Germline Missense Variants in CDC20 Result in Aberrant Mitotic Progression and Familial Cancer.

CDC20 is a coactivator of the anaphase promoting complex/cyclosome (APC/C) and is essential for mitotic progression. APC/CCDC20 is inhibited by the spindle assembly checkpoint (SAC), which prevents premature separation of sister chromatids and aneuploidy in daughter cells. Although overexpression of CDC20 is common in many cancers, oncogenic mutations have never been identified in humans. Using whole-exome sequencing, we identified heterozygous missense CDC20 variants (L151R and N331K) that segregate with ovarian germ cell tumors in two families. Functional characterization showed these mutants retain APC/C activation activity but have impaired binding to BUBR1, a component of the SAC. Expression of L151R and N331K variants promoted mitotic slippage in HeLa cells and primary skin fibroblasts derived from carriers. Generation of mice carrying the N331K variant using CRISPR-Cas9 showed that, although homozygous N331K mice were nonviable, heterozygotes displayed accelerated oncogenicity of Myc-driven cancers. These findings highlight an unappreciated role for CDC20 variants as tumor-promoting genes. SIGNIFICANCE: Two germline CDC20 missense variants that segregate with cancer in two families compromise the spindle assembly checkpoint and lead to aberrant mitotic progression, which could predispose cells to transformation. See related commentary by Villarroya-Beltri and Malumbres, p. 3432.

Investigating the causal role of MRE11A p.E506* in breast and ovarian cancer.

The nuclease MRE11A is often included in genetic test panels for hereditary breast and ovarian cancer (HBOC) due to its BRCA1-related molecular function in the DNA repair pathway. However, whether MRE11A is a true predisposition gene for HBOC is still questionable. We determined to investigate this notion by dissecting the molecular genetics of the c.1516G > T;p.E506* truncating MRE11A variant, that we pinpointed in two unrelated French-Canadian (FC) HBOC patients. We performed a case-control study for the variant in ~ 2500 breast, ovarian, and endometrial cancer patients from the founder FC population of Quebec. Furthermore, we looked for the presence of second somatic alterations in the MRE11A gene in the tumors of the carriers. In summary, these investigations suggested that the identified variant is not associated with an increased risk of developing breast or ovarian cancer. We finally performed a systematic review for all the previously reported MRE11A variants in breast and ovarian cancer. We found that MRE11A germline variants annotated as pathogenic on ClinVar often lacked evidence for such classification, hence misleading the clinical management for affected patients. In summary, our report suggests the lack of clinical utility of MRE11A testing in HBOC, at least in the White/Caucasian populations.

Methodologies for Transcript Profiling Using Long-Read Technologies.

RNA sequencing using next-generation sequencing technologies (NGS) is currently the standard approach for gene expression profiling, particularly for large-scale high-throughput studies. NGS technologies comprise high throughput, cost efficient short-read RNA-Seq, while emerging single molecule, long-read RNA-Seq technologies have enabled new approaches to study the transcriptome and its function. The emerging single molecule, long-read technologies are currently commercially available by Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT), while new methodologies based on short-read sequencing approaches are also being developed in order to provide long range single molecule level information-for example, the ones represented by the 10x Genomics linked read methodology. The shift toward long-read sequencing technologies for transcriptome characterization is based on current increases in throughput and decreases in cost, making these attractive for de novo transcriptome assembly, isoform expression quantification, and in-depth RNA species analysis. These types of analyses were challenging with standard short sequencing approaches, due to the complex nature of the transcriptome, which consists of variable lengths of transcripts and multiple alternatively spliced isoforms for most genes, as well as the high sequence similarity of highly abundant species of RNA, such as rRNAs. Here we aim to focus on single molecule level sequencing technologies and single-cell technologies that, combined with perturbation tools, allow the analysis of complete RNA species, whether short or long, at high resolution. In parallel, these tools have opened new ways in understanding gene functions at the tissue, network, and pathway levels, as well as their detailed functional characterization. Analysis of the epi-transcriptome, including RNA methylation and modification and the effects of such modifications on biological systems is now enabled through direct RNA sequencing instead of classical indirect approaches. However, many difficulties and challenges remain, such as methodologies to generate full-length RNA or cDNA libraries from all different species of RNAs, not only poly-A containing transcripts, and the identification of allele-specific transcripts due to current error rates of single molecule technologies, while the bioinformatics analysis on long-read data for accurate identification of 5' and 3' UTRs is still in development.

