Whole Exome Sequencing In Patients Research Articles

Genetic and phenotypic analysis of 225 Chinese children with developmental delay and/or intellectual disability using whole-exome sequencing

Developmental delay (DD), or intellectual disability (ID) is a very large group of early onset disorders that affects 1–2% of children worldwide, which have diverse genetic causes that should be identified. Genetic studies can elucidate the pathogenesis underlying DD/ID. In this study, whole-exome sequencing (WES) was performed on 225 Chinese DD/ID children (208 cases were sequenced as proband-parent trio) who were classified into seven phenotype subgroups. The phenotype and genomic data of patients with DD/ID were further retrospectively analyzed. There were 96/225 (42.67%; 95% confidence interval [CI] 36.15–49.18%) patients were found to have causative single nucleotide variants (SNVs) and small insertions/deletions (Indels) associated with DD/ID based on WES data. The diagnostic yields among the seven subgroups ranged from 31.25 to 71.43%. Three specific clinical features, hearing loss, visual loss, and facial dysmorphism, can significantly increase the diagnostic yield of WES in patients with DD/ID (P = 0.005, P = 0.005, and P = 0.039, respectively). Of note, hearing loss (odds ratio [OR] = 1.86%; 95% CI = 1.00-3.46, P = 0.046) or abnormal brainstem auditory evoked potential (BAEP) (OR = 1.91, 95% CI = 1.02–3.50, P = 0.042) was independently associated with causative genetic variants in DD/ID children. Our findings enrich the variation spectrums of SNVs/Indels associated with DD/ID, highlight the value genetic testing for DD/ID children, stress the importance of BAEP screen in DD/ID children, and help to facilitate early diagnose, clinical management and reproductive decisions, improve therapeutic response to medical treatment.

Whole Exome Sequencing of Patients with Adult-Onset Evans Syndrome: A Cohort of 80 Patients

Introduction Loss of tolerance toward several self-antigens suggests an underlying genetic predisposition. A previous study (1) conducted in children with Evans syndrome revealed a high prevalence of pathogenic mutations in genes involved in primary immunodeficiencies. Whether these findings are restricted to children is unknown. In this study, we assessed the prevalence of constitutional genetic variants in a cohort of patients with adult onset Evans syndrome. Patients and methods We consecutively included from November 2021 to June 2023 patients that had been diagnosed with adult onset (≥18 years) Evans syndrome (association of immune thrombocytopenia (ITP), warm autoimmune hemolytic anemia (wAIHA) and/or autoimmune neutropenia (AIN) according to international guidelines (2,3)), and identified in France throughout the network of the national reference center for adult' autoimmune cytopenia. We excluded patients with a systemic lupus (according to Systemic Lupus International Collaborating Clinics criteria (4)), and patients with indolent B cell malignancies at Evans syndrome onset. Clinical and biological data were retrospectively collected using a standardized form. Patients underwent whole-exome sequencing after written and informed consent was obtained. Gene variants already published and/or with in vitro confirmed consequences were considered as genetic diagnoses. Clinical interpretation of variants was performed according to the ACMG guidelines (5). A patient was classified as having a genetic diagnosis if carryinghemizygous/heterozygous pathogenic or likely pathogenic variant (for X-linked or autosomal dominant conditions), or 2 pathogenic or likely pathogenic variants in the same gene (for autosomal recessive conditions). Results We identified 80 patients (40% female, median age 31 years at Evans syndrome diagnosis [range 18-76]) with adult-onset Evans syndrome from 19 centers. Median age at first cytopenia was 28 years [range 1-73], and 13 patients (16%) were under 18 years at first cytopenia. 78 patients (96%) had ITP (chronic in 87%), 59 (73%) had wAIHA (with at least one relapse in 58%), and 38 (47%) had AIN (chronic in 74%). Median delay between first and second cytopenia was 18 months (range 0 - 329) and 28 patients (35%) had synchronous onset of cytopenia. Twenty-four patients (30%) had associated immune manifestation (involving endocrine system in 9%, lung in 9%, gastrointestinal tract in 6%, skin in 5%, central nervous system in 4%) and 28% had antinuclear antibody titers ≥ 1/160. Lymphoproliferation was found in 56%, including lymphadenopathy in 41% and splenomegaly in 35%. Nine patients (11%) had lymphoma or cancer history. Twenty patients (25%) had humoral immune defect, including 13 (16%) with a common variable immunodeficiency (CVID)-like phenotype. In the whole cohort, 9 patients (11.25%) had a genetic diagnosis that could be linked to Evans syndrome, with heterozygous variants in CTLA4, STAT3, NFKB1, CYBB, TNFAIP3, DDX41, MYH9 and homozygous variant in PIK3CG. Twenty (25%) patients had genetic testing prior to this study (targeted Sanger sequencing [N=3], or NGS panel [N=17]), resulting in 2 genetic diagnoses ( CYBB and TNFAIP3). A family history of immune manifestations in first-degree relatives was found in 56% of the patients with genetic diagnosis versus 20% of patients without (P=0.0308). However, the proportion of patients with CVID-like phenotype, associated immune disorder, lymphoproliferation, cancer/lymphoma, positive antinuclear antibodies, or pediatric onset of first cytopenia was not significantly increased in patients with genetic diagnosis. Discussion As expected, the proportion of genetic diagnoses in patients with adult onset Evans syndrome was lower that what was previously described in children, but led to a significant impact on clinical management in some patients. We did not find clinical or biological features associated with a genetic diagnosis, suggesting that genetic testing should be considered in every adult patient diagnosed with Evans syndrome in the absence of any obvious underlying cause or condition. References (1) Hadjadj J et al., Blood 2019; 134: 9-21. (2) Provan D et al., Blood Adv 2019; 3: 3780-817. (3) Jäger U et al., Blood Rev 2020; 41: 100648. (4) Petri M et al., Arthritis Rheum 2012; 64: 2677-86. (5) Richards S et al., Genet Med 2015; 17: 405-24.

