Exome sequencing reveals aberrant signalling pathways as hallmark of treatment-naive anal squamous cell carcinoma.

Anal squamous cell carcinomas (ASCC) are rare tumours in humans. The etiological role of HPV infection is now well established but little is known about the molecular landscape and signalling pathways involved in the pathogenesis of this cancer. Here we report the results from a whole exome sequencing of a homogeneous group of 20 treatment-naive ASCC. A total of 2422 somatic single nucleotide variations (SNV) were found, with an overall moderate rate of somatic mutations per tumour (median: 105 relevant SNV per tumour) but a high mutational load in 3 tumours. The mutational signatures associated with age and APOBEC were observed in 100% and 60% of tumours respectively. The most frequently mutated genes were PIK3CA (25%) followed by FBXW7 (15%), FAT1 (15%), and TRIP12 (15%), the two last ones having never been described in ASCC. The main copy number alterations were gains of chromosome 3q (affecting PIK3CA) and losses of chromosome 11q (affecting ATM). The combined analysis of somatic mutations and copy number alterations show that recurrent alterations of the PI3K/AKT/mTOR pathway are frequent (60%) in these tumours, as well as potentially targetable alterations of other signalling pathways that have never been described in ASCC such as chromatin remodelling (45%) and ubiquitin mediated proteolysis (35%). These results highlight the possible implication of these aberrant signalling pathways in anal carcinogenesis and suggest promising new therapeutic approaches in ASCC. The high somatic mutation burden found in some tumours, suggesting an elevated neoantigen load could also predict sensitivity of ASCC to immunotherapy.

Chimeric EWSR1-FLI1 regulates the Ewing sarcoma susceptibility gene EGR2 via a GGAA microsatellite.

Deciphering the ways in which somatic mutations and germline susceptibility variants cooperate to promote cancer is challenging. Ewing sarcoma is characterized by fusions between EWSR1 and members of the ETS gene family, usually EWSR1-FLI1, leading to the generation of oncogenic transcription factors that bind DNA at GGAA motifs. A recent genome-wide association study identified susceptibility variants near EGR2. Here we found that EGR2 knockdown inhibited proliferation, clonogenicity and spheroidal growth in vitro and induced regression of Ewing sarcoma xenografts. Targeted germline deep sequencing of the EGR2 locus in affected subjects and controls identified 291 Ewing-associated SNPs. At rs79965208, the A risk allele connected adjacent GGAA repeats by converting an interspaced GGAT motif into a GGAA motif, thereby increasing the number of consecutive GGAA motifs and thus the EWSR1-FLI1-dependent enhancer activity of this sequence, with epigenetic characteristics of an active regulatory element. EWSR1-FLI1 preferentially bound to the A risk allele, which increased global and allele-specific EGR2 expression. Collectively, our findings establish cooperation between a dominant oncogene and a susceptibility variant that regulates a major driver of Ewing sarcomagenesis.

Deep Sequencing Reveals Occurrence of Subclonal ALK Mutations in Neuroblastoma at Diagnosis.

PURPOSE: In neuroblastoma, activating ALK receptor tyrosine kinase point mutations play a major role in oncogenesis. We explored the potential occurrence of ALK mutations at a subclonal level using targeted deep sequencing. EXPERIMENTAL DESIGN: In a clinically representative series of 276 diagnostic neuroblastoma samples, exons 23 and 25 of the ALK gene, containing the F1174 and R1275 mutation hotspots, respectively, were resequenced with an extremely high depth of coverage. RESULTS: At the F1174 hotspot (exon 23), mutations were observed in 15 of 277 samples (range of fraction of mutated allele per sample: 0.562%-40.409%). At the R1275 hotspot (exon 25), ALK mutations were detected in 12 of 276 samples (range of fraction of mutated allele: 0.811%-73.001%). Altogether, subclonal events with a mutated allele fraction below 20% were observed in 15/27 ALK-mutated samples. The presence of an ALK mutation was associated with poorer 5-year overall survival (OS: 75% vs. 57%, P = 0.0212 log-rank test), with a strong correlation between F1174 ALK mutations and MYCN amplification being observed. CONCLUSIONS: In this series, deep sequencing allows the detection of F1174 and R1275 ALK mutational events at diagnosis in 10% of cases, with subclonal events in more than half of these, which would have gone undetected by Sanger sequencing. These findings are of clinical importance given the potential role of ALK mutations in clonal evolution and relapse. These findings also demonstrate the importance of deep sequencing techniques for the identification of patients especially when considering targeted therapy.

