Use Of Whole Exome Sequencing Research Articles

Recent advances in understanding the molecular genetic basis of mitochondrial disease.

Mitochondrial disease is hugely diverse with respect to associated clinical presentations and underlying genetic causes, with pathogenic variants in over 300 disease genes currently described. Approximately half of these have been discovered in the last decade due to the increasingly widespread application of next generation sequencing technologies, in particular unbiased, whole exome—and latterly, whole genome sequencing. These technologies allow more genetic data to be collected from patients with mitochondrial disorders, continually improving the diagnostic success rate in a clinical setting. Despite these significant advances, some patients still remain without a definitive genetic diagnosis. Large datasets containing many variants of unknown significance have become a major challenge with next generation sequencing strategies and these require significant functional validation to confirm pathogenicity. This interface between diagnostics and research is critical in continuing to expand the list of known pathogenic variants and concomitantly enhance our knowledge of mitochondrial biology. The increasing use of whole exome sequencing, whole genome sequencing and other “omics” techniques such as transcriptomics and proteomics will generate even more data and allow further interrogation and validation of genetic causes, including those outside of coding regions. This will improve diagnostic yields still further and emphasizes the integral role that functional assessment of variant causality plays in this process—the overarching focus of this review.

Clinical utility of genomic analysis in adults with idiopathic liver disease

Adult patients suffering from liver disease of unknown cause represent an understudied and underserved population. The use of whole-exome sequencing (WES) for the assessment of a broader spectrum of non-oncological diseases, among adults, remains poorly studied. We assessed the utility of WES in the diagnosis and management of adults with unexplained liver disease despite comprehensive evaluation by a hepatologist and with no history of alcohol overuse. We performed WES and deep phenotyping of 19 unrelated adult patients with idiopathic liver disease recruited at a tertiary academic health care center in the US. Analysis of the exome in 19 cases identified 4 monogenic disorders in 5 unrelated adults. Patient 1 suffered for 18 years from devastating complications of undiagnosed type 3 familial partial lipodystrophy due to a deleterious heterozygous variant in PPARG. Molecular diagnosis enabled initiation of leptin replacement therapy with subsequent normalization of liver aminotransferases, amelioration of dyslipidemia, and decreases in daily insulin requirements. Patients 2 and 3 were diagnosed with MDR3 deficiency due to recessive mutations in ABCB4. Patient 4 with a prior diagnosis of non-alcoholic steatohepatitis was found to harbor a mitochondrial disorder due to a homozygous pathogenic variant in NDUFB3; this finding enabled initiation of disease preventive measures including supplementation with antioxidants. Patient 5 is a lean patient with hepatic steatosis of unknown etiology who was found to have a damaging heterozygous variant in APOB. Genomic analysis yielded an actionable diagnosis in a substantial number (∼25%) of selected adult patients with chronic liver disease of unknown etiology. This study supports the use of WES in the evaluation and management of adults with idiopathic liver disease in clinical practice. We performed whole-exome sequencing in 19 adult patients with unexplained liver disease after an unrevealing conventional work-up performed by a hepatologist. In 5 cases, genomic analysis led to a diagnosis and informed treatment and management of the disease. Therefore, we suggest using whole-exome sequencing in the evaluation and management of adults with unexplained liver disease.

Use of whole exome sequencing in the NICU: Case of an extremely low birth weight infant with syndromic features

Single gene (Mendelian) disorders are one of the leading causes of neonatal morbidity and mortality. However, in the setting of preterm birth phenotypic features of genetic diseases are often undifferentiated and are clinically very difficult to interpret based on the wide range of differential diagnoses. We report an extremely low birth weight infant (ELBW) born prematurely at 23 + 0 gestational weeks after twin pregnancy with a novel clinical manifestation with persistent hyperglycaemia as well as the known manifestations of disease-associated hypokinesia, renal salt wasting, and multifocal atrial tachycardia. The patient died of heart failure on the 72nd day of life. Whole exome sequencing (WES) revealed a previously well established, disease-causing heterozygous likely pathogenic variant in the Harvey rat sarcoma viral oncogene homolog (HRAS)-gene (c.35G > C, p. G12A, rs104894230), which implied the clinical diagnosis of Costello syndrome (CS; OMIM#190020.0004). The twin brother merely had complications related to preterm birth and did not show any CS symptoms.In conclusion, our case illustrated that CS should be considered in ELBW infants showing a life-threatening combination of complex cardiac arrhythmia and hypokinesia. If a syndromic disorder is suspected in the neonatal intensive care unit (NICU) setting, rapid WES is a useful, non-invasive diagnostic tool in critically ill ELBW infants.

