Chromosomal instability by mutations in the novel minor spliceosome component CENATAC.

Aneuploidy is the leading cause of miscarriage and congenital birth defects, and a hallmark of cancer. Despite this strong association with human disease, the genetic causes of aneuploidy remain largely unknown. Through exome sequencing of patients with constitutional mosaic aneuploidy, we identified biallelic truncating mutations in CENATAC (CCDC84). We show that CENATAC is a novel component of the minor (U12-dependent) spliceosome that promotes splicing of a specific, rare minor intron subtype. This subtype is characterized by AT-AN splice sites and relatively high basal levels of intron retention. CENATAC depletion or expression of disease mutants resulted in excessive retention of AT-AN minor introns in ˜ 100 genes enriched for nucleocytoplasmic transport and cell cycle regulators, and caused chromosome segregation errors. Our findings reveal selectivity in minor intron splicing and suggest a link between minor spliceosome defects and constitutional aneuploidy in humans.

Identification of new Wilms tumour predisposition genes: an exome sequencing study.

BACKGROUND: Wilms tumour is the most common childhood renal cancer and is genetically heterogeneous. While several Wilms tumour predisposition genes have been identified, there is strong evidence that further predisposition genes are likely to exist. Our study aim was to identify new predisposition genes for Wilms tumour. METHODS: In this exome sequencing study, we analysed lymphocyte DNA from 890 individuals with Wilms tumour, including 91 affected individuals from 49 familial Wilms tumour pedigrees. We used the protein-truncating variant prioritisation method to prioritise potential disease-associated genes for further assessment. We evaluated new predisposition genes in exome sequencing data that we generated in 334 individuals with 27 other childhood cancers and in exome data from The Cancer Genome Atlas obtained from 7632 individuals with 28 adult cancers. FINDINGS: We identified constitutional cancer-predisposing mutations in 33 individuals with childhood cancer. The three identified genes with the strongest signal in the protein-truncating variant prioritisation analyses were TRIM28, FBXW7, and NYNRIN. 21 of 33 individuals had a mutation in TRIM28; there was a strong parent-of-origin effect, with all ten inherited mutations being maternally transmitted (p=0·00098). We also found a strong association with the rare epithelial subtype of Wilms tumour, with 14 of 16 tumours being epithelial or epithelial predominant. There were no TRIM28 mutations in individuals with other childhood or adult cancers. We identified truncating FBXW7 mutations in four individuals with Wilms tumour and a de-novo non-synonymous FBXW7 mutation in a child with a rhabdoid tumour. Biallelic truncating mutations in NYNRIN were identified in three individuals with Wilms tumour, which is highly unlikely to have occurred by chance (p<0·0001). Finally, we identified two de-novo KDM3B mutations, supporting the role of KDM3B as a childhood cancer predisposition gene. INTERPRETATION: The four new Wilms tumour predisposition genes identified-TRIM28, FBXW7, NYNRIN, and KDM3B-are involved in diverse biological processes and, together with the other 17 known Wilms tumour predisposition genes, account for about 10% of Wilms tumour cases. The overlap between these 21 constitutionally mutated predisposition genes and 20 genes somatically mutated in Wilms tumour is limited, consisting of only four genes. We recommend that all individuals with Wilms tumour should be offered genetic testing and particularly, those with epithelial Wilms tumour should be offered TRIM28 genetic testing. Only a third of the familial Wilms tumour clusters we analysed were attributable to known genes, indicating that further Wilms tumour predisposition factors await discovery. FUNDING: Wellcome Trust.

Correction: Germline mutations in the oncogene EZH2 cause Weaver syndrome and increased human height.

[This corrects the article DOI: 10.18632/oncotarget.385.].

Biallelic TRIP13 mutations predispose to Wilms tumor and chromosome missegregation.

Through exome sequencing, we identified six individuals with biallelic loss-of-function mutations in TRIP13. All six developed Wilms tumor. Constitutional mosaic aneuploidies, microcephaly, developmental delay and seizures, which are features of mosaic variegated aneuploidy (MVA) syndrome, were more variably present. Through functional studies, we show that TRIP13-mutant patient cells have no detectable TRIP13 and have substantial impairment of the spindle assembly checkpoint (SAC), leading to a high rate of chromosome missegregation. Accurate segregation, as well as SAC proficiency, is rescued by restoring TRIP13 function. Individuals with biallelic TRIP13 or BUB1B mutations have a high risk of embryonal tumors, and here we show that their cells display severe SAC impairment. MVA due to biallelic CEP57 mutations, or of unknown cause, is not associated with embryonal tumors and cells from these individuals show minimal SAC deficiency. These data provide insights into the complex relationships between aneuploidy and carcinogenesis.

