Combined Whole-exome Sequencing Research Articles

Decreased TAX1BP1 participates in systemic lupus erythematosus by regulating monocyte/macrophage function.

Systemic lupus erythematosus (SLE) involves disorders of innate and adaptive immune pathways. Tax1-binding protein 1 (TAX1BP1) modulates the production of antibodies in B cells and the T-cell cycle by regulating the NF-κB signaling pathway. However, the potential association of TAX1BP1 with SLE and its role in monocytes/macrophages have not been fully elucidated. In this study, we utilized whole-exome sequencing (WES) in combination with Sanger sequencing and identified 16 gene mutations, including in TAX1BP1, in an SLE family. TAX1BP1 protein expression with western blotting detection was reduced in SLE patients and correlated with disease activity negatively. Furthermore, RNA sequencing and 4D Label-Free Phosphoproteomic analysis were employed to characterize the transcriptome and phosphoproteome profiles in THP-1 and THP-1-differentiated M1 macrophages with TAX1BP1 knockdown. Silencing of TAX1BP1 in THP-1 and THP-1-differentiated M1 macrophages led to an increase in cluster of differentiation 80 (CD80) expression and differential changes in CD14 and CD16 expression, as assessed by flow cytometry. Additionally, western blot analysis showed that knockdown of TAX1BP1 led to a reduction in TRAF6 and p-p65 in THP-1-differentiated macrophages, with or without lipopolysaccharide (LPS) or tumor necrosis factor (TNF)-α stimulation. Taken together, our findings suggest that TAX1BP1 participates in SLE activity by regulating antigen presentation in monocytes and inflammatory responses in M1 macrophages.

Identification of pathogenic variants in three Chinese patients with McCune-Albright syndrome

To explore the genetic basis for three Chinese patients with McCune-Albright syndrome (MAS). Three children who had respectively presented at Shandong Provincial Hospital in April 2019 and Peking Union Medical College Hospital in August 2020 and May 2021 were selected as the research subjects. Peripheral blood samples of the probands and their family members were taken for the extraction of genomic DNA. Potential variants were screened by whole exome sequencing (WES), and candidate variants were validated by Sanger sequencing of the patients and their family members. The proband from family 1 was found to harbor a heterozygous c.601C>T (p.R201C) missense variant in exon 8 of the GNAS gene, whilst the probands from families 2 and 3 were both found to harbor a heterozygous c.602G>A (p.R201H) missense variant in exon 8 of the GNAS gene. Both variants were known to be pathogenic, and all probands were found to be mosaics for the corresponding variants but with various degrees. WES can effectively diagnose MAS and other somatic genetic disorders. In this study, the combined WES and Sanger sequencing have verified the degree of mosaicisms of pathogenic variants in the three MAS patients, albeit no apparent correlation was found between the degree of mosaicisms and the phenotype of patients. Above finding has provided a basis for genetic counseling and prenatal diagnosis for the affected families.

Novel mutation of IFT140 in an infant with Mainzer-Saldino syndrome presenting with retinal dystrophy

A seven-month-old girl presented with bilateral roving nystagmus, hyperopia, and retinal dystrophy, and was brought to our ophthalmology clinic. Visual-evoked potentials (VEPs) were non-recordable in both the eyes. No other systemic symptoms or abnormalities were observed. Whole exome sequencing (WES) identified a compound heterozygous mutation in the IFT140 gene: c.1990G > A (p. Glu664Lys) and c.2214_2217del (p.Asp738GlufsTer47). The genetic results support a diagnosis of Mainzer-Saldino syndrome (MSS). Importantly, c.2214_2217del is a novel mutation in the IFT140 gene. Although the patient presents with isolated retinal dystrophy, it is crucial to monitor renal function overtime. Taken together, our results reinforce the role of IFT140 in syndromic ciliopathies. This report also highlights the role of combined WES approaches in identifying underlying mutations in infants presenting with isolated retinal dystrophy, considering MSS may present differently over time.

A linkage and exome study of multiplex families with bipolar disorder implicates rare coding variants of ANK3 and additional rare alleles at 10q11-q21.

