Exome Analysis Research Articles

Advancing diagnosis and management of liver disease in adults through exome sequencing

Whole-exome sequencing (WES) is an effective tool for diagnosis in patients who remain undiagnosed despite a comprehensive clinical work-up. While WES is being used increasingly in pediatrics and oncology, it remains underutilized in non-oncological adult medicine, including in patients with liver disease, in part based on the faulty premise that adults are unlikely to harbor rare genetic variants with large effect size. Here, we aim to assess the burden of rare genetic variants underlying liver disease in adults at two major tertiary referral academic medical centers. WES analysis paired with comprehensive clinical evaluation was performed in fifty-two adult patients with liver disease of unknown etiology evaluated at two US tertiary academic health care centers. Exome analysis uncovered a definitive or presumed diagnosis in 33% of patients (17/52) providing insight into their disease pathogenesis, with most of these patients (12/17) not having a known family history of liver disease. Our data shows that over two-thirds of undiagnosed liver disease patients attaining a genetic diagnosis were being evaluated for cholestasis or hepatic steatosis of unknown etiology. This study reveals an underappreciated incidence and spectrum of genetic diseases presenting in adulthood and underscores the clinical value of incorporating exome sequencing in the evaluation and management of adults with liver disease of unknown etiology. S.V. is supported by the NIH/NIDDK (K08 DK113109 and R01 DK131033-01A1) and the Doris Duke Charitable Foundation Grant #2019081. This work was supported in part by NIH-funded Yale Liver Center, P30 DK34989.

NOTCH3 C201R variant causes cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) that can be confused with early-onset Alzheimer's disease

BackgroundNOTCH3 is the causative gene for autosomal dominant cerebral arteriopathy with subcortical infarctions and leukoencephalopathy (CADASIL) which is associated with both stroke and dementia. When CADASIL presents primarily as dementia it can be difficult to distinguish from Alzheimer's disease (AD) at both the clinical and neuropathological levels. MethodsWe performed exome sequencing of several affected individuals from a large family affected with AD. PCR amplification and direct Sanger sequencing were used to verify variants detected by exome analysis and to screen family members at-risk to carry those variants. Neuropathologic brain evaluation by immunohistochemistry and MRI were performed for the carriers of the NOTCH3 variant. ResultsIn a three-generation family with AD, we found a c.601 T > C p.Cys201Arg variant in the NOTCH3 gene that caused clinical and neuropathological manifestations of CADASIL. These features included earlier onset of dementia accompanied by behavioral abnormalities in the father and son and white matter abnormalities in the asymptomatic grandson. The family is one branch of a large pedigree studied by the Alzheimer's Disease Sequencing Project (ADSP). As part of the ADSP linkage analysis and whole genome sequencing endeavor, an ABCA1 variant, p.Ala937Val, was previously found associated with AD in this pedigree. ConclusionsOur findings, together with other reported pathogenic missense variants of the C201 codon in NOTCH3, support the role of cysteine 201 as a mutation hotspot for CADASIL and highlight the genetic complexity both clinically and pathologically of AD and related dementia.

Genetic research of inherited peripheral neuropathies

AbstractBackgroundOur department has been conducting genetic research on hereditary neuropathies, with a focus on the Charcot–Marie–Tooth (CMT) disease and other inherited peripheral neuropathies for the past 20 years. The CMT disease is an untreatable disease characterized by progressive muscle weakness, atrophy, and sensory impairment, primarily affecting the peripheral nerves.Materials and MethodsIn our institution, we collected blood samples from patients with the CMT disease, nationwide, and performed comprehensive genetic testing to elucidate its underlying causes.ResultUsing next‐generation sequencing (NGS), we successfully discovered two novel causative genes of the CMT disease, membrane metalloendopeptidase (MME), and cytochrome c oxidase assembly factor 7, via whole exome analysis of previously unidentified cases. These findings, combined with the comprehensive analysis enabled by NGS, have significantly improved the diagnostic rate of this disease.ConclusionNotably, the MME gene has been reported as the most frequent causative gene for autosomal recessive CMT disease, attracting global attention and subsequent research efforts. Accurate genetic diagnosis for each patient serves as a crucial first step toward understanding hereditary diseases. The identification of new causes of the CMT disease can assist in elucidating the molecular mechanisms involved completely, further contributing to the development of effective treatment approaches, including gene therapies.

