Size matters: Large copy number losses in Hirschsprung disease patients reveal genes involved in enteric nervous system development.

Hirschsprung disease (HSCR) is a complex genetic disease characterized by absence of ganglia in the intestine. HSCR etiology can be explained by a unique combination of genetic alterations: rare coding variants, predisposing haplotypes and Copy Number Variation (CNV). Approximately 18% of patients have additional anatomical malformations or neurological symptoms (HSCR-AAM). Pinpointing the responsible culprits within a CNV is challenging as often many genes are affected. Therefore, we selected candidate genes based on gene enrichment strategies using mouse enteric nervous system transcriptomes and constraint metrics. Next, we used a zebrafish model to investigate whether loss of these genes affects enteric neuron development in vivo. This study included three groups of patients, two groups without coding variants in disease associated genes: HSCR-AAM and HSCR patients without associated anomalies (HSCR-isolated). The third group consisted of all HSCR patients in which a confirmed pathogenic rare coding variant was identified. We compared these patient groups to unaffected controls. Predisposing haplotypes were determined, confirming that every HSCR subgroup had increased contributions of predisposing haplotypes, but their contribution was highest in isolated HSCR patients without RET coding variants. CNV profiling proved that specifically HSCR-AAM patients had larger Copy Number (CN) losses. Gene enrichment strategies using mouse enteric nervous system transcriptomes and constraint metrics were used to determine plausible candidate genes located within CN losses. Validation in zebrafish using CRISPR/Cas9 targeting confirmed the contribution of UFD1L, TBX2, SLC8A1, and MAPK8 to ENS development. In addition, we revealed epistasis between reduced Ret and Gnl1 expression and between reduced Ret and Tubb5 expression in vivo. Rare large CN losses-often de novo-contribute to HSCR in HSCR-AAM patients. We proved the involvement of six genes in enteric nervous system development and Hirschsprung disease.

Atypical 3q26/MECOM rearrangements genocopy inv(3)/t(3;3) in acute myeloid leukemia.

Acute myeloid leukemia (AML) with inv(3)/t(3;3)(q21q26) is a distinct World Health Organization recognized entity, characterized by its aggressive course and poor prognosis. In this subtype of AML, the translocation of a GATA2 enhancer (3q21) to MECOM (3q26) results in overexpression of the MECOM isoform EVI1 and monoallelic expression of GATA2 from the unaffected allele. The full-length MECOM transcript, MDS1-EVI1, is not expressed as the result of the 3q26 rearrangement. Besides the classical inv(3)/t(3;3), a number of other 3q26/MECOM rearrangements with poor treatment response have been reported in AML. Here, we demonstrate, in a group of 33 AML patients with atypical 3q26 rearrangements, MECOM involvement with EVI1 overexpression but no or low MDS1-EVI1 levels. Moreover, the 3q26 translocations in these AML patients often involve superenhancers of genes active in myeloid development (eg, CD164, PROM1, CDK6, or MYC). In >50% of these cases, allele-specific GATA2 expression was observed, either by copy-number loss or by an unexplained allelic imbalance. Altogether, atypical 3q26 recapitulate the main leukemic mechanism of inv(3)/t(3;3) AML, namely EVI1 overexpression driven by enhancer hijacking, absent MDS1-EVI1 expression and potential GATA2 involvement. Therefore, we conclude that both atypical 3q26/MECOM and inv(3)/t(3;3) can be classified as a single entity of 3q26-rearranged AMLs. Routine analyses determining MECOM rearrangements and EVI1 and MDS1-EVI1 expression are required to recognize 3q-rearranged AML cases.

Non-invasive prenatal diagnosis for translocation carriers-YES please or NO go?