The ICR142 NGS validation series: a resource for orthogonal assessment of NGS analysis.

To provide a useful community resource for orthogonal assessment of NGS analysis software, we present the ICR142 NGS validation series. The dataset includes high-quality exome sequence data from 142 samples together with Sanger sequence data at 704 sites; 416 sites with variants and 288 sites at which variants were called by an NGS analysis tool, but no variant is present in the corresponding Sanger sequence. The dataset includes 293 indel variants and 247 negative indel sites, and thus the ICR142 validation dataset is of particular utility in evaluating indel calling performance. The FASTQ files and Sanger sequence results can be accessed in the European Genome-phenome Archive under the accession number EGAS00001001332.

Mutations in the transcriptional repressor REST predispose to Wilms tumor.

Wilms tumor is the most common childhood renal cancer. To identify mutations that predispose to Wilms tumor, we are conducting exome sequencing studies. Here we describe 11 different inactivating mutations in the REST gene (encoding RE1-silencing transcription factor) in four familial Wilms tumor pedigrees and nine non-familial cases. Notably, no similar mutations were identified in the ICR1000 control series (13/558 versus 0/993; P < 0.0001) or in the ExAC series (13/558 versus 0/61,312; P < 0.0001). We identified a second mutational event in two tumors, suggesting that REST may act as a tumor-suppressor gene in Wilms tumor pathogenesis. REST is a zinc-finger transcription factor that functions in cellular differentiation and embryonic development. Notably, ten of 11 mutations clustered within the portion of REST encoding the DNA-binding domain, and functional analyses showed that these mutations compromise REST transcriptional repression. These data establish REST as a Wilms tumor predisposition gene accounting for ∼2% of Wilms tumor.

The ICR1000 UK exome series: a resource of gene variation in an outbred population.

To enhance knowledge of gene variation in outbred populations, and to provide a dataset with utility in research and clinical genomics, we performed exome sequencing of 1,000 UK individuals from the general population and applied a high-quality analysis pipeline that includes high sensitivity and specificity for indel detection. Each UK individual has, on average, 21,978 gene variants including 160 rare (0.1%) variants not present in any other individual in the series. These data provide a baseline expectation for gene variation in an outbred population. Summary data of all 295,391 variants we detected are included here and the individual exome sequences are available from the European Genome-phenome Archive as the ICR1000 UK exome series. Furthermore, samples and other phenotype and experimental data for these individuals are obtainable through application to the 1958 Birth Cohort committee.

Germline mutations in the PAF1 complex gene CTR9 predispose to Wilms tumour.

Wilms tumour is a childhood kidney cancer. Here we identify inactivating CTR9 mutations in 3 of 35 Wilms tumour families, through exome and Sanger sequencing. By contrast, no similar mutations are present in 1,000 population controls (P<0.0001). Each mutation segregates with Wilms tumour in the family and a second mutational event is present in available tumours. CTR9 is a key component of the polymerase-associated factor 1 complex which has multiple roles in RNA polymerase II regulation and is implicated in embryonic organogenesis and maintenance of embryonic stem cell pluripotency. These data establish CTR9 as a Wilms tumour predisposition gene and suggest it acts as a tumour suppressor gene.

Mutations in the DNA methyltransferase gene DNMT3A cause an overgrowth syndrome with intellectual disability.

Overgrowth disorders are a heterogeneous group of conditions characterized by increased growth parameters and other variable clinical features such as intellectual disability and facial dysmorphism. To identify new causes of human overgrowth, we performed exome sequencing in ten proband-parent trios and detected two de novo DNMT3A mutations. We identified 11 additional de novo mutations by sequencing DNMT3A in a further 142 individuals with overgrowth. The mutations alter residues in functional DNMT3A domains, and protein modeling suggests that they interfere with domain-domain interactions and histone binding. Similar mutations were not present in 1,000 UK population controls (13/152 cases versus 0/1,000 controls; P < 0.0001). Mutation carriers had a distinctive facial appearance, intellectual disability and greater height. DNMT3A encodes a DNA methyltransferase essential for establishing methylation during embryogenesis and is commonly somatically mutated in acute myeloid leukemia. Thus, DNMT3A joins an emerging group of epigenetic DNA- and histone-modifying genes associated with both developmental growth disorders and hematological malignancies.