STK10 Mutation Block Erythropoiesis in Acquired Pure Red Cell Aplasia Via Down-Regulated the Ribosome Biosynthesis

Background: Acquired pure red cell aplasia (PRCA) is anemia associated with the absence of erythroblasts and characterized by persistent and easy recurrence. However, the underline mechanisms of acquired PRCA remain obscure, and the role of gene mutation in the pathogenesis of acquired PRCA was not elucidated yet. Aims: To identify gene mutations in acquired PRCA patients and their possible effects on the origin of the disease. Methods: Blood and buccal genome DNA extracted from thirty newly diagnosed patients with acquired PRCA were detected with whole exome sequencing. The candidate genes with high frequency in acquired PRCA but the low frequency in 1000 normal genomes which may affect protein function were selected. The pGreen-CMV-puro system was employed to generate lentivirus expressing short hairpin RNAs to target the candidate genes, and stable transfected K562 cell lines with silenced candidate genes were constructed. The erythroid and megakaryocytic differentiation was evaluated for the transfected K562 cell lines either by benzidine staining hemoglobin and CD235a expression or CD41 expression on the cell surface. STK10 gene was selected which affects the erythropoiesis. The influence of the STK10 gene on the expression of p53 mRNA/ protein was also detected. The RNA sequencing in STK10 silenced K562 cells was performed and KEGG enrichment was analyzed. Next, ribosome RNA synthesis was detected, and ribosome proteins and p53 signaling pathway were also detected by western blotting. Results: Through whole exome sequencing in patients with acquired PRCA, we confirmed that STK10 gene mutation is common in acquired PRCA patients, the mRNA/protein expression of STK10 was reduced and p53 increased in the bone marrow of the patients in which the gene mutated. Furthermore, the silence of the STK10 gene through the lentiviral vector harboring short hairpin RNAs in K562 cells could inhibit the erythropoiesis after being induced by Hemin. Whereas, CD41 expression was similar in STK10 silenced K562 cells to control K562 cells after being inducted by phorbol 12-myristate 13-acetate. We performed RNA sequencing in STK10 silenced K562 cells. KEGG enrichment analysis of differentially expressed ribosome biosynthesis pathway and p53 signaling pathway was affected. 28S and 18S in ribosome RNA synthesis impaired in these STK10 silenced K562 cells through RNA electrophoresis. Further, through the western blotting test in STK10 silenced K562 cells, we found ribosome proteins expression down-regulated and p53, phospho-p53 and p21 expression up-regulated due to STK10 mutated. Conclusion: STK10 gene mutation is common in patients with acquired PRCA and causes the reduction of mRNAs and protein expression in the mutated patients. The underlying research revealed STK10 gene mutation could affect the ribosome biosynthesis pathway and down-regulated the ribosome protein level, contributing to the abnormal erythropoiesis. This research elucidated the pathogenesis of PRCA, which may provide evidence for precise diagnosis and exploring potential therapeutic targets in patients with acquired PRCA. Keywords: pure red cell aplasia, mutation, erythropoiesis, STK10, ribosome biosynthesis