INTRODUCTION: The presence of an unbalanced familial translocation can be reliably assessed in the cytotrophoblast of chorionic villi. However, carriers of a balanced translocation often decline invasive testing. This study aimed to investigate whether an unbalanced translocation can also be diagnosed in cell free DNA by whole-genome non-invasive prenatal screening (NIPS). MATERIAL AND METHODS: Pregnant women carrying a fetus with an unbalanced familial translocation, for whom NIPS as well as microarray data were available, were included in this retrospective assessment. NIPS was performed in the course of the TRIDENT study. RESULTS: In 12 cases, both NIPS and microarray data were available. In 10 of 12 cases the unbalanced translocation was correctly identified by NIPS without prior knowledge on parental translocation. One was missed because the fetal fraction was too low. One was missed because of technical restrictions in calling 16p gains. CONCLUSIONS: This study supports the hypothesis that routine NIPS may be used for prenatal diagnosis of unbalanced inheritance of familial translocations, especially with prior knowledge of the translocation allowing focused examination of the involved chromosomal regions. Our study showed that routine shallow sequencing designed for aneuploidy detection in cell free DNA may be sufficient for higher resolution NIPS, if specialized copy number software is used and if sufficient fetal fraction is present.

Complete APTX deletion in a patient with ataxia with oculomotor apraxia type 1.

BACKGROUND: Ataxia with oculomotor apraxia type 1 is an autosomal-recessive neurodegenerative disorder characterized by a childhood onset of slowly progressive cerebellar ataxia, followed by oculomotor apraxia and a severe primary motor peripheral axonal motor neuropathy. Ataxia with oculomotor apraxia type 1 is caused by bi-allelic mutations in APTX (chromosome 9p21.1). CASE PRESENTATION: Our patient has a clinical presentation that is typical for ataxia with oculomotor apraxia type 1 with no particularly severe phenotype. Multiplex Ligation-dependent Probe Amplification analysis resulted in the identification of a homozygous deletion of all coding APTX exons (3 to 9). SNP array analysis using the Illumina Infinium CytoSNP-850 K microarray indicated that the deletion was about 62 kb. Based on the SNP array results, the breakpoints were found using direct sequence analysis: c.-5 + 1225_*44991del67512, p.0?. Both parents were heterozygous for the deletion. Homozygous complete APTX deletions have been described in literature for two other patients. We obtained a sample from one of these two patients and characterized the deletion (156 kb) as c.-23729_*115366del155489, p.0?, including the non-coding exons 1A and 2 of APTX. The more severe phenotype reported for this patient is not observed in our patient. It remains unclear whether the larger size of the deletion (156 kb vs 62 kb) plays a role in the phenotype (no extra genes are deleted). CONCLUSION: Here we described an ataxia with oculomotor apraxia type 1 patient who has a homozygous deletion of the complete coding region of APTX. In contrast to the patient with the large deletion, our patient does not have a severe phenotype. More patients with deletions of APTX are required to investigate a genotype-phenotype effect.

Intrinsic Cellular Susceptibility to Barrett's Esophagus in Adults Born with Esophageal Atresia.

The prevalence of Barrett's esophagus (BE) in adults born with esophageal atresia (EA) is four times higher than in the general population and presents at a younger age (34 vs. 60 years). This is (partly) a consequence of chronic gastroesophageal reflux. Given the overlap between genes and pathways involved in foregut and BE development, we hypothesized that EA patients have an intrinsic predisposition to develop BE. Transcriptomes of Esophageal biopsies of EA patients with BE (n = 19, EA/BE); EA patients without BE (n = 44, EA-only) and BE patients without EA (n = 10, BE-only) were compared by RNA expression profiling. Subsequently, we simulated a reflux episode by exposing fibroblasts of 3 EA patients and 3 controls to acidic conditions. Transcriptome responses were compared to the differential expressed transcripts in the biopsies. Predisposing single nucleotide polymorphisms, associated with BE, were slightly increased in EA/BE versus BE-only patients. RNA expression profiling and pathway enrichment analysis revealed differences in retinoic acid metabolism and downstream signaling pathways and inflammatory, stress response and oncological processes. There was a similar effect on retinoic acid signaling and immune response in EA patients upon acid exposure. These results indicate that epithelial tissue homeostasis in EA patients is more prone to acidic disturbances.

