Nimbus: a design-driven analyses suite for amplicon-based NGS data.

Motivation: PCR-based DNA enrichment followed by massively parallel sequencing is a straightforward and cost effective method to sequence genes up to high depth. The full potential of amplicon-based sequencing assays is currently not achieved as analysis methods do not take into account the source amplicons of the detected variants. Tracking the source amplicons has the potential to identify systematic biases, enhance variant calling and improve the designs of future assays. Results: We present Nimbus, a software suite for the analysis of amplicon-based sequencing data. Nimbus includes tools for data pre-processing, alignment, single nucleotide polymorphism (SNP), insertion and deletion calling, quality control and visualization. Nimbus can detect SNPs in its alignment seeds and reduces alignment issues by the usage of decoy amplicons. Tracking the amplicons throughout analysis allows easy and fast design optimization by amplicon performance comparison. It enables detection of probable false positive variants present in a single amplicon from real variants present in multiple amplicons and provides multiple sample visualization. Nimbus was tested using HaloPlex Exome datasets and outperforms other callers for low-frequency variants. The variants called by Nimbus were highly concordant between twin samples and SNP-arrays. The Nimbus suite provides an end-to-end solution for variant calling, design optimization and visualization of amplicon-derived next-generation sequencing datasets. Availability and implementation: https://github.com/erasmus-center-for-biomics/Nimbus. Supplementary information: Supplementary data are available at Bioinformatics online.

Germline variant in MSX1 identified in a Dutch family with clustering of Barrett's esophagus and esophageal adenocarcinoma.

The vast majority of esophageal adenocarcinoma cases are sporadic and caused by somatic mutations. However, over the last decades several families have been identified with clustering of Barrett's esophagus and esophageal adenocarcinoma. This observation suggests that one or more hereditary factors may play a role in the initiation of Barrett's esophagus and esophageal adenocarcinoma in these families. A Dutch family with clustering of Barrett's esophagus and esophageal adenocarcinoma was identified. Normal DNA obtained from the proband diagnosed with Barrett's esophagus was analyzed with SNP array and exome sequencing. A custom-made panel consisting of potential germline variants was verified in the normal DNA of the affected family members. In addition, the respective tumors were analyzed for somatic loss of the wild type allele or the presence of an inactivating somatic mutation in the wild type allele. Exome sequencing revealed 244 candidate variants in the normal DNA of the proband, of which 212 variants were verified successfully. After the normal DNA of the affected family members was analyzed for the presence of the 212 potential germline variants and subsequently the respective tumors, only one potential germline variant in MSX1 (chr4: 4861985 T > G, c.359T > G, p.V120G, NM_002448) showed loss of the wild type allele in the tumor DNAs of the affected family members. A germline variant in MSX1 was identified in a Dutch family with clustering of Barrett's esophagus and esophageal adenocarcinoma. This finding indicates that the germline defect in MSX1 may be associated with Barrett's esophagus and cancer in this particular family.

Next-generation sequencing reveals novel rare fusion events with functional implication in prostate cancer.

Gene fusions, mainly between TMPRSS2 and ERG, are frequent early genomic rearrangements in prostate cancer (PCa). In order to discover novel genomic fusion events, we applied whole-genome paired-end sequencing to identify structural alterations present in a primary PCa patient (G089) and in a PCa cell line (PC346C). Overall, we identified over 3800 genomic rearrangements in each of the two samples as compared with the reference genome. Correcting these structural variations for polymorphisms using whole-genome sequences of 46 normal samples, the numbers of cancer-related rearrangements were 674 and 387 for G089 and PC346C, respectively. From these, 192 in G089 and 106 in PC346C affected gene structures. Exclusion of small intronic deletions left 33 intergenic breaks in G089 and 14 in PC346C. Out of these, 12 and 9 reassembled genes with the same orientation, capable of generating a feasible fusion transcript. Using PCR we validated all the reliable predicted gene fusions. Two gene fusions were in-frame: MPP5-FAM71D in PC346C and ARHGEF3-C8ORF38 in G089. Downregulation of FAM71D and MPP5-FAM71D transcripts in PC346C cells decreased proliferation; however, no effect was observed in the RWPE-1-immortalized normal prostate epithelial cells. Together, our data showed that gene rearrangements frequently occur in PCa genomes but result in a limited number of fusion transcripts. Most of these fusion transcripts do not encode in-frame fusion proteins. The unique in-frame MPP5-FAM71D fusion product is important for proliferation of PC346C cells.

Patient survival in uveal melanoma is not affected by oncogenic mutations in GNAQ and GNA11.