Whole-exome sequencing in the evaluation of fetal structural anomalies: a prospective cohort study

Identification of chromosomal aneuploidies and copy number variants that are associated with fetal structural anomalies has substantial value. Although whole-exome sequencing (WES) has been applied to case series of a few selected prenatal cases, its value in routine clinical settings has not been prospectively assessed in a large unselected cohort of fetuses with structural anomalies. We therefore aimed to determine the incremental diagnostic yield (ie, the added value) of WES following uninformative results of standard investigations with karyotype testing and chromosomal microarray in an unselected cohort of sequential pregnancies showing fetal structural anomalies. In this prospective cohort study, the parents of fetuses who were found to have a structural anomaly in a prenatal ultrasound were screened for possible participation in the study. These participants were predominantly identified in or were referred to the Columbia University Carmen and John Thain Center for Prenatal Pediatrics (New York, NY, USA). Fetuses with confirmed aneuploidy or a causal pathogenic copy number variant were excluded from WES analyses. By use of WES of the fetuses and parents (parent-fetus trios), we identified genetic variants that indicated an underlying cause (diagnostic genetic variants) and genetic variants that met the criteria of bioinformatic signatures that had previously been described to be significantly enriched among diagnostic genetic variants. Between April 24, 2015, and April 19, 2017, 517 sequentially identified pregnant women found to have fetuses with a structural anomaly were screened for their eligibility for inclusion in our study. 71 (14%) couples declined testing, 87 (17%) trios were missing at least one DNA sample (from either parent or the fetus), 69 (13%) trios had a clinically relevant abnormal karyotype or chromosomal microarray finding, 51 (10%) couples did not consent to WES or withdrew consent, and five (1%) samples were not of good enough quality for analysis. DNA samples from 234 (45%) eligible trios were therefore used for analysis of the primary outcome. By use of trio sequence data, we identified diagnostic genetic variants in 24 (10%) families. Mutations with bioinformatic signatures that were indicative of pathogenicity but with insufficient evidence to be considered diagnostic were also evaluated; 46 (20%) of the 234 fetuses assessed were found to have such signatures. Our analysis of WES data in a prospective cohort of unselected fetuses with structural anomalies shows the value added by WES following the use of routine genetic tests. Our findings suggest that, in cases of fetal anomalies in which assessment with karyotype testing and chromosomal microarray fail to determine the underlying cause of a structural anomaly, WES can add clinically relevant information that could assist current management of a pregnancy. The unique challenges of WES-based prenatal diagnostics require analysis by a multidisciplinary team of perinatal practitioners and laboratory specialists. Institute for Genomic Medicine (Columbia University Irving Medical Center).

The utility of whole exome sequencing in diagnosing neurological disorders in adults from a highly consanguineous population

There is increasing evidence that whole exome sequencing (WES) has a high diagnostic yield and is cost-efficient for individuals with neurological phenotypes. However, there is limited data on the use of WES in non-Western populations, including populations with a high rate of consanguinity. Retrospective chart review was performed on 24 adults with undiagnosed neurological symptoms evaluated in genetics and neurology clinics in a tertiary care facility on the Arabian Peninsula, and had WES between 2014 and 2016. Definitive diagnoses were made in 13/24 (54%) of cases. Of these, 5/13 (38%) revealed novel pathogenic variants. Of the known 19/24 (79%) consanguineous cases, diagnostic rate was slightly higher, 11/19 (58%) as compared to 2/5 (40%) among non-consanguineous cases. Autosomal recessive disorders comprised 10/13 (77%) of molecular diagnoses, all found to be due to homozygous pathogenic variants among consanguineous cases. WES in this cohort of adults with neurological symptoms had a high diagnostic rate likely due to high consanguinity rates in this population, as evidenced by the high diagnostic rate of homozygous pathogenic variants.

