A T-cell antigen atlas for meningioma: novel options for immunotherapy.

Meningiomas are the most common primary intracranial tumors. Although most symptomatic cases can be managed by surgery and/or radiotherapy, a relevant number of patients experience an unfavorable clinical course and additional treatment options are needed. As meningiomas are often perfused by dural branches of the external carotid artery, which is located outside the blood-brain barrier, they might be an accessible target for immunotherapy. However, the landscape of naturally presented tumor antigens in meningioma is unknown. We here provide a T-cell antigen atlas for meningioma by in-depth profiling of the naturally presented immunopeptidome using LC-MS/MS. Candidate target antigens were selected based on a comparative approach using an extensive immunopeptidome data set of normal tissues. Meningioma-exclusive antigens for HLA class I and II are described here for the first time. Top-ranking targets were further functionally characterized by showing their immunogenicity through in vitro T-cell priming assays. Thus, we provide an atlas of meningioma T-cell antigens which will be publicly available for further research. In addition, we have identified novel actionable targets that warrant further investigation as an immunotherapy option for meningioma.

The whole-genome landscape of medulloblastoma subtypes.

Current therapies for medulloblastoma, a highly malignant childhood brain tumour, impose debilitating effects on the developing child, and highlight the need for molecularly targeted treatments with reduced toxicity. Previous studies have been unable to identify the full spectrum of driver genes and molecular processes that operate in medulloblastoma subgroups. Here we analyse the somatic landscape across 491 sequenced medulloblastoma samples and the molecular heterogeneity among 1,256 epigenetically analysed cases, and identify subgroup-specific driver alterations that include previously undiscovered actionable targets. Driver mutations were confidently assigned to most patients belonging to Group 3 and Group 4 medulloblastoma subgroups, greatly enhancing previous knowledge. New molecular subtypes were differentially enriched for specific driver events, including hotspot in-frame insertions that target KBTBD4 and 'enhancer hijacking' events that activate PRDM6. Thus, the application of integrative genomics to an extensive cohort of clinical samples derived from a single childhood cancer entity revealed a series of cancer genes and biologically relevant subtype diversity that represent attractive therapeutic targets for the treatment of patients with medulloblastoma.

A complementary study approach unravels novel players in the pathoetiology of Hirschsprung disease.

Hirschsprung disease (HSCR, OMIM 142623) involves congenital intestinal obstruction caused by dysfunction of neural crest cells and their progeny during enteric nervous system (ENS) development. HSCR is a multifactorial disorder; pathogenetic variants accounting for disease phenotype are identified only in a minority of cases, and the identification of novel disease-relevant genes remains challenging. In order to identify and to validate a potential disease-causing relevance of novel HSCR candidate genes, we established a complementary study approach, combining whole exome sequencing (WES) with transcriptome analysis of murine embryonic ENS-related tissues, literature and database searches, in silico network analyses, and functional readouts using candidate gene-specific genome-edited cell clones. WES datasets of two patients with HSCR and their non-affected parents were analysed, and four novel HSCR candidate genes could be identified: ATP7A, SREBF1, ABCD1 and PIAS2. Further rare variants in these genes were identified in additional HSCR patients, suggesting disease relevance. Transcriptomics revealed that these genes are expressed in embryonic and fetal gastrointestinal tissues. Knockout of these genes in neuronal cells demonstrated impaired cell differentiation, proliferation and/or survival. Our approach identified and validated candidate HSCR genes and provided further insight into the underlying pathomechanisms of HSCR.

Whole exome sequencing identifies novel germline variants of SLC15A4 gene as potentially cancer predisposing in familial colorectal cancer.

