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The majority of studies assessing the contribution of pathogenic germline variants (PGVs) to cancer predisposition have focused on patients with single cancers. We analyzed 45 known cancer predisposition genes (CPGs) in germline samples of 202 patients with hematological malignancies (HMs) plus one or more other independent cancer managed at major tertiary medical centers on two different continents. This included 120 patients with therapy-related myeloid neoplasms (t-MNs), where the HM occurred after cytotoxic treatment for a first malignancy, and 82 patients with multiple cancers in which the HM was not preceded by cytotoxic therapy (MC-HM). Using American College of Medical Genetics/Association for Molecular Pathology variant classification guidelines, 13% of patients had PGVs, most frequently identified in CHEK2 (17% of PGVs), BRCA1 (13%), DDX41 (13%), and TP53 (7%). The frequency of PGVs in MC-HM was higher than in t-MN, although not statistically significant (18 vs. 9%; p  = 0.085). The frequency of PGVs in lymphoid and myeloid HM patients was similar (19 vs. 17.5%; p  > 0.9). Critically, patients with PGVs in BRCA1 , BRCA2 or TP53 did not satisfy current clinical phenotypic criteria for germline testing. Our data suggest that a personal history of multiple cancers, one being a HM, should trigger screening for PGVs.

Therapy-related myeloid neoplasms (T-MN) are poorly characterized secondary hematological malignancies following chemotherapy/radiotherapy exposure. We compared the clinical and mutational characteristics of T-MN ( n  = 129) and primary myelodysplastic syndrome (P-MDS, n  = 108) patients. Although the somatic mutation frequency was similar between T-MN and P-MDS patients (93% in both groups), the pattern was distinct. TP53 mutations were more frequent in T-MN (29.5 vs. 7%), while spliceosomal complex mutations were more common in P-MDS (56.5 vs. 25.6%). In contrast to P-MDS, the ring sideroblasts (RS) phenotype was not associated with better survival in T-MN, most probably due to genetic association with TP53 mutations. SF3B1 was mutated in 96% of P-MDS with ≥15% RS, but in only 32% T-MN. TP53 mutations were detected in 92% T-MN with ≥15% RS and SF3B1 wild-type cases. Interestingly, T-MN and P-MDS patients with “Very low” or “Low” Revised International Prognostic Scoring System (IPSS-R) showed similar biological and clinical characteristics. In a Cox regression analysis, TP53 mutation was a poor prognostic factor in T-MN, independent of IPSS-R cytogenetics, disease-modifying therapy, and NRAS mutation. Our data have direct implications for T-MN management and provide evidence that, in addition to conventional disease parameters, mutational analysis should be incorporated in T-MN risk stratification.

BackgroundPseudodiastrophic dysplasia (PDD) is a severe skeletal dysplasia associated with prenatal manifestation and early lethality. Clinically, PDD is classified as a ‘dysplasia with multiple joint dislocations’; however, the molecular aetiology of the disorder is currently unknown.MethodsWhole exome sequencing (WES) was performed on three patients from two unrelated families, clinically diagnosed with PDD, in order to identify the underlying genetic cause. The functional effects of the identified variants were characterised using primary cells and human cell-based overexpression assays.ResultsWES resulted in the identification of biallelic variants in the established skeletal dysplasia genes, B3GAT3 (family 1) and CANT1 (family 2). Mutations in these genes have previously been reported to cause ‘multiple joint dislocations, short stature, and craniofacial dysmorphism with or without congenital heart defects’ (‘JDSCD’; B3GAT3) and Desbuquois dysplasia 1 (CANT1), disorders in the same nosological group as PDD. Follow-up of the B3GAT3 variants demonstrated significantly reduced B3GAT3/GlcAT-I expression. Downstream in vitro functional analysis revealed abolished biosynthesis of glycosaminoglycan side chains on proteoglycans. Functional evaluation of the CANT1 variant showed impaired nucleotidase activity, which results in inhibition of glycosaminoglycan synthesis through accumulation of uridine diphosphate.ConclusionFor the families described in this study, the PDD phenotype was caused by mutations in the known skeletal dysplasia genes B3GAT3 and CANT1, demonstrating the advantage of genomic analyses in delineating the molecular diagnosis of skeletal dysplasias. This finding expands the phenotypic spectrum of B3GAT3-related and CANT1-related skeletal dysplasias to include PDD and highlights the significant phenotypic overlap of conditions within the proteoglycan biosynthesis pathway.

