High-throughput sequencing contributes to the diagnosis of tubulopathies and familial hypercalcemia hypocalciuria in adults.

Hereditary tubulopathies are rare diseases with unknown prevalence in adults. Often diagnosed in childhood, hereditary tubulopathies can nevertheless be evoked in adults. Precise diagnosis can be difficult or delayed due to insidious development of symptoms, comorbidities and polypharmacy. Here we evaluated the diagnostic value of a specific panel of known genes implicated in tubulopathies in adult patients and compared to our data obtained in children. To do this we analyzed 1033 non-related adult patients of which 744 had a clinical diagnosis of tubulopathy and 289 had a diagnosis of familial hypercalcemia with hypocalciuria recruited by three European reference centers. Three-quarters of our tubulopathies cohort included individuals with clinical suspicion of Gitelman syndrome, kidney hypophosphatemia and kidney tubular acidosis. We detected pathogenic variants in 26 different genes confirming a genetic diagnosis of tubulopathy in 29% of cases. In 16 cases (2.1%) the genetic testing changed the clinical diagnosis. The diagnosis of familial hypercalcemia with hypocalciuria was confirmed in 12% of cases. Thus, our work demonstrates the genetic origin of tubulopathies in one out of three adult patients, half of the rate observed in children. Hence, establishing a precise diagnosis is crucial for patients, in order to guide care, to survey and prevent chronic complications, and for genetic counselling. At the same time, this work enhances our understanding of complex phenotypes and enriches the database with the causal variants described.

TP53 Outperforms Other Androgen Receptor Biomarkers to Predict Abiraterone or Enzalutamide Outcome in Metastatic Castration-Resistant Prostate Cancer.

PURPOSE: To infer the prognostic value of simultaneous androgen receptor (AR) and TP53 profiling in liquid biopsies from patients with metastatic castration-resistant prostate cancer (mCRPC) starting a new line of AR signaling inhibitors (ARSi).Experimental Design: Between March 2014 and April 2017, we recruited patients with mCRPC (n = 168) prior to ARSi in a cohort study encompassing 10 European centers. Blood samples were collected for comprehensive profiling of CellSearch-enriched circulating tumor cells (CTC) and circulating tumor DNA (ctDNA). Targeted CTC RNA sequencing (RNA-seq) allowed the detection of eight AR splice variants (ARV). Low-pass whole-genome and targeted gene-body sequencing of AR and TP53 was applied to identify amplifications, loss of heterozygosity, mutations, and structural rearrangements in ctDNA. Clinical or radiologic progression-free survival (PFS) was estimated by Kaplan-Meier analysis, and independent associations were determined using multivariable Cox regression models. RESULTS: Overall, no single AR perturbation remained associated with adverse prognosis after multivariable analysis. Instead, tumor burden estimates (CTC counts, ctDNA fraction, and visceral metastases) were significantly associated with PFS. TP53 inactivation harbored independent prognostic value [HR 1.88; 95% confidence interval (CI), 1.18-3.00; P = 0.008], and outperformed ARV expression and detection of genomic AR alterations. Using Cox coefficient analysis of clinical parameters and TP53 status, we identified three prognostic groups with differing PFS estimates (median, 14.7 vs. 7.51 vs. 2.62 months; P < 0.0001), which was validated in an independent mCRPC cohort (n = 202) starting first-line ARSi (median, 14.3 vs. 6.39 vs. 2.23 months; P < 0.0001). CONCLUSIONS: In an all-comer cohort, tumor burden estimates and TP53 outperform any AR perturbation to infer prognosis.See related commentary by Rebello et al., p. 1699.

Comprehensive Profiling of the Androgen Receptor in Liquid Biopsies from Castration-resistant Prostate Cancer Reveals Novel Intra-AR Structural Variation and Splice Variant Expression Patterns.