Mechanical induction of the tumorigenic ß-catenin pathway by tumour growth pressure.

The tumour microenvironment may contribute to tumorigenesis owing to mechanical forces such as fibrotic stiffness or mechanical pressure caused by the expansion of hyper-proliferative cells. Here we explore the contribution of the mechanical pressure exerted by tumour growth onto non-tumorous adjacent epithelium. In the early stage of mouse colon tumour development in the Notch(+)Apc(+/1638N) mouse model, we observed mechanistic pressure stress in the non-tumorous epithelial cells caused by hyper-proliferative adjacent crypts overexpressing active Notch, which is associated with increased Ret and ß-catenin signalling. We thus developed a method that allows the delivery of a defined mechanical pressure in vivo, by subcutaneously inserting a magnet close to the mouse colon. The implanted magnet generated a magnetic force on ultra-magnetic liposomes, stabilized in the mesenchymal cells of the connective tissue surrounding colonic crypts after intravenous injection. The magnetically induced pressure quantitatively mimicked the endogenous early tumour growth stress in the order of 1,200 Pa, without affecting tissue stiffness, as monitored by ultrasound strain imaging and shear wave elastography. The exertion of pressure mimicking that of tumour growth led to rapid Ret activation and downstream phosphorylation of ß-catenin on Tyr654, imparing its interaction with the E-cadherin in adherens junctions, and which was followed by ß-catenin nuclear translocation after 15 days. As a consequence, increased expression of ß-catenin-target genes was observed at 1 month, together with crypt enlargement accompanying the formation of early tumorous aberrant crypt foci. Mechanical activation of the tumorigenic ß-catenin pathway suggests unexplored modes of tumour propagation based on mechanical signalling pathways in healthy epithelial cells surrounding the tumour, which may contribute to tumour heterogeneity.

Circulating tumor DNA as a non-invasive substitute to metastasis biopsy for tumor genotyping and personalized medicine in a prospective trial across all tumor types.

Cell-free tumor DNA (ctDNA) has the potential to enable non-invasive diagnostic tests for personalized medicine in providing similar molecular information as that derived from invasive tumor biopsies. The histology-independent phase II SHIVA trial matches patients with targeted therapeutics based on previous screening of multiple somatic mutations using metastatic biopsies. To evaluate the utility of ctDNA in this trial, as an ancillary study we performed de novo detection of somatic mutations using plasma DNA compared to metastasis biopsies in 34 patients covering 18 different tumor types, scanning 46 genes and more than 6800 COSMIC mutations with a multiplexed next-generation sequencing panel. In 27 patients, 28 of 29 mutations identified in metastasis biopsies (97%) were detected in matched ctDNA. Among these 27 patients, one additional mutation was found in ctDNA only. In the seven other patients, mutation detection from metastasis biopsy failed due to inadequate biopsy material, but was successful in all plasma DNA samples providing three more potential actionable mutations. These results suggest that ctDNA analysis is a potential alternative and/or replacement to analyses using costly, harmful and lengthy tissue biopsies of metastasis, irrespective of cancer type and metastatic site, for multiplexed mutation detection in selecting personalized therapies based on the patient's tumor genetic content.

Circulating tumor DNA and circulating tumor cells in metastatic triple negative breast cancer patients.