Combined Afirma Genomic Sequencing Classifier and TERT promoter mutation detection in molecular assessment of Bethesda III-VI thyroid nodules.

Molecular analysis of thyroid nodules can provide diagnostic and prognostic information as well as indicate opportunities for targeted therapy. Whole RNA exome analysis offers information that can be leveraged to predict tumor behavior.

Spectrum of congenital anomalies of the kidney and urinary tract (CAKUT) including renal parenchymal malformations during fetal life and the implementation of prenatal exome sequencing (WES)

Objectives and backgroundCongenital malformations of the kidney and urinary tract (CAKUT) have a prevalence of 4–60 in 10,000 livebirths and constitute for 40–50% of all end stage pediatric kidney disease. CAKUT can have a genetic background due to monogenetic inherited disease, such as PKD or ciliopathies. They can also be found in combination with extra-renal findings as part of a syndrome. Upon detection of genitourinary malformations during the fetal anomaly scan the question arises if further genetic testing is required. The purpose of this study was to determine the phenotypic presentation of CAKUT cases and the results of exome analysis (WES).MethodsThis is a retrospective analysis of 63 fetal cases with a diagnosis of CAKUT or DSD at a single center between August 2018 and December 2022.ResultsA total of 63 cases (5.6%) out of 1123 matched CAKUT phenotypes including renal parenchyma malformations. In 15 out of 63 WES analysis a pathogenic variant was detected (23.8%). In fetuses with isolated CAKUT the rate of detecting a pathogenic variant on exome sequencing was five out of 44 (11.4%). Ten out of 19 fetuses (52.6%) that displayed extra-renal findings in combination with CAKUT were diagnosed with a pathogenic variant.ConclusionsWES provides an increase in diagnosing pathogenic variants in cases of prenatally detected CAKUT. Especially in fetuses with extra-renal malformations, WES facilitates a gain in information on the fetal genotype to enhance prenatal counselling and management.

Exome Survey and Candidate Gene Re-Sequencing Identifies Novel Exstrophy Candidate Genes and Implicates LZTR1 in Disease Formation.

The bladder exstrophy-epispadias complex (BEEC) is a spectrum of congenital abnormalities that involves the abdominal wall, the bony pelvis, the urinary tract, the external genitalia, and, in severe cases, the gastrointestinal tract as well. Herein, we performed an exome analysis of case-parent trios with cloacal exstrophy (CE), the most severe form of the BEEC. Furthermore, we surveyed the exome of a sib-pair presenting with classic bladder exstrophy (CBE) and epispadias (E) only. Moreover, we performed large-scale re-sequencing of CBE individuals for novel candidate genes that were derived from the current exome analysis, as well as for previously reported candidate genes within the CBE phenocritical region, 22q11.2. The exome survey in the CE case-parent trios identified two candidate genes harboring de novo variants (NR1H2, GKAP1), four candidate genes with autosomal-recessive biallelic variants (AKR1B10, CLSTN3, NDST4, PLEKHB1) and one candidate gene with suggestive uniparental disomy (SVEP1). However, re-sequencing did not identify any additional variant carriers in these candidate genes. Analysis of the affected sib-pair revealed no candidate gene. Re-sequencing of the genes within the 22q11.2 CBE phenocritical region identified two highly conserved frameshift variants that led to early termination in two independent CBE males, in LZTR1 (c.978_985del, p.Ser327fster6) and in SLC7A4 (c.1087delC, p.Arg363fster68). According to previous studies, our study further implicates LZTR1 in CBE formation. Exome analysis-derived candidate genes from CE individuals may not represent a frequent indicator for other BEEC phenotypes and warrant molecular analysis before their involvement in disease formation can be assumed.

Prognostic survival biomarkers of tumor-fused dendritic cell vaccine therapy in patients with newly diagnosed glioblastoma