BackgroundBipolar disorder is a highly heritable psychiatric condition for which specific genetic factors remain largely unknown. In the present study, we used combined whole-exome sequencing and linkage analysis to identify risk loci and dissect the contribution of common and rare variants in families with a high density of illness.MethodsOverall, 117 participants from 15 Australian extended families with bipolar disorder (72 with affective disorder, including 50 with bipolar disorder type I or II, 13 with schizoaffective disorder–manic type and 9 with recurrent unipolar disorder) underwent whole-exome sequencing. We performed genome-wide linkage analysis using MERLIN and conditional linkage analysis using LAMP. We assessed the contribution of potentially functional rare variants using a gene-based segregation test.ResultsWe identified a significant linkage peak on chromosome 10q11-q21 (maximal single nucleotide polymorphism = rs10761725; exponential logarithm of the odds [LODexp] = 3.03; empirical p = 0.046). The linkage interval spanned 36 protein-coding genes, including a gene associated with bipolar disorder, ankyrin 3 (ANK3). Conditional linkage analysis showed that common ANK3 risk variants previously identified in genome-wide association studies — or variants in linkage disequilibrium with those variants — did not explain the linkage signal (rs10994397 LOD = 0.63; rs9804190 LOD = 0.04). A family-based segregation test with 34 rare variants from 14 genes under the linkage interval suggested rare variant contributions of 3 brain-expressed genes: NRBF2 (p = 0.005), PCDH15 (p = 0.002) and ANK3 (p = 0.014).LimitationsWe did not examine non-coding variants, but they may explain the remaining linkage signal.ConclusionCombining family-based linkage analysis with next-generation sequencing data is effective for identifying putative disease genes and specific risk variants in complex disorders. We identified rare missense variants in ANK3, PCDH15 and NRBF2 that could confer disease risk, providing valuable targets for functional characterization.

Diagnostic utility of exome sequencing for inherited peripheral neuropathies

Introduction. Hereditary motor and sensory neuropathies, a highly genetic heterogeneous group of disorders, have a phenotype caused by peripheral nerve damage.Purpose of the study – to assess the extent of genetic heterogeneity of hereditary motor and sensory neuropathies in Russian patients and to evaluate the diagnostic effectiveness of using full-exome research methods to find the genetic cause of hereditary motor and sensory neuropathies.Materials and methods. The material for the study was DNA samples from 51 patients and their family members referred for whole exome sequencing to the DNA-diagnostics laboratory of Research Centre for Medical Genetics in 2017–2019. Methods: whole exome sequencing, Sanger sequencing, restriction fragment length polymorphism.Results. Whole exome sequencing in combination with segregation analysis of the pathogenic variants in families allowed to determine the cause of the disease in 41 % of cases. In another 16 % of cases, candidate genetic variants as a possible cause of the disease were revealed, but additional studies are needed to confirm it. The most frequently mutated gene was MFN2 caused neuropathy in 6 unrelated families. MPZ gene mutations were detected in two families, AARS gene mutations were revealed in another two families, and mutations in GJB1, HINT1, INF2, LRSAM1, LITAF, MME, NEFL, WWOX were detected once. Among the causal variants, mutations in B4GALNT1 caused spastic paraplegia, in COL6A1 led to Bethlem’s congenital muscular dystrophy, and in SYT2 caused congenital myasthenic syndrome indicating difficulties in differential diagnosis of inherited neuromuscular disorders. A PMP22 duplication was detected in 2 families prior to whole exome sequencing.Conclusion. Whole exome sequencing is very important for finding the molecular cause of hereditary motor and sensory neuropathies. In most cases, additional methods should be used to clarify the pathogenicity of variants detected by whole exome sequencing. However, it is necessary to remember that the most common cause of the disease is a large duplication of the region 17p11.2.

