Application Of Whole Exome Sequencing Research Articles

Molecular Genetic Study of Congenital Ichthyosiform Erythroderma (CIE) Reveals a Novel Likely Pathogenic Variant in an Iranian Pedigree

Introduction: Congenital ichthyosiform erythroderma (CIE) is a subtype of autosomal recessive congenital ichthyosis (ARCI), a group of ineffective keratinization disorders, which mainly results from missense mutations in the transglutaminase 1 (TGM1) gene. Case Presentation: Herein, a 9-year-old male case of CIE is presented, for whom we conducted genetic testing to uncover the underlying molecular cause of his condition. We performed whole-exome sequencing (WES) on the DNA extracted from blood, and the data was analyzed for checking pathogenic variants. Analysis of the WES data identified a novel missense variant, c.1165C >T (p. Arg389Cys), in the TGM1 gene. Evaluation of this variant via in silico tools showed its detrimental consequences on the stability and function of the encoded protein. The variant was characterized as likely pathogenic based on the American College of Medical Genetics and Genomics guidelines for variant interpretation. Analysis of all available family members confirmed the co-segregation of this novel variant with the CIE disease within the family. Conclusions: This study reported the successful application of WES and bioinformatics analysis to identify a novel mutation in a well-established ARCI-causing residue in an Iranian patient.

Progress in the genetic studies of spermatogenesis abnormalities.

About 15% couples suffer from infertility, half of which are caused by male factors. Male infertility usually manifests as teratozoospermia, oligospermia and/or asthenospermia, of which the most severe form is azoospermia. In this review, we summarize the recent progress in the study of genetic factors involved in nonobstructive azoospermia and teratozoospermia, Recently, with the rapid development of high-throughput chips and sequencing technologies, many genetic factors of spermatogenesis have been discovered and analyzed. For the nonobstructive azoospermia, genome-wide association studies (GWAS) and high-throughput sequencing revealed many risk loci of nonobstructive azoospermia. For the teratozoospermia, the application of whole-exome sequencing (WES) revealed a series of disease-causing genes, greatly enriching our knowledge of teratozoospermia including multiple morphological abnormalities of the flagella (MMAF). The discovery of lots of disease genes helped the characterization of the pathological mechanisms of male infertility. Therefore, a comprehensive and in-depth understanding of genetic factors in spermatogenesis abnormalities will play important roles in the clinical diagnosis, treatment and genetic counseling of male infertility.

A ZFYVE19 gene mutation associated with neonatal cholestasis and cilia dysfunction: case report with a novel pathogenic variant

BackgroundZFYVE19 (Zinc Finger FYVE-Type Containing 19) mutations have most recently been associated to a novel type of high gamma-glutamyl transpeptidase (GGT), non-syndromic, neonatal-onset intrahepatic chronic cholestasis possibly associated to cilia dysfunction. Herein, we report a new case with further studies of whole exome sequencing (WES) and immunofluorescence in primary cilia of her cultured fibroblasts which confirm the observation.ResultsA now 5-year-old girl born to clinically healthy consanguineous Moroccan parents was assessed at 59 days of life due to severe cholestatic jaundice with increased serum bile acids and GGT, and preserved hepatocellular synthetic function. Despite fibrosis/cirrhosis and biliary ducts proliferation on liver biopsy suggested an extrahepatic biliary obstacle, normal intra-operatory cholangiography excluded biliary atresia. Under choleretic treatment, she maintained a clinically stable anicteric cholestasis but developped hyperlipidemia. After exclusion of the main causes of cholestasis by multiple tests, abnormal concentrations of sterols and WES led to a diagnosis of hereditary sitosterolemia (OMIM #618666), likely unrelated to her cholestasis. Further sequencing investigation revealed a homozygous non-sense mutation (p.Arg223Ter) in ZFYVE19 leading to a 222 aa truncated protein and present in both heterozygous parents. Immunofluorescence analysis of primary cilia on cultured skin fibroblasts showed a ciliary phenotype mainly defined by fragmented cilia and centrioles abnormalities.ConclusionsOur findings are consistent with and expands the recent evidence linking ZFYVE19 to a novel, likely non-syndromic, high GGT-PFIC phenotype with neonatal onset. Due to the possible role of ZFYVE19 in cilia function and the unprecedented coexistence of a coincidental hereditary sterol disorder in our case, continuous monitoring will be necessary to substantiate type of liver disease progression and/or possible emergence of a multisystemic involvement. What mentioned above confirms that the application of WES in children with undiagnosed cholestasis may lead to the identification of new causative genes, widening the knowledge on the pathophysiology.