Weaver syndrome and EZH2 mutations: Clarifying the clinical phenotype.

Weaver syndrome, first described in 1974, is characterized by tall stature, a typical facial appearance, and variable intellectual disability. In 2011, mutations in the histone methyltransferase, EZH2, were shown to cause Weaver syndrome. To date, we have identified 48 individuals with EZH2 mutations. The mutations were primarily missense mutations occurring throughout the gene, with some clustering in the SET domain (12/48). Truncating mutations were uncommon (4/48) and only identified in the final exon, after the SET domain. Through analyses of clinical data and facial photographs of EZH2 mutation-positive individuals, we have shown that the facial features can be subtle and the clinical diagnosis of Weaver syndrome is thus challenging, especially in older individuals. However, tall stature is very common, reported in >90% of affected individuals. Intellectual disability is also common, present in ~80%, but is highly variable and frequently mild. Additional clinical features which may help in stratifying individuals to EZH2 mutation testing include camptodactyly, soft, doughy skin, umbilical hernia, and a low, hoarse cry. Considerable phenotypic overlap between Sotos and Weaver syndromes is also evident. The identification of an EZH2 mutation can therefore provide an objective means of confirming a subtle presentation of Weaver syndrome and/or distinguishing Weaver and Sotos syndromes. As mutation testing becomes increasingly accessible and larger numbers of EZH2 mutation-positive individuals are identified, knowledge of the clinical spectrum and prognostic implications of EZH2 mutations should improve.

Mosaic PPM1D mutations are associated with predisposition to breast and ovarian cancer.

Improved sequencing technologies offer unprecedented opportunities for investigating the role of rare genetic variation in common disease. However, there are considerable challenges with respect to study design, data analysis and replication. Using pooled next-generation sequencing of 507 genes implicated in the repair of DNA in 1,150 samples, an analytical strategy focused on protein-truncating variants (PTVs) and a large-scale sequencing case-control replication experiment in 13,642 individuals, here we show that rare PTVs in the p53-inducible protein phosphatase PPM1D are associated with predisposition to breast cancer and ovarian cancer. PPM1D PTV mutations were present in 25 out of 7,781 cases versus 1 out of 5,861 controls (P = 1.12 × 10(-5)), including 18 mutations in 6,912 individuals with breast cancer (P = 2.42 × 10(-4)) and 12 mutations in 1,121 individuals with ovarian cancer (P = 3.10 × 10(-9)). Notably, all of the identified PPM1D PTVs were mosaic in lymphocyte DNA and clustered within a 370-base-pair region in the final exon of the gene, carboxy-terminal to the phosphatase catalytic domain. Functional studies demonstrate that the mutations result in enhanced suppression of p53 in response to ionizing radiation exposure, suggesting that the mutant alleles encode hyperactive PPM1D isoforms. Thus, although the mutations cause premature protein truncation, they do not result in the simple loss-of-function effect typically associated with this class of variant, but instead probably have a gain-of-function effect. Our results have implications for the detection and management of breast and ovarian cancer risk. More generally, these data provide new insights into the role of rare and of mosaic genetic variants in common conditions, and the use of sequencing in their identification.

A genome-wide association study identifies susceptibility loci for Wilms tumor.

Wilms tumor is the most common renal malignancy of childhood. To identify common variants that confer susceptibility to Wilms tumor, we conducted a genome-wide association study in 757 individuals with Wilms tumor (cases) and 1,879 controls. We evaluated ten SNPs in regions significantly associated at P < 5 × 10(-5) in two independent replication series from the UK (769 cases and 2,814 controls) and the United States (719 cases and 1,037 controls). We identified clear significant associations at 2p24 (rs3755132, P = 1.03 × 10(-14); rs807624, P = 1.32 × 10(-14)) and 11q14 (rs790356, P = 4.25 × 10(-15)). Both regions contain genes that are plausibly related to Wilms tumorigenesis. We also identified candidate association signals at 5q14, 22q12 and Xp22.