Circulating tumor DNA (ctDNA) is a new circulating tumor biomarker which might be used as a prognostic biomarker in a way similar to circulating tumor cells (CTCs). Here, we used the high prevalence of TP53 mutations in triple negative breast cancer (TNBC) to compare ctDNA and CTC detection rates and prognostic value in metastatic TNBC patients. Forty patients were enrolled before starting a new line of treatment. TP53 mutations were characterized in archived tumor tissues and in plasma DNA using two next generation sequencing (NGS) platforms in parallel. Archived tumor tissue was sequenced successfully for 31/40 patients. TP53 mutations were found in 26/31 (84%) of tumor samples. The same mutation was detected in the matched plasma of 21/26 (81%) patients with an additional mutation found only in the plasma for one patient. Mutated allele fractions ranged from 2 to 70% (median 5%). The observed correlation between the two NGS approaches (R(2) = 0.903) suggested that ctDNA levels data were quantitative. Among the 27 patients with TP53 mutations, CTC count was ≥1 in 19 patients (70%) and ≥5 in 14 patients (52%). ctDNA levels had no prognostic impact on time to progression (TTP) or overall survival (OS), whereas CTC numbers were correlated with OS (p = 0.04) and marginally with TTP (p = 0.06). Performance status and elevated LDH also had significant prognostic impact. Here, absence of prognostic impact of baseline ctDNA level suggests that mechanisms of ctDNA release in metastatic TNBC may involve, beyond tumor burden, biological features that do not dramatically affect patient outcome.

Genomic landscape of Ewing sarcoma defines an aggressive subtype with co-association of STAG2 and TP53 mutations.

UNLABELLED: Ewing sarcoma is a primary bone tumor initiated by EWSR1-ETS gene fusions. To identify secondary genetic lesions that contribute to tumor progression, we performed whole-genome sequencing of 112 Ewing sarcoma samples and matched germline DNA. Overall, Ewing sarcoma tumors had relatively few single-nucleotide variants, indels, structural variants, and copy-number alterations. Apart from whole chromosome arm copy-number changes, the most common somatic mutations were detected in STAG2 (17%), CDKN2A (12%), TP53 (7%), EZH2, BCOR, and ZMYM3 (2.7% each). Strikingly, STAG2 mutations and CDKN2A deletions were mutually exclusive, as confirmed in Ewing sarcoma cell lines. In an expanded cohort of 299 patients with clinical data, we discovered that STAG2 and TP53 mutations are often concurrent and are associated with poor outcome. Finally, we detected subclonal STAG2 mutations in diagnostic tumors and expansion of STAG2-immunonegative cells in relapsed tumors as compared with matched diagnostic samples. SIGNIFICANCE: Whole-genome sequencing reveals that the somatic mutation rate in Ewing sarcoma is low. Tumors that harbor STAG2 and TP53 mutations have a particularly dismal prognosis with current treatments and require alternative therapies. Novel drugs that target epigenetic regulators may constitute viable therapeutic strategies in a subset of patients with mutations in chromatin modifiers.

Emergence of new ALK mutations at relapse of neuroblastoma.

PURPOSE: In neuroblastoma, the ALK receptor tyrosine kinase is activated by point mutations. We investigated the potential role of ALK mutations in neuroblastoma clonal evolution. METHODS: We analyzed ALK mutations in 54 paired diagnosis-relapse neuroblastoma samples using Sanger sequencing. When an ALK mutation was observed in one paired sample, a minor mutated component in the other sample was searched for by more than 100,000× deep sequencing of the relevant hotspot, with a sensitivity of 0.17%. RESULTS: All nine ALK-mutated cases at diagnosis demonstrated the same mutation at relapse, in one case in only one of several relapse nodules. In five additional cases, the mutation seemed to be relapse specific, four of which were investigated by deep sequencing. In two cases, no mutation evidence was observed at diagnosis. In one case, the mutation was present at a subclonal level (0.798%) at diagnosis, whereas in another case, two different mutations resulting in identical amino acid changes were detected, one only at diagnosis and the other only at relapse. Further evidence of clonal evolution of ALK-mutated cells was provided by establishment of a fully ALK-mutated cell line from a primary sample with an ALK-mutated cell population at subclonal level (6.6%). CONCLUSION: In neuroblastoma, subclonal ALK mutations can be present at diagnosis with subsequent clonal expansion at relapse. Given the potential of ALK-targeted therapy, the significant spatiotemporal variation of ALK mutations is of utmost importance, highlighting the potential of deep sequencing for detection of subclonal mutations with a sensitivity 100-fold that of Sanger sequencing and the importance of serial samplings for therapeutic decisions.