A novel GNRHR gene mutation causing congenital hypogonadotrophic hypogonadism in a Brazilian kindred.

Congenital hypogonadotrophic hypogonadism (CHH) is a challenging inherited endocrine disorder characterised by absent or incomplete pubertal development and infertility as a result of the low action/secretion of the hypothalamic gonadotrophin-releasing hormone (GnRH). Given a growing list of gene mutations accounting for CHH, the application of massively parallel sequencing comprises an excellent molecular diagnostic approach because it enables the simultaneous evaluation of many genes. The present study proposes the use of whole exome sequencing (WES) to identify causative and modifying mutations based on a phenotype-genotype CHH analysis using an in-house exome pipeline. Based on 44 known genes related to CHH in humans, we were able to identify a novel homozygous gonadotrophin-releasing hormone receptor (GNRHR) p.Thr269Met mutant, which segregates with the CHH kindred and was predicted to be deleterious by in silico analysis. A functional study measuring intracellular inositol phosphate (IP) when stimulated with GnRH on COS-7 cells confirmed that the p.Thr269Met GnRHR mutant performed greatly diminished IP accumulation relative to the transfected wild-type GnRHR. Additionally, the proband carries three heterozygous variants in CCDC141 and one homozygous in SEMA3A gene, although their effects with respect to modifying the phenotype are uncertain. Because they do not segregate with reproductive phenotype in family members, we advocate they do not contribute to CHH oligogenicity. WES proved to be useful for CHH molecular diagnosis and reinforced its benefit with respect to identifying heterogeneous genetic disorders. Our findings expand the GnRHR mutation spectrum and phenotype-genotype correlation in CHH.

Prenatal Diagnosis by Whole Exome Sequencing in Fetuses with Ultrasound Abnormalities.

Whole-exome sequencing (WES) has been used as a standard of care for postnatal diagnosis in the clinical setting in the past few years for children and adults with undiagnosed disease. Many rare disorders have been diagnosed through WES, which is less expensive than the traditional serial genetic testing where patients had previously spent years on an uninformative diagnostic odyssey. Seeking a diagnosis often entails enduring time consuming, and sometimes invasive procedures which may be associated with medical risks that are stressful for families and impose a heavy burden on the health-care system. However, the use of WES is considered impractical in the prenatal and neonatal testing period because of the technical and computational challenges of performing genomic sequencing from small amounts of genetic material, and the need for faster turnaround time (TAT) than the current 6-8weeks TAT provided by most clinical labs offering postnatal testing. With the rapidly evolving methods of sequence analysis, there are clinical challenges such as the constantly increasing number of genes being identified which are not yet fully phenotypically characterized, especially when ascertained prenatally or neonatally before all the clinical features may be evident. Despite these challenges, there are many clinical benefits to acquiring genomic information in the prenatal and neonatal period. These include superior prognostic information which allows for prenatal planning of mode of delivery and hospital for delivery and optimized neonatal management. We have developed a clinical WES assay using small amounts of DNA with a TAT of 10days for use in the prenatal or neonatal setting. This test is used to detect small nucleotide variants and indels in fetuses and neonates with structural abnormalities.