Whole exome sequencing of patients with varicella-zoster virus and herpes simplex virus induced acute retinal necrosis reveals rare disease-associated genetic variants.

Herpes simplex virus (HSV) and varicella-zoster virus (VZV) are neurotropic human alphaherpesviruses endemic worldwide. Upon primary infection, both viruses establish lifelong latency in neurons and reactivate intermittently to cause a variety of mild to severe diseases. Acute retinal necrosis (ARN) is a rare, sight-threatening eye disease induced by ocular VZV or HSV infection. The virus and host factors involved in ARN pathogenesis remain incompletely described. We hypothesize an underlying genetic defect in at least part of ARN cases. We collected blood from 17 patients with HSV-or VZV-induced ARN, isolated DNA and performed Whole Exome Sequencing by Illumina followed by analysis in Varseq with criteria of CADD score > 15 and frequency in GnomAD < 0.1% combined with biological filters. Gene modifications relative to healthy control genomes were filtered according to high quality and read-depth, low frequency, high deleteriousness predictions and biological relevance. We identified a total of 50 potentially disease-causing genetic variants, including missense, frameshift and splice site variants and on in-frame deletion in 16 of the 17 patients. The vast majority of these genes are involved in innate immunity, followed by adaptive immunity, autophagy, and apoptosis; in several instances variants within a given gene or pathway was identified in several patients. We propose that the identified variants may contribute to insufficient viral control and increased necrosis ocular disease presentation in the patients and serve as a knowledge base and starting point for the development of improved diagnostic, prophylactic, and therapeutic applications.

Loss of zebrafish pkd1l1 causes biliary defects that have implications for biliary atresia splenic malformation.

Biliary atresia is a fibroinflammatory neonatal disease with no effective therapies. A subset of cases (10-20%) is associated with laterality defects - labeled biliary atresia splenic malformation (BASM) syndrome. Recently, whole-exome sequencing of patients with BASM identified deleterious variants in PKD1L1. PKD1L1 is involved in left-right axis determination; however, its role in cholangiocytes is unknown. We generated the pkd1l1hsc117 allele using CRISPR/Cas9 mutagenesis in zebrafish to determine the role of Pkd1l1 in biliary development and function. Wild-type and mutant larvae were assessed for laterality defects, biliary function and biliary tree architecture at 5 days post fertilization. pkd1l1hsc117 mutant larvae exhibited early left-right patterning defects. The gallbladder was positioned on the left in 47% of mutants compared to 4% of wild-type larvae. Accumulation of PED6 in the gallbladder, an indicator of hepatobiliary function, was significantly reduced in pkd1l1hsc117 mutants (46%) compared to wild-type larvae (4%). pkd1l1hsc117 larvae exhibited fewer biliary epithelial cells and reduced density of the intrahepatic biliary network compared to those in wild-type larvae. These data highlight the essential role of pkd1l1 in normal development and function of the zebrafish biliary system, supporting a role for this gene as a cause of BASM.

Proteomic and genetic analyses of influenza A viruses identify pan-viral host targets

Influenza A Virus (IAV) is a recurring respiratory virus with limited availability of antiviral therapies. Understanding host proteins essential for IAV infection can identify targets for alternative host-directed therapies (HDTs). Using affinity purification-mass spectrometry and global phosphoproteomic and protein abundance analyses using three IAV strains (pH1N1, H3N2, H5N1) in three human cell types (A549, NHBE, THP-1), we map 332 IAV-human protein-protein interactions and identify 13 IAV-modulated kinases. Whole exome sequencing of patients who experienced severe influenza reveals several genes, including scaffold protein AHNAK, with predicted loss-of-function variants that are also identified in our proteomic analyses. Of our identified host factors, 54 significantly alter IAV infection upon siRNA knockdown, and two factors, AHNAK and coatomer subunit COPB1, are also essential for productive infection by SARS-CoV-2. Finally, 16 compounds targeting our identified host factors suppress IAV replication, with two targeting CDK2 and FLT3 showing pan-antiviral activity across influenza and coronavirus families. This study provides a comprehensive network model of IAV infection in human cells, identifying functional host targets for pan-viral HDT.