About 15% of colorectal cancer (CRC) patients have first-degree relatives affected by the same malignancy. However, for most families the cause of familial aggregation of CRC is unknown. To identify novel high-to-moderate-penetrance germline variants underlying CRC susceptibility, we performed whole exome sequencing (WES) on four CRC cases and two unaffected members of a Polish family without any mutation in known CRC predisposition genes. After WES, we used our in-house developed Familial Cancer Variant Prioritization Pipeline and identified two novel variants in the solute carrier family 15 member 4 (SLC15A4) gene. The heterozygous missense variant, p. Y444C, was predicted to affect the phylogenetically conserved PTR2/POT domain and to have a deleterious effect on the function of the encoded peptide/histidine transporter. The other variant was located in the upstream region of the same gene (GRCh37.p13, 12_129308531_C_T; 43 bp upstream of transcription start site, ENST00000266771.5) and it was annotated to affect the promoter region of SLC15A4 as well as binding sites of 17 different transcription factors. Our findings of two distinct variants in the same gene may indicate a synergistic up-regulation of SLC15A4 as the underlying genetic cause and implicate this gene for the first time in genetic inheritance of familial CRC.

Comprehensive genomic analysis of refractory multiple myeloma reveals a complex mutational landscape associated with drug resistance and novel therapeutic vulnerabilities.

The outcomes of patients with multiple myeloma (MM) refractory to immunomodulatory agents (IMiDs) and proteasome inhibitors (PIs) remain poor. In this study, we performed whole genome and transcriptome sequencing of 39 heavily pretreated relapsed/refractory MM (RRMM) patients to identify mechanisms of resistance and potential therapeutic targets. We observed a high mutational load and indications of increased genomic instability. Recurrently mutated genes in RRMM, which had not been previously reported or only observed at a lower frequency in newly diagnosed MM, included NRAS, BRAF, TP53, SLC4A7, MLLT4, EWSR1, HCFC2, and COPS3. We found multiple genomic regions with bi-allelic events affecting tumor suppressor genes and demonstrated a significant adverse impact of bi-allelic TP53 alterations on survival. With regard to potentially resistance conferring mutations, recurrently mutated gene networks included genes with relevance for PI and IMiD activity; the latter particularly affecting members of the Cereblon and the COP9 signalosome complex. We observed a major impact of signatures associated with exposure to melphalan or impaired DNA double-strand break homologous recombination repair in RRMM. The latter coincided with mutations in genes associated with PARP inhibitor sensitivity in 49% of RRMM patients; a finding with potential therapeutic implications. In conclusion, this comprehensive genomic characterization revealed a complex mutational and structural landscape in RRMM and highlights potential implications for therapeutic strategies.

Whole-Exome Sequencing Identifies a Novel Germline Variant in PTK7 Gene in Familial Colorectal Cancer.

Colorectal cancer (CRC) is the third most frequently diagnosed malignancy worldwide. Only 5% of all CRC cases are due to germline mutations in known predisposition genes, and the remaining genetic burden still has to be discovered. In this study, we performed whole-exome sequencing on six members of a Polish family diagnosed with CRC and identified a novel germline variant in the protein tyrosine kinase 7 (inactive) gene (PTK7, ENST00000230419, V354M). Targeted screening of the variant in 1705 familial CRC cases and 1674 healthy elderly individuals identified the variant in an additional familial CRC case. Introduction of this variant in HT-29 cells resulted in increased cell proliferation, migration, and invasion; it also caused down-regulation of CREB, p21 and p53 mRNA and protein levels, and increased AKT phosphorylation. These changes indicated inhibition of apoptosis pathways and activation of AKT signaling. Our study confirmed the oncogenic function of PTK7 and supported its role in genetic predisposition of familial CRC.

Recurrent Germline Variant in RAD21 Predisposes Children to Lymphoblastic Leukemia or Lymphoma.

Somatic loss of function mutations in cohesin genes are frequently associated with various cancer types, while cohesin disruption in the germline causes cohesinopathies such as Cornelia-de-Lange syndrome (CdLS). Here, we present the discovery of a recurrent heterozygous RAD21 germline aberration at amino acid position 298 (p.P298S/A) identified in three children with lymphoblastic leukemia or lymphoma in a total dataset of 482 pediatric cancer patients. While RAD21 p.P298S/A did not disrupt the formation of the cohesin complex, it altered RAD21 gene expression, DNA damage response and primary patient fibroblasts showed increased G2/M arrest after irradiation and Mitomycin-C treatment. Subsequent single-cell RNA-sequencing analysis of healthy human bone marrow confirmed the upregulation of distinct cohesin gene patterns during hematopoiesis, highlighting the importance of RAD21 expression within proliferating B- and T-cells. Our clinical and functional data therefore suggest that RAD21 germline variants can predispose to childhood lymphoblastic leukemia or lymphoma without displaying a CdLS phenotype.