Genetic diagnosis of infantile‐onset epilepsy in the clinic: Application of whole‐exome sequencing following epilepsy gene panel testing

This study aimed to evaluate the clinical utility of whole-exome sequencing in a group of infantile-onset epilepsy patients who tested negative for epilepsy using a gene panel test. Whole-exome sequencing was performed on 59 patients who tested negative on customized epilepsy gene panel testing. We identified eight pathogenic or likely pathogenic sequence variants in eight different genes (FARS2, YWHAG, KCNC1, DYRK1A, SMC1A, PIGA, OGT, and FGF12), one pathogenic structural variant (8.6 Mb-sized deletion on chromosome X [140 994 419-149 630 805]), and three putative low-frequency mosaic variants from three different genes (GABBR2, MTOR, and CUX1). Subsequent whole-exome sequencing revealed an additional 8% of diagnostic yield with genetic confirmation of epilepsy in 55.4% (62/112) of our cohort. Three genes (YWHAG, KCNC1, and FGF12) were identified as epilepsy-causing genes after the original gene panel was designed. The others were initially linked with mitochondrial encephalopathy or different neurodevelopmental disorders, although an epilepsy phenotype was listed as one of the clinical features. Application of whole-exome sequencing following epilepsy gene panel testing provided 8% of additional diagnostic yield in an infantile-onset epilepsy cohort. Whole-exome sequencing could provide an opportunity to reanalyze newly recognized epilepsy-linked genes without updating the gene panel design.

Chapter 2 - Pituitary stalk interruption syndrome

Pituitary stalk interruption syndrome (PSIS) is a distinct developmental defect of the pituitary gland identified by magnetic resonance imaging and characterized by a thin, interrupted, attenuated or absent pituitary stalk, hypoplasia or aplasia of the adenohypophysis, and an ectopic posterior pituitary. The precise etiology of PSIS still remains elusive or incompletely confirmed in most cases. Adverse perinatal events, including breech delivery and hypoxia, were initially proposed as the underlying mechanism affecting the hypothalamic-pituitary axis. Nevertheless, recent findings have uncovered a wide variety of PSIS-associated molecular defects in genes involved in pituitary development, holoprosencephaly (HPE), neural development, and other important cellular processes such as cilia function. The application of whole exome sequencing (WES) in relatively large cohorts has identified an expanded pool of potential candidate genes, mostly related to the Wnt, Notch, and sonic hedgehog signaling pathways that regulate pituitary growth and development during embryogenesis. Importantly, WES has revealed coexisting pathogenic variants in a significant number of patients; therefore, pointing to a multigenic origin and inheritance pattern of PSIS. The disorder is characterized by inter- and intrafamilial variability and incomplete or variable penetrance. Overall, PSIS is currently viewed as a mild form of an expanded HPE spectrum. The wide and complex clinical manifestations include evolving pituitary hormone deficiencies (with variable timing of onset and progression) and extrapituitary malformations. Severe and life-threatening symptomatology is observed in a subset of patients with complete pituitary hormone deficiency during the neonatal period. Nevertheless, most patients are referred later in childhood for growth retardation. Prompt and appropriate hormone substitution therapy constitutes the cornerstone of treatment. Further studies are needed to uncover the etiopathogenesis of PSIS.

Application of WES Towards Molecular Investigation of Congenital Cataracts: Identification of Novel Alleles and Genes in a Hospital-Based Cohort of South India.

Congenital cataracts are the prime cause for irreversible blindness in children. The global incidence of congenital cataract is 2.2–13.6 per 10,000 births, with the highest prevalence in Asia. Nearly half of the congenital cataracts are of familial nature, with a predominant autosomal dominant pattern of inheritance. Over 38 of the 45 mapped loci for isolated congenital or infantile cataracts have been associated with a mutation in a specific gene. The clinical and genetic heterogeneity of congenital cataracts makes the molecular diagnosis a bit of a complicated task. Hence, whole exome sequencing (WES) was utilized to concurrently screen all known cataract genes and to examine novel candidate factors for a disease-causing mutation in probands from 11 pedigrees affected with familial congenital cataracts. Analysis of the WES data for known cataract genes identified causative mutations in six pedigrees (55%) in PAX6, FYCO1 (two variants), EPHA2, P3H2, TDRD7 and an additional likely causative mutation in a novel gene NCOA6, which represents the first dominant mutation in this gene. This study identifies a novel cataract gene not yet linked to human disease. NCOA6 is a transcriptional coactivator that interacts with nuclear hormone receptors to enhance their transcriptional activator function.