Novel V1551L Mutation in SAMD9L Inhibits Cell Cycle Progression and Results in Pancytopenia That Progresses to MDS with Monosomy 7

Germline SAMD9L mutation is a recently recognized cause of constitutional bone marrow failure with a unique propensity for clonal evolution to myelodysplastic syndrome (MDS) with monosomy 7. It is now known that a clinical syndrome originally described in the 1970s as Ataxia-Pancytopenia Syndrome is caused by missense mutation in SAMD9L, and subsequent case series have demonstrated diverse clinical outcomes in patients with germline SAMD9L mutations. SAMD9L is located on chromosome 7, and gain-of-function mutations in SAMD9L is thought to decrease cell proliferation by inhibiting the normal cell cycle. In patients with SAMD9L mutations, the development of a monosomy 7 clone then rescues the inhibitory effect of mutant SAMD9L but can result in progression to acute leukemia. Interestingly, there are also reports of transient monosomy 7 due to uniparental disomy occurring in the monosomy 7 clone resulting in normal hematopoiesis.We describe the case of a male patient who presented at age 8-months of age with fever, hypotension, and pancytopenia. Bone marrow biopsy was hypocellular with debris-laden histiocytes. Diagnostic evaluation included normal chromosome stability testing (DEB and MMC), normal telomere length testing, and normal gene panels for inherited bone marrow failure and primary HLH. Patient, then, had spontaneous count recovery on day 41 of illness with progressive improvement becoming transfusion independent without any further infections. Repeat bone marrow biopsy 1 month after count recovery demonstrated dysplasia and cytogenetic evaluation revealed monosomy 7 in 11 out of 20 metaphases. Whole exome sequencing demonstrated a novel mutation in SAMD9L. Parents were analyzed for the SAMD9L mutation which were negative. Repeat bone marrow evaluation at 4, 7, and 10 months after presentation continued to demonstrate dysplasia and monosomy 7. After 12 months of observation, decision was made to perform a bone marrow transplant but the patient died from diffuse alveolar hemorrhage shortly after engraftment.EdU incorporation assay showed that 293T cells expressing SAMD9L V1551L mutation exhibit a cell cycle arrest phenotype as shown by the lack of cells in S phase compared to cells expressing the normal SAMD9L protein or empty vector. Furthermore, Digital droplet PCR (ddPCR) with wild type- and mutation-specific primers was used to quantify the percent of mutant allele in the patients' fibroblasts, whole blood, granulocytes, lymphocytes and lymphocyte cell cultures over 12 weeks. The patient's whole blood DNA had a low fraction of the mutation (26.8%), supporting the cytogenetic finding of mosaic monosomy 7. Interestingly, lymphocyte cell line generated from the patient's blood showed a higher fraction of mutation in about 40-45% similar to what is seen in fibroblasts which suggests that the patient's monosomy 7 clone which rescued the neutrophil count seems to preferentially contribute to the myeloid lineage versus the lymphocyte lineage. We have further developed induced pluripotent stem cells from this patient to further explore this question.In summary, our case describes a novel SAMD9L heterozygous missense mutation (p.Val1551Leu) resulting in gain-of-function mutation which inhibits cellular proliferation resulting in bone marrow failure. This case highlights the new insights offered through the use of whole exome sequencing and also the difficulty of making the clinical decision to treat with bone marrow transplant in the setting of multiple reports of disease remission via self-correction. If we are able to identify the clone giving rise to mature cells and quantify it over time, we may be able to provide a more informed recommendation regarding bone marrow transplant versus observation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Intrafamilial variability of XYLT2-related spondyloocular syndrome

Spondyloocular syndrome is characterized by generalized osteoporosis, multiple fractures and severe ocular findings. The causative XYLT2 mutations have recently been identified with the use of whole exome sequencing. We report on two siblings with spondyloocular syndrome who presented with varying clinical severity. A novel XYLT2 missense mutation was detected in a region evolutionary conserved across the species. This report along with the previous reports demonstrates that variable expressivity may be possible even within the same family. These two siblings with a novel mutation further expand the clinical and mutational spectrum of spondyloocular syndrome.

