Predicting the impact of rare variants on RNA splicing in CAGI6.

Variants which disrupt splicing are a frequent cause of rare disease that have been under-ascertained clinically. Accurate and efficient methods to predict a variant's impact on splicing are needed to interpret the growing number of variants of unknown significance (VUS) identified by exome and genome sequencing. Here, we present the results of the CAGI6 Splicing VUS challenge, which invited predictions of the splicing impact of 56 variants ascertained clinically and functionally validated to determine splicing impact. The performance of 12 prediction methods, along with SpliceAI and CADD, was compared on the 56 functionally validated variants. The maximum accuracy achieved was 82% from two different approaches, one weighting SpliceAI scores by minor allele frequency, and one applying the recently published Splicing Prediction Pipeline (SPiP). SPiP performed optimally in terms of sensitivity, while an ensemble method combining multiple prediction tools and information from databases exceeded all others for specificity. Several challenge methods equalled or exceeded the performance of SpliceAI, with ultimate choice of prediction method likely to depend on experimental or clinical aims. One quarter of the variants were incorrectly predicted by at least 50% of the methods, highlighting the need for further improvements to splicing prediction methods for successful clinical application.

SMAD4 mosaicism in juvenile polyposis: Essential contribution of somatic analysis in diagnosis.

Juvenile polyposis syndrome (JPS) is a rare disease characterized by multiple hamartomatous polyps in the gastrointestinal tract, associated with pathogenic variants of BMPR1A and SMAD4. We present the description of SMAD4 mosaicism in a 30-year-old man who had caecum adenocarcinoma, 11 juvenile colon polyps and epistaxis since childhood. We conducted NGS polyposis and CRC panel analysis on DNA extracted from two polyps, revealing a likely pathogenic SMAD4 variant: NM_005359.5:c. 1600C>T, p.(Gln534*). This variant was then identified at a very low frequency on blood and normal colonic tissue, by targeted visualization of previously obtained NGS data. These findings support the presence of a likely pathogenic mosaic SMAD4 variant that aligns with the patient's phenotype. Given the relatively frequent occurrence of de novo SMAD4 mutations, somatic mosaicism could account for a significant proportion of sporadic JPS patients with unidentified pathogenic variants. This case underscores the diagnosis challenge of detecting mosaicism and emphasizes the importance of somatic analyses.

Germline HPF1 retrogene insertion in RB1 gene involved in cancer predisposition.

About half of the human genome is composed of repeated sequences derived from mobile elements, mainly retrotransposons, generally without pathogenic effect. Familial forms of retinoblastoma are caused by germline pathogenic variants in RB1 gene. Here, we describe a family with retinoblastoma affecting a father and his son. No pathogenic variant was identified after DNA analysis of RB1 gene coding sequence and exon-intron junctions. However, RB1 mRNA analysis showed a chimeric transcript with insertion of 114 nucleotides from HPF1 gene inside RB1 gene. This chimeric transcript led to an insertion of 38 amino acids in functional domain of retinoblastoma protein. Subsequent DNA analysis in RB1 intron 17 revealed the presence of a full-length HPF1 retrogene insertion in opposite orientation. Functional assay shows that this insertion has a deleterious impact on retinoblastoma protein function. This is the first report of a full-length retrogene insertion involved in human Mendelian disease leading to a chimeric transcript and a non-functional chimeric protein. Some retrogene insertions may be missed by standard diagnostic genetic testing, so contribution of retrogene insertions to human disease may be underestimated. The increasing use of whole genome sequencing in diagnostic settings will help to get a more comprehensive view of retrogenes.

SPiP: Splicing Prediction Pipeline, a machine learning tool for massive detection of exonic and intronic variant effects on mRNA splicing.