A novel pathogenic missense ADAMTS17 variant that impairs secretion causes Weill-Marchesani Syndrome with variably dysmorphic hand features.

Weill-Marchesani syndrome (WMS) is a rare disorder displaying short stature, brachydactyly and joint stiffness, and ocular features including microspherophakia and ectopia lentis. Brachydactyly and joint stiffness appear less commonly in patients with WMS4 caused by pathogenic ADAMTS17 variants. Here, we investigated a large family with WMS from Newfoundland, Canada. These patients displayed core WMS features, but with proportionate hands that were clinically equivocal for brachydactyly. Whole exome sequencing and autozygosity mapping unveiled a novel pathogenic missense ADAMTS17 variant (c.3068 G > A, p.C1023Y). Sanger sequencing demonstrated variant co-segregation with WMS, and absence in 150 population matched controls. Given ADAMTS17 involvement, we performed deep phenotyping of the patients' hands. Anthropometrics applied to hand roentgenograms showed that metacarpophalangeal measurements of affected patients were smaller than expected for their age and sex, and when compared to their unaffected sibling. Furthermore, we found a possible sub-clinical phenotype involving markedly shortened metacarpophalangeal bones with intrafamilial variability. Transfection of the variant ADAMTS17 into HEK293T cells revealed significantly reduced secretion into the extracellular medium compared to wild-type. This work expands understanding of the molecular pathogenesis of ADAMTS17, clarifies the variable hand phenotype, and underscores a role for anthropometrics in characterizing sub-clinical brachydactyly in these patients.

Poorly differentiated thyroid carcinoma of childhood and adolescence: a distinct entity characterized by DICER1 mutations.

Poorly differentiated thyroid carcinomas (PDTC) in young individuals are rare and their clinical and histopathologic features, genetic mechanisms, and outcomes remain largely unknown. Here, we report a detailed characterization of a series of six PDTC in patients ≤21 years old defined by Turin diagnostic criteria studied for mutations and gene fusions characteristic of thyroid cancer using targeted next-generation sequencing (NGS) and whole-exome sequencing (WES). All tumors had solid, insular, or trabecular growth pattern and high mitotic rate, and five out of six tumors showed tumor necrosis. Targeted NGS assay identified somatic mutations in the DICER1 gene in five of six (83%) tumors, all of which were "hotspot" mutations encoding the metal-ion binding sites of the RNase IIIb domain of DICER1. WES was performed in five cases which confirmed all hotspot mutations and detected two tumors with additional inactivating DICER1 alterations. Of these two, one was a germline pathogenic DICER1 variant and the other had loss of heterozygosity for DICER1. No other mutations or gene fusions characteristic of adult well-differentiated thyroid cancer and PDTC (BRAF, RAS, TERT, RET/PTC, and other) were detected. On follow-up, available for five patients, three patients died of disease 8-24 months after diagnosis, whereas two were alive with no disease. The results of our study demonstrate that childhood- and adolescent-onset PDTC are genetically distinct from adult-onset PDTC in that they are strongly associated with DICER1 mutations and may herald DICER1 syndrome in a minority. As such, all young persons with PDTC may benefit from genetic counseling. Furthermore, their clinically aggressive behavior contrasts sharply with the indolent nature of the great majority of thyroid tumors with DICER1 mutations reported to date.

DGCR8 microprocessor defect characterizes familial multinodular goiter with schwannomatosis.