Infantile hypertrophic pyloric stenosis in patients with esophageal atresia.

BACKGROUND: Patients born with esophageal atresia (EA) have a higher incidence of infantile hypertrophic pyloric stenosis (IHPS), suggestive of a relationship. A shared etiology makes sense from a developmental perspective as both affected structures are foregut derived. A genetic component has been described for both conditions as single entities and EA and IHPS are variable components in several monogenetic syndromes. We hypothesized that defects disturbing foregut morphogenesis are responsible for this combination of malformations. METHODS: We investigated the genetic variation of 15 patients with both EA and IHPS with unaffected parents using exome sequencing and SNP array-based genotyping, and compared the results to mouse transcriptome data of the developing foregut. RESULTS: We did not identify putatively deleterious de novo mutations or recessive variants. However, we detected rare inherited variants in EA or IHPS disease genes or in genes important in foregut morphogenesis, expressed at the proper developmental time-points. Two pathways were significantly enriched (p < 1 × 10-5 ): proliferation and differentiation of smooth muscle cells and self-renewal of satellite cells. CONCLUSIONS: None of our findings could fully explain the combination of abnormalities on its own, which makes complex inheritance the most plausible genetic explanation, most likely in combination with mechanical and/or environmental factors. As we did not find one defining monogenetic cause for the EA/IHPS phenotype, the impact of the corrective surgery could should be further investigated.

Update of the Pompe disease mutation database with 107 sequence variants and a format for severity rating.

Pompe disease was named after the Dutch pathologist Dr JC Pompe who reported about a deceased infant with idiopathic hypertrophy of the heart. The clinical findings were failure to thrive, generalized muscle weakness and cardio-respiratory failure. The key pathologic finding was massive storage of glycogen in heart, skeletal muscle and many other tissues. The disease was classified as glycogen storage disease type II and decades later shown to be a lysosomal disorder caused by acid alpha-glucosidase deficiency. The clinical spectrum of Pompe disease appeared much broader than originally recognized. Adults with the same enzyme deficiency, alternatively named acid maltase deficiency, were reported to have slowly progressive skeletal muscle weakness and respiratory problems, but no cardiac involvement. The clinical heterogeneity is largely explained by the kind and severity of mutations in the acid alpha-glucosidase gene (GAA), but secondary factors, as yet unknown, have a substantial impact. The Pompe disease mutation database aims to list all GAA sequence variations and describe their effect. This update with 107 sequence variations (95 being novel) brings the number of published variations to 289, the number of non-pathogenic mutations to 67 and the number of proven pathogenic mutations to 197. Further, this article introduces a tool to rate the various mutations by severity, which will improve understanding of the genotype-phenotype correlation and facilitate the diagnosis and prognosis in Pompe disease.

Genetic characterization of Polish ccRCC patients: somatic mutation analysis of PBRM1, BAP1 and KDMC5, genomic SNP array analysis in tumor biopsy and preliminary results of chromosome aberrations analysis in plasma cell free DNA.

BACKGROUND: Mutation analysis and cytogenetic testing in clear cell renal cell carcinoma (ccRCC) is not yet implemented in a routine diagnostics of ccRCC. MATERIAL AND METHODS: We characterized the chromosomal alterations in 83 ccRCC tumors from Polish patients using whole genome SNP genotyping assay. Moreover, the utility of next generation sequencing of cell free DNA (cfDNA) in patients plasma as a potential tool for non-invasive cytogenetic analysis was tested. Additionally, tumor specific somatic mutations in PBRM1, BAP1 and KDM5C were determined. RESULTS: We confirmed a correlation between deletions at 9p and higher tumor size, and deletion of chromosome 20 and the survival time. In Fuhrman grade 1, only aberrations of 3p and 8p deletion, gain of 5q and 13q and gains of chromosome 7 and 16 were present. The number of aberrations increased with Fuhrman grade, all chromosomes displayed cytogenetic changes in G3 and G4. ccRCC specific chromosome aberrations were observed in cfDNA, although discrepancies were found between cfDNA and tumor samples. In total 12 common and 94 rare variants were detected in PBRM1, BAP1 and KDM5C, with four potentially pathogenic variants. We observed markedly lower mutation load in PBRM1. CONCLUSIONS: Cytogenetic analysis of cfDNA may allow more accurate diagnosis of tumor aberrations and therefore the correlation between the chromosome aberrations in cfDNA and clinical outcome should be studied in larger cohorts. The functional studies on in BAP1, KDM5C, PBRM1 mutations in large, independent sample set would be necessary for the assessment of their prognostic and diagnostic potential.