Multi-factor data normalization enables the detection of copy number aberrations in amplicon sequencing data.

MOTIVATION: Because of its low cost, amplicon sequencing, also known as ultra-deep targeted sequencing, is now becoming widely used in oncology for detection of actionable mutations, i.e. mutations influencing cell sensitivity to targeted therapies. Amplicon sequencing is based on the polymerase chain reaction amplification of the regions of interest, a process that considerably distorts the information on copy numbers initially present in the tumor DNA. Therefore, additional experiments such as single nucleotide polymorphism (SNP) or comparative genomic hybridization (CGH) arrays often complement amplicon sequencing in clinics to identify copy number status of genes whose amplification or deletion has direct consequences on the efficacy of a particular cancer treatment. So far, there has been no proven method to extract the information on gene copy number aberrations based solely on amplicon sequencing. RESULTS: Here we present ONCOCNV, a method that includes a multifactor normalization and annotation technique enabling the detection of large copy number changes from amplicon sequencing data. We validated our approach on high and low amplicon density datasets and demonstrated that ONCOCNV can achieve a precision comparable with that of array CGH techniques in detecting copy number aberrations. Thus, ONCOCNV applied on amplicon sequencing data would make the use of additional array CGH or SNP array experiments unnecessary.

A scoring strategy combining statistics and functional genomics supports a possible role for common polygenic variation in autism.

Autism spectrum disorders (ASD) are highly heritable complex neurodevelopmental disorders with a 4:1 male: female ratio. Common genetic variation could explain 40-60% of the variance in liability to autism. Because of their small effect, genome-wide association studies (GWASs) have only identified a small number of individual single-nucleotide polymorphisms (SNPs). To increase the power of GWASs in complex disorders, methods like convergent functional genomics (CFG) have emerged to extract true association signals from noise and to identify and prioritize genes from SNPs using a scoring strategy combining statistics and functional genomics. We adapted and applied this approach to analyze data from a GWAS performed on families with multiple children affected with autism from Autism Speaks Autism Genetic Resource Exchange (AGRE). We identified a set of 133 candidate markers that were localized in or close to genes with functional relevance in ASD from a discovery population (545 multiplex families); a gender specific genetic score (GS) based on these common variants explained 1% (P = 0.01 in males) and 5% (P = 8.7 × 10(-7) in females) of genetic variance in an independent sample of multiplex families. Overall, our work demonstrates that prioritization of GWAS data based on functional genomics identified common variants associated with autism and provided additional support for a common polygenic background in autism.

Streamlined ion torrent PGM-based diagnostics: BRCA1 and BRCA2 genes as a model.

To meet challenges in terms of throughput and turnaround time, many diagnostic laboratories are shifting from Sanger sequencing to higher throughput next-generation sequencing (NGS) platforms. Bearing in mind that the performance and quality criteria expected from NGS in diagnostic or research settings are strikingly different, we have developed an Ion Torrent's PGM-based routine diagnostic procedure for BRCA1/2 sequencing. The procedure was first tested on a training set of 62 control samples, and then blindly validated on 77 samples in parallel with our routine technique. The training set was composed of difficult cases, for example, insertions and/or deletions of various sizes, large-scale rearrangements and, obviously, mutations occurring in homopolymer regions. We also compared two bioinformatic solutions in this diagnostic context, an in-house academic pipeline and the commercially available NextGene software (Softgenetics). NextGene analysis provided higher sensitivity, as four previously undetected single-nucleotide variations were found. Regarding specificity, an average of 1.5 confirmatory Sanger sequencings per patient was needed for complete BRCA1/2 screening. Large-scale rearrangements were identified by two distinct analyses, that is, bioinformatics and fragment analysis with electrophoresis profile comparison. Turnaround time was enhanced, as a series of 30 patients were sequenced by one technician, making the results available for the clinician in 10 working days following blood sampling. BRCA1/2 genes are a good model, representative of the difficulties commonly encountered in diagnostic settings, which is why we believe our findings are of interest for the whole community, and the pipeline described can be adapted by any user of PGM for diagnostic purposes.