Predisposition gene identification in common cancers by exome sequencing: insights from familial breast cancer.

The genetic component of breast cancer predisposition remains largely unexplained. Candidate gene case-control resequencing has identified predisposition genes characterised by rare, protein truncating mutations that confer moderate risks of disease. In theory, exome sequencing should yield additional genes of this class. Here, we explore the feasibility and design considerations of this approach. We performed exome sequencing in 50 individuals with familial breast cancer, applying frequency and protein function filters to identify variants most likely to be pathogenic. We identified 867,378 variants that passed the call quality filters of which 1,296 variants passed the frequency and protein truncation filters. The median number of validated, rare, protein truncating variants was 10 in individuals with, and without, mutations in known genes. The functional candidacy of mutated genes was similar in both groups. Without prior knowledge, the known genes would not have been recognisable as breast cancer predisposition genes. Everyone carries multiple rare mutations that are plausibly related to disease. Exome sequencing in common conditions will therefore require intelligent sample and variant prioritisation strategies in large case-control studies to deliver robust genetic evidence of disease association.

Germline mutations in the oncogene EZH2 cause Weaver syndrome and increased human height.

The biological processes controlling human growth are diverse, complex and poorly understood. Genetic factors are important and human height has been shown to be a highly polygenic trait to which common and rare genetic variation contributes. Weaver syndrome is a human overgrowth condition characterised by tall stature, dysmorphic facial features, learning disability and variable additional features. We performed exome sequencing in four individuals with Weaver syndrome, identifying a mutation in the histone methyltransferase, EZH2, in each case. Sequencing of EZH2 in additional individuals with overgrowth identified a further 15 mutations. The EZH2 mutation spectrum in Weaver syndrome shows considerable overlap with the inactivating somatic EZH2 mutations recently reported in myeloid malignancies. Our data establish EZH2 mutations as the cause of Weaver syndrome and provide further links between histone modifications and regulation of human growth.

Mutations in CEP57 cause mosaic variegated aneuploidy syndrome.

Using exome sequencing and a variant prioritization strategy that focuses on loss-of-function variants, we identified biallelic, loss-of-function CEP57 mutations as a cause of constitutional mosaic aneuploidies. CEP57 is a centrosomal protein and is involved in nucleating and stabilizing microtubules. Our findings indicate that these and/or additional functions of CEP57 are crucial for maintaining correct chromosomal number during cell division.

DICER1 syndrome: clarifying the diagnosis, clinical features and management implications of a pleiotropic tumour predisposition syndrome.

BACKGROUND: Constitutional DICER1 mutations were recently reported to cause familial pleuropulmonary blastoma (PPB). AIM: To investigate the contribution and phenotypic spectrum of constitutional and somatic DICER1 mutations to cancer. METHODS AND RESULTS: The authors sequenced DICER1 in constitutional DNA from 823 unrelated patients with a variety of tumours and in 781 cancer cell lines. Constitutional DICER1 mutations were identified in 19 families including 11/14 with PPB, 2/3 with cystic nephroma, 4/7 with ovarian Sertoli-Leydig-type tumours, 1/243 with Wilms tumour (this patient also had a Sertoli-Leydig tumour), 1/1 with intraocular medulloepithelioma (this patient also had PPB), 1/86 with medulloblastoma/infratentorial primitive neuroectodermal tumour, and 1/172 with germ cell tumour. The inheritance was investigated in 17 families. DICER1 mutations were identified in 25 relatives: 17 were unaffected, one mother had ovarian Sertoli-Leydig tumour, one half-sibling had cystic nephroma, and six relatives had non-toxic thyroid cysts/goitre. Analysis of eight tumours from DICER1 mutation-positive patients showed universal retention of the wild-type allele. DICER1 truncating mutations were identified in 4/781 cancer cell lines; all were in microsatellite unstable lines and therefore unlikely to be driver mutations. CONCLUSION: Constitutional DICER1 haploinsufficiency predisposes to a broad range of tumours, making a substantial contribution to PPB, cystic nephroma and ovarian Sertoli-Leydig tumours, but a smaller contribution to other tumours. Most mutation carriers are unaffected, indicating that tumour risk is modest. The authors define the clinical contexts in which DICER1 mutation testing should be considered, the associated tumour risks, and the implications for at-risk individuals. They have termed this condition 'DICER1 syndrome'. ACCESSION NUMBERS: The cDNA Genbank accession number for the DICER1 sequence reported in this paper is NM_030621.2.