Genetic characterization of non-5q proximal spinal muscular atrophy in a French cohort: the place of whole exome sequencing

Proximal spinal muscular atrophy (SMA) is defined by a degeneration of the anterior horn cells resulting in muscle weakness predominantly in the proximal lower limbs. While most patients carry a biallelic deletion in the SMN1 gene (localized in chromosome 5q), little is known regarding patients without SMN1-mutation, and a genetic diagnosis is not always possible. Here, we report a cohort of 24 French patients with non-5q proximal SMA from five neuromuscular centers who all, except two, had next-generation sequencing (NGS) gene panel, followed by whole exome sequencing (WES) if gene panel showed a negative result. The two remaining patients benefited directly from WES or whole genome sequencing (WGS). A total of ten patients with causative variants were identified, nine of whom were index cases (9/23 families = 39%). Eight variants were identified by gene panel: five variants in DYNC1H1, and three in BICD2. Compound heterozygous causative variants in ASAH1 were identified directly by WES, and one variant in DYNC1H1 was identified directly by WGS. No causative variant was found using WES in patients with a previous panel with negative results (14 cases). We thus recommend using primarily NGS panels in patients with non-5q-SMA and using WES, especially when several members of the same family are affected and/or when trio analyses are possible, or WGS as second-line testing if available.

NLRP7 participates in the human subcortical maternal complex and its variants cause female infertility characterized by early embryo arrest.

Successful human reproduction requires normal oocyte maturation, fertilization, and early embryo development. Early embryo arrest is a common phenomenon leading to female infertility, but the genetic basis is largely unknown. NLR family pyrin domain-containing 7 (NLRP7) is a member of the NLRP subfamily. Previous studies have shown that variants of NLRP7 are one of the crucial causes of female recurrent hydatidiform mole, but whether NLRP7 variants can directly affect early embryo development is unclear. We performed whole-exome sequencing in patients who experienced early embryo arrest, and five heterozygous variants (c.251G > A, c.1258G > A, c.1441G > A, c. 2227G > A, c.2323C > T) of NLRP7 were identified in affected individuals. Plasmids of NLRP7 and subcortical maternal complex components were overexpressed in 293T cells, and Co-IP experiments showed that NLRP7 interacted with NLRP5, TLE6, PADI6, NLRP2, KHDC3L, OOEP, and ZBED3. Injecting complementary RNAs in mouse oocytes and early embryos showed that NLRP7 variants influenced the oocyte quality and some of the variants significantly affected early embryo development. These findings contribute to our understanding of the role of NLRP7 in human early embryo development and provide a new genetic marker for clinical early embryo arrest patients. KEY MESSAGES: Five heterozygous variants of NLRP7 (c.1441G > A; 2227G > A; c.251G > A; c.1258G > A; c.2323C > T) were identified in five infertile patients who experienced early embryo arrest. NLRP7 is a component of human subcortical maternal complex. NLRP7 variants lead to poor quality of oocytes and early embryo development arrest. This study provides a new genetic marker for clinical early embryo arrest patients.

Combined whole exome sequencing and chromosomal microarray analysis improve clinical interpretation of genomic variants in patients with intellectual disability