NIMG-46. RADIOGENOMIC FEATURES PREDICT CLINICALLY RELEVANT GENOME-WIDE ALTERATION SIGNATURES IN GLIOBLASTOMA

Abstract BACKGROUND Previously, we have shown that combined whole-exome sequencing (WES) and genome-wide somatic copy number alteration (SCNA) information can separate IDH1/2-wildtype glioblastoma into two prognostic molecular subtypes (Group 1 and Group 2) and that these subtypes cannot be distinguished by epigenetic or clinical features. However, the potential for radiographic features to discriminate between these molecular subtypes has not been established. METHODS Radiogenomic features (n=35,400) were extracted from 46 multiparametric, pre-operative magnetic resonance imaging (MRI) of IDH1/2-wildtype glioblastoma patients from The Cancer Imaging Archive, all of whom have corresponding WES and SCNA data in The Cancer Genome Atlas. We developed a novel feature selection method that leverages the structure of extracted radiogenomic MRI features to mitigate the dimensionality challenge posed by the disparity between the number of features and patients in our cohort. Seven traditional machine learning classifiers were trained to distinguish Group 1 versus Group 2 using our feature selection method. Our feature selection was compared to lasso feature selection, recursive feature elimination, and variance thresholding. RESULTS We are able to classify Group 1 versus Group 2 glioblastomas with a cross-validated area under the curve (AUC) score of 0.82 using ridge logistic regression and our proposed feature selection method, which reduces the size of our feature set from 35,400 to 288. An interrogation of the selected features suggests that features describing contours in the T2 abnormality region on the FLAIR MRI modality may best distinguish these two groups from one another. CONCLUSIONS We successfully trained a machine learning model that allows for relevant targeted feature extraction from standard MRI to accurately predict molecularly-defined risk-stratifying IDH1/2-wildtype glioblastoma patient groups. This algorithm may be applied to future prospective studies to assess the utility of MRI as a surrogate for costly prognostic genomic studies.

A comprehensive bioinformatic analysis of 126 patients with an inherited platelet disorder to identify both sequence and copy number genetic variants.

Inherited bleeding disorders (IBDs) comprise an extremely heterogeneous group of diseases that reflect abnormalities of blood vessels, coagulation proteins, and platelets. Previously the UK-GAPP study has used whole-exome sequencing in combination with deep platelet phenotyping to identify pathogenic genetic variants in both known and novel genes in approximately 40% of the patients. To interrogate the remaining "unknown" cohort and improve this detection rate, we employed an IBD-specific gene panel of 119 genes using the Congenica Clinical Interpretation Platform to detect both single-nucleotide variants and copy number variants in 126 patients. In total, 135 different heterozygous variants in genes implicated in bleeding disorders were identified. Of which, 22 were classified pathogenic, 26 likely pathogenic, and the remaining were of uncertain significance. There were marked differences in the number of reported variants in individuals between the four patient groups: platelet count (35), platelet function (43), combined platelet count and function (59), and normal count (17). Additionally, we report three novel copy number variations (CNVs) not previously detected. We show that a combined single-nucleotide variation (SNV)/CNV analysis using the Congenica platform not only improves detection rates for IBDs, suggesting that such an approach can be applied to other genetic disorders where there is a high degree of heterogeneity.

Abstract 3875: Investigating tumor-immune interactions in adult sarcomas of the extremities

Abstract Introduction: Sarcoma is a heterogeneous group of connective tissue cancers with over 60 different subtypes. Curative treatment relies on surgical resection combined with radiation. In the event of metastasis, chemotherapy is commonly used but generally only with palliative intent. Given the heterogeneity of sarcomas, targeted therapies are limited and this has contributed to the relatively stagnant survival rates seen in the past decades. Objective: With poorly defined cancer antigens and genetic mutations, more personalized treatments, such as immunotherapy, and in particular adoptive cell transfer therapy (ACT), may be a more viable and promising option for select sarcoma patients than currently available palliative chemotherapy. As such, our study aims to create patient specific models to investigate the interactions between tumor-infiltrating lymphocytes (TILs) and autologous tumor cells. Methods: Over 250 tissue and blood samples from untreated sarcoma patients have been collected. The tissue samples are dissected and used for in vitro expansion of TILs or tumor cells. TIL cultures are characterized via flow cytometry and TIL responses are quantified using an IFNy ELISA. Tumor cultures are validated with RT-qPCR and dd-PCR to ensure tumor-specific mutations are retained. After characterizing and validating the cultures, autologous tumor cells and TILs are co-cultured to examine tumor-immune interactions. Outcomes of immune activity in the presence of tumor cells and tumorigenicity are evaluated to better understand the mechanisms that may be implicated in sarcoma immunotherapy. Results: At the end of a 3-week expansion period, of the 93 primary tumor samples cultured for TILs, only 7 did not yield any TIL growth; however, the final TIL yield varied between different patient samples, ranging from 0.005 - 83M cells. Both TIL cultures expanded from different samples of the same tumor and from tumors of different patients demonstrated variable cell proportions of CD4+ and CD8+ TILs. Some cultures also contained a proportion of CD56+ cells. ELISA documented TIL responses to stimulation in all collected cultures, indicating a viable and functional population. Of the 24 primary tumor samples cultured, 17 (8 of 10 undifferentiated pleomorphic sarcomas; 7 of 10 myxofibrosarcomas; 2 of 4 osteosarcomas) generated viable tumor cell cultures as defined by the presentation of stable growth beyond passage 5. Conclusion: Tumor cells and functional TILs can be isolated and expanded from most sarcomas; however, the methods must be optimized for each case. Further tumor culture validation by whole exome sequencing in combination with ddPCR is ongoing. A benchmark of an effective and clinically significant TIL yield will need to be better defined going forward. TILs are active in vitro and vary in population composition. Future experiments will focus on evaluating the killing capacity and tumor specificity of TILs. Citation Format: Alice Ko, Victoria Coward, Minji Lee, Kayley Xu, Nalan Gokgoz, Brendan Dickson, Jay Wunder, Irene L. Andrulis. Investigating tumor-immune interactions in adult sarcomas of the extremities [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3875.