Dendritic cell (DC)-based immunotherapy has been applied to glioblastoma (GBM); however, biomarkers informing response remain poorly understood. We conducted a phase I/IIa clinical trial investigating tumor-fused DC (TFDC) immunotherapy following temozolomide-based chemoradiotherapy in patients with newly diagnosed GBM and determined prognostic factors in patients receiving TFDC immunotherapy. Twenty-eight adult patients with GBM isocitrate dehydrogenase (IDH) wild-type (IDH-WT) were enrolled; 127 TFDC vaccine injections (4.5 ± 2.6 times/patient) were administered. Patients with GBM IDH-WT had a respectable 5-year survival rate (24%), verifying the clinical activity of TFDC immunotherapy, particularly against O6-methylguanine-DNA methyltransferase (MGMT) unmethylated GBM (5-year survival rate: 33%). To identify novel factors influencing overall survival (OS) in GBM IDH-WT treated with TFDC immunotherapy, clinical parameters were assessed and comprehensive molecular profiling involving transcriptome and exome analyses was performed. MGMT promoter methylation status, extent of tumor resection, and vaccine parameters (administration frequency, DC and tumor cell numbers, and fusion ratio) were not associated with survival following TFDC immunotherapy. Old age and pre- and post-operative Karnofsky performance status were significantly correlated with OS. Low HLA-A expression and lack of CCDC88A, KRT4, TACC2, and TONSL mutations in tumor cells were correlated with better prognosis. We validated the activity of TFDC immunotherapy against GBM IDH-WT, including chemoresistant, MGMT promoter unmethylated cases. The identification of molecular biomarkers predictive of TFDC immunotherapy efficacy in GBM IDH-WT will facilitate the design of and patient stratification in a phase-3 trial to maximize treatment benefits.

Sequence Variants in the WNT10B Underlying Non-Syndromic Split-Hand/Foot Malformation

Introduction: Split hand and foot malformation (SHFM) or ectrodactyly is a rare limb deformity characterized by median cleft of the hand and foot with impaired or missing central rays. It can occur as an isolated anomaly or in association with abnormalities of other body parts. Methods: After delineating the clinical features of two families (A–B), with non-syndromic SHFM, exome and Sanger sequencing were employed to search for the disease-causing variants. Results: Analysis of exome and Sanger sequencing data revealed two causative variants in the WNT10B gene in affected members of the two families. This included a novel missense change [c.338G>C; p.(Gly113Ala)] in family A and a previously reported frameshift variant [c.884-896delTCCAGCCCCGTCT; p.(Phe295Cysfs*87)] in family B. Conclusion: Our findings add a novel variant in WNT10B gene as the underlying cause of SHFM. The finding adds to the growing body of knowledge about the genetic basis of developmental disorders and provides valuable insights into the molecular mechanisms that regulate limb development.

Two new cases of brazilian boys presenting a Rett-like phenotype due to FOXG1: case report and literature review

Rett syndrome (RTT) is a rare neurodevelopmental disorder usually affecting females. Most typical forms of RTT patients are hemizygous for pathogenic variants in MECP2. FOXG1 syndrome is a rare and severe neurodevelopmental disorder with a broad spectrum of clinical features which could be described as RTT-like. In this paper we describe two non related patients who presented with developmental delay, microcephaly and hyperkinetic movements. One of them had epilepsy. Diagnosis was made by exome analysis and highlights an uncommon differential diagnosis in developmental delay in children.