Molecular profiling of pediatric meningiomas shows tumor characteristics distinct from adult meningiomas.

In contrast to adults, meningiomas are uncommon tumors in childhood and adolescence. Whether adult and pediatric meningiomas differ on a molecular level is unclear. Here we report detailed genomic analyses of 37 pediatric meningiomas by sequencing and DNA methylation profiling. Histologically, the series was dominated by meningioma subtypes with aggressive behavior, with 70% of patients suffering from WHO grade II or III meningiomas. The most frequent cytogenetic aberrations were loss of chromosomes 22 (23/37 [62%]), 1 (9/37 [24%]), 18 (7/37 [19%]), and 14 (5/37 [14%]). Tumors with NF2 alterations exhibited overall increased chromosomal instability. Unsupervised clustering of DNA methylation profiles revealed separation into three groups: designated group 1 composed of clear cell and papillary meningiomas, whereas group 2A comprised predominantly atypical meningiomas and group 2B enriched for rare high-grade subtypes (rhabdoid, chordoid). Meningiomas from NF2 patients clustered exclusively within groups 1 and 2A. When compared with a dataset of 105 adult meningiomas, the pediatric meningiomas largely grouped separately. Targeted panel DNA sequencing of 34 tumors revealed frequent NF2 alterations, while other typical alterations found in adult non-NF2 tumors were absent. These data demonstrate that pediatric meningiomas are characterized by molecular features distinct from adult tumors.

A rare large duplication of MLH1 identified in Lynch syndrome.

BACKGROUND: The most frequently identified strong cancer predisposition mutations for colorectal cancer (CRC) are those in the mismatch repair (MMR) genes in Lynch syndrome. Laboratory diagnostics include testing tumors for immunohistochemical staining (IHC) of the Lynch syndrome-associated DNA MMR proteins and/or for microsatellite instability (MSI) followed by sequencing or other techniques, such as denaturing high performance liquid chromatography (DHPLC), to identify the mutation. METHODS: In an ongoing project focusing on finding Mendelian cancer syndromes we applied whole-exome/whole-genome sequencing (WES/WGS) to 19 CRC families. RESULTS: Three families were identified with a pathogenic/likely pathogenic germline variant in a MMR gene that had previously tested negative in DHPLC gene variant screening. All families had a history of CRC in several family members across multiple generations. Tumor analysis showed loss of the MMR protein IHC staining corresponding to the mutated genes, as well as MSI. In family A, a structural variant, a duplication of exons 4 to 13, was identified in MLH1. The duplication was predicted to lead to a frameshift at amino acid 520 and a premature stop codon at amino acid 539. In family B, a 1 base pair deletion was found in MLH1, resulting in a frameshift and a stop codon at amino acid 491. In family C, we identified a splice site variant in MSH2, which was predicted to lead loss of a splice donor site. CONCLUSIONS: We identified altogether three pathogenic/likely pathogenic variants in the MMR genes in three of the 19 sequenced families. The MLH1 variants, a duplication of exons 4 to 13 and a frameshift variant, were novel, based on the InSiGHT and ClinVar databases; the MSH2 splice site variant was reported by a single submitter in ClinVar. As a variant class, duplications have rarely been reported in the MMR gene literature, particularly those covering several exons.

Whole Exome Sequencing Identifies APCDD1 and HDAC5 Genes as Potentially Cancer Predisposing in Familial Colorectal Cancer.