Clinical Application of Whole Exome Sequencing to Identify Rare but Remediable Neurologic Disorders.

Background: The aim of this study was to describe the application of whole exome sequencing (WES) in the accurate genetic diagnosis and personalized treatment of extremely rare neurogenetic disorders. Methods: From 2017 to 2019, children with neurodevelopmental symptoms were evaluated using WES in the pediatric neurology clinic and medical genetics center. The clinical presentation, laboratory findings including the genetic results from WES, and diagnosis-based treatment and outcomes of the four patients are discussed. Results: A total of 376 children with neurodevelopmental symptom were evaluated by WES, and four patients (1.1%) were diagnosed with treatable neurologic disorders. Patient 1 (Pt 1) showed global muscle hypotonia, dysmorphic facial features, and multiple anomalies beginning in the perinatal period. Pt 1 was diagnosed with congenital myasthenic syndrome 22 of PREPL deficiency. Pt 2 presented with hypotonia and developmental arrest and was diagnosed with autosomal recessive dopa-responsive dystonia due to TH deficiency. Pt 3, who suffered from intractable epilepsy and progressive cognitive decline, was diagnosed with epileptic encephalopathy 47 with a heterozygous FGF12 mutation. Pt 4 presented with motor delay and episodic ataxia and was diagnosed with episodic ataxia type II (heterozygous CACNA1A mutation). The patients’ major neurologic symptoms were remarkably relieved with pyridostigmine (Pt 1), levodopa (Pt 2), sodium channel blocker (Pt 3), and acetazolamide (Pt 4), and most patients regained developmental milestones in the follow-up period (0.4 to 3 years). Conclusions: The early application of WES helps in the identification of extremely rare genetic diseases, for which effective treatment modalities exist. Ultimately, WES resulted in optimal clinical outcomes of affected patients.

Recent advances in sarcoidosis genomics: epigenetics, gene expression, and gene by environment (G × E) interaction studies.

We aim to review the most recent findings in genomics of sarcoidosis and highlight the gaps in the field. Original explorations of sarcoidosis subphenotypes, including cases associated with the World Trade Center and ocular sarcoidosis, have identified novel risk loci. Innovative gene--environment interaction studies utilizing modern analytical techniques have discovered risk loci associated with smoking and insecticide exposure. The application of whole-exome sequencing has identified genetic variants associated with persistent sarcoidosis and rare functional variations. A single epigenomics study has provided background knowledge of DNA methylation mechanisms in comparison with gene expression data. The application of machine-learning techniques has suggested new drug repositioning for the treatment of sarcoidosis. Several gene expression studies have identified prominent inflammatory pathways enriched in the affected tissue. Certainly, sarcoidosis research has recently advanced in the exploration of disease subphenotypes, utilizing novel analytical techniques, and including measures of clinical variation. Nevertheless, large-scale and diverse cohorts investigated with advanced sequencing methods, such as whole-genome and single-cell RNA sequencing, epigenomics, and meta-analysis coupled with cutting-edge analytic approaches, when employed, will broaden and translate genomics findings into clinical applications, and ultimately open venues for personalized medicine.

Expanded genetic insight and clinical experience of DNMT1-complex disorder.