#2850 EFFICIENCY OF THE GENETIC STUDY IN THE DIAGNOSIS OF AUTOSOMAL DOMINANT TUBULOINTERSTITIAL NEPHROPATHY

Abstract Background and Aims Autosomal dominant tubulointerstitial nephropathy (ADTKD) is a rare underdiagnosed cause of chronic kidney disease (CKD). ADTKD is a renal disease with a non-specific phenotypic expression that cause a slow progression over time. Given the great variability in its inter-familial presentation and among the same family members carrying the same mutation, its diagnosis is a challenge. Five genes have been identified to date such as Uromodulin (UMOD), Renin (REN), Hepatocyte Nuclear Factor 1b (HNF1β), Mucin-1 (MUC1) and, more recently, new genes, SEC61A1(encoding protein transport protein Sec61 subunit α and atypical (DNAJB11). Our main objective is to analyze the diagnostic yield of the genetic study in cases of clinical suspicion of ADTKD in patients with CKD of unknown etiology. We present a case series of patients from our center with CKD of unknown etiology and clinical suspicion of ADTKD. Genetic analysis of the gene SEC61A1 and DNAJB11 was not available at that time. Method This is a descriptive observational retrospective study from 2016 to 2021 at the Virgen del Rocio University Hospital in Seville, Spain. A total of 26 cases had phenotypic presentation of ADTKD. We collected clinical, laboratory, radiologic, and pathologic data. We performed in all the cases genetic testing for UMOD, MUC1, REN and HNF1β mutations. We collected data in a database, including age of presentation of kidney disease, sex, family history of kidney disease, the presence of hypertension at early age (defined as under 40 years of age), hyperuricemia (defined as uric acid >7.5 mg/dl) at early age (<40 years), hemoglobin at the time of diagnosis, age at last follow up with renal function and estimated glomerular filtration rate (eGFR) using the CKD-EPI (CKD Epidemiology Collaboration), urine elemental, renal ultrasound and renal biopsy if it had been performed. Descriptive results of quantitative variables are expressed as median. For categorical data, frequency and percentage are reported. Results Of the 26 cases with suspected ADTKD, 4 cases presented a genetic study that confirmed the diagnosis, that is, 15% of the cases. The median age at the time of the diagnosis was 41 years (IQR 32.7–48). 84% of the cases were male; 34% of cases had a family history of CKD of unknown etiology. 65% percent of the cases had a history of hyperuricemia at early age (<40 years), with a personal history of hypertension <40 years in 30% of the cases; 44% of the cases had renal lithiasis. The median glomerular filtration rate (CKD-EPI) at the time of diagnosis was 70 ml/min/1.73 m2 (46.4–70.7). In most of the patients, the urine elemental was normal. Renal cysts were found on ultrasound in 46% of patients. In 5 of the twenty-six patients a renal biopsy was performed, in which 60% presented data compatible with ADTKD with interstitial fibrosis and tubular atrophy. 4 of the patients with clinical suspicion of ADTKD obtained a genetic study that confirmed the diagnosis. The most frequently identified gene was HNF1β, being present in two of the four patients. The other genes identified were UMOD and MUC. The results are summarized in Table 1. Conclusion Adequate selection of patients based on clinical suspicion is essential to give greater profitability to the genetic study of this entity. The negativity of the study cannot exclude the diagnosis, since at the moment not all the causative genes of this pathology have been identified and it is necessary to use massive sequencing techniques or even the analysis of the complete exome to see the possibility of other variants of the disease.

Clinical and bi-genomic DNA findings of patients suspected to have mitochondrial diseases.

Background: Mitochondrial diseases are the most common group of inherited metabolic disorders, causing difficulties in definite diagnosis due to clinical and genetic heterogeneity. Clinical components are predominantly associated with pathogenic variants shown in nuclear or mitochondrial genomes that affect vital respiratory chain function. The development of high-throughput sequencing technologies has accelerated the elucidation of the genetic etiology of many genetic diseases that previously remained undiagnosed. Methods: Thirty affected patients from 24 unrelated families with clinical, radiological, biochemical, and histopathological evaluations considered for mitochondrial diseases were investigated. DNA isolated from the peripheral blood samples of probands was sequenced for nuclear exome and mitochondrial DNA (mtDNA) analyses. MtDNA sequencing was also performed from the muscle biopsy material in one patient. For segregation, Sanger sequencing is performed for pathogenic alterations in five other affected family members and healthy parents. Results: Exome sequencing revealed 14 different pathogenic variants in nine genes encoding mitochondrial function peptides (AARS2, EARS2, ECHS1, FBXL4, MICOS13, NDUFAF6, OXCT1, POLG, and TK2) in 12 patients from nine families and four variants in genes encoding important for muscle structure (CAPN3, DYSF, and TCAP) in six patients from four families. Three probands carried pathogenic mtDNA variations in two genes (MT-ATP6 and MT-TL1). Nine variants in five genes are reported for the first time with disease association: (AARS2: c.277C>T/p.(R93*), c.845C>G/p.(S282C); EARS2: c.319C>T/p.(R107C), c.1283delC/p.(P428Lfs*); ECHS1: c.161G>A/p.(R54His); c.202G>A/p.(E68Lys); NDUFAF6: c.479delA/p.(N162Ifs*27); and OXCT1: c.1370C>T/p.(T457I), c.1173-139G>T/p.(?). Conclusion: Bi-genomic DNA sequencing clarified genetic etiology in 67% (16/24) of the families. Diagnostic utility by mtDNA sequencing in 13% (3/24) and exome sequencing in 54% (13/24) of the families prioritized searching for nuclear genome pathologies for the first-tier test. Weakness and muscle wasting observed in 17% (4/24) of the families underlined that limb-girdle muscular dystrophy, similar to mitochondrial myopathy, is an essential point for differential diagnosis. The correct diagnosis is crucial for comprehensive genetic counseling of families. Also, it contributes to making treatment-helpful referrals, such as ensuring early access to medication for patients with mutations in the TK2 gene.