Modeling splicing is essential for tackling the challenge of variant interpretation as each nucleotide variation can be pathogenic by affecting pre-mRNA splicing via disruption/creation of splicing motifs such as 5'/3' splice sites, branch sites, or splicing regulatory elements. Unfortunately, most in silico tools focus on a specific type of splicing motif, which is why we developed the Splicing Prediction Pipeline (SPiP) to perform, in one single bioinformatic analysis based on a machine learning approach, a comprehensive assessment of the variant effect on different splicing motifs. We gathered a curated set of 4616 variants scattered all along the sequence of 227 genes, with their corresponding splicing studies. The Bayesian analysis provided us with the number of control variants, that is, variants without impact on splicing, to mimic the deluge of variants from high-throughput sequencing data. Results show that SPiP can deal with the diversity of splicing alterations, with 83.13% sensitivity and 99% specificity to detect spliceogenic variants. Overall performance as measured by area under the receiving operator curve was 0.986, better than SpliceAI and SQUIRLS (0.965 and 0.766) for the same data set. SPiP lends itself to a unique suite for comprehensive prediction of spliceogenicity in the genomic medicine era. SPiP is available at: https://sourceforge.net/projects/splicing-prediction-pipeline/.

Assessment of Multiplex Digital Droplet RT-PCR as a Diagnostic Tool for SARS-CoV-2 Detection in Nasopharyngeal Swabs and Saliva Samples.

BACKGROUND: Reverse transcription-quantitative PCR on nasopharyngeal swabs is currently the reference COVID-19 diagnosis method but exhibits imperfect sensitivity. METHODS: We developed a multiplex reverse transcription-digital droplet PCR (RT-ddPCR) assay, targeting 6 SARS-CoV-2 genomic regions, and evaluated it on nasopharyngeal swabs and saliva samples collected from 130 COVID-19 positive or negative ambulatory individuals, who presented symptoms suggestive of mild or moderate SARS-CoV2 infection. RESULTS: For the nasopharyngeal swab samples, the results obtained using the 6-plex RT-ddPCR and RT-qPCR assays were all concordant. The 6-plex RT-ddPCR assay was more sensitive than RT-qPCR (85% versus 62%) on saliva samples from patients with positive nasopharyngeal swabs. CONCLUSION: Multiplex RT-ddPCR represents an alternative and complementary tool for the diagnosis of COVID-19, in particular to control RT-qPCR ambiguous results. It can also be applied to saliva for repetitive sampling and testing individuals for whom nasopharyngeal swabbing is not possible.

Further delineation of the NTHL1 associated syndrome: A report from the French Oncogenetic Consortium.

Biallelic pathogenic variants in the NTHL1 (Nth like DNA glycosylase 1) gene cause a recently identified autosomal recessive hereditary cancer syndrome predisposing to adenomatous polyposis and colorectal cancer. Half of biallelic carriers also display multiple colonic or extra-colonic primary tumors, mainly breast, endometrium, urothelium, and brain tumors. Published data designate NTHL1 as an important contributor to hereditary cancers but also underline the scarcity of available informations. Thanks to the French oncogenetic consortium (Groupe Génétique et Cancer), we collected NTHL1 variants from 7765 patients attending for hereditary colorectal cancer or polyposis (n = 3936) or other hereditary cancers (n = 3829). Here, we describe 10 patients with pathogenic biallelic NTHL1 germline variants, that is, the second largest NTHL1 series. All carriers were from the "colorectal cancer or polyposis" series. All nine biallelic carriers who underwent colonoscopy presented adenomatous polyps. For digestive cancers, average age at diagnosis was 56.2 and we reported colorectal, duodenal, caecal, and pancreatic cancers. Extra-digestive malignancies included sarcoma, basal cell carcinoma, breast cancer, urothelial carcinoma, and melanoma. Although tumor risks remain to be precisely defined, these novel data support NTHL1 inclusion in diagnostic panel testing. Colonic surveillance should be conducted based on MUTYH recommendations while extra-colonic surveillance has to be defined.

Assessment of branch point prediction tools to predict physiological branch points and their alteration by variants.