BACKGROUND: Mutations in GNAQ and GNA11, encoding the oncogenic G-protein alpha subunit q and 11, respectively, occur frequently in the majority of uveal melanomas. METHODS: Exons 4 and 5 from GNAQ and GNA11 were amplified and sequenced from 92 ciliary body and choroidal melanomas. The mutation status was correlated with disease-free survival (DFS) and other parameters. RESULTS: None of the tumours harboured a GNAQ exon 4 mutation. A GNAQ mutation in exon 5 codon 209 was found in 46 out of 92 (50.0%) of the tumours. Only 1 out of 92 (1.1%) melanomas showed a mutation in GNA11 exon 4 codon 183, whereas 39 out of 92 (42.4%) harboured a mutation in exon 5 of GNA11 codon 209. Six tumours did not show any mutations in exons 4 and 5 of these genes. Univariate analyses showed no correlation between DFS and the mutation status. CONCLUSION: GNAQ and GNA11 mutations are, in equal matter, not associated with patient outcome.

VACTERL Association Etiology: The Impact of de novo and Rare Copy Number Variations.

Copy number variations (CNVs), either DNA gains or losses, have been found at common regions throughout the human genome. Most CNVs neither have a pathogenic significance nor result in disease-related phenotypes but, instead, reflect the normal population variance. However, larger CNVs, which often arise de novo, are frequently associated with human disease. A genetic contribution has long been suspected in VACTERL (Vertebral, Anal, Cardiac, TracheoEsophageal fistula, Renal and Limb anomalies) association. The anomalies observed in this association overlap with several monogenetic conditions associated with mutations in specific genes, e.g. Townes Brocks (SALL1), Feingold syndrome (MYCN) or Fanconi anemia. So far VACTERL association has typically been considered a diagnosis of exclusion. Identifying recurrent or de novo genomic variations in individuals with VACTERL association could make it easier to distinguish VACTERL association from other syndromes and could provide insight into disease mechanisms. Sporadically, de novo CNVs associated with VACTERL are described in literature. In addition to this literature review of genomic variation in published VACTERL association patients, we describe CNVs present in 68 VACTERL association patients collected in our institution. De novo variations (>30 kb) are absent in our VACTERL association cohort. However, we identified recurrent rare CNVs which, although inherited, could point to mechanisms or biological processes contributing to this constellation of developmental defects.

Etiological and pathogenic factors in congenital diaphragmatic hernia.

Congenital diaphragmatic hernia (CDH) is a congenital anomaly associated with an increased mortality and morbidity. In this article, we review the currently known etiological and pathogenic factors in CDH.

Developmental and genetic aspects of congenital diaphragmatic hernia.

Congenital diaphragmatic hernia (CDH) is a frequent occurring cause of neonatal respiratory distress and occurs 1 in every 3,000 liveborns. Ventilatory support and pharmaceutical treatment of the co-occurring lung hypoplasia and pulmonary hypertension are insufficient in, respectively, 20% of isolated cases and 60% of complex ones leading to early perinatal death. The exact cause of CDH remains to be identified in the majority of human CDH patients and prognostic factors predicting treatment refraction are largely unknown. Their identification is hampered by the multifactorial and heterogenic nature of this congenital anomaly. However, application of high-resolution molecular cytogenetic techniques to patients' DNA now enables detection of chromosomal aberrations in 30% of the patients. Furthermore, recent insights in rodent embryogenesis pointed to a specific disruption of the early mesenchymal structures in the primordial diaphragm of CDH-induced offspring. Together, these data allowed for the introduction of new hypotheses on CDH pathogenesis, although many issues remain to be resolved. In this review, we have combined these new insights and remaining questions on diaphragm pathogenesis with a concise overview of the clinical, embryological, and genetic data available.

Chromosomal aberrations in iris melanomas.

BACKGROUND: Uveal melanomas can develop in the choroid, ciliary body and iris. In choroidal and ciliary body melanomas, specific chromosomal changes correlate with metastatic disease. Iris melanomas have a better prognosis than choroidal melanomas, and it would be interesting to know if they share chromosomal changes. In addition, iris melanomas might harbour UV-induced mutations of tumour suppressor genes, such as PTEN and CDKN2A. METHODS: Twenty iris melanomas were analysed for chromosome 1p, 3, 6, 8, 9p and 10q abnormalities using fluorescence in situ hybridisation. These results were correlated to clinical follow-up data using statistical analyses. RESULTS: (Partial) loss of chromosome 3 was observed in nine iris melanomas, and gain of 8q was present in seven tumours. Loss of chromosome 9p was demonstrated in seven tumours, but no deletions of the PTEN region on chromosome 10 were found. Three patients died of metastatic disease, and one patient developed liver metastases, but is still alive. Univariate analysis indicated a lower disease-free survival for patients with diffuse growing melanomas (p=0.01), melanomas that lost a copy of chromosome 3 (p=0.03), or invading the ciliary body (p=0.01). In a multivariate analysis, none of the correlations were significant. CONCLUSION: Loss of chromosome 3 as well as loss of chromosomal region 9p21 (that entails tumour suppressor gene CDKN2A) plays a role in iris melanoma. A firm correlation with disease-free survival could not be established, possibly due to the small sample size.