BACKGROUNDDICER1 is the only miRNA biogenesis component associated with an inherited tumor syndrome, featuring multinodular goiter (MNG) and rare pediatric-onset lesions. Other susceptibility genes for familial forms of MNG likely exist.METHODSWhole-exome sequencing of a kindred with early-onset MNG and schwannomatosis was followed by investigation of germline pathogenic variants that fully segregated with the disease. Genome-wide analyses were performed on 13 tissue samples from familial and nonfamilial DGCR8-E518K-positive tumors, including MNG, schwannomas, papillary thyroid cancers (PTCs), and Wilms tumors. miRNA profiles of 4 tissue types were compared, and sequencing of miRNA, pre-miRNA, and mRNA was performed in a subset of 9 schwannomas, 4 of which harbor DGCR8-E518K.RESULTSWe identified c.1552G>A;p.E518K in DGCR8, a microprocessor component located in 22q, in the kindred. The variant identified is a somatic hotspot in Wilms tumors and has been identified in 2 PTCs. Copy number loss of chromosome 22q, leading to loss of heterozygosity at the DGCR8 locus, was found in all 13 samples harboring c.1552G>A;p.E518K. miRNA profiling of PTCs, MNG, schwannomas, and Wilms tumors revealed a common profile among E518K hemizygous tumors. In vitro cleavage demonstrated improper processing of pre-miRNA by DGCR8-E518K. MicroRNA and RNA profiling show that this variant disrupts precursor microRNA production, impacting populations of canonical microRNAs and mirtrons.CONCLUSIONWe identified DGCR8 as the cause of an unreported autosomal dominant mendelian tumor susceptibility syndrome: familial multinodular goiter with schwannomatosis.FUNDINGCanadian Institutes of Health Research, Compute Canada, Alex's Lemonade Stand Foundation, the Mia Neri Foundation for Childhood Cancer, Cassa di Sovvenzioni e Risparmio fra il Personale della Banca d'Italia, and the KinderKrebsInitiative Buchholz/Holm-Seppensen.

Ovarian small cell carcinoma in one of a pair of monozygous twins.

One of a pair of monozygous twins was diagnosed and died of small cell carcinoma of the ovary of hypercalcemic type (SCCOHT) at the age of 30 years. Her sister remained unaffected and was very concerned about her risk for developing SCCOHT. By performing comprehensive molecular analysis using whole exome sequencing (WES) approach, we showed that the deceased twin's tumour has bi-allelic somatic genetic defects (a pathogenic frameshift deletion in SMARCA4 and LOH on chr19p). Results of WES of constitutional DNA from her unaffected sister were confirmatory. Based on our findings, we concluded that the living twin is not at risk for SCCOHT and does not need to consider preventive oophorectomy.

Causative Mutations and Mechanism of Androgenetic Hydatidiform Moles.

Androgenetic complete hydatidiform moles are human pregnancies with no embryos and affect 1 in every 1,400 pregnancies. They have mostly androgenetic monospermic genomes with all the chromosomes originating from a haploid sperm and no maternal chromosomes. Androgenetic complete hydatidiform moles were described in 1977, but how they occur has remained an open question. We identified bi-allelic deleterious mutations in MEI1, TOP6BL/C11orf80, and REC114, with roles in meiotic double-strand breaks formation in women with recurrent androgenetic complete hydatidiform moles. We investigated the occurrence of androgenesis in Mei1-deficient female mice and discovered that 8% of their oocytes lose all their chromosomes by extruding them with the spindles into the first polar body. We demonstrate that Mei1-/- oocytes are capable of fertilization and 5% produce androgenetic zygotes. Thus, we uncover a meiotic abnormality in mammals and a mechanism for the genesis of androgenetic zygotes that is the extrusion of all maternal chromosomes and their spindles into the first polar body.

Transcript Profiling Using Long-Read Sequencing Technologies.

RNA sequencing using next-generation sequencing (NGS, RNA-Seq) technologies is currently the standard approach for gene expression profiling, particularly for large-scale high-throughput studies. NGS technologies comprise short-read RNA-Seq (dominated by Illumina) and long-read RNA-Seq technologies provided by Pacific Bioscience (PacBio) and Oxford Nanopore Technologies (ONT). Although short-read sequencing technologies are the most widely used, long-read technologies are increasingly becoming the standard approach for de novo transcriptome assembly and isoform expression quantification due to the complex nature of the transcriptome which consists of variable lengths of transcripts and multiple alternatively spliced isoforms for most genes. In this chapter, we describe experimental procedures for library preparation, sequencing, and associated data analysis approaches for PacBio and ONT with a major focus on full length cDNA synthesis, de novo transcriptome assembly, and isoform quantification.

Current and Future Methods for mRNA Analysis: A Drive Toward Single Molecule Sequencing.