Identifying the genetic causes for prenatally diagnosed structural congenital anomalies (SCAs) by whole-exome sequencing (WES)

BackgroundWhole-exome sequencing (WES) has become an invaluable tool for genetic diagnosis in paediatrics. However, it has not been widely adopted in the prenatal setting. This study evaluated the use of WES in prenatal genetic diagnosis in fetuses with structural congenital anomalies (SCAs) detected on prenatal ultrasound.MethodThirty-three families with fetal SCAs on prenatal ultrasonography and normal chromosomal microarray results were recruited. Genomic DNA was extracted from various fetal samples including amniotic fluid, chorionic villi, and placental tissue. Parental DNA was extracted from peripheral blood when available. We used WES to sequence the coding regions of parental-fetal trios and to identify the causal variants based on the ultrasonographic features of the fetus.ResultsPathogenic mutations were identified in three families (n = 3/33, 9.1%), including mutations in DNAH11, RAF1 and CHD7, which were associated with primary ciliary dyskinesia, Noonan syndrome, and CHARGE syndrome, respectively. In addition, variants of unknown significance (VUSs) were detected in six families (18.2%), in which genetic changes only partly explained prenatal features.ConclusionWES identified pathogenic mutations in 9.1% of fetuses with SCAs and normal chromosomal microarray results. Databases for fetal genotype-phenotype correlations and standardized guidelines for variant interpretation in prenatal diagnosis need to be established to facilitate the use of WES for routine testing in prenatal diagnosis.

Coverage and diagnostic yield of Whole Exome Sequencing for the Evaluation of Cases with Dilated and Hypertrophic Cardiomyopathy

Targeted next generation sequencing of gene panels has become a popular tool for the genetic diagnosis of hypertrophic (HCM) and dilated cardiomyopathy (DCM). However, it is uncertain whether the use of Whole Exome Sequencing (WES) represents a more effective approach for diagnosis of cases with HCM and DCM. In this study, we performed indirect comparisons of the coverage and diagnostic yield of WES on genes and variants related to HCM and DCM versus 4 different commercial gene panels using 40 HCM and DCM patients, assuming perfect coverage in those panels. We identified 6 pathogenic or likely pathogenic among 14 HCM patients (diagnostic yield 43%). 3 pathogenic or likely pathogenic were found among the 26 DCM patients (diagnostic yield 12%). The coverage was similar to that of four existing commercial gene panels due to the clustering of mutation within MYH7, MYBPC3, TPM1, TNT2, and TTN. Moreover, the coverage of WES appeared inadequate for TNNI3 and PLN. We conclude that most of the pathogenic variants for HCM and DCM can be found within a small number of genes which were covered by all the commercial gene panels, and the application of WES did not increase diagnostic yield.

Compound heterozygous variants in POR gene identified by whole-exome sequencing in a Chinese pedigree with cytochrome P450 oxidoreductase deficiency.

ImportanceCytochrome P450 oxidoreductase deficiency (PORD) is a rare disease exhibiting a variety of clinical manifestations. This condition specifically leads to disordered steroidogenesis, which can affect the development of the reproductive system, skeleton, and other parts of the body. The severe form of PORD is difficult to differentiate with Antley‐Bixler syndrome (ABS). The genetic characters and clinical evaluation of PORD are still unclear in China.ObjectiveTo perform an exome analysis and identify the pathogenic cause in order to assist clinicians to obtain a proper evaluation on the genetic condition.MethodsThe proband underwent detailed physical evaluations. DNA of the proband and his parents was isolated and whole‐exome sequencing (WES) was performed. Variants were analyzed and evaluation according to the ACMG guideline.ResultsA 1‐year‐old Chinese boy with midface hypoplasia, choanal stenosis, multiple joint contractures, micropenis and right cryptorchidism was misdiagnosed with Crouzon syndrome. By trio‐whole‐exome sequencing, we identified an unreported compound heterozygous mutation (c.667C>T, p.R223* and c.1370G>A, p.R457H) in POR in the proband. This mutation was inherited from healthy heterozygous parents, supporting the diagnosis of PORD, which was further confirmed by biochemical characteristics.InterpretationWe have identified a pathogenic variant with an unreported compound heterozygous POR mutation, which expands the clinical and genetic spectra of PORD and emphasizes the usefulness of WES for genetic diagnosis.