BACKGROUND: Branch points (BPs) map within short motifs upstream of acceptor splice sites (3'ss) and are essential for splicing of pre-mature mRNA. Several BP-dedicated bioinformatics tools, including HSF, SVM-BPfinder, BPP, Branchpointer, LaBranchoR and RNABPS were developed during the last decade. Here, we evaluated their capability to detect the position of BPs, and also to predict the impact on splicing of variants occurring upstream of 3'ss. RESULTS: We used a large set of constitutive and alternative human 3'ss collected from Ensembl (n = 264,787 3'ss) and from in-house RNAseq experiments (n = 51,986 3'ss). We also gathered an unprecedented collection of functional splicing data for 120 variants (62 unpublished) occurring in BP areas of disease-causing genes. Branchpointer showed the best performance to detect the relevant BPs upstream of constitutive and alternative 3'ss (99.48 and 65.84% accuracies, respectively). For variants occurring in a BP area, BPP emerged as having the best performance to predict effects on mRNA splicing, with an accuracy of 89.17%. CONCLUSIONS: Our investigations revealed that Branchpointer was optimal to detect BPs upstream of 3'ss, and that BPP was most relevant to predict splicing alteration due to variants in the BP area.

Novel diagnostic tool for prediction of variant spliceogenicity derived from a set of 395 combined in silico/in vitro studies: an international collaborative effort.

Variant interpretation is the key issue in molecular diagnosis. Spliceogenic variants exemplify this issue as each nucleotide variant can be deleterious via disruption or creation of splice site consensus sequences. Consequently, reliable in silico prediction of variant spliceogenicity would be a major improvement. Thanks to an international effort, a set of 395 variants studied at the mRNA level and occurring in 5' and 3' consensus regions (defined as the 11 and 14 bases surrounding the exon/intron junction, respectively) was collected for 11 different genes, including BRCA1, BRCA2, CFTR and RHD, and used to train and validate a new prediction protocol named Splicing Prediction in Consensus Elements (SPiCE). SPiCE combines in silico predictions from SpliceSiteFinder-like and MaxEntScan and uses logistic regression to define optimal decision thresholds. It revealed an unprecedented sensitivity and specificity of 99.5 and 95.2%, respectively, and the impact on splicing was correctly predicted for 98.8% of variants. We therefore propose SPiCE as the new tool for predicting variant spliceogenicity. It could be easily implemented in any diagnostic laboratory as a routine decision making tool to help geneticists to face the deluge of variants in the next-generation sequencing era. SPiCE is accessible at (https://sourceforge.net/projects/spicev2-1/).

First estimate of the scale of canonical 5' splice site GT>GC variants capable of generating wild-type transcripts.

It has long been known that canonical 5' splice site (5'SS) GT>GC variants may be compatible with normal splicing. However, to date, the actual scale of canonical 5'SSs capable of generating wild-type transcripts in the case of GT>GC substitutions remains unknown. Herein, combining data derived from a meta-analysis of 45 human disease-causing 5'SS GT>GC variants and a cell culture-based full-length gene splicing assay of 103 5'SS GT>GC substitutions, we estimate that ~15-18% of canonical GT 5'SSs retain their capacity to generate between 1% and 84% normal transcripts when GT is substituted by GC. We further demonstrate that the canonical 5'SSs in which substitution of GT by GC-generated normal transcripts exhibit stronger complementarity to the 5' end of U1 snRNA than those sites whose substitutions of GT by GC did not lead to the generation of normal transcripts. We also observed a correlation between the generation of wild-type transcripts and a milder than expected clinical phenotype but found that none of the available splicing prediction tools were capable of reliably distinguishing 5'SS GT>GC variants that generated wild-type transcripts from those that did not. Our findings imply that 5'SS GT>GC variants in human disease genes may not invariably be pathogenic.

A Parent-of-Origin Effect Impacts the Phenotype in Low Penetrance Retinoblastoma Families Segregating the c.1981C>T/p.Arg661Trp Mutation of RB1.