Abstract CT046: Predictive biomarkers and pharmacodynamic changes in tumor RNA expression in a phase I study of anti PD-1 monoclonal antibody PF-06801591

Abstract Introduction: PF-06801591 is a humanized IgG4 monoclonal antibody that binds to programmed cell death (PD-1) receptor, blocking its interaction with PD-1 ligands. In a phase I study (NCT02573259; dose-escalation) of 40 patients (pts) with multiple solid tumor types treated with PF-06891591 intravenously (IV) or subcutaneously (SC), 7 pts showed partial response. PF-06801591 was well tolerated at all doses. Biopsy samples were evaluated for predictive biomarkers and pharmacodynamic effects. Methods: Biopsy samples at baseline and on-treatment (IV:day 29; SC:day 36) were collected from 4 dose cohorts (IV:1, 3 and 10 mg/kg; SC:300 mg). Whole-exome sequencing (WES) and RNA-seq of biopsy tissue were used to estimate tumor mutation burden (TMB) and gene expression. Regression analysis was used to identify TMB/genes/pathways potentially associated with response in baseline tissue. Differential analysis of baseline and on-treatment biopsies identified genes/pathways potentially upregulated by PF-06801591. PD-L1 (clone SP-263; Ventana) expression was evaluated in tumor biopsies by immunohistochemistry (IHC) and pathologist scoring. Results: In a combined WES analysis of samples from baseline biopsies (all doses, n=24 pts), higher TMB was significantly associated with improved objective response (R^2=0.215, P=0.013). From RNA-seq analysis, genes positively associated with response were involved in interferon-gamma (IFNG) and PD-1 signaling, and cell cycle; CTLA4 was among the genes most significantly associated with response (P<0.001). Response was better correlated with levels of PD-L1 RNA (P=0.028) than PD-L1 protein (IHC; P=0.39 to 0.84). RNA-seq analysis of paired baseline and on-treatment biopsies (all doses) showed increased expression of genes involved in adaptive immune response pathways, including chemokines and chemokine receptors such as CXCL9. Decreased expression of cell cycle genes such as CDKN1B was observed in on-treatment biopsies. Conclusion: Based on a mixed set of tumor types with possible sensitivity to anti-PD-1, high TMB, IFNG signaling, and PD-L1 were associated with response to PF-06801591, as reported for other anti-PD-1 antibodies. Albeit based on a small sample size and diverse indication profile, baseline expression of CTLA4 appears to be highly associated with response and PD-L1 RNA levels may be better associated with response than protein expression by IHC. Increased expression of genes/pathways associated with adaptive immune activation in on-treatment biopsies indicates an active, immunomodulatory mechanism with anti-PD-1 therapy. This ongoing study will evaluate predictive biomarkers in dose-expansion cohorts of PF-06801591 in non-small cell lung cancer and urothelial cancer. Acknowledgments Study sponsor: Pfizer. Medical writing support: Chu Kong Liew (Engage Scientific Solutions), funded by Pfizer. Citation Format: Siwen Hu-Lieskovan, Fadi Braiteh, Juneko E. Grilley-Olson, Shobha Potluri, Xiao Wang, Alison Forgie, Vinicius Bonato, Jeffrey Chou, Melissa L. Johnson. Predictive biomarkers and pharmacodynamic changes in tumor RNA expression in a phase I study of anti PD-1 monoclonal antibody PF-06801591 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT046.