The transcriptome encompasses a range of species including messenger RNA, and other noncoding RNA such as rRNA, tRNA, and short and long noncoding RNAs. Due to the huge role played by mRNA in development and disease, several methods have been developed to sequence and characterize mRNA, with RNA sequencing (RNA-Seq) emerging as the current method of choice particularly for large high-throughput studies. Short-read RNA-Seq which involves sequencing of short cDNA fragments and computationally assembling them to reconstruct the transcriptome, or aligning them to a reference is the most widely used approach. However, due to inherent limitations of this approach in de novo transcriptome assembly and isoform quantification, long-read RNA-Seq approaches, which also happen to be single molecule sequencing approaches, are increasingly becoming the standard for de novo transcriptome assembly and isoform quantification. In this chapter, we review the technical aspects of the current methods of RNA-Seq, both short and long-read approaches, and data analysis methods available. We discuss recent advances in single-cell RNA-Seq and direct RNA-Seq approaches, which perhaps will dominate the future of RNA-Seq.

Atypical tuberous sclerosis complex presenting as familial renal cell carcinoma with leiomyomatous stroma.

We report an atypical tuberous sclerosis complex (TSC) phenotype presenting as familial multiple renal cell carcinomas (RCCs) with (angio)leiomyomatous stroma (RCCLS) (5/7 familial RCCs) on a background of multiple angiomyolipomas, hypopigmented skin macules, and absence of neurological anomalies. In the index case and three relatives, germline genetic testing identified a heterozygous TSC2 missense pathogenic variant [c.2714 G > A, (p.Arg905Gln)], a rare TSC-associated alteration which has previously been associated with a milder TSC phenotype. Whole-exome sequencing of five RCCs from the index case and one RCC from his mother demonstrated either unique tumour-specific deleterious second hits in TSC2 or significant allelic imbalance at the TSC2 gene locus (5/6 RCCs). This study confirms the key tumourigenic role of tumour-specific TSC2 second hits in TSC-associated RCCs and supports the notion that RCCLS may be strongly related to abnormalities of the mTOR pathway.

DICER1 Mutations Are Frequent in Adolescent-Onset Papillary Thyroid Carcinoma.

Context: Papillary thyroid carcinoma (PTC) is a common malignancy in adolescence and is molecularly and clinically distinct from adult PTC. Mutations in the DICER1 gene are associated with thyroid abnormalities, including multinodular goiter and differentiated thyroid carcinoma. Objective: In this study, we sought to characterize the prevalence of DICER1 variants in pediatric PTC, specifically in tumors without conventional PTC oncogenic alterations. Patients: Patients (N = 40) who underwent partial or total thyroidectomy and who were <18 years of age at the time of surgery were selected. Design: The 40 consecutive thyroidectomy specimens (30 malignant, 10 benign) underwent genotyping for 17 PTC-associated variants, as well as full sequencing of the exons and exon-intron boundaries of DICER1. Results: Conventional alterations were found in 12 of 30 (40%) PTCs (five BRAFV600E, three RET/PTC1, four RET/PTC3). Pathogenic DICER1 variants were identified in 3 of 30 (10%) PTCs and in 2 of 10 (20%) benign nodules, all of which lacked conventional alterations and did not recur during follow-up. DICER1 alterations thus constituted 3 of 18 (16.7%) PTCs without conventional alterations. The three DICER1-mutated carcinomas each had two somatic DICER1 alterations, whereas two follicular-nodular lesions arose in those with germline DICER1 mutations and harbored characteristic second somatic RNase IIIb "hotspot" mutations. Conclusions: DICER1 is a driver of pediatric thyroid nodules, and DICER1-mutated PTC may represent a distinct class of low-risk malignancies. Given the prevalence of variants in children, we advocate for inclusion of DICER1 sequencing and gene dosage determination in molecular analysis of pediatric thyroid specimens.

Anaplastic sarcomas of the kidney are characterized by DICER1 mutations.