Background Prenatal presentation of polycystic kidney disease (PKD), characterized by bilateral renal cysts and enlarged echogenic kidneys on ultrasound, often results in perinatal death. Prenatal manifestations of PKD are generally associated with autosomal recessive PKD, most commonly a result of pathogenic variants in PKHD1 , but in rare cases can also be driven by bi-allelic inheritance of pathogenic variants in genes more commonly associated with autosomal dominant PKD such as PKD1 . Diagnosing the underlying cause of prenatal PKD can be complicated by atypical histology, and/or a prenatal phenotype that does not align with family history. In this study, five cases of prenatal PKD with atypical or inconclusive features identified during post-mortem investigations underwent trio exome or genome sequencing, termed a genomic autopsy. Results Genomic autopsy was able to delineate the genetic basis of prenatal PKD in all five families. Conclusion Our findings demonstrate the diagnostic utility of a genomic autopsy in providing a genetic diagnosis for fetal PKD cases post-mortem, particularly in atypical presentations. A genetic diagnosis is highly beneficial for future family planning, including the use of reproductive technologies, as well as identifying presymptomatic parents who are likely to develop PKD in the future.

Background Periventricular nodular heterotopia (PNH) is a malformation of cortical development characterized by nodules of abnormally migrated neurons. The cause of posteriorly placed PNH is not well characterised and we present a case that provides insights into the cause of posterior PNH. Case presentation We report a fetus with extensive posterior PNH in association with biallelic variants in LAMC3 . LAMC3 mutations have previously been shown to cause polymicrogyria and pachygyria in the occipital cortex, but not PNH. The occipital location of PNH in our case and the proposed function of LAMC3 in cortical development suggest that the identified LAMC3 variants may be causal of PNH in this fetus. Conclusion We hypothesise that this finding extends the cortical phenotype associated with LAMC3 and provides valuable insight into genetic cause of posterior PNH.

Background We report a large family with four successive generations, presenting with a complex phenotype of severe congenital neutropenia (SCN), partially penetrant monocytosis, and hearing loss of varying severity. Methods We performed whole exome sequencing to identify the causative variants. Sanger sequencing was used to perform segregation analyses on remaining family members. Results We identified and classified a pathogenic GFI1 variant and a likely pathogenic variant in MYO6 which together explain the complex phenotypes seen in this family. Conclusions We present a case illustrating the benefits of a broad screening approach that allows identification of oligogenic determinants of complex human phenotypes which may have been missed if the screening was limited to a targeted gene panel with the assumption of a syndromic disorder. This is important for correct genetic diagnosis of families and disentangling the range and severity of phenotypes associated with high impact variants.

Hamish Scott and colleagues report that germline mutations in GATA2 segregate with myelodysplastic syndrome and acute myeloid leukemia in four pedigrees. The resulting alterations occur in a conserved zinc finger DNA-binding domain of GATA2. We report the discovery of GATA2 as a new myelodysplastic syndrome (MDS)-acute myeloid leukemia (AML) predisposition gene. We found the same, previously unidentified heterozygous c.1061C>T (p.Thr354Met) missense mutation in the GATA2 transcription factor gene segregating with the multigenerational transmission of MDS-AML in three families and a GATA2 c.1063_1065delACA (p.Thr355del) mutation at an adjacent codon in a fourth MDS family. The resulting alterations reside within the second zinc finger of GATA2, which mediates DNA-binding and protein-protein interactions. We show differential effects of the mutations on the transactivation of target genes, cellular differentiation, apoptosis and global gene expression. Identification of such predisposing genes to familial forms of MDS and AML is critical for more effective diagnosis and prognosis, counseling, selection of related bone marrow transplant donors and development of therapies.

Biological and biomedical research relies on comprehensive understanding of protein-coding transcripts. However, the total number of human proteins is still unknown due to the prevalence of alternative splicing. In this paper, we detected 31,566 novel transcripts with coding potential by filtering our ab initio predictions with 50 RNA-seq datasets from diverse tissues/cell lines. PCR followed by MiSeq sequencing showed that at least 84.1% of these predicted novel splice sites could be validated. In contrast to known transcripts, the expression of these novel transcripts were highly tissue-specific. Based on these novel transcripts, at least 36 novel proteins were detected from shotgun proteomics data of 41 breast samples. We also showed L1 retrotransposons have a more significant impact on the origin of new transcripts/genes than previously thought. Furthermore, we found that alternative splicing is extraordinarily widespread for genes involved in specific biological functions like protein binding, nucleoside binding, neuron projection, membrane organization and cell adhesion. In the end, the total number of human transcripts with protein-coding potential was estimated to be at least 204,950.