PHP83 - Are whole Exome and whole Genome Sequencing Approaches Cost-Effective? A Systematic Review of the Literature

We conducted a systematic literature review to summarize the current health economic evidence for whole-exome sequencing (WES) and whole-genome sequencing (WGS). Relevant studies were identified in the EMBASE, MEDLINE, Cochrane Library, EconLit and University of York Centre for Reviews and Dissemination databases from January 2005 to July 2016. Publications were included in the review if they were economic evaluations, cost studies, or outcome studies. Thirty-six studies met our inclusion criteria. These publications investigated the use of WES and WGS in a variety of genetic conditions in clinical practice, the most common being neurological or neurodevelopmental disorders. Study sample size varied from a single child to 2,000 patients. Cost estimates for a single test ranged from $555 to $5,169 for WES and from $1,906 to $24,810 for WGS. Few cost analyses presented data transparently and many publications did not state which components were included in cost estimates. The current health economic evidence base to support the more widespread use of WES and WGS in clinical practice is very limited. Studies that carefully evaluate the costs, effectiveness, and cost-effectiveness of these tests are urgently needed to support their translation into clinical practice.

Mucopolysaccharidosis IIIB and mild skeletal anomalies: coexistence of NAGLU and CYP26B1 missense variations in the same patient in a Chinese family

BackgroundSanfilippo type B syndrome (mucopolysac-charidosis type IIIB; MPS IIIB) is an autosomal recessive lysosomal storage disorder. It is caused by a critically reduced α-2-acetamido-2-deoxy-D-glucoside acetamidodeoxy glucohydrolase (α-N-acetylglucosaminidase or NAGLU) activity. Recently, an autosomal recessive disorder of skeletal dysplasia associated with CYP26B1 was reported in three families, in which the patients were all homozygous variations. However, the co-occurrence of two rare diseases in a person is very rare. Here, we reported one patient with two novel pathogenic missense variations in NAGLU and CYP26B1.Case presentationWe found an infant with biallelic variation both in NAGLU-compound heterozygous c.1843C > T (p. R615C) and c.1224C > A (p. H408Q) as well as in CYP26B1-compound heterozygous c.529G > A (p. E177K) and c.525C > A (p. H175Q). All variations were novel but predicted pathogenicity according to American College of Medical Genetics and Genomics (ACMG) guidelines. The main phenotypes of the infant were quite different from those previously reported, and some were combinations of the two rare diseases, including epilepsy, early onset epileptic encephalopathy, hypermyotonia, skull deformity, dilatation of the lateral ventricles and premature closure of fontanel. His NAGLU enzyme activity was significantly decreased.ConclusionsNAGLU and CYP26B1 mutations were related to MPS IIIB and skeletal dysplasia, respectively. Here, we first reported the pathogenic mutations of two genes concurrent in one patient, which not only expands the phenotype and genotype spectra of NAGLU and CYP26B1, but more importantly indicates the possibility of simultaneous occurrence of two rare diseases in one patient. This interesting finding should be attributed to the use of whole exome sequencing (WES), which indicates that we should be aware of the importance of WES in diagnosing rare diseases.

Effectiveness of whole exome sequencing in unsolved patients with a clinical suspicion of a mitochondrial disorder in Estonia

ObjectiveReaching a genetic diagnosis of mitochondrial disorders (MDs) is challenging due to their broad phenotypic and genotypic heterogeneity. However, there is growing evidence that the use of whole exome sequencing (WES) for diagnosing patients with a clinical suspicion of an MD is effective (39–60%). We aimed to study the effectiveness of WES in clinical practice in Estonia, in patients with an unsolved, but suspected MD. We also show our first results of mtDNA analysis obtained from standard WES reads. MethodsRetrospective cases were selected from a database of 181 patients whose fibroblast cell cultures had been stored from 2003 to 2013. Prospective cases were selected during the period of 2014–2016 from patients referred to a clinical geneticist in whom an MD was suspected. We scored each patient according to the mitochondrial disease criteria (MDC) (Morava et al., 2006) after re-evaluation of their clinical data, and then performed WES analysis. ResultsA total of 28 patients were selected to the study group. A disease-causing variant was found in 16 patients (57%) using WES. An MD was diagnosed in four patients (14%), with variants in the SLC25A4, POLG, SPATA5, and NDUFB11 genes. Other variants found were associated with a neuromuscular disease (SMN1, MYH2, and LMNA genes), neurodegenerative disorder (TSPOAP1, CACNA1A, ALS2, and SCN2A genes), multisystemic disease (EPG5, NKX1–2, ATRX, and ABCC6 genes), and one in an isolated cardiomyopathy causing gene (MYBPC3). The mtDNA point mutation was found in the MT-ATP6 gene of one patient upon mtDNA analysis. ConclusionsThe diagnostic yield of WES in our cohort was 57%, proving to be a very good effectiveness. However, MDs were found in only 14% of the patients. We suggest WES analysis as a first-tier method in clinical genetic practice for children with any multisystem, neurological, and/or neuromuscular problem, as nuclear DNA variants are more common in children with MDs; a large number of patients harbor disease-causing variants in genes other than the mitochondria-related ones, and the clinical presentation might not always point towards an MD. We have also successfully conducted analysis of mtDNA from standard WES reads, providing further evidence that this method could be routinely used in the future.