Only one beer can be mortal: a case report of two sisters with cardiac arrest due to a homozygous mutation in PPA2 gene

We report the long way to the correct diagnosis in two teenage sisters who developed a cardiac arrest after consuming minimal amounts of alcohol. The older girl dramatically survived two cardiac arrests at the age of 14 and 15 years. She underwent an extensive examination that revealed isolated cardiac abnormalities including fibrosis, dilated cardiomyopathy and inflammation. The younger girl also had a cardiac arrest at the age of 15 and died suddenly after consuming 1–2 beers, 3 years after her sister´s first incident. Autopsy of the heart revealed acute myocarditis without structural alterations. Multigene panel analysis (not including PPA2) showed SCN5A and CACNA1D variants in both sisters and their healthy mother. Six years later duo exome allowed the diagnosis of an autosomal recessive PPA2-related mitochondriopathy. We discuss the molecular results and clinical picture of our patients compared to other PPA2-related cases. We highlight the diagnostic contribution of multigene panels and exome analysis. The genetic diagnosis is important for medical care and for everyday life, specifically because alcohol intake can result in cardiac arrest and should be strictly avoided. Conclusion: Duo exome sequencing clarified the diagnosis of PPA2-related mitochondriopathy in two sisters with isolated cardiac features and sudden cardiac arrest triggered by minimal amounts of alcohol.What is Known:• Multigene-Panel or exome analysis is a valuable tool to identify genetic causes of hereditary cardiac arrhythmias.• Variants of unknown significance can lead to misinterpretation. PPA2-related mitochondriopathy is a very rare autosomal recessive condition that is normally fatal in infancy.What is New:• Duo exome analysis in two teeenage sisters with cardiac arrest revealed a homozygous mild PPA2 mutation as the underlying pathology restricted to the heart muscle

Association of C-MYC, MYC target gene, and unfolded protein response (UPR) expression with clinical benefit from the oral aurora kinase A (AURKA) inhibitor, alisertib (A), in combination with paclitaxel (P) compared with P alone in patients (Pts) with HER2-negative metastatic breast cancer (MBC).

1037 Background: AURKA is a key regulator of the mitotic spindle, G2/M transition and epithelial-mesenchymal transition. AURKA is amplified and/or overexpressed in breast cancer and is associated with therapy resistance and worse survival. A randomized phase II trial in hormone receptor (HR)-positive, HER2-negative and triple negative (TN) MBC pts showed that addition of A to weekly P significantly improved progression-free survival (PFS) compared with P alone (O’Shaughnessy J et al. JAMA N etwork Open, 2021). Pts’ primary or metastatic disease tissues were analyzed for biomarkers associated with clinical benefit from A. Methods: Retrospective analysis of tumor whole exome and whole transcriptome sequencing of formalin-fixed paraffin embedded pre-treatment tissues from 96 pts (77 HR+, 19 TN) was performed. Clinical benefit from A+P or P was defined as having PFS of at least 6 mos and lack of benefit as PFS less than 6 mos. Enrichment for cancer gene mutations was assessed using Fisher's exact test. Transcriptome data were evaluated for differential expression with DeSeq2 and gene set enrichment analysis (GSEA) and compared to cancer hallmark gene sets. Molecular features were compared between A+P responders and non-responders, P responders and non-responders, and A+P and P responders. P-values <0.05 were considered significant. Results: PIK3CA, TP53, and CDH1 were altered in 41%, 39%, and 13% of tumors, respectively, and these and other known cancer genes were not associated with A+P benefit. Alterations in AKT1, PIK3CA, ERBB2, CDH1, TP53, and KMT2C/ KMT2D, as well as amplifications of CCND1, MYB, and MDM2, did not preclude benefit from A+P. RNA expression of AURKA, or its upstream regulator, FOXM1, was not significantly different based on treatment group or benefit from A+P or P. Increased C-MYC RNA expression (p=0.008), and enrichment for MYC targets and UPR by GSEA were enriched in pts with lack of benefit from P compared to those with benefit from P (p<0.05), but not in pts with lack of benefit from A+P. Wnt/Beta-catenin signaling was enriched in pts with lack of benefit from P compared to those with benefit from P (p=0.03), and with lack of benefit from A+P compared to those with benefit from A+P (p=0.086). Benefit from A+P was associated with enrichment for MYC targets and UPR compared with benefit from P (p=0.024), suggesting activity of A+P, but not P, with high MYC activation and UPR. Conclusions: A added to P improved PFS in HR+ HER2- and TN MBC pts compared with P alone. Pts whose breast cancers had increased C-MYC expression, high MYC activation and increased UPR on RNA expression derived greater clinical benefit from A+P than from P alone. Clinical trial information: NCT02187991 .