Breakpoint features of genomic rearrangements in neuroblastoma with unbalanced translocations and chromothripsis.

Neuroblastoma is a pediatric cancer of the peripheral nervous system in which structural chromosome aberrations are emblematic of aggressive tumors. In this study, we performed an in-depth analysis of somatic rearrangements in two neuroblastoma cell lines and two primary tumors using paired-end sequencing of mate-pair libraries and RNA-seq. The cell lines presented with typical genetic alterations of neuroblastoma and the two tumors belong to the group of neuroblastoma exhibiting a profile of chromothripsis. Inter and intra-chromosomal rearrangements were identified in the four samples, allowing in particular characterization of unbalanced translocations at high resolution. Using complementary experiments, we further characterized 51 rearrangements at the base pair resolution that revealed 59 DNA junctions. In a subset of cases, complex rearrangements were observed with templated insertion of fragments of nearby sequences. Although we did not identify known particular motifs in the local environment of the breakpoints, we documented frequent microhomologies at the junctions in both chromothripsis and non-chromothripsis associated breakpoints. RNA-seq experiments confirmed expression of several predicted chimeric genes and genes with disrupted exon structure including ALK, NBAS, FHIT, PTPRD and ODZ4. Our study therefore indicates that both non-homologous end joining-mediated repair and replicative processes may account for genomic rearrangements in neuroblastoma. RNA-seq analysis allows the identification of the subset of abnormal transcripts expressed from genomic rearrangements that may be involved in neuroblastoma oncogenesis.

Designs and challenges for personalized medicine studies in oncology: focus on the SHIVA trial.

Personalized medicine is defined by the National Cancer Institute as "a form of medicine that uses information about a person's genes, proteins, and environment to prevent, diagnose, and treat disease." In oncology, the term "personalized medicine" arose with the emergence of molecularly targeted agents. The prescription of approved molecularly targeted agents to cancer patients currently relies on the primary tumor location and histological subtype. Predictive biomarkers of efficacy of these modern agents have been exclusively validated in specific tumor types. A major concern today is to determine whether the prescription of molecularly targeted therapies based on tumor molecular abnormalities, independently of primary tumor location and histology, would improve the outcome of cancer patients. This new paradigm requires prospective validation before being implemented in clinical practice. In this paper, we will first review different designs, including observational cohorts, as well as nonrandomized and randomized clinical trials, that have been recently proposed to evaluate the relevance of this approach, and further discuss their advantages and drawbacks. The design of the SHIVA trial, a randomized proof-of-concept phase II trial comparing therapy based on tumor molecular profiling versus conventional therapy in patients with refractory cancer will be detailed. Finally, we will discuss the multiple challenges associated with the implementation of personalized medicine in oncology, as well as perspectives for the future.

DICER1 mutations in familial multinodular goiter with and without ovarian Sertoli-Leydig cell tumors.

CONTEXT: Nontoxic multinodular goiter (MNG) is frequently observed in the general population, but little is known about the underlying genetic susceptibility to this disease. Familial cases of MNG have been reported, and published reports describe 5 families that also contain at least 1 individual with a Sertoli-Leydig cell tumor of the ovary (SLCT). Germline mutations in DICER1, a gene that codes for an RNase III endoribonuclease, have been identified in families affected by pleuropulmonary blastoma (PPB), some of whom include cases of MNG and gonadal tumors such as SLCTs. OBJECTIVE: To determine whether familial MNG with or without SLCT in the absence of PPB was associated with mutations in DICER1. DESIGN, SETTING, AND PATIENTS: From September 2009 to September 2010, we screened 53 individuals from 2 MNG and 3 MNG/SLCT families at McGill University for mutations in DICER1. We investigated blood lymphocytes and MNG and SLCT tissue from family members for loss of the wild-type DICER1 allele (loss of heterozygosity), DICER1 expression, and microRNA (miRNA) dysregulation. MAIN OUTCOME MEASURE: Detection of germline DICER1 gene mutations in familial MNG with and without SLCT. RESULTS: We identified and characterized germline DICER1 mutations in 37 individuals from 5 families. Two mutations were predicted to be protein truncating, 2 resulted in in-frame deletions, and 1 was a missense mutation. Molecular analysis of the 3 SLCTs showed no loss of heterozygosity of DICER1, and immunohistochemical analysis in 2 samples showed strong expression of DICER1 in Sertoli cells but weak staining of Leydig cells. miRNA profiling of RNA from lymphoblastoid cell lines from both affected and unaffected members of the familial MNG cases revealed miRNA perturbations in DICER1 mutation carriers. CONCLUSIONS: DICER1 mutations are associated with both familial MNG and MNG with SLCT, independent of PPB. These germline DICER1 mutations are associated with dysregulation of miRNA expression patterns.