BACKGROUND: Expression of the androgen receptor splice variant 7 (AR-V7) is associated with poor response to second-line endocrine therapy in castration-resistant prostate cancer (CRPC). However, a large fraction of nonresponding patients are AR-V7-negative. OBJECTIVE: To investigate if a comprehensive liquid biopsy-based AR profile may improve patient stratification in the context of second-line endocrine therapy. DESIGN, SETTING, AND PARTICIPANTS: Peripheral blood was collected from patients with CRPC (n=30) before initiation of a new line of systemic therapy. We performed profiling of circulating tumour DNA via low-pass whole-genome sequencing and targeted sequencing of the entire AR gene, including introns. Targeted RNA sequencing was performed on enriched circulating tumour cell fractions to assess the expression levels of seven AR splice variants (ARVs). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Somatic AR variations, including copy-number alterations, structural variations, and point mutations, were combined with ARV expression patterns and correlated to clinicopathologic parameters. RESULTS AND LIMITATIONS: Collectively, any AR perturbation, including ARV, was detected in 25/30 patients. Surprisingly, intra-AR structural variation was present in 15/30 patients, of whom 14 expressed ARVs. The majority of ARV-positive patients expressed multiple ARVs, with AR-V3 the most abundantly expressed. The presence of any ARV was associated with progression-free survival after second-line endocrine treatment (hazard ratio 4.53, 95% confidence interval 1.424-14.41; p=0.0105). Six out of 17 poor responders were AR-V7-negative, but four carried other AR perturbations. CONCLUSIONS: Comprehensive AR profiling, which is feasible using liquid biopsies, is necessary to increase our understanding of the mechanisms underpinning resistance to endocrine treatment. PATIENT SUMMARY: Alterations in the androgen receptor are associated with endocrine treatment outcomes. This study demonstrates that it is possible to identify different types of alterations via simple blood draws. Follow-up studies are needed to determine the effect of such alterations on hormonal therapy.

Performance of multiplicom's BRCA MASTR Dx kit on the detection of BRCA1 and BRCA2 mutations in fresh frozen ovarian and breast tumor samples.

Next-generation sequencing (NGS) has enabled new approaches for detection of mutations in the BRCA1 and BRCA2 genes responsible for hereditary breast and ovarian cancer (HBOC). The search for germline mutations in the BRCA1 and BRCA2 genes is of importance with respect to oncogenetic and surgical (bilateral mastectomy, ovariectomy) counselling. Testing tumor material for BRCA mutations is of increasing importance for therapeutic decision making as the poly ADP ribose polymerase (PARP) inhibitor, olaparib, is now available to treat patients with specific forms of ovarian cancer and BRCA mutations. Molecular genetics laboratories should develop reliable and sensitive techniques for the complete analysis of the BRCA1 and BRCA2 genes. This is a challenge due to the size of the coding sequence of the BRCA1/2 genes, the absence of hot spot mutations, and particularly by the lower DNA quality obtained from Formalin-Fixed Paraffin-Embedded (FFPE) tissue. As a result, a number of analyses are uninterpretable and do not always provide a result to the clinician, limiting the optimal therapeutic management of patients. The availability of Fresh Frozen Tissue (FFT) for some laboratories and the excellent quality of the DNA extracted from it offers an alternative. For this reason, we evaluated Multiplicom's BRCA MASTR Dx assay on a set of 97 FFT derived DNA samples, in combination with the MID for Illumina MiSeq for BRCA1 and BRCA2 mutation detection. We obtained interpretable NGS results for all tested samples and showed > 99,7% sensitivity, specificity and accuracy.

Schizophrenia-Associated MIR204 Regulates Noncoding RNAs and Affects Neurotransmitter and Ion Channel Gene Sets.

As regulators of gene expression, microRNAs (miRNAs) are likely to play an important role in the development of disease. In this study we present a large-scale strategy to identify miRNAs with a role in the regulation of neuronal processes. Thereby we found variant rs7861254 located near the MIR204 gene to be significantly associated with schizophrenia. This variant resulted in reduced expression of miR-204 in neuronal-like SH-SY5Y cells. Analysis of the consequences of the altered miR-204 expression on the transcriptome of these cells uncovered a new mode of action for miR-204, being the regulation of noncoding RNAs (ncRNAs), including several miRNAs, such as MIR296. Furthermore, pathway analysis showed downstream effects of miR-204 on neurotransmitter and ion channel related gene sets, potentially mediated by miRNAs regulated through miR-204.