IntroductionaCGH determines pathogenic copy number variations (CNVs) in about 10% of patients with intellectual disability (ID). In another 20% of patients, probably pathogenic CNVs or variants with uncertain clinical significance are detected. It may be variants that do not fully explain the patient’s symptoms, aberrations with reduced penetrance or inherited from healthy parents. The use of a sequencing method for such cases is advisable.ObjectivesImprovement of diagnosis of intellectual disability.MethodsaCGH with 60K Agilent microarrays, qPCR, targeted sequencing, whole exome sequencing (WES).Results Six patients with ID and inherited deletions/duplications detected by aCGH and their parents if available were further examined by sequencing. Four patients had maternal CNVs: (1) del1q41 (SPATA17, LINC00210, RRP15), (2) del7q35 (TCAF2, exon 8), (3) dup8p22p21.3 (PSD3, exons 1-11), and (4) del12p11.1 (SYT10, exons 1-2). Two patients had paternal CNVs: (5) dup1q44 (SMYD3, exons 2-5) and (6) del15q11.2 (TUBGCP5, CYFIP1, NIPA1, NIPA2, LOC283683). The severe phenotype of patient (5) with dup1q44 could not be explained by the paternally inherited disruption of the single SMYD3 gene. WES determined probably pathogenic SNV in the MID1 gene associated with Opitz GBBB syndrome (OMIM 300000), which corresponds better to the patient’s phenotype and is likely to be the cause of the disease. Although del1q41 is included in the region of chromosome 1q41-q42 deletion syndrome (OMIM 612530) the phenotype of the patient (1) is much milder; WES in the patient detected two pathogenic (MPO, MAN2C1) and one probably pathogenic (ARID1B) SNVs. In patient (6) with del15q11.2 pat WES detected additional pathogenic SNV in exon 7 of the ARSE gene. In patient (3) with dup8p22p21.3 WES determined two SNVs with uncertain significance in the KIDINS220, FOXG1 genes. No SNVs were detected by WES in patient (2) with del7q35. For patient (4) with del12p11.1 targeted SYT10 sequencing revealed no pathogenic SNVs as well.ConclusionsSometimes aCGH-analysis is sufficient to identify the causes of ID, however, in the case of detection of CNVs with uncertain clinical significance and/or inherited from healthy parents, it may be necessary to further examine the patient using sequencing methods. So, the accurate diagnosis was made by WES for one patient of eight. For another two patients the combination of CNVs and SNPs should be considered. For the last three patients the described aberrations could not explain the phenotype and whole genome sequencing may be the solution.This study was supported by the Russian Science Foundation, grant 21-65-00017, https://rscf.ru/project/21-65-00017/Disclosure of InterestNone Declared

Thalidomide as an Effective Treatment in Sideroblastic Anemia, Immunodeficiency, Periodic Fevers, and Developmental Delay (SIFD)

PurposeSideroblastic anemia, immunodeficiency, periodic fevers, and developmental delay (SIFD) is an autosomal recessive syndrome caused by biallelic loss-of-function variant of tRNA nucleotidyl transferase 1 (TRNT1). Efficacious methods to treat SIFD are lacking. We identified two novel mutations in TRNT1 and an efficacious and novel therapy for SIFD.MethodsWe retrospectively summarized the clinical records of two patients with SIFD from different families and reviewed all published cases of SIFD.ResultsBoth patients had periodic fever, developmental delay, rash, microcytic anemia, and B cell lymphopenia with infections. Whole-exome sequencing of patient 1 identified a previously unreported homozygous mutation of TRNT1 (c.706G > A/p.Glu236Lys). He received intravenous immunoglobulin (IVIG) replacement and antibiotics, but died at 1 year of age. Gene testing in patient 2 revealed compound heterozygous mutations (c.907C > G/p.Gln303Glu and c.88A > G/p.Met30Val) in TRNT1, the former of which is a novel mutation. Periodic fever was controlled in the first month after adalimumab therapy and IVIG replacement, but recurred in the second month. Adalimumab was discontinued and replaced with thalidomide, which controlled the periodic fever and normalized inflammatory markers effectively. A retrospective analysis of reported cases revealed 69 patients with SIFD carrying 46 mutations. The male: female ratio was 1: 1, and the mean age of onset was 3.0 months. The most common clinical manifestations in patients with SIFD were microcytic anemia (82.6%), hypogammaglobulinemia/B cell lymphopenia (75.4%), periodic fever (66.7%), and developmental delay (60.0%). In addition to the typical tetralogy, SIFD features several heterogeneous symptoms involving multiple systems. Corticosteroids, immunosuppressants, and anakinra have low efficacy, whereas etanercept suppressed fever and improved anemia in reports. Bone-marrow transplantation can be used to treat severe SIFD, but carries a high risk. In total, 28.2% (20/71) of reported patients died, mainly because of multi-organ failure. Biallelic mutations located in exon1-intron5 lead to more severe phenotypes and higher mortality. Furthermore, 15.5% (11/71) patients survived to adulthood. The symptoms could be resolved spontaneously in five patients.ConclusionsThalidomide can control the inflammation of SIFD and represents a new treatment for SIFD.