ObjectiveTo report novel causal mutations, expanded clinical phenotypes, and clinical management of DNA methyltransferase 1 (DNMT1)-complex disorder.MethodsNeurophysiologic testing, imaging, and genetic findings were summarized in clinical context for 5 cases with DNMT1-complex disorder.ResultsWe identified 2 novel DNMT1 mutations (p.E510K and p.P1546A) by whole-exome sequencing (WES). Case 1 (p.E510K) presented with childhood ataxia, treatment-refractory seizures, and rapid cognitive decline in his 50s. Case 2 also had childhood onset and presented with seizures, language regression, hearing loss, narcolepsy with cataplexy symptoms, optic atrophy, sensory neuropathy, and hypogammaglobulinemia requiring IV immunoglobulin. Case 2 (p.P1546A) was identified with a de novo and the first mutation residing outside the targeting sequence domain. Case 3 (p.A570V) had paralytic asymmetric onset attacks triggered by emotionality and lasting sometimes for weeks. Neuropsychological testing showed executive dysfunction localizing to frontosubcortical and frontoparietal structures. He gradually developed left predominant brain atrophy. MRI showed T2 hyperintense lesions that enhanced on T1 postgadolinium images, and brain PET showed hypometabolism in atrophied regions. Case 4 (p.T497P) underwent left cochlear implant, resulting in significant hearing improvements at all tested frequencies (250–6,000 Hz). Case 5 (p.Y511H) had profound gait ataxia with posterior column atrophy of the spinal cord and abnormal evoked potentials primarily affecting the fasciculus gracilis.ConclusionsBroader application of WES further expands genotype-phenotype correlations of DNMT1-complex disorder. Two mutations are identified with early childhood onsets. The expanded new phenotypes include asymmetric brain hemiatrophy with parenchymal gadolinium enhancement, spinal cord atrophy, prolonged cataplectic spells, and hypogammaglobulinemia. Hearing loss treatment by cochlear implantation is helpful and should be considered.

A novel pathogenic variant in the LRTOMT gene causes autosomal recessive non-syndromic hearing loss in an Iranian family

BackgroundHearing loss (HL) is the most common sensorineural disorder with high phenotypic and genotypic heterogeneity, which negatively affects life quality. Autosomal recessive non-syndromic hearing loss (ARNSHL) constitutes a major share of HL cases. In the present study, Whole exome sequencing (WES) was applied to investigate the underlying etiology of HL in an Iranian patient with ARNSHL.MethodsA proband from an Iranian consanguineous family was examined via WES, following GJB2 sequencing. WES was utilized to find possible genetic etiology of the disease. Various Bioinformatics tools were used to assess the pathogenicity of the variants. Co-segregation analysis of the candidate variant was carried out. Interpretation of variants was performed according to the American College of Medical Genetics and Genomics (ACMG) guidelines.ResultsWES results showed a novel frameshift (16 bp deletion) variant (p.Ala170Alafs*20) in the LRTOMT gene. This variant, which resides in exon 6, was found to be co-segregating in the family. It fulfils the criteria set by the ACMG guidelines of being pathogenic.ConclusionHere, we report successful application of WES to identify the molecular pathogenesis of ARNSHL, which is a genetically heterogeneous disorder, in a patient with ARNSHL.

Application of Whole-Exome Sequencing in Detecting Copy Number Variants in Patients with Developmental Delay and/or Multiple Congenital Malformations

Overcoming challenges for the unambiguous detection of copy number variations is essential to broaden our understanding of the role of genomic variants in the clinical phenotype. With the improvement of software and databases, whole-exome sequencing quickly can become an excellent strategy in the routine diagnosis of patients with a developmental delay and/or multiple congenital malformations. However, even after a detailed analysis of pathogenic single-nucleotide variants and indels in known disease genes, using whole-exome sequencing, some patients with suspected syndromic conditions are left without a conclusive diagnosis. These negative results could be the result of different factors including nongenetic etiologies, lack of knowledge about the genes that cause different disease phenotypes, or, in some cases, a deletion or duplication of genomic information not routinely detectable by whole-exome sequencing variant calling. Although copy number variant detection is possible using whole-exome sequencing data, such analysis presents significant challenges and cannot yet be used to replace chromosomal arrays for identification of deletions or duplications.

Identification of two novel mutations in three Chinese families with Kallmann syndrome using whole exome sequencing.

Kallmann syndrome (KS) is a rare developmental disorder that manifests as congenital hypogonadotropic hypogonadism with anosmia. More than 19 genes have been found to be associated with KS. However, approximately 70% of the causes of KS remain unclear. Here, we studied seven KS patients, from three families, who had delayed puberty and olfactory bulb dysplasia. However, the families of these patients showed a range of other unique clinical features, including hearing loss, anosmia (to varying degrees) and unilateral renal agenesis. We performed whole exome sequencing and copy number variation (CNV) sequencing on samples acquired from these patients. We identified two novel mutations (c.844delC in ANOS1, c.475C>T in SOX10) and a novel trigenic pattern, PROKR2/CHD7/FEZF1 (c.337T>C in PROKR2, c.748C>G in FEZF1, c.8773G>A in CHD7). The c.844delC mutation in the ANOS1 gene was predicted to generate a truncated form of the anosmin-1 protein. SIFT and PolyPhen-2 predicted that the c.475C>T mutation in SOX10 had a damaging effect. The PROKR2 mutation (c.337T>C) was previously reported as harmful. No pathogenic copy number alterations were detected. Our study expands the genotypic and phenotypic spectrum of KS, a disease that shows considerable clinical and genetic heterogeneity. The application of whole exome sequencing could facilitate our understanding of the pathogenesis of KS.