Germline mutations in predisposition genes account for only 20% of all familial colorectal cancers (CRC) and the remaining genetic burden may be due to rare high- to moderate-penetrance germline variants that are not explored. With the aim of identifying such potential cancer-predisposing variants, we performed whole exome sequencing on three CRC cases and three unaffected members of a Polish family and identified two novel heterozygous variants: a coding variant in APC downregulated 1 gene (APCDD1, p.R299H) and a non-coding variant in the 5' untranslated region (UTR) of histone deacetylase 5 gene (HDAC5). Sanger sequencing confirmed the variants segregating with the disease and Taqman assays revealed 8 additional APCDD1 variants in a cohort of 1705 familial CRC patients and no further HDAC5 variants. Proliferation assays indicated an insignificant proliferative impact for the APCDD1 variant. Luciferase reporter assays using the HDAC5 variant resulted in an enhanced promoter activity. Targeting of transcription factor binding sites of SNAI-2 and TCF4 interrupted by the HDAC5 variant showed a significant impact of TCF4 on promoter activity of mutated HDAC5. Our findings contribute not only to the identification of unrecognized genetic causes of familial CRC but also underline the importance of 5'UTR variants affecting transcriptional regulation and the pathogenesis of complex disorders.

Mutational patterns and regulatory networks in epigenetic subgroups of meningioma.

DNA methylation patterns delineate clinically relevant subgroups of meningioma. We previously established the six meningioma methylation classes (MC) benign 1-3, intermediate A and B, and malignant. Here, we set out to identify subgroup-specific mutational patterns and gene regulation. Whole genome sequencing was performed on 62 samples across all MCs and WHO grades from 62 patients with matched blood control, including 40 sporadic meningiomas and 22 meningiomas arising after radiation (Mrad). RNA sequencing was added for 18 of these cases and chromatin-immunoprecipitation for histone H3 lysine 27 acetylation (H3K27ac) followed by sequencing (ChIP-seq) for 16 samples. Besides the known mutations in meningioma, structural variants were found as the mechanism of NF2 inactivation in a small subset (5%) of sporadic meningiomas, similar to previous reports for Mrad. Aberrations of DMD were found to be enriched in MCs with NF2 mutations, and DMD was among the most differentially upregulated genes in NF2 mutant compared to NF2 wild-type cases. The mutational signature AC3, which has been associated with defects in homologous recombination repair (HRR), was detected in both sporadic meningioma and Mrad, but widely distributed across the genome in sporadic cases and enriched near genomic breakpoints in Mrad. Compared to the other MCs, the number of single nucleotide variants matching the AC3 pattern was significantly higher in the malignant MC, which also exhibited higher genomic instability, determined by the numbers of both large segments affected by copy number alterations and breakpoints between large segments. ChIP-seq analysis for H3K27ac revealed a specific activation of genes regulated by the transcription factor FOXM1 in the malignant MC. This analysis also revealed a super enhancer near the HOXD gene cluster in this MC, which, together with general upregulation of HOX genes in the malignant MC, indicates a role of HOX genes in meningioma aggressiveness. This data elucidates the biological mechanisms rendering different epigenetic subgroups of meningiomas, and suggests leveraging HRR as a novel therapeutic target.

TALEN/CRISPR-mediated engineering of a promoterless anti-viral RNAi hairpin into an endogenous miRNA locus.

Successful RNAi applications depend on strategies allowing robust and persistent expression of minimal gene silencing triggers without perturbing endogenous gene expression. Here, we propose a novel avenue which is integration of a promoterless shmiRNA, i.e. a shRNA embedded in a micro-RNA (miRNA) scaffold, into an engineered genomic miRNA locus. For proof-of-concept, we used TALE or CRISPR/Cas9 nucleases to site-specifically integrate an anti-hepatitis C virus (HCV) shmiRNA into the liver-specific miR-122/hcr locus in hepatoma cells, with the aim to obtain cellular clones that are genetically protected against HCV infection. Using reporter assays, Northern blotting and qRT-PCR, we confirmed anti-HCV shmiRNA expression as well as miR-122 integrity and functionality in selected cellular progeny. Moreover, we employed a comprehensive battery of PCR, cDNA/miRNA profiling and whole genome sequencing analyses to validate targeted integration of a single shmiRNA molecule at the expected position, and to rule out deleterious effects on the genomes or transcriptomes of the engineered cells. Importantly, a subgenomic HCV replicon and a full-length reporter virus, but not a Dengue virus control, were significantly impaired in the modified cells. Our original combination of DNA engineering and RNAi expression technologies benefits numerous applications, from miRNA, genome and transgenesis research, to human gene therapy.