Chronic Fatigue Syndrome and DNA Hypomethylation of the Glucocorticoid Receptor Gene Promoter 1F Region: Associations With HPA Axis Hypofunction and Childhood Trauma.

OBJECTIVES: Chronic fatigue syndrome (CFS) has been associated with hypothalamic-pituitary-adrenal axis hypofunction and enhanced glucocorticoid receptor (GR) sensitivity. In addition, childhood trauma is considered a major risk factor for the syndrome. This study examines DNA methylation of the GR gene (NR3C1) in CFS and associations with childhood sexual and physical trauma. METHODS: Quantification of DNA methylation within the 1F promoter region of NR3C1 was performed in 76 female patients (46 with no/mild and 30 with moderate/severe childhood trauma) and 19 healthy controls by using Sequenom EpiTYPER. Further, we examined the association of NR3C1-1F promoter methylation with the outcomes of the low-dose (0.5 mg) dexamethasone/corticotropin-releasing factor test in a subset of the study population. Mann-Whitney U tests and Spearman correlations were used for statistical analyses. RESULTS: Overall NR3C1-1F DNA methylation was lower in patients with CFS than in controls. After cytosine guanine dinucleotide (CpG)-specific analysis, CpG_1.5 remained significant after Bonferroni correction (adjusted p = .0014). Within the CFS group, overall methylation (ρ = 0.477, p = .016) and selective CpG units (CpG_1.5: ρ = 0.538, p = .007; CpG_12.13: ρ = 0.448, p = .025) were positively correlated with salivary cortisol after dexamethasone administration. There was no significant difference in NR3C1-1F methylation between traumatized and nontraumatized patients. CONCLUSIONS: We found evidence of NR3C1 promoter hypomethylation in female patients with CFS and the functional relevance of these differences was consistent with the hypothalamic-pituitary-adrenalaxis hypofunction hypothesis (GR hypersuppression). However, we found no evidence of an additional effect of childhood trauma on CFS via alterations in NR3C1 methylation.

No evidence for GNAS copy number variants in patients with features of Albright's hereditary osteodystrophy and abnormal platelet Gs activity.

Albright's hereditary osteodystrophy (AHO) is characterized by short stature, round face, calcifications, obesity, brachydactyly and intellectual disability. AHO without hormone resistance is called pseudopseudohypoparathyroidism (PPHP), a rare clinical condition difficult to diagnose with highly variable features. PPHP is caused by paternally inherited loss-of-function mutations in the GNAS. Patients with 2q37 microdeletions or HDAC4 mutations are also defined as having an AHO-like phenotype with normal stimulatory G (Gs) function. We have studied 256 patients with AHO features but no other diagnosis. Their platelet Gs activity was determined via the aggregation-inhibition test showing Gs hypo- or hyperfuncton in 24% and 15% of the patients, respectively. Before initiating with detailed (epi)genetic GNAS studies, we here wanted to excluded copy number variants (CNVs) in GNAS as cause of AHO with a novel large-scale screening technique. Multiplex amplicon quantification (MAQ) for CNVs screening was developed for the 20q13.3 region including GNAS and potential long-range imprinting control elements such as STX16. This is the first large-scale GNAS CNV study in patients with common AHO features but no CNVs were detected. In conclusion, CNVs in the GNAS region are not likely to cause an AHO-like phenotype with or without abnormal platelet Gs activity. Future studies will be undertaken to find out whether these AHO patients with abnormal Gs function are characterized by GNAS coding or methylation defects.

Optimized filtering reduces the error rate in detecting genomic variants by short-read sequencing.

