc.     

COL1-related overlap disorder: A novel connective tissue disorder incorporating the osteogenesis imperfecta/Ehlers-Danlos syndrome overlap

The 2017 classification of Ehlers-Danlos syndromes (EDS) identifies three types associated with causative variants in COL1A1/COL1A2 and distinct from osteogenesis imperfecta (OI). Previously, patients have been described with variable features of both disorders, and causative variants in COL1A1/COL1A2; but this phenotype has not been included in the current classification. Here, we expand and re-define this OI/EDS overlap as a missing EDS type. Twenty-one individuals from 13 families were reported, in whom COL1A1/COL1A2 variants were found after a suspicion of EDS. None of them could be classified as affected by OI or by any of the three recognized EDS variants associated with COL1A1/COL1A2. This phenotype is dominated by EDS-related features. OI-related features were limited to mildly reduced bone mass, occasional fractures and short stature. Eight COL1A1/COL1A2 variants were novel and five recurrent with a predominance of glycine substitutions affecting residues within the procollagen N-proteinase cleavage site of α1(I) and α2(I) procollagens. Selected variants were investigated by biochemical, ultrastructural and immunofluorescence studies. The pattern of observed changes in the dermis and in vitro for selected variants was more typical of EDS rather than OI. Our findings indicate the existence of a wider recognizable spectrum associated with COL1A1/COL1A2.     

Alternative splicing of the human luteal LH receptor during luteolysis and maternal recognition of pregnancy

Deletion of exon 10 of the human LH receptor impairs LH but not hCG action. Other splice variants of the LH receptor impair both LH and hCG action in other species. We hypothesized that alternatively spliced LH receptors are involved in luteolysis and luteal rescue with hCG in women. mRNA was extracted from human luteinized granulosa cells and from corpora lutea from across the luteal phase and after luteal rescue in vivo with exogenous hCG. Splice variants were detected by RT-PCR using carefully designed primer pairs. Products were visualized on agarose gels, extracted, purified and sequenced. Three splice variants of the human LH receptor were detected and characterized. These demonstrate a region of multiple splicing between exons 8 and 11 of the receptor. A naturally occurring splice variant with exon 10 alone removed was not identified. There was no obvious change in the pattern of splice variants across the luteal phase in the presence or absence of hCG. These data do not support the hypothesis that qualitative changes in LH receptor splicing have a role in luteolysis or that a naturally occurring LH receptor lacking exon 10 has a role in maternal recognition of pregnancy.