Identification of SLC20A1 and SLC15A4 among other genes as potential risk factors for combined pituitary hormone deficiency.

PURPOSE: Combined pituitary hormone deficiency (CPHD) is characterized by a malformed or underdeveloped pituitary gland resulting in an impaired pituitary hormone secretion. Several transcription factors have been described in its etiology, but defects in known genes account for only a small proportion of cases. METHODS: To identify novel genetic causes for congenital hypopituitarism, we performed exome-sequencing studies on 10 patients with CPHD and their unaffected parents. Two candidate genes were sequenced in further 200 patients. Genotype data of known hypopituitary genes are reviewed. RESULTS: We discovered 51 likely damaging variants in 38 genes; 12 of the 51 variants represent de novo events (24%); 11 of the 38 genes (29%) were present in the E12.5/E14.5 pituitary transcriptome. Targeted sequencing of two candidate genes, SLC20A1 and SLC15A4, of the solute carrier membrane transport protein family in 200 additional patients demonstrated two further variants predicted as damaging. We also found combinations of de novo (SLC20A1/SLC15A4) and transmitted variants (GLI2/LHX3) in the same individuals, leading to the full-blown CPHD phenotype. CONCLUSION: These data expand the pituitary target genes repertoire for diagnostics and further functional studies. Exome sequencing has identified a combination of rare variants in different genes that might explain incomplete penetrance in CPHD.

Cutis laxa, exocrine pancreatic insufficiency and altered cellular metabolomics as additional symptoms in a new patient with ATP6AP1-CDG.

Congenital disorders of glycosylation (CDG) are genetic defects in the glycoconjugate biosynthesis. >100 types of CDG are known, most of them cause multi-organ diseases. Here we describe a boy whose leading symptoms comprise cutis laxa, pancreatic insufficiency and hepatosplenomegaly. Whole exome sequencing identified the novel hemizygous mutation c.542T>G (p.L181R) in the X-linked ATP6AP1, an accessory protein of the mammalian vacuolar H+-ATPase, which led to a general N-glycosylation deficiency. Studies of serum N-glycans revealed reduction of complex sialylated and appearance of truncated diantennary structures. Proliferation of the patient's fibroblasts was significantly reduced and doubling time prolonged. Additionally, there were alterations in the fibroblasts' amino acid levels and the acylcarnitine composition. Especially, short-chain species were reduced, whereas several medium- to long-chain acylcarnitines (C14-OH to C18) were elevated. Investigation of the main lipid classes revealed that total cholesterol was significantly enriched in the patient's fibroblasts at the expense of phophatidylcholine and phosphatidylethanolamine. Within the minor lipid species, hexosylceramide was reduced, while its immediate precursor ceramide was increased. Since catalase activity and ACOX3 expression in peroxisomes were reduced, we assume an ATP6AP1-dependent impact on the ß-oxidation of fatty acids. These results help to understand the complex clinical characteristics of this new patient.

Impact of clinical exomes in neurodevelopmental and neurometabolic disorders.