The use of fetal exome sequencing in prenatal diagnosis: a points to consider document of the American College of Medical Genetics and Genomics (ACMG)

The use of fetal exome sequencing in prenatal diagnosis: a points to consider document of the American College of Medical Genetics and Genomics (ACMG)

Tiered analysis of whole-exome sequencing for epilepsy diagnosis.

It is thought that despite highly variable phenotypic expression, 70-80% of all epileptic cases are caused by one or more genetic mutations. Next generation sequencing technologies, such as whole exome sequencing (WES), can be used in a diagnostic or research setting to identify genetic mutations which may have significant prognostic implications for patients and their families. In this study, 398 genes associated with epilepsy or recurrent seizures were stratified into tiers based on genotype-phenotype concordance, tissue gene expression, frequency of affected individuals with mutations and evidence from functional and family studies. WES was completed on 14 DNA samples (2 with known mutations in SCN1A and 12 with no known mutations) from individuals diagnosed with epilepsy using an Ion AmpliSeq approach. WES confirmed positive SCN1A mutations in two samples. In n = 5/12 samples (S-3 to -14) we identified potentially causative mutations across five different genes. S-5 was identified to have a novel missense mutation in CCM2; S-6 a novel frameshift mutation identified in ADGRV1; S-10 had a previously reported pathogenic mutation in PCDH19, whilst a novel missense mutation in PCDH19 was shown in S-12; and S-13 identified to have separate missense mutations in KCNA2 and NPRL3. The application of WES followed by a targeted variant prioritization approach allowed for the discovery of potentially causative mutations in our cohort of previously undiagnosed epilepsy patients.

The application of whole exome sequencing in different metastatic potential of heptaocellular carcinoma

Objective Genome-wide exome sequencing was used to analyze DNA of hepatocellular carcinoma (HCC) tissues and cell lines with different metastatic potentials to explore the mutation genes and sites related to the HCC metastasis. Methods Genomic DNA extracted from twelve HCC tumors and its adjacent tissues and six HCC cells (HCC-LM3, MHCC-97L, MHCC-97H, HepG2, SMCC-7721 and PLC-RFP-5) with different metastatic potentials were analyzed by whole exome sequencing. Bioinformatics analysis and screening of mutations was used to investigated mutation genes and sites potentially associated with HCC metastasis. Results Comparing high and low metastatic potential HCC, high metastatic potential HCC group had more than three gene mutations including UIMC1 (rs3733876 C/T), SEMA3C (rs1527482 G/A), SAMD9 (rs10279499 G/T), RECK (rs16932912 G/A), PDLIM4 (rs4877 G/T, LIMK2 (rs3747154 A/G), CRIM1 (rs3821169 G/T) which was associated with HCC metastasis. Mutate genes and its sites such as CAPZB (rs79308175C/T), CSF1R (rs10079250A/G), HSPA5 (rs143920039A/T), SORBS3 (rs2449331T/C, rs3758036C/T) only occured in high metastatic potential HCC cells (HCC-LM3, MHCC-97L and MHCC-97H), while mutation sites RASAL3 (rs146624357G/A, rs142945276 C/T) only appeared in three low metastatic potential cells (HepG2, SMCC-7721 and PLC-RFP-5). The DNA comparative analysis of three homologous high metastatic potential HCC cell lines (HCC-LM3, MHCC-97Land MHCC-97H) showed that only EHBP1 (rs77403174T/C) was presented in HCC-LM3, while FAM189B (rs150296362C/T), SERPINB13 (rs144903442G/A) and ARHGEF1 (rs2303797C/T) only presented in MHCC-97L and MHCC-97H. Conclusion Mutations and its sites such as UIMC1 (rs3733876C/T), SEMA3C (rs1527482G/A), SAMD9 (rs10279499G/T) was detected to potentially related to the HCC metastasis by genome-wide exome sequencing. Key words: Whole exome sequencing; Hepatocellular carcinoma; Mutations; Metastasis

Whole-Exome Sequencing (WES) for Illumina Short Read SequencersUsing Solution-Based Capture.