Retinoblastoma (Rb), the most common pediatric intraocular neoplasm, results from inactivation of both alleles of the RB1 tumor suppressor gene. The second allele is most commonly lost, as demonstrated by loss of heterozygosity studies. RB1 germline carriers usually develop bilateral tumors, but some Rb families display low penetrance and variable expressivity. In order to decipher the underlying mechanisms, 23 unrelated low penetrance pedigrees segregating the common c.1981C>T/p.Arg661Trp mutation and other low penetrance mutations were studied. In families segregating the c.1981C>T mutation, we demonstrated, for the first time, a correlation between the gender of the transmitting carrier and penetrance, as evidenced by Fisher's exact test: the probability of being unaffected is 90.3% and 32.5% when the mutation is inherited from the mother and the father, respectively (p-value = 7.10(-7). Interestingly, a similar correlation was observed in families segregating other low penetrance alleles. Consequently, we investigated the putative involvement of an imprinted, modifier gene in low penetrance Rb. We first ruled out a MED4-driven mechanism by MED4 methylation and expression analyses. We then focused on the differentially methylated CpG85 island located in intron 2 of RB1 and showing parent-of-origin-specific DNA methylation. This differential methylation promotes expression of the maternal c.1981C>T allele. We propose that the maternally inherited c.1981C>T/p.Arg661Trp allele retains sufficient tumor suppressor activity to prevent retinoblastoma development. In contrast, when the mutation is paternally transmitted, the low residual activity would mimic a null mutation and subsequently lead to retinoblastoma. This implies that the c.1981C>T mutation is not deleterious per se but needs to be destabilized in order to reach pRb haploinsufficiency and initiate tumorigenesis. We suggest that this phenomenon might be a general mechanism to explain phenotypic differences in low penetrance Rb families.

Functional Assessment of Genetic Variants with Outcomes Adapted to Clinical Decision-Making.

Understanding the medical effect of an ever-growing number of human variants detected is a long term challenge in genetic counseling. Functional assays, based on in vitro or in vivo evaluations of the variant effects, provide essential information, but they require robust statistical validation, as well as adapted outputs, to be implemented in the clinical decision-making process. Here, we assessed 25 pathogenic and 15 neutral missense variants of the BRCA1 breast/ovarian cancer susceptibility gene in four BRCA1 functional assays. Next, we developed a novel approach that refines the variant ranking in these functional assays. Lastly, we developed a computational system that provides a probabilistic classification of variants, adapted to clinical interpretation. Using this system, the best functional assay exhibits a variant classification accuracy estimated at 93%. Additional theoretical simulations highlight the benefit of this ready-to-use system in the classification of variants after functional assessment, which should facilitate the consideration of functional evidences in the decision-making process after genetic testing. Finally, we demonstrate the versatility of the system with the classification of siRNAs tested for human cell growth inhibition in high throughput screening.

Combined genetic and splicing analysis of BRCA1 c.[594-2A>C; 641A>G] highlights the relevance of naturally occurring in-frame transcripts for developing disease gene variant classification algorithms.

A recent analysis using family history weighting and co-observation classification modeling indicated that BRCA1 c.594-2A > C (IVS9-2A > C), previously described to cause exon 10 skipping (a truncating alteration), displays characteristics inconsistent with those of a high risk pathogenic BRCA1 variant. We used large-scale genetic and clinical resources from the ENIGMA, CIMBA and BCAC consortia to assess pathogenicity of c.594-2A > C. The combined odds for causality considering case-control, segregation and breast tumor pathology information was 3.23 × 10-8 Our data indicate that c.594-2A > C is always in cis with c.641A > G. The spliceogenic effect of c.[594-2A > C;641A > G] was characterized using RNA analysis of human samples and splicing minigenes. As expected, c.[594-2A > C; 641A > G] caused exon 10 skipping, albeit not due to c.594-2A > C impairing the acceptor site but rather by c.641A > G modifying exon 10 splicing regulatory element(s). Multiple blood-based RNA assays indicated that the variant allele did not produce detectable levels of full-length transcripts, with a per allele BRCA1 expression profile composed of ≈70-80% truncating transcripts, and ≈20-30% of in-frame Δ9,10 transcripts predicted to encode a BRCA1 protein with tumor suppression function.We confirm that BRCA1c.[594-2A > C;641A > G] should not be considered a high-risk pathogenic variant. Importantly, results from our detailed mRNA analysis suggest that BRCA-associated cancer risk is likely not markedly increased for individuals who carry a truncating variant in BRCA1 exons 9 or 10, or any other BRCA1 allele that permits 20-30% of tumor suppressor function. More generally, our findings highlight the importance of assessing naturally occurring alternative splicing for clinical evaluation of variants in disease-causing genes.