Copy number variations in 375 patients with oesophageal atresia and/or tracheoesophageal fistula.

Oesophageal atresia (OA) with or without tracheoesophageal fistula (TOF) are rare anatomical congenital malformations whose cause is unknown in over 90% of patients. A genetic background is suggested, and among the reported genetic defects are copy number variations (CNVs). We hypothesized that CNVs contribute to OA/TOF development. Quantifying their prevalence could aid in genetic diagnosis and clinical care strategies. Therefore, we profiled 375 patients in a combined Dutch, American and German cohort via genomic microarray and compared the CNV profiles with their unaffected parents and published control cohorts. We identified 167 rare CNVs containing genes (frequency<0.0005 in our in-house cohort). Eight rare CNVs - in six patients - were de novo, including one CNV previously associated with oesophageal disease. (hg19 chr7:g.(143820444_143839360)_(159119486_159138663)del) 1.55% of isolated OA/TOF patients and 1.62% of patients with additional congenital anomalies had de novo CNVs. Furthermore, three (15q13.3, 16p13.3 and 22q11.2) susceptibility loci were identified based on their overlap with known OA/TOF-associated CNV syndromes and overlap with loci in published CNV association case-control studies in developmental delay. Our study suggests that CNVs contribute to OA/TOF development. In addition to the identified likely deleterious de novo CNVs, we detected 167 rare CNVs. Although not directly disease-causing, these CNVs might be of interest, as they can act as a modifier in a multiple hit model, or as the second hit in a recessive condition.

Mutations in a TGF-ß ligand, TGFB3, cause syndromic aortic aneurysms and dissections.

BACKGROUND: Aneurysms affecting the aorta are a common condition associated with high mortality as a result of aortic dissection or rupture. Investigations of the pathogenic mechanisms involved in syndromic types of thoracic aortic aneurysms, such as Marfan and Loeys-Dietz syndromes, have revealed an important contribution of disturbed transforming growth factor (TGF)-ß signaling. OBJECTIVES: This study sought to discover a novel gene causing syndromic aortic aneurysms in order to unravel the underlying pathogenesis. METHODS: We combined genome-wide linkage analysis, exome sequencing, and candidate gene Sanger sequencing in a total of 470 index cases with thoracic aortic aneurysms. Extensive cardiological examination, including physical examination, electrocardiography, and transthoracic echocardiography was performed. In adults, imaging of the entire aorta using computed tomography or magnetic resonance imaging was done. RESULTS: Here, we report on 43 patients from 11 families with syndromic presentations of aortic aneurysms caused by TGFB3 mutations. We demonstrate that TGFB3 mutations are associated with significant cardiovascular involvement, including thoracic/abdominal aortic aneurysm and dissection, and mitral valve disease. Other systemic features overlap clinically with Loeys-Dietz, Shprintzen-Goldberg, and Marfan syndromes, including cleft palate, bifid uvula, skeletal overgrowth, cervical spine instability and clubfoot deformity. In line with previous observations in aortic wall tissues of patients with mutations in effectors of TGF-ß signaling (TGFBR1/2, SMAD3, and TGFB2), we confirm a paradoxical up-regulation of both canonical and noncanonical TGF-ß signaling in association with up-regulation of the expression of TGF-ß ligands. CONCLUSIONS: Our findings emphasize the broad clinical variability associated with TGFB3 mutations and highlight the importance of early recognition of the disease because of high cardiovascular risk.