The use of whole exome sequencing and murine patient derived xenografts as a method of chemosensitivity testing in sarcoma

BackgroundSoft tissue and bone sarcoma represent a broad spectrum of different pathology and genetic variance. Current chemotherapy regimens are derived from randomised trials and represent empirical treatment. Chemosensitivity testing and whole exome sequencing (WES) may offer personalized chemotherapy treatment based on genetic mutations.MethodsA pilot, prospective, non-randomised control experimental study was conducted. Twelve patients with metastatic bone or soft tissue sarcoma that had failed first line chemotherapy treatment were enrolled for this study. Human tissue taken at surgical biopsy under general anaesthetic was divided between two arms of the trial. Subsections of the tumour were used for WES and the remainder was implanted subcutaneously in immunodeficient mice (PDX). Results of WES were analysed using a bioinformatics pipeline to identify mutations conferring susceptibility to kinase inhibitors and common chemotherapeutic agents. PDX models exhibiting successful growth underwent WES of the tumour and subsequent chemosensitivity testing.ResultsWES was successful in all 12 patients, with successful establishment PDX tumours models in seven patients. WES identified potential actionable therapeutics in all patients. Significant variation in predicted therapeutics was demonstrated between three PDX samples and their matched tumour samples.ConclusionAnalysis of WES of fresh tumour specimens via a bioinformatics pipeline may identify potential actionable chemotherapy agents. Further research into this field may lead to the development of personalized cancer therapy for sarcoma.

Novel Genetic Variants of Sporadic Atrial Septal Defect (ASD) in a Chinese Population Identified by Whole-Exome Sequencing (WES).

BackgroundRecently, mutations in several genes have been described to be associated with sporadic ASD, but some genetic variants remain to be identified. The aim of this study was to use whole-exome sequencing (WES) combined with bioinformatics analysis to identify novel genetic variants in cases of sporadic congenital ASD, followed by validation by Sanger sequencing.Material/MethodsFive Han patients with secundum ASD were recruited, and their tissue samples were analyzed by WES, followed by verification by Sanger sequencing of tissue and blood samples. Further evaluation using blood samples included 452 additional patients with sporadic secundum ASD (212 male and 240 female patients) and 519 healthy subjects (252 male and 267 female subjects) for further verification by a multiplexed MassARRAY system. Bioinformatic analyses were performed to identify novel genetic variants associated with sporadic ASD.ResultsFrom five patients with sporadic ASD, a total of 181,762 genomic variants in 33 exon loci, validated by Sanger sequencing, were selected and underwent MassARRAY analysis in 452 patients with ASD and 519 healthy subjects. Three loci with high mutation frequencies, the 138665410 FOXL2 gene variant, the 23862952 MYH6 gene variant, and the 71098693 HYDIN gene variant were found to be significantly associated with sporadic ASD (P<0.05); variants in FOXL2 and MYH6 were found in patients with isolated, sporadic ASD (P<5×10−4).ConclusionsThis was the first study that demonstrated variants in FOXL2 and HYDIN associated with sporadic ASD, and supported the use of WES and bioinformatics analysis to identify disease-associated mutations.