Whole exome sequencing (WES) is well established in research and is now being introduced into clinically indicated diagnostics (so-called clinical exomes). We evaluated the diagnostic yield and clinical implications of WES in 72 patients from 60 families with undiagnosed neurodevelopmental disorders (NDD), neurometabolic disorders, and dystonias. Pathogenic or likely pathogenic variants leading to a molecular diagnosis could be identified in 21 of the 60 families (overall 35%, in 36% of patients with NDD, in 43% of patients with neurometabolic disorders, in 25% of patients with dystonias). In one family two coexisting autosomal recessive diseases caused by homozygous pathogenic variants in two different genes were diagnosed. In another family, a homozygous frameshift variant in STRADA was found to cause a severe NDD with early onset epilepsy, brain anomalies, hypotonia, heart defect, nephrocalcinosis, macrocephaly and distinctive facies so far designated as PMSE (polyhydramnios, megalencephaly, symptomatic epilepsy) syndrome. In 7 of the 21 families with a molecular diagnosis the pathogenic variants were only identified by clinical follow-up, manual reevaluation of the literature, a change of filter setting, and/or reconsideration of inheritance pattern. Most importantly, clinical implications included management changes in 8 cases and impact on family planning in 20 families with a molecular diagnosis. This study shows that reevaluation and follow-up can improve the diagnostic rate and that WES results have important implications on medical management and family planning. Furthermore, we could confirm STRADA as a gene associated with syndromic ID but find it questionable if the current designation as PMSE depicts the most important clinical features.

Diagnosis of CoPAN by whole exome sequencing: Waking up a sleeping tiger's eye.

Neurodegeneration with brain iron accumulation (NBIA) is a group of neurodegenerative disorders characterized by iron accumulation in the basal ganglia. Recently, mutations in CoA synthase (COASY) have been identified as a cause of a novel NBIA subtype (COASY Protein-Associated Neurodegeneration, CoPAN) in two patients with dystonic paraparesis, parkinsonian features, cognitive impairment, behavior abnormalities, and axonal neuropathy. COASY encodes an enzyme required for Coenzyme A (CoA) biosynthesis. Using whole exome sequencing (WES) we identified compound heterozygous COASY mutations in two siblings with intellectual disability, ataxic gait, progressive spasticity, and obsessive-compulsive behavior. The "eye-of-the tiger-sign," a characteristic hypointense spot within the hyperintense globi pallidi on MRI found in the most common subtype of NBIA (Pantothenate Kinase-Associated Neurodegeneration, PKAN), was not present. Instead, bilateral hyperintensity and swelling of caudate nucleus, putamen, and thalamus were found. In addition, our patients showed a small corpus callosum and frontotemporal and parietal white matter changes, expanding the brain phenotype of patients with CoPAN. Metabolic investigations showed increased free carnitine and decreased acylcarnitines in the patients dried blood samples. Carnitine palmitoyl transferase 1 (CPT1) deficiency was excluded by further enzymatic and metabolic investigations. As CoA and its derivate Acetyl-CoA play an essential role in fatty acid metabolism, we assume that abnormal acylcarnitine profiles are a result of the COASY mutations. This report not only illustrates that WES is a powerful tool to elucidate the etiology of rare genetic diseases, but also identifies unique neuroimaging and metabolic findings that may be key features for an early diagnosis of CoPAN.

DDX3X mutations in two girls with a phenotype overlapping Toriello-Carey syndrome.

Recently, de novo heterozygous variants in DDX3X have been reported in about 1.5% of 2659 females with previously unexplained intellectual disability (ID). We report on the identification of DDX3X variants in two unrelated girls with clinical features of Toriello-Carey Syndrome (T-CS). In patient 1, the recurrent variant c.1703C>T; p.(P568L) was identified when reconsidering X-linked de novo heterozygous variants in exome sequencing data. In patient 2, the DDX3X variant c.1600C>G; p.(R534G) was also detected by exome sequencing. Based on these data, de novo heterozygous DDX3X variants should be considered not only in females with unexplained ID, but also in individuals with a clinical diagnosis of T-CS.

Homozygous missense mutation in the LMAN2L gene segregates with intellectual disability in a large consanguineous Pakistani family.