Homozygous BUB1B mutation and susceptibility to gastrointestinal neoplasia.

A patient received a diagnosis of adenocarcinoma of the ampulla of Vater at 34 years of age. Two decades later, adenomatous polyps were found, followed by multiple primary invasive adenocarcinomas of both the colon and the stomach. Premature chromatid separation and mosaic variegated aneuploidy, combined with structural chromosomal abnormalities, were detected in his cells. We identified a germline homozygous intronic mutation, c.2386-11A→G, in the spindle-assembly checkpoint gene BUB1B, which creates a de novo splice site that is favored over the authentic (i.e., preferentially used) site. Our findings expand the phenotype associated with BUB1B mutations and the mosaic variegated aneuploidy syndrome to include common adult-onset cancers and provide evidence for the interdependency of the APC protein (encoded by the adenomatous polyposis coli gene) and the BUBR1 protein (encoded by BUB1B) in humans. (Funded by the Turner Family Cancer Research Fund and others.).

Germline DICER1 mutations and familial cystic nephroma.

BACKGROUND: Multilocular cystic nephroma (CN) is a benign kidney tumour and is part of a family of kidney neoplasms including cystic partially differentiated nephroblastoma and Wilms tumour (WT). CN is rarely familial or bilateral, but it occurs in about 10% of families where pleuropulmonary blastoma (PPB) is present. Recently, germline mutations in DICER1 were found in familial PPB. OBJECTIVE: To search for DICER1 mutations in two families with familial CN; PPB was present in one family. Additionally, to test germline DNA from 50 children with sporadic WT for DICER1 mutations. RESULTS: Both families with multiple CN were found to have mutations in DICER1 leading to premature stop codons, predicted to result in loss of the ribonuclease and dsRNA binding domains. These domains are essential to the function of DICER1. No germline mutations were found in any of the 50 children who had developed WT. CONCLUSION: It has been established that DICER1 mutations cause familial CN and may be implicated in bilateral CN. No germline mutations were found in the patients with WT, suggesting that DICER1 mutations are unlikely to have a major role in the aetiology of sporadic WT. These results provide further evidence implicating miRNA dysregulation in tumourigenesis.

Mutation analysis of the gene encoding the PALB2-binding protein MRG15 in BRCA1/2-negative breast cancer families.

MRG15 is a recently identified member of the BRCA multiprotein complex, essential for the maintenance of the genome integrity and DNA repair. The functional relationship between PALB2 and MRG15 makes MRG15 a strong candidate breast cancer susceptibility gene. We screened affected probands from 232 BRCA1/2-negative breast cancer families for mutations in MRG15. We identified seven previously unreported variants but in silico analyses revealed that none of these variants appears to modify the function of MRG15. Thus, it seems unlikely that any constitutional changes in MRG15 confer an increased risk for breast cancer.

Ultra high throughput sequencing excludes MDH1 as candidate gene for RP28-linked retinitis pigmentosa.