Distinctive Phenotypic Abnormalities Associated with Submicroscopic 21q22 Deletion Including DYRK1A.

Partial monosomy 21 has been reported, but the phenotypes described are variable with location and size of the deletion. We present 2 patients with a partially overlapping microdeletion of 21q22 and a striking phenotypic resemblance. They both presented with severe psychomotor delay, behavioral problems, no speech, microcephaly, feeding problems with frequent regurgitation, idiopathic thrombocytopenia, obesity, deep set eyes, down turned corners of the mouth, dysplastic ears, and small chin. Brain MRI showed cerebral atrophy mostly evident in frontal and temporal lobes, widened ventricles and thin corpus callosum in both cases, and in one patient evidence of a migration disorder. The first patient also presented with epilepsy and a ventricular septum defect. The second patient had a unilateral Peters anomaly. Microarray analysis showed a partially overlapping microdeletion spanning about 2.5 Mb in the 21q22.1-q22.2 region including the DYRK1A gene and excluding RUNX1. These patients present with a recognizable phenotype specific for this 21q22.1-q22.2 locus. We searched the literature for patients with overlapping deletions including the DYRK1A gene, in order to define other genes responsible for this presentation.

Pre- and postnatal diagnosis and outcome of fetuses and neonates with esophageal atresia and tracheoesophageal fistula.

OBJECTIVES: Clinical symptoms and ultrasound signs during pregnancy could suggest the presence of esophageal atresia (EA). However, most often EA is diagnosed postnatally. The aim of our study is to evaluate the course and outcome for prenatally and postnatally diagnosed EA. In addition, we studied the outcome of isolated versus nonisolated EA. METHODS: In a retrospective data analysis, ultrasound characteristics, maternal and neonatal variables as well as clinical outcome were compared for fetuses/neonates with prenatal (n = 30) or postnatal (n = 49) diagnosis of EA. Clinical outcome in terms of morbidity and mortality of isolated EA was compared with that of EA complicated by chromosomal or structural anomalies. RESULTS: Prenatally diagnosed children were born 2 weeks earlier than postnatally diagnosed children (36.4 weeks vs 38.2 weeks; P = 0.02). The former had higher mortality rates (30 vs 12%; P = 0.05) and more associated anomalies (80 vs 59%; P = 0.04). In both subsets, there was a high morbidity rate in the survivors (not significant). Nonisolated EA was associated with greater occurrence of polyhydramnios (53 vs 27%; P = 0.04) and higher mortality rate (28 vs 0%; P = 0.002). CONCLUSIONS: Mortality was significantly higher in prenatally diagnosed infants and in infants with additional congenital anomalies. Isolated EA is associated with good outcome.

Phenotype-genotype correlation in a familial IGF1R microdeletion case.

BACKGROUND: IGF1R (insulin-like growth factor 1 receptor) haploinsufficiency is a rare event causing difficulties in defining clear genotype-phenotype correlations, although short stature is its well established hallmark. Several pure 15q26 monosomies (n=22) have been described in the literature, including those with breakpoints proximal to the IGF1R gene. Clinical heterogeneity is characteristic for these mainly de novo telomeric deletions and is illustrated by the involvement of several different organ systems such as the heart, diaphragm, lungs, kidneys and limbs, besides growth failure in the patient's phenotype. The clinical variability in these patients could be explained by the haploinsufficiency of multiple genes besides the IGF1R gene. In comparison, the six different IGF1R mutations revealed to date exhibit some variance in their clinical features as well, probably because different parts of the downstream IGF1R signalling cascade were affected. METHODS AND RESULTS: Using the recently developed technique multiplex ligation dependent probe amplification (MLPA), a chromosome 15q26.3 microdeletion harbouring part of the IGF1R gene was identified in a Dutch family. This deletion segregated with short height in seven out of 14 relatives across three generations. Metaphase fluorescence in situ hybridisation (FISH) and Affymetrix 250k single nucleotide polymorphism (SNP) microarray were used to characterise the deletion into more detail and showed that exons 11-21 of the IGF1R and a small hypothetical protein (LOC 145814) were deleted. CONCLUSION: Clinical work-up of this newly identified family, which constitutes the smallest (0.095 Mb) pure 15q26.3 interstitial deletion to date, confirms that disruption of the IGF1R gene does not induce major organ malformation or severe mental retardation.