Massively parallel sequencing of tenosynovial giant cell tumors reveals novel CSF1 fusion transcripts and novel somatic CBL mutations.

Tenosynovial giant cell tumor (TSGCT) is a rare neoplasm. Although surgical resection is the widely accepted primary treatment for TSGCT, recurrences are frequent, and patients' joint function may be severely compromised. Previous studies reported that CSF1-COL6A3 fusion genes were identified in approximately 30% of TSGCTs. The aim of our study was to comprehensively clarify the genomic abnormalities in TSGCTs. We performed whole exome sequencing in combination with target sequence validation on 34 TSGCT samples. RNA sequencing was also performed on 18 samples. RNA sequencing revealed fusion transcripts involving CSF1, including novel CSF1-VCAM1, CSF1-FN1 and CSF1-CDH1 fusions, in 13/18 (72%) cases. These fusion genes were validated by chromogenic in situ hybridization. All CSF1 fusions resulted in the deletion of CSF1 exon 9, which was previously shown to be an important negative regulator of CSF1 expression. We also found that 12 (35%) of the 34 TSGCT samples harbored CBL missense mutations. All mutations were detected in exons 8 or 9, which encode the linker and RING finger domain. Among these mutations, C404Y, L380P and R420Q were recurrent. CBL-mutated cases showed higher JAK2 expression than wild-type CBL cases (p = 0.013). CSF1 fusion genes and CBL mutations were not mutually exclusive, and both alterations were detected in six of the 18 (33%) tumors. The frequent deletion of CSF1 exon 9 in the fusion transcripts suggested the importance of this event in the etiology of TSGCT. Our results may contribute to the development of new targeted therapies using JAK2 inhibitors for CBL-mutated TSGCT.

Infectious Disease Risk in Dialysis Patients: A Transdisciplinary Approach.

Purpose of review:Infections are a major contributor to morbidity and mortality in end-stage renal disease (ESRD) patients. A better understanding of the interplay between infectious processes and ESRD may eventually lead to the development of targeted treatment strategies aimed at lowering overall disease morbidity and mortality. Monogenic causes are a major contributor to the development of adult chronic kidney disease (CKD). Recent studies identified a genetic cause in 10% to 20% of adults with CKD. With the introduction of whole-exome sequencing (WES) into clinical mainstay, this proportion is expected to increase in the future. Once patients develop CKD/ESRD due to a genetic cause, secondary changes, such as a compromised immune status, affect overall disease progression and clinical outcomes. Stratification according to genotype may enable us to study its effects on secondary disease outcomes, such as infectious risk. Moreover, this knowledge will enable us to better understand the molecular interplay between primary disease and secondary disease outcomes.Sources of information:We conducted a literature review using search engines such as PubMed, PubMed central, and Medline, as well as cumulative knowledge from our respective areas of expertise.Methods:This is a transdisciplinary perspective on infectious complications in ESRD due to monogenic causes, such as autosomal dominant polycystic kidney disease (ADPKD), combining expertise in genomics and immunology.Key findings:In ADPKD, infection is a frequent complication manifesting primarily as lower urinary tract infection and less frequently as renal infection. Infectious episodes may be a direct consequence of a specific underlying structural abnormality, for example the characteristic cysts, among others. However, evidence suggests that infectious disease risk is also increased in ESRD due to secondary not-well-understood disease mechanisms. These disease mechanisms may vary depending on the underlying nature of the primary disease. While the infectious disease risk is well documented in ADPKD, there are currently insufficient data on the risk in other monogenic causes of ESRD. WES in combination with novel technologies, such as RNA sequencing and single-cell RNA sequencing, can provide insight into the molecular mechanisms of disease progression in different monogenic causes of CKD/ESRD and may lead to the development of novel risk-stratification profiles in the future.Limitations:This is not a systematic review of the literature and the proposed perspective is tainted by the authors’ point of view on the topic.Implications:WES in combination with novel technologies such as RNA sequencing may enable us to fully unravel underlying disease mechanisms and secondary disease outcomes in monogenic causes of CKD and better characterize individual risk profiles. This understanding will hopefully facilitate the development of novel targeted therapies.