Use of CLIA accredited multi-omic platforms to correlate clinical significance of WES/WTS testing with functional protein/phosphoprotein drug target activity.

e15150 Background: Targeted cancer therapies almost exclusively disrupt protein function and phosphorylation, but genomics-based biomarkers underpin the vast majority of oncology molecular profiling. The reverse phase protein array (RPPA) technology has been used in the clinical setting to identify potential drug targets and to monitor the effects of treatment on protein expression and phosphorylation. Here, we investigated the Theralink RPPA assay, a highly sensitive CAP/CLIA accredited protein/phosphoprotein profiling technology, across a pan-tumor cohort of 188 samples which had undergone whole exome sequencing and transcriptomics. Methods: Using the Theralink RPPA assay, 32 key protein and phosphoprotein cancer therapy targets were quantified from formalin-fixed, laser capture microdissected (LCM) tumor epithelium across a pan-tumor cohort of 188 cancer cases. All samples had concomitant CLIA-based whole exome and whole transcriptome analysis performed. Most of these tumors were head and neck cancer (34%), uterine cancer (23%), and ovarian/fallopian tube cancer (17%), with the remaining 26% made up of various cancer types, including skin, breast, lung, brain, and other cancers. Results: Of the pan-tumor cohort, 95% of cases had at least one actionable protein target identified, compared to 86% of cases with clinically relevant genomic alterations. Altered genes and Theralink RPPA assay protein targets were broadly classified into 10 biological pathways. The pathway most frequently identified as actionable via Theralink’s RPPA assay was HER-family protein signaling (85% of cases) with concurrent genomic alterations in HER-family genes found for 9% of cases. In contrast, mTOR signaling was found to be the most frequent clinically relevant genomically altered pathway (48% of cases). When using a clinical breast cancer reference for HER2 protein/phosphorylation levels that is guiding HER2-targeted therapy in the unamplified setting, actionable total and phospho-HER2 levels were found in 82% of the pan-tumor cases, overlapping with ERBB2 genomic alterations in 4% of cases. The gene found to be altered the most was TP53 (101 cases; 54%), which was not part of the targeted Theralink RPPA assay. Conclusions: Our data suggest that quantification of key cancer proteins and phosphoproteins using RPPA extend the identification of actionable drug targets beyond what was found with WES/WTS testing alone. Especially the quantification of total and phospho-HER2 identified a significant pan-tumor subpopulation that may benefit from HER2-targeted therapies, including trastuzumab deruxtecan (T-Dxd).

Genomic analysis in patients with soft tissue sarcoma to examine gene mutations involved in chromosome segregation, mitotic spindle assembly, and degradation.