Mutiple DICER1-related lesions associated with a germline deep intronic mutation.

Germline DICER1 pathogenic variants predispose to numerous benign and malignant tumors. In this report, we describe DICER1 gene analysis in an adolescent diagnosed with multinodular goiter, ovarian Sertoli-Leydig cell tumor, and lung cyst. DICER1 mutational screening at the DNA level failed to detect any pathogenic variant. Subsequent messenger RNA (mRNA) analysis revealed a 132 nucleotide intronic sequence exonization. This truncating event was caused by a deep intronic mutation generating a de novo acceptor splice site. This study demonstrates that some undetected DICER1 mutations should be investigated at the mRNA level.

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.

Extensive Variation in the Mutation Rate Between and Within Human Genes Associated with Mendelian Disease.

We have investigated whether the mutation rate varies between genes and sites using de novo mutations (DNMs) from three genes associated with Mendelian diseases (RB1, NF1, and MECP2). We show that the relative frequency of mutations at CpG dinucleotides relative to non-CpG sites varies between genes and relative to the genomic average. In particular we show that the rate of transition mutation at CpG sites relative to the rate of non-CpG transversion is substantially higher in our disease genes than amongst DNMs in general; the rate of CpG transition can be several hundred-fold greater than the rate of non-CpG transversion. We also show that the mutation rate varies significantly between sites of a particular mutational type, such as non-CpG transversion, within a gene. We estimate that for all categories of sites, except CpG transitions, there is at least a 30-fold difference in the mutation rate between the 10% of sites with the highest and lowest mutation rates. However, our best estimate is that the mutation rate varies by several hundred-fold variation. We suggest that the presence of hypermutable sites may be one reason certain genes are associated with disease.

BRCA Share: A Collection of Clinical BRCA Gene Variants.

As next-generation sequencing increases access to human genetic variation, the challenge of determining clinical significance of variants becomes ever more acute. Germline variants in the BRCA1 and BRCA2 genes can confer substantial lifetime risk of breast and ovarian cancer. Assessment of variant pathogenicity is a vital part of clinical genetic testing for these genes. A database of clinical observations of BRCA variants is a critical resource in that process. This article describes BRCA Share™, a database created by a unique international alliance of academic centers and commercial testing laboratories. By integrating the content of the Universal Mutation Database generated by the French Unicancer Genetic Group with the testing results of two large commercial laboratories, Quest Diagnostics and Laboratory Corporation of America (LabCorp), BRCA Share™ has assembled one of the largest publicly accessible collections of BRCA variants currently available. Although access is available to academic researchers without charge, commercial participants in the project are required to pay a support fee and contribute their data. The fees fund the ongoing curation effort, as well as planned experiments to functionally characterize variants of uncertain significance. BRCA Share™ databases can therefore be considered as models of successful data sharing between private companies and the academic world.

Genomic hallmarks of homologous recombination deficiency in invasive breast carcinomas.