Somatic GNAS and USP8 mutations have been implicated in sporadic somatotrophinomas and corticotrophinomas, respectively. However, no genes are known to be recurrently mutated in sporadic prolactinomas. The prevalence of copy number variants (CNV), which is emerging as a mechanism of tumorigenesis in sporadic pituitary adenomas in general, is also unclear in prolactinomas. To characterize the genetic events underpinning sporadic prolactinomas, we performed whole exome sequencing of paired tumor and germline DNA from 12 prolactinoma patients. We observed recurrent large-scale CNV, most commonly in the form of copy number gains. We also identified sequence variants of interest in 15 genes. This included the DRD2, PRL, TMEM67, and MLH3 genes with plausible links to prolactinoma formation. Of the 15 genes of interest, CNV was seen at the gene locus in the corresponding tumor in 10 cases, and pituitary expression of eight genes was in the top 10% of tissues. However, none of our shortlisted somatic variants appeared to be classical driver mutations as no variant was found in more than one tumor. Future directions of research include mechanistic studies to investigate how CNV may contribute to prolactinoma formation, larger studies of relevant prolactinoma subsets according to clinical characteristics, and additional genetic investigations for aberrations not captured by whole exome sequencing.

Introduction: Germline mutations in the succinate dehydrogenase genes (SDHA/B/C/D, SDHAF2 - collectively, SDHx) have been implicated in paraganglioma (PGL), renal cell carcinoma (RCC), gastrointestinal stromal tumor (GIST) and pituitary adenoma (PA). Negative SDHB tumor staining is indicative of SDH-deficient tumors, and thereby germline SDHx mutations (Evenepoel et al Genet Med 2015; De Sousa et al Eur J Endocrinol 2017). As for most Sanger and next generation sequencing (NGS) tests, SDHx genetic testing targets exons and ≤20bp of flanking intronic regions. Deep intronic mutations thus represent a potentially missed cause of various heritable disorders. Methods: We investigated a family with novel co-occurrence of all four SDH-related tumors: PGL in two siblings; GIST in a third sibling; PA in the fourth sibling; and RCC in their deceased mother. Despite negative SDHB staining of the PGLs, no germline mutations were found in SDHx or other PGL genes after 12 yr of extensive testing. We proceeded to whole exome sequencing (WES; Illumina NextSeq 500) using germline DNA from the four siblings and tumor DNA from their available formalin-fixed operative specimens. Transcriptome analysis (RNAseq; Illumina TruSeq LT) was performed using whole blood from the proband. Local genomic pathology and clinical genetic databases were searched for similar cases. Results: Amongst 130 germline WES variants of interest, we found a novel, highly conserved, deep intronic variant in a known PGL gene, SDHC, in all four affected siblings. RNAseq revealed aberrant SDHC splicing with 71% of mRNA reads extending 75bp into intron 1 and terminating at the site of the familial variant. The retained intronic segment introduced a premature stop codon immediately after exon 1. Tumor WES of the available PGL specimen demonstrated Chr1 deletion with loss of the wild-type SDHC allele, consistent with the two-hit model of tumor suppressor genes. Amongst other local patients with SDH-deficient tumors, 9/27 (33%) also lacked SDHx mutations by standard testing. Re-analysis of five patients with available raw data from prior NGS tests revealed another patient with the same SDHC variant as the index family. NGS haplotype analysis showed that 7% of Chr1 was identical between this patient and the index family, suggesting cryptic relatedness. WGS is underway in another 12 unsolved cases of SDH-deficient tumors to identify other SDHx deep intronic mutations. Conclusions: This is the first report of a deep intronic SDHx mutation, highlighting a unique mechanism of SDH-related tumorigenesis and explaining at least some previously unsolved cases of SDH-deficient tumors. More broadly, this study demonstrates how deep intronic mutations may be a cause of false negative genetic test results and illustrates the expanding utility of NGS methodologies across heritable disorders.