The role of recessive inheritance in early-onset epileptic encephalopathies: a combined whole-exome sequencing and copy number study

Early-onset epileptic encephalopathy (EE) and combined developmental and epileptic encephalopathies (DEE) are clinically and genetically heterogeneous severely devastating conditions. Recent studies emphasized de novo variants as major underlying cause suggesting a generally low-recurrence risk. In order to better understand the full genetic landscape of EE and DEE, we performed high-resolution chromosomal microarray analysis in combination with whole-exome sequencing in 63 deeply phenotyped independent patients. After bioinformatic filtering for rare variants, diagnostic yield was improved for recessive disorders by manual data curation as well as molecular modeling of missense variants and untargeted plasma-metabolomics in selected patients. In total, we yielded a diagnosis in ∼42% of cases with causative copy number variants in 6 patients (∼10%) and causative sequence variants in 16 established disease genes in 20 patients (∼32%), including compound heterozygosity for causative sequence and copy number variants in one patient. In total, 38% of diagnosed cases were caused by recessive genes, of which two cases escaped automatic calling due to one allele occurring de novo. Notably, we found the recessive gene SPATA5 causative in as much as 3% of our cohort, indicating that it may have been underdiagnosed in previous studies. We further support candidacy for neurodevelopmental disorders of four previously described genes (PIK3AP1, GTF3C3, UFC1, and WRAP53), three of which also followed a recessive inheritance pattern. Our results therefore confirm the importance of de novo causative gene variants in EE/DEE, but additionally illustrate the major role of mostly compound heterozygous or hemizygous recessive inheritance and consequently high-recurrence risk.

Combined CNV, haplotyping and whole exome sequencing enable identification of two distinct novel EYS mutations causing RP in a single inbred tribe.

Whole exome sequencing (WES) has become routine in clinical practice, especially in studies of recessive hereditary diseases in inbred consanguineous families, where homozygosity of a founder mutation is assumed. Multiple members of two consanguineous families of a single Bedouin tribe were diagnosed with apparently autosomal recessive/pseudo-dominant retinitis pigmentosa (RP). Affected individuals exhibited severe visual impairment with nyctalopia, marked constriction of visual fields, markedly reduced and delayed responses on electro-retinography (ERG) and eventual loss of central vision. Combined copy-number variant (CNV) analysis, haplotype reconstruction and WES of the kindred identified two distinct novel mutations in EYS (RP25): a p.(W1817*) nonsense mutation (identified through WES) and a large deletion encompassing 9 of the 43 exons, that was missed by WES and was identified through microarray CNV analysis. Segregation analysis of both mutations demonstrated that all affected individuals were either homozygous for one of the mutations, or compound heterozygous for both. The two mutations are predicted to cause loss of function of the encoded protein and were not present in screening of 200 ethnically-matched controls. Our findings of two distinct mutations in the same gene in a single inbred kindred, identified only through combined WES and microarray CNV analysis, highlight the limitations of either CNV or WES alone, as the heterozygous deletion had normal WES read-depth values. Moreover, they demonstrate pitfalls in homozygosity mapping for disease-causing variant identification in inbred communities.

Validating Comprehensive Next-Generation Sequencing Results for Precision Oncology: The NCT/DKTK Molecularly Aided Stratification for Tumor Eradication Research Experience.