PURPOSE: Mutations in IDH3B, an enzyme participating in the Krebs cycle, have recently been found to cause autosomal recessive retinitis pigmentosa (arRP). The MDH1 gene maps within the RP28 arRP linkage interval and encodes cytoplasmic malate dehydrogenase, an enzyme functionally related to IDH3B. As a proof of concept for candidate gene screening to be routinely performed by ultra high throughput sequencing (UHTs), we analyzed MDH1 in a patient from each of the two families described so far to show linkage between arRP and RP28. METHODS: With genomic long-range PCR, we amplified all introns and exons of the MDH1 gene (23.4 kb). PCR products were then sequenced by short-read UHTs with no further processing. Computer-based mapping of the reads and mutation detection were performed by three independent software packages. RESULTS: Despite the intrinsic complexity of human genome sequences, reads were easily mapped and analyzed, and all algorithms used provided the same results. The two patients were homozygous for all DNA variants identified in the region, which confirms previous linkage and homozygosity mapping results, but had different haplotypes, indicating genetic or allelic heterogeneity. None of the DNA changes detected could be associated with the disease. CONCLUSIONS: The MDH1 gene is not the cause of RP28-linked arRP. Our experimental strategy shows that long-range genomic PCR followed by UHTs provides an excellent system to perform a thorough screening of candidate genes for hereditary retinal degeneration.

A single-base substitution within an intronic repetitive element causes dominant retinitis pigmentosa with reduced penetrance.

We report the study of a large American family displaying autosomal dominant retinitis pigmentosa with reduced penetrance, a form of hereditary retinal degeneration. Although the inheritance pattern and previous linkage mapping pointed to the involvement of the PRPF31 gene, extensive screening of all its exons and their boundaries failed in the past to reveal any mutation. In this work, we sequenced the entire PRPF31 genomic region by both the classical Sanger method and ultrahigh throughput (UHT) sequencing. Among the many variants identified, a single-base substitution (c.1374+654C>G) located deep within intron 13 and inside a repetitive DNA element was common to all patients and obligate asymptomatic carriers. This change created a new splice donor site leading to the synthesis of two mutant PRPF31 isoforms, degraded by nonsense-mediated mRNA decay. As a consequence, amounts of PRPF31 mRNA derived from the mutant allele were very reduced, with no evidence of mutant proteins being synthesized. Our results indicate that c.1374+654C>G causes retinitis pigmentosa via haploinsufficiency, similar to the vast majority of PRPF31 mutations described so far. We discuss the potential of UHT sequencing technologies in mutation screening and the continued identification of pathogenic splicing mutations buried deep within intronic regions.

Two trans-acting eQTLs modulate the penetrance of PRPF31 mutations.

Dominant mutations in the gene encoding the ubiquitously-expressed splicing factor PRPF31 cause retinitis pigmentosa, a form of hereditary retinal degeneration, with reduced penetrance. We and others have previously shown that penetrance is tightly correlated with PRPF31 expression, as lymphoblastoid cell lines (LCLs) from affected patients produce less abundant PRPF31 transcripts than LCLs from their unaffected relatives carrying the same mutation. We have investigated the genetic elements determining the variable expression of PRPF31, and therefore possibly influencing the penetrance of its mutations, by quantifying PRPF31 mRNA levels in LCLs derived from 15 CEPH families (200 individuals), representative of the general population. We found that PRPF31 transcript abundance was a highly variable and highly heritable character. Moreover, by linkage analysis we showed that PRPF31 expression was significantly associated with at least one expression quantitative trait locus (eQTL), spanning a 8.2-Mb region on chromosome 14q21-23. We also investigated a previously mapped penetrance factor located near PRPF31 itself in LCLs from individuals belonging to selected families segregating PRPF31 mutations that displayed reduced penetrance. Our results indicate that, despite its constant association with the non-mutant allele, this factor was able to modulate the expression of both PRPF31 alleles. Furthermore, we showed that LCLs from affected patients have less PRPF31 RNA than those of asymptomatic patients, even at the pre-splicing stage. Altogether, these data demonstrate that PRPF31 mRNA expression and consequently the penetrance of PRPF31 mutations is managed by diffusible compounds encoded by at least two modifiers, acting in a co-regulatory system on both PRPF31 alleles during transcription.