Next-generation sequencing (NGS) is transforming clinical research and diagnostics, vastly enhancing our ability to identify novel disease-causing genetic mutations and perform comprehensive diagnostic testing in the clinic. Whole-exome sequencing (WES) is a commonly used method which captures the majority of coding regions of the genome for sequencing, as these regions contain the majority of disease-causing mutations. The clinical applications of WES are not limited to diagnosis; the technique can be employed to help determine an optimal therapeutic strategy for a patient considering their mutation profile. WES may also be used to predict a patient's risk of developing a disease, e.g., type 2 diabetes (T2D), and can therefore be used to tailor advice for the patient about lifestyle choices that could mitigate those risks. Thus, genome sequencing strategies, such as WES, underpin the emerging field of personalized medicine. Initiatives also exist for sharing WES data in public repositories, e.g., the Exome Aggregation Consortium (ExAC) database. In time, by mining these valuable data resources, we will acquire a better understanding of the roles of both single rare mutations and specific combinations of common mutations (mutation signatures) in the pathology of complex diseases such as diabetes.Herein, we describe a protocol for performing WES on genomic DNA extracted from blood or saliva. Starting with gDNA extraction, we document preparation of a library for sequencing on Illumina instruments and the enrichment of the protein-coding regions from the library using the Roche NimbleGen SeqCap EZ Exome v3 kit; a solution-based capture method. We include details of how to efficiently purify the products of each step using the AMPure XP System and describe how to use qPCR to test the efficiency of capture, and thus determine finished library quality.

Aberrant Function of the C-Terminal Tail of HIST1H1E Accelerates Cellular Senescence and Causes Premature Aging

Histones mediate dynamic packaging of nuclear DNA in chromatin, a process that is precisely controlled to guarantee efficient compaction of the genome and proper chromosomal segregation during cell division and to accomplish DNA replication, transcription, and repair. Due to the important structural and regulatory roles played by histones, it is not surprising that histone functional dysregulation or aberrant levels of histones can have severe consequences for multiple cellular processes and ultimately might affect development or contribute to cell transformation. Recently, germline frameshift mutations involving the C-terminal tail of HIST1H1E, which is a widely expressed member of the linker histone family and facilitates higher-order chromatin folding, have been causally linked to an as-yet poorly defined syndrome that includes intellectual disability. We report that these mutations result in stable proteins that reside in the nucleus, bind to chromatin, disrupt proper compaction of DNA, and are associated with a specific methylation pattern. Cells expressing these mutant proteins have a dramatically reduced proliferation rate and competence, hardly enter into the S phase, and undergo accelerated senescence. Remarkably, clinical assessment of a relatively large cohort of subjects sharing these mutations revealed a premature aging phenotype as a previously unrecognized feature of the disorder. Our findings identify a direct link between aberrant chromatin remodeling, cellular senescence, and accelerated aging.

Abstract LB-226: Utilizing 486 gene assay on a NGS platform to validate guideline-adherent tumor mutational burden (TMB) module to calculate TMB