Heterogeneity of familial medulloblastoma and contribution of germline PTCH1 and SUFU mutations to sporadic medulloblastoma.

PTCH1 and SUFU are both regulators of the sonic hedgehog signalling pathway. Germline inactivating mutations in both genes are associated with multisystem phenotypes including medulloblastoma. Somatic inactivating mutations in PTCH1 and SUFU each occur in approximately 10% of medulloblastomas. Recently, SUFU mutations were reported in familial medulloblastoma pedigrees without additional phenotypic features. We sought to further investigate the contribution of germline PTCH1 and SUFU mutations to familial and sporadic medulloblastoma. We performed full-gene mutational analysis of both PTCH1 and SUFU in three familial medulloblastoma pedigrees and 83 individuals with sporadic non-familial medulloblastoma. We identified no mutations in PTCH1 or SUFU in the three familial medulloblastoma pedigrees. We identified no PTCH1 mutations and two SUFU mutations that cause premature protein truncating in the series of sporadic non-familial medulloblastomas. The SUFU mutations were identified in two of the 16 individuals with desmoplastic medulloblastomas. These data indicate that familial medulloblastoma is a genetically heterogeneous disorder with at least one further susceptibility gene to be discovered. Furthermore, although both PTCH1 and SUFU play a key role in the sonic hedgehog signalling pathway, PTCH1 does not make an appreciable contribution to non-familial sporadic medulloblastoma, whereas inactivating germline mutations of SUFU cause ~2-3% of sporadic medulloblastomas and > 10% of desmoplastic medulloblastomas.

Generation of trisomies in cancer cells by multipolar mitosis and incomplete cytokinesis.

One extra chromosome copy (i.e., trisomy) is the most common type of chromosome aberration in cancer cells. The mechanisms behind the generation of trisomies in tumor cells are largely unknown, although it has been suggested that dysfunction of the spindle assembly checkpoint (SAC) leads to an accumulation of trisomies through failure to correctly segregate sister chromatids in successive cell divisions. By using Wilms tumor as a model for cancers with trisomies, we now show that trisomic cells can form even in the presence of a functional SAC through tripolar cell divisions in which sister chromatid separation proceeds in a regular fashion, but cytokinesis failure nevertheless leads to an asymmetrical segregation of chromosomes into two daughter cells. A model for the generation of trisomies by such asymmetrical cell division accurately predicted several features of clones having extra chromosomes in vivo, including the ratio between trisomies and tetrasomies and the observation that different trisomies found in the same tumor occupy identical proportions of cells and colocalize in tumor tissue. Our findings provide an experimentally validated model explaining how multiple trisomies can occur in tumor cells that still maintain accurate sister chromatid separation at metaphase-anaphase transition and thereby physiologically satisfy the SAC.

Molecular causes for BUBR1 dysfunction in the human cancer predisposition syndrome mosaic variegated aneuploidy.

Genetic mutations in the mitotic regulatory kinase BUBR1 are associated with the cancer-susceptible disorder mosaic variegated aneuploidy (MVA). In patients with biallelic mutations, a missense mutation pairs with a truncating mutation. Here, we show that cell lines derived from MVA patients with biallelic mutations have an impaired mitotic checkpoint, chromosome alignment defects, and low overall BUBR1 abundance. Ectopic expression of BUBR1 restored mitotic checkpoint activity, proving that BUBR1 dysfunction causes chromosome segregation errors in the patients. Combined analysis of patient cells and functional protein replacement shows that all MVA mutations fall in two distinct classes: those that impose specific defects in checkpoint activity or microtubule attachment and those that lower BUBR1 protein abundance. Low protein abundance is the direct result of the absence of transcripts from truncating mutants combined with high protein turnover of missense mutants. In this group of missense mutants, the amino acid change consistently occurs in or near the BUBR1 kinase domain. Our findings provide a molecular explanation for chromosomal instability in patients with biallelic genetic mutations in BUBR1.