BACKGROUND: Intellectual disability (ID) is a neurodevelopmental disorder affecting 1%-3% of the population worldwide. It is characterised by high phenotypic and genetic heterogeneity and in most cases the underlying cause of the disorder is unknown. In our study we investigated a large consanguineous family from Baluchistan, Pakistan, comprising seven affected individuals with a severe form of autosomal recessive ID (ARID) and epilepsy, to elucidate a putative genetic cause. METHODS AND RESULTS: Whole exome sequencing (WES) of a trio, including a child with ID and epilepsy and its healthy parents that were part of this large family, revealed a homozygous missense variant p.R53Q in the lectin mannose-binding 2-like (LMAN2L) gene. This homozygous variant was co-segregating in the family with the phenotype of severe ID and infantile epilepsy; unaffected family members were heterozygous variant carriers. The variant was predicted to be pathogenic by five different in silico programmes and further three-dimensional structure modelling of the protein suggests that variant p.R53Q may impair protein-protein interaction. LMAN2L (OMIM: 609552) encodes for the lectin, mannose-binding 2-like protein which is a cargo receptor in the endoplasmic reticulum important for glycoprotein transport. Genome-wide association studies have identified an association of LMAN2L to different neuropsychiatric disorders. CONCLUSION: This is the first report linking LMAN2L to a phenotype of severe ARID and seizures, indicating that the deleterious homozygous p.R53Q variant very likely causes the disorder.

Exome sequencing reveals a novel CWF19L1 mutation associated with intellectual disability and cerebellar atrophy.

Intellectual disability (ID) with cerebellar ataxia comprises a genetically heterogeneous group of neurodevelopmental disorders. We identified a homozygous frameshift mutation in CWF19L1 (c.467delC; p.(P156Hfs*33)) by a combination of linkage analysis and Whole Exome Sequencing in a consanguineous Turkish family with a 9-year-old boy affected by early onset cerebellar ataxia and mild ID. Serial MRI showed mildly progressive cerebellar atrophy. Absent C19L1 protein expression in lymphoblastoid cell lines strongly suggested that c.467delC is a disease-causing alteration. One further pregnancy of the mother had been terminated at 22 weeks of gestation because of a small cerebellum and agenesis of corpus callosum. The homozygous CWF19L1 variant was also present in the fetus. Postmortem examination of the fetus in addition showed unilateral hexadactyly and vertebral malformations. These features have not been reported and may represent an expansion of the CWF19L1-related phenotypic spectrum, but could also be due to another, possibly autosomal recessive disorder. The exact function of the evolutionarily highly conserved C19L1 protein is unknown. So far, homozygous or compound heterozygous mutations in CWF19L1 have been identified in two Turkish siblings and a Dutch girl, respectively, affected by cerebellar ataxia and ID. A zebrafish model showed that CWF19L1 loss-of-function mutations result in abnormal cerebellar morphology and movement disorders. Our report corroborates that loss-of-function mutations in CWF19Ll lead to early onset cerebellar ataxia and (progressive) cerebellar atrophy. © 2016 Wiley Periodicals, Inc.

Pedigree based DNA sequencing pipeline for germline genomes of cancer families.

BACKGROUND: In the course of our whole-genome sequencing efforts, we have developed a pipeline for analyzing germline genomes from Mendelian types of cancer pedigrees (familial cancer variant prioritization pipeline, FCVPP). RESULTS: The variant calling step distinguishes two types of genomic variants: single nucleotide variants (SNVs) and indels, which undergo technical quality control. Mendelian types of variants are assumed to be rare and variants with frequencies higher that 0.1 % are screened out using human 1000 Genomes (Phase 3) and non-TCGA ExAC population data. Segregation in the pedigree allows variants to be present in affected family members and not in old, unaffected ones. The effectiveness of variant segregation depends on the number and relatedness of the family members: if over 5 third-degree (or more distant) relatives are available, the experience has shown that the number of likely variants is reduced from many hundreds to a few tens. These are then subjected to bioinformatics analysis, starting with the combined annotation dependent depletion (CADD) tool, which predicts the likelihood of the variant being deleterious. Different sets of individual tools are used for further evaluation of the deleteriousness of coding variants, 5' and 3' untranslated regions (UTRs), and intergenic variants. CONLUSIONS: The likelihood of success of the present genomic pipeline in finding novel high- or medium-penetrant genes depends on many steps but first and foremost, the pedigree needs to be reasonably large and the assignments and diagnoses among the members need to be correct.