Abstract Introduction: The quantification of TMB has emerged as a highly promising biomarker in predicting immunotherapeutic responses. The application of whole exome sequencing to measure TMB has shown to predict response to immunotherapy. Multiple studies have shown that TMB quantification using large panels are comparable to whole exome sequencing and are more practical for implementation in clinical care. The QIAseq TMB panel is a new NGS assay designed to detect genetic alterations in 486 genes, along with MSI and TMB score. Methods: The validation was guided by the joint consensus recommendation for validation of NGS assays by the AMP & CAP and NYSDOH. The validation included evaluations of precision, analytic sensitivity, analytic specificity, accuracy, reportable range, and reference range for TMB. DNA from of 60 samples which were characterized on Foundation CGP assay were used during the validation. Libraries were prepared using the QIAseq TMB panel kit and sequenced on NextSeq 550. The TMB module used for calculation of TMB in this validation was designed and developed by QIAGEN. TMB was calculated as the number of somatic mutations (SNVs and Indels only) per megabase (Mb) of the target region (1.2 Mb for QIAseq TMB panel). Results: In silico analysis using the TCGA data by QIAGEN QCI pipeline showed that the QIAseq TMB panel estimates TMB at a high level of concordance with whole exome sequencing (WES). The data show a high correlation (r2 = 0.98) between the TMB values determined using WES data and TMB using the QIAseq TMB panel data. Our validation data shows significant correlation (r2 = 0.94) among TMB values on the QIAseq panel and Foundation one CGP. Conclusions: In this validation we demonstrate that the QIAseq TMB assay can be used to reliably predict TMB across a wide range of cancer types using the QIAGEN QCI TMB module. Our lab’s experience provides an example for others that may wish to implement TMB testing by NGS on a comprehensive panel. Citation Format: PANKAJ K. AHLUWALIA, ASHIS MONDAL, SUDHA ANANTH, SALEH HENEIDI, VAMSI KOTA, RAVINDRA KOLHE. Utilizing 486 gene assay on a NGS platform to validate guideline-adherent tumor mutational burden (TMB) module to calculate TMB [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 LB-226.

Application of Whole Exome Sequencing in Congenital Secretory Diarrhea Diagnosis

Application of Whole Exome Sequencing in Congenital Secretory Diarrhea Diagnosis

OR08-4 A Novel Mutation in the Follicle-Stimulating Hormone Receptor (Ile423Thr) in a Woman with Normal Pubertal Development and Primary Amenorrhea: In Vivo, In Vitro, and In Silico Studies

Women with inactivating mutations in the FSHR may exhibit an array of phenotypes, from lack of pubertal development, primary amenorrhea, and complete insensitivity to FSH, to secondary amenorrhea and premature ovarian failure. In contrast to the LHCGR, only 19 inactivating mutations in the FSHR have been described to date. We report herein the case of a 22 year old women who presented with normal pubertal development but primary amenorrhea, associated with elevated serum FSH and LH levels (27-30 IU/L), low serum estradiol (E2) (21-40 pg/ml) and normal serum AMH (2.2-2.5 ng/ml) levels. After administration of recombinant FSH (total dose 1425 IU/9 days) serum E2 levels rose from 20 to 65 pg/ml with a parallel increase in number (from 5 to 17 follicles) and size (from 3 to 10 mm diameter) of follicles, and ovarian volumes (from 4 to 10 cm3). Sequencing of the FSHR revealed a new homozygous mutation in codon 423, where ATC was changed to ACC, generating the Ile423Thr substitution at the junction of the transmembrane domain (TM) 2 and the exo-loop (EL) 1 of the FSHR. The mother and two half-brothers were heterozygous for the same mutation. The molecular physiopathogenesis of the novel Ile423Thr mutation was analyzed by a series of in silico and in vitro biochemical studies in HEK293 cells transiently expressing either the wild-type (Wt) FSHR or the Ile423Thr mutant cloned into the pSG5 vector, and the results were compared with those obtained in cells transfected with the expression-deficient Asp408Tyr mutant, which leads to a more severe form of hypergonadotropic hypogonadism (1). Compared with the Wt FSHR, the plasma membrane expression of the mature (~80 kDa) form of the Ile423Thr mutant was slightly reduced, while that of the Asp408Tyr mutant was remarkably low, as disclosed by immunoblotting. A dissociation in the responses to FSH-stimulated signaling between the mutant receptors was found: compared with the Wt FSHR, FSH-stimulated cAMP production and ERK phosphorylation were reduced by 67% and 27% in the Ile423Thr mutant and by 52% and 43% in the Asp408Tyr FSHR, respectively. In silico molecular dynamics simulations of the FSHRs in an explicit membrane environment unveiled important differences in the behavior of both mutants vs the Wt receptor, particularly in the interhelical interactions of Thr423 with TM7 and EL2, and Tyr408 with TMs 5 and 7. Concurrently, these data suggest that conformational differences during the inactive (particularly during trafficking of the receptor proteins from the endoplasmic reticulum to the plasma membrane) and active (agonist-occupied receptor) states, account for the distinct expression levels and differential signaling exhibited by the Ile423Thr and Asp408Tyr mutant FSHRs. (1) Bramble MS, et al. A novel follicle-stimulating hormone receptor mutation causing primary ovarian failure: a fertility application of whole exome sequencing. Hum. Reprod. 2016; 31;905-914.