Can we improve outcome of congenital diaphragmatic hernia?

This review gives an overview of the disease spectrum of congenital diaphragmatic hernia (CDH). Etiological factors, prenatal predictors of survival, new treatment strategies and long-term morbidity are described. Early recognition of problems and improvement of treatment strategies in CDH patients may increase survival and prevent secondary morbidity. Multidisciplinary healthcare is necessary to improve healthcare for CDH patients. Absence of international therapy guidelines, lack of evidence of many therapeutic modalities and the relative low number of CDH patients calls for cooperation between centers with an expertise in the treatment of CDH patients. The international CDH Euro-Consortium is an example of such a collaborative network, which enhances exchange of knowledge, future research and development of treatment protocols.

Oncogenic GNAQ mutations are not correlated with disease-free survival in uveal melanoma.

BACKGROUND: Recently, oncogenic G protein alpha subunit q (GNAQ) mutations have been described in about 50% of uveal melanomas and in the blue nevi of the skin. METHODS: GNAQ exon 5 was amplified from 75 ciliary body and choroidal melanoma DNAs and sequenced directly. GNAQ mutation status was correlated with disease-free survival (DFS), as well as other clinical and histopathological factors, and with chromosomal variations detected by FISH and CGH. RESULTS: Of the 75 tumour DNA samples analysed, 40 (53.3%) harboured oncogenic mutations in GNAQ codon 209. Univariate and multivariate analysis showed that GNAQ mutation status was not significantly correlated with DFS. CONCLUSION: The GNAQ mutation status is not suitable to predict DFS. However, the high frequency of GNAQ mutations may render it a promising target for therapeutic intervention.

Genetic and environmental factors in the etiology of esophageal atresia and/or tracheoesophageal fistula: an overview of the current concepts.

Esophageal atresia and/or tracheoesophageal fistula (EA/TEF) are severe congenital anomalies. Although recent years have brought significant improvement in clinical treatment, our understanding of the etiology of these defects is lagging. Many genes and genetic pathways have been implicated in the development of EA/TEF, but only a few genes have been shown to be involved in humans, in animals, or in both. Extrapolating data from animal models to humans is not always straightforward. Environmental factors may also carry a risk, but the mechanisms are yet to be elucidated. This review gives an overview of the current state of knowledge about both genetic and environmental risk factors in the etiology of EA/TEF.

The etiology of congenital diaphragmatic hernia: still largely unknown?

Congenital diaphragmatic hernia (CDH) is a severe birth defect characterized by a defect in the diaphragm associated with pulmonary hypoplasia and postnatal pulmonary hypertension. Half of the cases present with other non-pulmonary congenital anomalies (so called non-isolated CDH) and in 5-10% of cases there is a chromosomal etiology. The clinical aspects of CDH are well documented but knowledge on the etiology of CDH is largely lacking. Worldwide many researchers have focused research efforts on CDH. Their findings have led to several hypotheses proposing roles for genetic and environmental factors. In this review we have combined these findings with our own research on the genetics of CDH in results from recent literature and propose a theory on the etiology of CDH. We also propose a protocol for the CDH patient that will help clinicians and researchers to obtain maximal success out of their collaborations that will eventually lead to unravelling the etiology of this intriguing birth defect.

TACI mutations and disease susceptibility in patients with common variable immunodeficiency.

The most prevalent primary immunodeficiency is common variable immunodeficiency (CVID). Mutations have been described in four genes, ICOS, CD19, BAFF-R and TNFRSF13B (encoding TACI), together associated with 10-15% of CVID cases. We investigated a family with CVID and identified the heterozygous C104R TNFRSF13B mutation in two of the three index-children with CVID, a mother with selective immunoglobulin A deficiency, a mother with recurrent infections and a healthy grandfather. Remarkably, we did not find the TNFRSF13B mutation in the third index-child with CVID, despite his hypogammaglobulinaemia and decreased response to unconjugated pneumococcal vaccine. This family illustrates that TNFRSF13B mutations induce disease susceptibility rather than cause disease directly. Apparently, other genetic or environmental factors, still to be identified, contributed to the development of CVID in this family. Consequently, TNFRSF13B mutations must be interpreted with caution in the clinical setting.

Mapping of 5q35 chromosomal rearrangements within a genomically unstable region.