Diagnostic challenge for the rare lysosomal storage disease: Late infantile GM1 gangliosidosis

BackgroundGM1 gangliosidosis is a rare lysosomal storage disorder caused by GLB1 mutations. Because of its extreme rarity and symptoms that overlap with other neurodegenerative diseases, its diagnosis is sometimes challenging, especially in the late infantile form with less severe phenotype. We aim to expand the clinical and genetic spectrum of late infantile GM1 gangliosidosis. MethodsWe confirmed a diagnosis of GM1 gangliosidosis based on GLB1 mutations and/or the deficiency of β-galactosidase activity. We identified the first two cases by whole-exome sequencing, and then the other six cases by direct sequencing of GLB1 with enzyme analysis. ResultsAll eight patients presented with developmental delay or regression during late infancy and later developed epilepsy, mostly intractable generalized tonic seizures. No clinical signs of storage disorders were noted except for skeletal abnormalities. Interestingly, we found aspartate transaminase (AST) elevations alone with normal alanine transaminase (ALT) levels in all patients. The recurrent mutation, p.D448V in GLB1, accounted for 50.0% of total alleles in our cohort. ConclusionsWith a high index of clinical suspicion, skeletal survey and AST level would be important for early diagnosis of GM1 gangliosidosis. In addition, we would highlight the clinical usefulness of whole-exome sequencing in the diagnosis of non-classical presentation of ultra-rare neurodegenerative disease in children.

Novel CARMIL2 Mutations in Patients with Variable Clinical Dermatitis, Infections, and Combined Immunodeficiency.

Combined immunodeficiencies are a heterogeneous collection of primary immune disorders that exhibit defects in T cell development or function, along with impaired B cell activity even in light of normal B cell maturation. CARMIL2 (RLTPR) is a protein involved in cytoskeletal organization and cell migration, which also plays a role in CD28 co-signaling of T cells. Mutations in this protein have recently been reported to cause a novel primary immunodeficiency disorder with variable phenotypic presentations. Here, we describe seven patients from three unrelated, consanguineous multiplex families that presented with dermatitis, esophagitis, and recurrent skin and chest infections with evidence of combined immunodeficiency. Through the use of whole exome sequencing and autozygome-guided analysis, we uncovered two mutations not previously reported (p.R50T and p.L846Sfs) in CARMIL2. Real-time PCR analysis revealed that the biallelic frameshift mutation is under negative selection, likely due to nonsense-mediated RNA decay and leading to loss of detectable protein upon immunoblotting. Protein loss was also observed for the missense mutation, and 3D modeling suggested a disturbance in structural stability due to an increase in the electrostatic energy for the affected amino acid and surrounding residues. Immunophenotyping revealed that patient Treg counts were significantly depressed, and that CD4+ T cells were heavily skewed towards the naïve status. CD3/CD28 signaling impairment was evidenced by reduced proliferative response to stimulation. This work broadens the allelic heterogeneity associated with CARMIL2 and highlights a deleterious missense alteration located outside the leucine-rich repeat of the protein, where all other missense mutations have been reported to date.

A novel RP1 mutation demonstrated in a Turkish family with autosomal recessive retinitis pigmentosa

Retinitis pigmentosa (RP) refers to a heterogeneous group of inherited ocular diseases that result in a progressive retinal degeneration. Due to the genetic heterogeneity of RP, 76 different forms have been described till now. Also, autosomal recessive, dominant and X-linked inheritance has also been reported. A major indication for use of whole exome sequencing (WES) in the molecular diagnosis of patients depends on the genetic heterogeneity.In this study, we performed WES for a patient diagnosed with RP. The results confirmed with Sanger sequencing revealed a novel frameshift homozygous mutation at exon 4 of RP1 gene: c.4743dupA indicating an autosomal recessive inheritance in this family.Genetic counseling is needed in such families diagnosed as retinitis pigmentosa. After finding the causative mutation in such families preimplantation genetic diagnosis can be advised for their future pregnancies.