e23523 Background: Soft tissue sarcoma (STS) is a rare cancer with few drug discovery. Recent genomic analysis of STS revealed a high degree of chromosomal instability (CIN) including genome-wide copy number alterations, aneuploidy, whole genome doubling and chromothripsis. In STS patients, we have reported extensive somatic LOH in tumors, a hallmark of CIN, represented by haploid of germline mutations/variants in cancer-related genes. CIN of STS implicates abnormalities in chromosome segregation, spindle assembly and degradation in mitosis but few studies have examined the impact of gene mutations involved in the pathological mitotic processes in STS. Methods: We recruited 155 patients with metastatic/recurrent STS (135 female and 20 male, mean age 51 at analysis, 100 LMS, 19 LPS, 4 ESS, 3 UPS, 3 AS, 3 MPT and others) with information of familial cancer burden. Whole exome sequencing and analysis were performed in both blood and tumor samples as described in 2018ASCO. Results: STS Patients with tumors harboring less than 33% somatic LOH (n = 54) in a total of somatic and LOH mutations/variants in 595 COSMIC genes have a better prognosis than those (34-66%, n = 49, 67-100%, n = 52) with more LOH genes (5-year OS; 72% vs 53% or 40%, p = 0.0499, p = 0.0023, respectively). LOH was associated with neither TMB nor MSI status but with a total number of somatic and germline mutations (R = 0.874). Of the genes involved in chromosome segregation and mitosis, we found that a family of ARHGAP genes which play a role in the spindle degradation and Aurora A kinase activation was most frequently mutated in tumor and germline genomes (n = 102 in a total of 155, n = 71 in 100 LMS). Damaging ARHGAP mutations/variants are correlated with higher LOH values (54.1±2.8%, n = 102 vs. 33.1±3.8%, n = 53, mean±SE, p < 0.001, for LMS, 63.3±2.9%, n = 71 vs. 36.7±5.4%, n = 29, mean±SE, p < 0.001) and poor prognosis of patients (5-year OS; 48% n = 102 vs. 71% n = 53, p = 0.0107). We also found damaging mutations/variants in a family of CAPN (calpain) genes involved in cleavage of the cohesion ring for chromosome separation (n = 73 in 155) and CENPT gene essential for bridging between kinetochore and microtubules via NDC80 (n = 43 in 155). For CENPT, loss of wild type allele at codons encoding functional Ser121 and Arg122 was found in 10 patients with LMS and DSRCT. Damaging CAPN mutations/variants are correlated with higher LOH values (60.6±3.0%, n = 73 vs. 34.8±3.2%, n = 82, mean±SE, p < 0.001). Conclusions: This study demonstrates extensive genomic abnormalities involved in the pathways of chromosome segregation and mitosis in STS. Recent studies, including ourselves, confirmed overexpression of KIF18A, NDC80 and NUF2 in STS, lethal targets for KIF18A and CHD (Calponin Homology Domain) inhibitors in CIN tumors. Together, our results will open a new avenue evolving treatment landscape of STS.

Parkinsonism in spinocerebellar ataxia with axonal neuropathy caused by adult-onset COA7 variants: a case report

BackgroundIndividuals with variants of cytochrome c oxidase assembly factor 7 (COA7), a mitochondrial functional-related gene, exhibit symptoms of spinocerebellar ataxia with axonal neuropathy before the age of 20. However, COA7 variants with parkinsonism or adult-onset type cases have not been described.Case presentationWe report the case of a patient who developed cerebellar symptoms and slowly progressive sensory and motor neuropathy in the extremities, similar to Charcot-Marie-Tooth disease, at age 30, followed by parkinsonism at age 58. Exome analysis revealed COA7 missense mutation in homozygotes (NM_023077.2:c.17A > G, NP_075565.2: p.Asp6Gly). Dopamine transporter single-photon emission computed tomography using a 123I-Ioflupane revealed clear hypo-accumulation in the bilateral striatum. However, 123I-metaiodobenzylguanidine myocardial scintigraphy showed normal sympathetic nerve function. Levodopa administration improved parkinsonism in this patient.ConclusionsCOA7 gene variants may have caused parkinsonism in this case because mitochondrial function-related genes, such as parkin and PINK1, are known causative genes in some familial Parkinson’s diseases.

Whole exome and transcriptome analysis revealed the activation of ERK and Akt signaling pathway in canine histiocytic sarcoma

Histiocytic sarcoma (HS) is an incurable aggressive tumor, and no consensus has been made on the treatment due to its rare occurrence. Since dogs spontaneously develop the disease and several cell lines are available, they have been advocated as translational animal models. In the present study, therefore, we explored gene mutations and aberrant molecular pathways in canine HS by next generation sequencing to identify molecular targets for treatment. Whole exome sequencing and RNA-sequencing revealed gene mutations related to receptor tyrosine kinase pathways and activation of ERK1/2, PI3K-AKT, and STAT3 pathways. Analysis by quantitative PCR and immunohistochemistry revealed that fibroblast growth factor receptor 1 (FGFR1) is over-expressed. Moreover, activation of ERK and Akt signaling were confirmed in all HS cell lines, and FGFR1 inhibitors showed dose-dependent growth inhibitory effects in two of the twelve canine HS cell lines. The findings obtained in the present study indicated that ERK and Akt signaling were activated in canine HS and drugs targeting FGFR1 might be effective in part of the cases. The present study provides translational evidence that leads to establishment of novel therapeutic strategies targeting ERK and Akt signaling in HS patients.

Whole-exome sequencing reveals the metastatic potential of hepatocellular carcinoma from the perspective of tumor and circulating tumor DNA.