BACKGROUND: Recent molecular studies of breakpoints of recurrent chromosome rearrangements revealed the role of genomic architecture in their formation. In particular, segmental duplications representing blocks of >1 kb with >90% sequence homology were shown to mediate non-allelic homologous recombination (NAHR). However, the occurrence of the majority of newly detected submicroscopic imbalances cannot be explained by the presence of segmental duplications. Therefore, further studies are needed to investigate whether architectural features other than segmental duplications mediate these rearrangements. METHODS: We analysed a series of patients with breakpoints clustering within chromosome band 5q35. Using high density arrays and subsequent quantitative polymerase chain reaction (qPCR), we characterised the breakpoints of four interstitial deletions (including one associated with an unbalanced paracentric inversion), a duplication and a familial reciprocal t(5;18)(q35;q22) translocation. RESULTS AND CONCLUSION: Five of the breakpoints were located within an interval of approximately 265 kb encompassing the RANBP17 and TLX3 genes. This region is also targeted by the recurrent cryptic t(5;14)(q35;q32) translocation, which occurs in approximately 20% of childhood T cell acute lymphoblastic leukaemia (T-ALL). In silico analysis indicated the architectural features most likely to contribute to the genomic instability of this region, which was supported by our molecular data. Of further interest, in two patients and the familial translocation, the delineated breakpoint regions encompassed highly homologous LINEs (long interspersed nuclear elements), suggesting that NAHR between these LINEs may have mediated these rearrangements.

Prenatal detection and outcome of congenital diaphragmatic hernia (CDH) associated with deletion of chromosome 15q26: two patients and review of the literature.

Congenital diaphragmatic hernia (CDH) is a severe birth defect characterized by a defect in the diaphragm with pulmonary hypoplasia and postnatal pulmonary hypertension. Approximately 50% of CDH cases are associated with other non-pulmonary congenital anomalies (so called non-isolated CDH) and in 5-10% of cases there is a chromosomal etiology. The majority of CDH cases are detected prenatally. In some cases prenatal chromosome analysis reveals a causative chromosomal anomaly, most often aneuploidy. Deletion of 15q26 is the most frequently described structural chromosomal aberration in patients with non-isolated CDH. In this paper we report on two patients with a deletion of 15q26 and phenotypes similar to other patients with CDH caused by 15q26 deletions. This phenotype consists of intra-uterine growth retardation, left-sided CDH, cardiac anomalies and characteristic facial features, similar to those seen in Fryns syndrome. We propose that when this combination of birth defects is identified, either pre- or postnatally, further investigations to confirm or exclude a deletion of 15q26 are indicated, since the diagnosis of this deletion will have major consequences for the prognosis and, therefore, can affect decision making.

Genetic factors in congenital diaphragmatic hernia.

Congenital diaphragmatic hernia (CDH) is a relatively common birth defect associated with high mortality and morbidity. Although the exact etiology of most cases of CDH remains unknown, there is a growing body of evidence that genetic factors play an important role in the development of CDH. In this review, we examine key findings that are likely to form the basis for future research in this field. Specific topics include a short overview of normal and abnormal diaphragm development, a discussion of syndromic forms of CDH, a detailed review of chromosomal regions recurrently altered in CDH, a description of the retinoid hypothesis of CDH, and evidence of the roles of specific genes in the development of CDH.

Congenital diaphragmatic hernia associated with duplication of 11q23-qter.

Congenital diaphragmatic hernia (CDH) is a relatively common birth defect with a high mortality. Although little is known about its etiology, there is increasing evidence for a strong genetic contribution. Both numerical and structural chromosomal abnormalities have been described in patients with CDH. Partial trisomy 11q and partial trisomy 22 associated with the common t(11;22) has been reported in several cases of CDH. It has been assumed that the diaphragmatic defect seen in these individuals was primarily due to duplication of material from chromosome 22q11. However, in this report we describe a family with a t(11;12) in which one of two brothers with partial trisomy 11q has a left sided posterolateral CDH. This is the second case of CDH in partial trisomy 11q due to an unbalanced translocation other than t(11;22). Using array-based comparative genomic hybridization and fluorescent in situ hybridization, we mapped the breakpoints in both brothers and their mother who is a balanced translocation carrier. Our results suggest that duplication of one or more genes on a approximately 19 Mb region of 11q23.3-qter predisposes to the development of CDH. These effects may be the primary cause of CDH in individuals t(11;22) or may be additive to effects from the duplication of chromosome 22 material. We also conclude that the partial trisomy 11q syndrome has a variable phenotype and that CDH should be added to the spectrum of anomalies that can be present in this syndrome.