Rapidly evolving genomics technologies, in particular comprehensive next-generation sequencing (NGS), have led to exponential growth in the understanding of cancer biology, shifting oncology toward personalized treatment strategies. However, comprehensive NGS approaches, such as whole-exome sequencing, have limitations that are related to the technology itself as well as to the input source. Hence, clinical implementation of comprehensive NGS in a quality-controlled diagnostic workflow requires both the standardization of sequencing procedures and continuous validation of sequencing results by orthogonal methods in an ongoing program to enable the determination of key test parameters and continuous improvement of NGS and bioinformatics pipelines. We present validation data on 220 patients who were enrolled between 2013 and 2016 in a multi-institutional, genomics-guided precision oncology program (Molecularly Aided Stratification for Tumor Eradication Research) of the National Center for Tumor Diseases Heidelberg and the German Cancer Consortium. More than 90% of clinically actionable genomic alterations identified by combined whole-exome sequencing and transcriptome sequencing were successfully validated, with varying frequencies of discordant results across different types of alterations (fusions, 3.7%; single-nucleotide variants, 2.6%; amplifications, 1.1%; overexpression, 0.9%; deletions, 0.6%). The implementation of new computational methods for NGS data analysis led to a substantial improvement of gene fusion calling over time. Collectively, these data demonstrate the value of a rigorous validation program that partners with comprehensive NGS to successfully implement and continuously improve cancer precision medicine in a clinical setting.

Early-onset Parkinson disease caused by a mutation in CHCHD2 and mitochondrial dysfunction

ObjectiveOur goal was to identify the gene(s) associated with an early-onset form of Parkinson disease (PD) and the molecular defects associated with this mutation.MethodsWe combined whole-exome sequencing and functional genomics to identify the genes associated with early-onset PD. We used fluorescence microscopy, cell, and mitochondrial biology measurements to identify the molecular defects resulting from the identified mutation.ResultsHere, we report an association of a homozygous variant in CHCHD2, encoding coiled-coil-helix-coiled-coil-helix domain containing protein 2, a mitochondrial protein of unknown function, with an early-onset form of PD in a 26-year-old Caucasian woman. The CHCHD2 mutation in PD patient fibroblasts causes fragmentation of the mitochondrial reticular morphology and results in reduced oxidative phosphorylation at complex I and complex IV. Although patient cells could maintain a proton motive force, reactive oxygen species production was increased, which correlated with an increased metabolic rate.ConclusionsOur findings implicate CHCHD2 in the pathogenesis of recessive early-onset PD, expanding the repertoire of mitochondrial proteins that play a direct role in this disease.

Identification of ANKDD1B variants in an ankylosing spondylitis pedigree and a sporadic patient

BackgroundAnkylosing spondylitis (AS) is a debilitating autoimmune disease affecting tens of millions of people in the world. The genetics of AS is unclear. Analysis of rare AS pedigrees might facilitate our understanding of AS pathogenesis.MethodsWe used genome-wide linkage analysis and whole-exome sequencing in combination with variant co-segregation verification and haplotype analysis to study an AS pedigree and a sporadic AS patient.ResultsWe identified a missense variant in the ankyrin repeat and death domain containing 1B gene ANKDD1B from a Han Chinese pedigree with dominantly inherited AS. This variant (p.L87V) co-segregates with all male patients of the pedigree. In females, the penetrance of the symptoms is incomplete with one identified patient out of 5 carriers, consistent with the reduced frequency of AS in females of the general population. We further identified a distinct missense variant affecting a conserved amino acid (p.R102L) of ANKDD1B in a male from 30 sporadic early onset AS patients. Both variants are absent in 500 normal controls. We determined the haplotypes of four major known AS risk loci, including HLA-B*27, 2p15, ERAP1 and IL23R, and found that only HLA-B*27 is strongly associated with patients in our cohort.ConclusionsTogether these results suggest that ANKDD1B variants might be associated with AS and genetic analyses of more AS patients are warranted to verify this association.

Identifying and Targeting Sporadic Oncogenic Genetic Aberrations in Mouse Models of Triple-Negative Breast Cancer.