Relapse of hepatocellular carcinoma (HCC) due to vascular invasion is common, but the genomic mechanisms remain unclear, and molecular determinants of high-risk relapse cases are lacking. We aimed to reveal the evolutionary trajectory of microvascular invasion (MVI) and develop a predictive signature for relapse in HCC. Whole-exome sequencing was performed on tumor and peritumor tissues, portal vein tumor thrombus (PVTT), and circulating tumor DNA (ctDNA) to compare the genomic profiles between 5 HCC patients with MVI and 5 patients without MVI. We conducted an integrated analysis of exome and transcriptome to develop and validate a prognostic signature in two public cohorts and one cohort from Zhongshan Hospital, Fudan University. Shared genomic landscapes and identical clonal origins among tumor, PVTT, and ctDNA were observed in MVI ( +) HCC, suggesting that genomic changes favoring metastasis occur at the primary tumor stage and are inherited in metastatic lesions and ctDNA. There was no clonal relatedness between the primary tumor and ctDNA in MVI ( - ) HCC. HCC had dynamic mutation alterations during MVI and exhibited genetic heterogeneity between primary and metastatic tumors, which can be comprehensively reflected by ctDNA. A relapse-related gene signature named RGSHCC was developed based on the significantly mutated genes associated with MVI and shown to be a robust classifier of HCC relapse. We characterized the genomic alterations during HCC vascular invasion and revealed a previously undescribed evolution pattern of ctDNA in HCC. A novel multiomics-based signature was developed to identify high-risk relapse populations.

Genomic and Transcriptomic Analyses of Malignant Pleural Mesothelioma (MPM) Samples Reveal Crucial Insights for Preclinical Testing

Cell lines are extensively used to study cancer biology. However, the use of highly passaged commercial cell lines has to be questioned, as they do not closely resemble the originating tumor. To understand the reliability of preclinical models for Malignant pleural mesothelioma (MPM) studies, we have performed whole transcriptome and whole exome analyses of fresh frozen MPM tumors and compared them to cell lines generated from these tumors, as well as commercial cell lines and a preclinical MPM mouse model. Patient-derived cell lines were generated from digested fresh tumors and whole exome sequencing was performed on DNA isolated from formalin-fixed, paraffin-embedded (FFPE) tumor samples, corresponding patient-derived cell lines, and normal tissue. RNA sequencing libraries were prepared from 10 fresh frozen tumor samples, the 10 corresponding patient-derived cell lines, and 7 commercial cell lines. Our results identified alterations in tumor suppressor genes such as FBXW7, CDKN2A, CDKN2B, and MTAP, all known to drive MPM tumorigenesis. Patient-derived cell lines correlate to a high degree with their originating tumor. Gene expressions involved in multiple pathways such as EMT, apoptosis, myogenesis, and angiogenesis are upregulated in tumor samples when compared to patient-derived cell lines; however, they are downregulated in commercial cell lines compared to patient-derived cell lines, indicating significant differences between the two model systems. Our results show that the genome and transcriptome of tumors correlate to a higher degree with patient-derived cell lines rather than commercial cell lines. These results are of major relevance for the scientific community in regard to using cell lines as an appropriate model, resembling the pathway of interest to avoid misleading results for clinical applications.

Analysis of Clinical Laboratory Detecting Challenging Variants from Exome Sequencing Using Simulated Patient–Parent Trio Sample: Pilot Study for Neurodevelopmental Disorder de Novo Variants

To date, there has been no systematic analysis for the clinical laboratory in detecting technically challenging variants using the trio-based exome sequencing (ES) approach. Here, we present an interlaboratory pilot proficiency testing study that used synthetic patient-parent specimens to assess the detection of challenging variants with de novo dominant inheritance modes for neurodevelopmental disorders using various trio-based ES. In total, 27 clinical laboratories that performed diagnostic exome analyses participated in the survey. One of the 26 challenging variants was identified by all laboratories, whereas all 26 variants were identified by only nine laboratories. The lack of identification of mosaic variants was often due to the bioinformatics analysis that excluded the variant. For missing intended heterozygous variants, probable root causes were related to the technical bioinformatics pipeline and variant interpretation and reporting. For each missing variant, there may be more than one probable reason from the different laboratories. There was considerable variation in interlaboratory performance for detecting challenging variants using trio-based ES. This finding may have important implications for the design and validation of tests for different variant types in clinical laboratories, especially for technically challenging variants, and necessary workflow modification can potentially improve trio-based ES performance.