Therapeutic strategies targeting Homologous Recombination Deficiency (HRD) in breast cancer requires patient stratification. The LST (Large-scale State Transitions) genomic signature previously validated for triple-negative breast carcinomas (TNBC) was evaluated as biomarker of HRD in luminal (hormone receptor positive) and HER2-overexpressing (HER2+) tumors. The LST genomic signature related to the number of large-scale chromosomal breakpoints in SNP-array tumor profile was applied to identify HRD in in-house and TCGA sets of breast tumors, in which the status of BRCA1/2 and other genes was also investigated. In the in-house dataset, HRD was predicted in 5% (20/385) of sporadic tumors luminal or HER2+ by the LST genomic signature and the inactivation of BRCA1, BRCA2 or RAD51C confirmed this prediction in 75% (12/16) of the tested cases. In 14% (6/43) of tumors occurring in BRCA1/2 mutant carriers, the corresponding wild-type allele was retained emphasizing the importance of determining the tumor status. In the TCGA luminal and HER2+ subtypes HRD incidence was estimated at 5% (18/329, 95%CI: 5-8%) and 2% (1/59, 95%CI: 2-9%), respectively. In TNBC cisplatin-based neo-adjuvant clinical trials, HRD is shown to be a necessary condition for cisplatin sensitivity. This analysis demonstrates the high performance of the LST genomic signature for HRD detection in breast cancers, which suggests its potential as a biomarker for genetic testing and patient stratification for clinical trials evaluating platinum salts and PARP inhibitors.

The survival gene MED4 explains low penetrance retinoblastoma in patients with large RB1 deletion.

Retinoblastoma is a non-hereditary as well as an inherited pediatric tumor of the developing retina resulting from the inactivation of both copies of the RB1 tumor suppressor gene. Familial retinoblastoma is a highly penetrant genetic disease that usually develops by carrying germline mutations that inactivate one allele of the RB1 gene, leading to multiple retinoblastomas. However, large and complete germline RB1 deletions are associated with low or no tumor risk for reasons that remain unknown. In this study, we define a minimal genomic region associated with this low penetrance. This region encompasses few genes including MED4 a subunit of the mediator complex. We further show that retinoblastoma RB1 -/- cells cannot survive in the absence of MED4, both in vitro and in orthotopic xenograft models in vivo, therefore identifying MED4 as a survival gene in retinoblastoma. We propose that the contiguous loss of the adjacent retinoblastoma gene, MED4, explains the low penetrance in patients with large deletions that include both RB1 and MED4. Our findings also point to another synthetic lethal target in tumors with inactivated RB1 and highlight the importance of collateral damage in carcinogenesis.

Comprehensive annotation of splice junctions supports pervasive alternative splicing at the BRCA1 locus: a report from the ENIGMA consortium.

Loss-of-function germline mutations in BRCA1 (MIM #113705) confer markedly increased risk of breast and ovarian cancer. The full-length transcript codifies for a protein involved in DNA repair pathways and cell-cycle checkpoints. Several BRCA1 splicing isoforms have been described in public domain databases, but the physiological role (if any) of BRCA1 alternative splicing remains to be established. An accurate description of 'naturally occurring' alternative splicing at this locus is a prerequisite to understand its biological significance. However, a systematic analysis of alternative splicing at the BRCA1 locus is yet to be conducted. Here, the Evidence-Based Network for the Interpretation of Germ-Line Mutant Alleles consortium combines RT-PCR, exon scanning, cloning, sequencing and relative semi-quantification to describe naturally occurring BRCA1 alternative splicing with unprecedented resolution. The study has been conducted in blood-related RNA sources, commonly used for clinical splicing assays, as well as in one healthy breast tissue. We have characterized a total of 63 BRCA1 alternative splicing events, including 35 novel findings. A minimum of 10 splicing events (Δ1Aq, Δ5, Δ5q, Δ8p, Δ9, Δ(9,10), Δ9_11, Δ11q, Δ13p and Δ14p) represent a substantial fraction of the full-length expression level (ranging from 5 to 100%). Remarkably, our data indicate that BRCA1 alternative splicing is similar in blood and breast, a finding supporting the clinical relevance of blood-based in vitro splicing assays. Overall, our data suggest an alternative splicing model in which most non-mutually exclusive alternative splicing events are randomly combined into individual mRNA molecules to produce hundreds of different BRCA1 isoforms.