Distinguishing single-nucleotide variants (SNVs) from errors in whole-genome sequences remains challenging. Here we describe a set of filters, together with a freely accessible software tool, that selectively reduce error rates and thereby facilitate variant detection in data from two short-read sequencing technologies, Complete Genomics and Illumina. By sequencing the nearly identical genomes from monozygotic twins and considering shared SNVs as 'true variants' and discordant SNVs as 'errors', we optimized thresholds for 12 individual filters and assessed which of the 1,048 filter combinations were effective in terms of sensitivity and specificity. Cumulative application of all effective filters reduced the error rate by 290-fold, facilitating the identification of genetic differences between monozygotic twins. We also applied an adapted, less stringent set of filters to reliably identify somatic mutations in a highly rearranged tumor and to identify variants in the NA19240 HapMap genome relative to a reference set of SNVs.

Dual association of a TRKA polymorphism with schizophrenia.

OBJECTIVE: An interaction between predisposing genes and environmental stressors is thought to underlie the neurodevelopmental disorder schizophrenia. In a targeted gene screening, we previously found that the minor allele of the single nucleotide polymorphism (SNP) rs6336 in the neurotrophic tyrosine kinase receptor 1 (NTRK1/TRKA) gene is associated with schizophrenia as a risk factor. METHODS: We genotyped the TRKA SNP in a total of eight independent Caucasian schizophrenia case-control groups. RESULT: Remarkably, although in five of the groups a higher frequency of the risk allele was indeed found in the patients compared with the controls, in the three other groups the SNP acted as a protective factor. CONCLUSION: An intriguing possibility is that this dual character of the TRKA SNP is caused by its interaction with endophenotypic and/or epistatic factors.

Resequencing of positional candidates identifies low frequency IL23R coding variants protecting against inflammatory bowel disease.

Genome-wide association studies (GWAS) have identified dozens of risk loci for many complex disorders, including Crohn's disease. However, common disease-associated SNPs explain at most ∼20% of the genetic variance for Crohn's disease. Several factors may account for this unexplained heritability, including rare risk variants not adequately tagged thus far in GWAS. That rare susceptibility variants indeed contribute to variation in multifactorial phenotypes has been demonstrated for colorectal cancer, plasma high-density lipoprotein cholesterol levels, blood pressure, type 1 diabetes, hypertriglyceridemia and, in the case of Crohn's disease, for NOD2 (refs. 14,15). Here we describe the use of high-throughput resequencing of DNA pools to search for rare coding variants influencing susceptibility to Crohn's disease in 63 GWAS-identified positional candidate genes. We identify low frequency coding variants conferring protection against inflammatory bowel disease in IL23R, but we conclude that rare coding variants in positional candidates do not make a large contribution to inherited predisposition to Crohn's disease.

Multiplex Amplicon Quantification (MAQ), a fast and efficient method for the simultaneous detection of copy number alterations in neuroblastoma.

BACKGROUND: Cancer genomes display characteristic patterns of chromosomal imbalances, often with diagnostic and prognostic relevance. Therefore assays for genome-wide copy number screening and simultaneous detection of copy number alterations in specific chromosomal regions are of increasing importance in the diagnostic work-up of tumors. RESULTS: We tested the performance of Multiplex Amplicon Quantification, a newly developed low-cost, closed-tube and high-throughput PCR-based technique for detection of copy number alterations in regions with prognostic relevance for neuroblastoma. Comparison with array CGH and the established Multiplex Ligation-dependent Probe Amplification method on 52 neuroblastoma tumors showed that Multiplex Amplicon Quantification can reliably detect the important genomic aberrations. CONCLUSION: Multiplex Amplicon Quantification is a low-cost and high-throughput PCR-based technique that can reliably detect copy number alterations in regions with prognostic relevance for neuroblastoma.

Relative contribution of simple mutations vs. copy number variations in five Parkinson disease genes in the Belgian population.