Triple-negative breast cancers (TNBC) are genetically characterized by aberrations in TP53 and a low rate of activating point mutations in common oncogenes, rendering it challenging in applying targeted therapies. We performed whole-exome sequencing (WES) and RNA sequencing (RNA-seq) to identify somatic genetic alterations in mouse models of TNBCs driven by loss of Trp53 alone or in combination with Brca1 Amplifications or translocations that resulted in elevated oncoprotein expression or oncoprotein-containing fusions, respectively, as well as frameshift mutations of tumor suppressors were identified in approximately 50% of the tumors evaluated. Although the spectrum of sporadic genetic alterations was diverse, the majority had in common the ability to activate the MAPK/PI3K pathways. Importantly, we demonstrated that approved or experimental drugs efficiently induce tumor regression specifically in tumors harboring somatic aberrations of the drug target. Our study suggests that the combination of WES and RNA-seq on human TNBC will lead to the identification of actionable therapeutic targets for precision medicine-guided TNBC treatment.Significance: Using combined WES and RNA-seq analyses, we identified sporadic oncogenic events in TNBC mouse models that share the capacity to activate the MAPK and/or PI3K pathways. Our data support a treatment tailored to the genetics of individual tumors that parallels the approaches being investigated in the ongoing NCI-MATCH, My Pathway Trial, and ESMART clinical trials. Cancer Discov; 8(3); 354-69. ©2017 AACR.See related commentary by Natrajan et al., p. 272See related article by Matissek et al., p. 336This article is highlighted in the In This Issue feature, p. 253.

A novel mutation of dipeptidyl aminopeptidase-like protein-6 in a family with suspicious idiopathic ventricular fibrillation.

Sudden cardiac death (SCD) occurs in a broad spectrum of cardiac pathologies and is an important cause of mortality in the general population. Idiopathic ventricular fibrillation (IVF) is a rare but important factor resulting in SCD. It is diagnosed in a resuscitated cardiac arrest victim underlying unknown cause, with documented ventricular fibrillation. Previous studies have demonstrated that mutations in dipeptidyl aminopeptidase-like protein-6 (DPP6) and cardiac sodium channel Nav1.5 (SCN5A) are the most important genetic factors involve in IVF. By using whole sequencing to identify the genetic lesion of a family with suspicious idiopathic ventricular fibrillation. Prospective genetic study. In this study, we employed whole-exome sequencing in combination with arrhythmia-related gene filtering to identify the genetic lesion for a family suffering from suspicious IVF, syncope and SCD. We then generated the plasmids of DPP6-pcDNA3.1+ (WT and c.1578G>C/p.Q526H). Kv4.3-pcDNA3.1+ was co-transfected together with/without DPP6-pcDNA3.1+ (WT and/or c.1578G>C/p.Q526H) into HEK293 cells to perform the patch clamp experiments. A novel missense mutation (c.1578G>C/p.Q526H) of DPP6 was identified and co-segregated with affected patients in this family. Patch clamp experiments suggested that this novel mutation might result in a gain of function and disturb the efflux of potassium ion. Our study not only reported the second missense mutation of DPP6 in heart disease and expanded the spectrum of DPP6 mutations, but also contribute to the genetic diagnosis and counseling of families with suspicious IVF, syncope and SCD.

Identification of a LMNA (c.646C>T) variant by whole-exome sequencing in combination with a dilated cardiomyopathy (DCM) related gene filter in a family with familiar DCM.

Identification of a LMNA (c.646C>T) variant by whole-exome sequencing in combination with a dilated cardiomyopathy (DCM) related gene filter in a family with familiar DCM.

Identification of MYLK3 mutations in familial dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a primary cause of heart failure, life-threatening arrhythmias, and cardiac death. Pathogenic mutations have been identified at the loci of more than 50 genes in approximately 50% of DCM cases, while the etiologies of the remainder have yet to be determined. In this study, we applied whole exome sequencing in combination with segregation analysis to one pedigree with familial DCM, and identified a read-through mutation (c.2459 A > C; p.*820Sext*19) in the myosin light chain kinase 3 gene (MYLK3). We then conducted MYLK3 gene screening of 15 DCM patients (7 familial and 8 sporadic) who were negative for mutation screening of the previously-reported cardiomyopathy-causing genes, and identified another case with a MYLK3 frameshift mutation (c.1879_1885del; p.L627fs*41). In vitro experiments and immunohistochemistry suggested that the MYLK3 mutations identified in this study result in markedly reduced levels of protein expression and myosin light chain 2 phosphorylation. This is the first report that MYLK3 mutations can cause DCM in humans. The clinical phenotypes of DCM patients were consistent with MYLK3 loss-of-function mouse and zebrafish models in which cardiac enlargement and heart failure are observed. Our findings highlight an essential role for cardiac myosin light chain kinase in the human heart.