Anaplastic sarcoma of the kidney is a rare tumor (≤25 reported cases) characterized by the presence of cysts, and solid areas composed of bundles of undifferentiated spindle cells, showing marked cellular anaplasia (usually accompanied by TP53 overexpression). These tumors often feature prominent areas of cartilage or chondroid material. Germline mutations in DICER1, encoding the microRNA (miRNA) processor DICER1, cause an eponymous syndrome. Recent reports suggest that anaplastic sarcoma of the kidney should be included in DICER1 syndrome as germline DICER1 mutations are associated with the occurrence of such tumors. Therefore, we sought to determine the following: (1) what proportion of anaplastic sarcoma of the kidney have DICER1 mutations; (2) whether the identified mutations affect both alleles of DICER1 (ie, are biallelic); (3) whether somatic missense mutations in the DICER1 RNase IIIb domain impact miRNA generation; and (4) whether TP53 alteration always occurs in these tumors. DICER1 mutations were evaluated by Sanger sequencing and next-generation sequencing in nine tumor/normal pairs. Impact of DICER1 mutations on miRNA generation was evaluated via an in vitro DICER1 cleavage assay. TP53 status was assessed by immunohistochemistry and next-generation sequencing. Eight of the nine cases had at least one RNase IIIb DICER1 mutation that impacted the generation of miRNAs. There were six tumors with truncating DICER1 mutations and in four of them, the mutation found in the tumor was also detected in adjacent normal tissue, and therefore was likely to be either mosaic or germline in origin. Analysis of mutation phase revealed that two of three tumors had biallelic DICER1 mutations. Six of nine anaplastic sarcomas of the kidney had aberrant TP53 immunohistochemisty with damaging TP53 mutations identified in three cases. Taken together, these data suggest that the great majority of anaplastic sarcomas of the kidney have DICER1 mutations and confirm that these tumors are part of the DICER1 syndrome.

Functionally Null RAD51D Missense Mutation Associates Strongly with Ovarian Carcinoma.

RAD51D is a key player in DNA repair by homologous recombination (HR), and RAD51D truncating variant carriers have an increased risk for ovarian cancer. However, the contribution of nontruncating RAD51D variants to cancer predisposition remains uncertain. Using deep sequencing and case-control genotyping studies, we show that in French Canadians, the missense RAD51D variant c.620C>T;p.S207L is highly prevalent and is associated with a significantly increased risk for ovarian high-grade serous carcinoma (HGSC; 3.8% cases vs. 0.2% controls). The frequency of the p.S207L variant did not significantly differ from that of controls in breast, endometrial, pancreas, or colorectal adenocarcinomas. Functionally, we show that this mutation impairs HR by disrupting the RAD51D-XRCC2 interaction and confers PARP inhibitor sensitivity. These results highlight the importance of a functional RAD51D-XRCC2 interaction to promote HR and prevent the development of HGSC. This study identifies c.620C>T;p.S207L as the first bona fide pathogenic RAD51D missense cancer susceptibility allele and supports the use of targeted PARP-inhibitor therapies in ovarian cancer patients carrying deleterious missense RAD51D variants. Cancer Res; 77(16); 4517-29. ©2017 AACR.

A truncating mutation in CEP55 is the likely cause of MARCH, a novel syndrome affecting neuronal mitosis.

BACKGROUND: Hydranencephaly is a congenital anomaly leading to replacement of the cerebral hemispheres with a fluid-filled cyst. The goals of this work are to describe a novel autosomal-recessive syndrome that includes hydranencephaly (multinucleated neurons, anhydramnios, renal dysplasia, cerebellar hypoplasia and hydranencephaly (MARCH)); to identify its genetic cause(s) and to provide functional insight into pathomechanism. METHODS: We used homozygosity mapping and exome sequencing to identify recessive mutations in a single family with three affected fetuses. Immunohistochemistry, RT-PCR and imaging in cell lines, and zebrafish models, were used to explore the function of the gene and the effect of the mutation. RESULTS: We identified a homozygous nonsense mutation in CEP55 segregating with MARCH. Testing the effect of this allele on patient-derived cells indicated both a reduction of the overall CEP55 message and the production of a message that likely gives rise to a truncated protein. Suppression or ablation of cep55l in zebrafish embryos recapitulated key features of MARCH, most notably renal dysplasia, cerebellar hypoplasia and craniofacial abnormalities. These phenotypes could be rescued by full-length but not truncated human CEP55 message. Finally, we expressed the truncated form of CEP55 in human cells, where we observed a failure of truncated protein to localise to the midbody, leading to abscission failure and multinucleated daughter cells. CONCLUSIONS: CEP55 loss of function mutations likely underlie MARCH, a novel multiple congenital anomaly syndrome. This association expands the involvement of centrosomal proteins in human genetic disorders by highlighting a role in midbody function.