The relative contribution of simple mutations and copy number variations (CNVs) in SNCA, PARK2, PINK1, PARK7, and LRRK2 to the genetic etiology of Parkinson disease (PD) is still unclear because most studies did not completely analyze each gene. In a large group of Belgian PD patients (N = 310) and control individuals (N = 270), we determined the mutation frequency of both simple mutations and CNVs in these five PD genes, using direct sequencing, multiplex amplicon quantification (MAQ), and real-time PCR assays. Overall, we identified 14 novel heterozygous variants, of which 11 were absent in control individuals. We observed eight PARK2 (multiple) exon multiplications in PD patients and one exon deletion in a control individual. Furthermore, we identified one SNCA whole-gene duplication. The PARK2 and LRRK2 mutation frequencies in Belgian PD patients were similar to those reported in other studies. However, at this stage the true pathogenic nature of some heterozygous mutations in recessive genes remains elusive. Furthermore, though mutations is SNCA, PINK1, and PARK7 are rare, our identification of a SNCA duplication confirmed that screening of these genes remains meaningful.

Mutations in SEPT9 cause hereditary neuralgic amyotrophy.

Hereditary neuralgic amyotrophy (HNA) is an autosomal dominant recurrent neuropathy affecting the brachial plexus. HNA is triggered by environmental factors such as infection or parturition. We report three mutations in the gene septin 9 (SEPT9) in six families with HNA linked to chromosome 17q25. HNA is the first monogenetic disease caused by mutations in a gene of the septin family. Septins are implicated in formation of the cytoskeleton, cell division and tumorigenesis.

novoSNP, a novel computational tool for sequence variation discovery.

Technological improvements shifted sequencing from low-throughput, work-intensive, gel-based systems to high-throughput capillary systems. This resulted in a broad use of genomic resequencing to identify sequence variations in genes and regulatory, as well as extended genomic regions. We describe a software package, novoSNP, that conscientiously discovers single nucleotide polymorphisms (SNPs) and insertion-deletion polymorphisms (INDELs) in sequence trace files in a fast, reliable, and user-friendly way. We compared the performance of novoSNP with that of PolyPhred and PolyBayes on two data sets. The first data set comprised 1028 sequence trace files obtained from diagnostic mutation analyses of SCN1A (neuronal voltage-gated sodium channel alpha-subunit type I gene). The second data set comprised 9062 sequence trace files from a genomic resequencing project aiming at the construction of a high-density SNP map of MAPT (microtubule-associated protein tau gene). Visual inspection of these data sets had identified 38 sequence variations for SCN1A and 488 for MAPT. novoSNP automatically identified all 38 SCN1A variations including five INDELs, while for MAPT only 15 of the 488 variations were not correctly marked. PolyPhred detected far fewer SNPs as compared to novoSNP and missed nearly all INDELs. PolyBayes, designed for the sequence analysis of cloned templates, detected only a limited number of the variations present in the data set. Besides the significant improvement in the automated detection of sequence variations both in diagnostic mutation analyses and in SNP discovery projects, novoSNP also offers a user-friendly interface for inspecting possible genetic variations.

VEGF is a modifier of amyotrophic lateral sclerosis in mice and humans and protects motoneurons against ischemic death.

Amyotrophic lateral sclerosis (ALS) is an incurable degenerative disorder of motoneurons. We recently reported that reduced expression of Vegfa causes ALS-like motoneuron degeneration in Vegfa(delta/delta) mice. In a meta-analysis of over 900 individuals from Sweden and over 1,000 individuals from Belgium and England, we now report that subjects homozygous with respect to the haplotypes -2,578A/-1,154A/-634G or -2,578A/-1,154G/-634G in the VEGF promoter/leader sequence had a 1.8 times greater risk of ALS (P = 0.00004). These 'at-risk' haplotypes lowered circulating VEGF levels in vivo and reduced VEGF gene transcription, IRES-mediated VEGF expression and translation of a novel large-VEGF isoform (L-VEGF) in vivo. Moreover, SOD1(G93A) mice crossbred with Vegfa(delta/delta) mice died earlier due to more severe motoneuron degeneration. Vegfa(delta/delta) mice were unusually susceptible to persistent paralysis after spinal cord ischemia, and treatment with Vegfa protected mice against ischemic motoneuron death. These findings indicate that VEGF is a modifier of motoneuron degeneration in human ALS and unveil a therapeutic potential of Vegfa for stressed motoneurons in mice.