Craniofacial dysmorphism, skeletal anomalies, and impaired intellectual development syndrome-1 in two new patients with the same homozygous TMCO1 variant and review of the literature

Craniofacial dysmorphism, skeletal anomalies, and impaired intellectual development syndrome-1 (CFSMR1; OMIM#213980) is a rare autosomal recessive disorder characterized by the clinical triad of developmental delay and/or intellectual disability, a typical facial gestalt with brachycephaly, highly-arched bushy eyebrows, synophrys, hypertelorism, wide nasal bridge, and short nose, as well as multiple vertebrae and rib malformations, such as bifid and fused ribs and abnormal vertebral segmentation and fusion. Biallelic loss-of-function variants in TMCO1 cause CFSMR1. We report on two unrelated Egyptian patients with a phenotype suggestive of CFSMR. Single whole-exome sequencing in patient 1 and Sanger sequencing of TMCO1 in patient 2 revealed the same homozygous TMCO1 nonsense variant c.187C > T/p.(Arg63*) in both affected individuals; patients’ healthy parents were heterozygous carriers of the variant. Congenital hearing loss in patients 1 and 2 is an occasional finding in individuals affected by CFSMR. Camptodactyly and syndactyly, which were noted in patient 2, have not or rarely been reported in CFSMR. Review of the literature revealed a total of 30 individuals with the clinically recognizable and unique phenotype of CFSMR1, including the patients reported here, who all carried biallelic TMCO1 variants. Six different TMCO1 variants have been reported in the 30 patients from 14 families, comprising three nonsense, two 2-bp deletions, and a splice donor site variant. All disease-associated TMCO1 variants likely represent null alleles resulting in absence of the encoded protein. TMCO1 has been proposed to act as a Ca2+ channel, while other data revealed TMCO1 as a mitochondrial protein and a component of the translocon at the endoplasmic reticulum, a cellular machinery important for the biogenesis of multi-pass membrane proteins. RAB5IF/C20orf24 has recently been identified as causative gene for craniofacial dysmorphism, skeletal anomalies, and impaired intellectual development syndrome-2 (CFSMR2; OMIM#616994). Heterodimerization of RAB5IF/C20orf24 and TMCO1 and their interdependence may suggest a pathophysiological role of ER-mitochondria interaction underlying CFSMR.

Identification of novel variants in retinitis pigmentosa genes by whole-exome sequencing

Retinitis pigmentosa is an inherited degenerative disorder causing severe retinal dystrophy and visual impairment, mainly with onset in the first or second decades. The next-generation sequencing has become an efficient tool to identify disease-causing mutations in retinitis pigmentosa. The aim of this retrospective study was to investigate novel gene variants and evaluate the utility of whole-exome sequencing in patients with retinitis pigmentosa. The medical records of 20 patients with retinitis pigmentosa at Eskişehir City Hospital between September 2019 and February 2022 were analyzed retrospectively. Peripheral venous blood was obtained, followed by the extraction of genomic DNAs. The medical and ophthalmic histories were collected, and ophthalmological examinations were performed. Whole-exome sequencing was performed to determine the genetic etiology of the patients. The proportion of genetically solved cases was 75% (15/20) in the patients with retinitis pigmentosa. Molecular genetic testing identified 13 biallelic and 4 monoallelic mutations in known retinitis pigmentosa genes, including 11 novel variants. According to in silico prediction tools, nine variants were predicted as pathogenic or possibly pathogenic. We identified six previously reported mutations to be associated with retinitis pigmentosa. The age of onset of the patients ranged from 3 to 19, with a mean age of onset of 11.6. All patients had a loss of central vision. As the first study of the application of whole-exome sequencing among patients with retinitis pigmentosa in a Turkish cohort, our results may contribute to the characterization of the spectrum of variants related to retinitis pigmentosa in the Turkish population. Future population-based studies will enable us to reveal the detailed genetic epidemiology of retinitis pigmentosa.

Applying bioinformatic analysis for prognostic assessment of the &lt;i&gt;HS3ST6&lt;/i&gt; missense mutations clinical significance in the development of hereditary angioedema

Hereditary angioedema (HAE) is a genetically determined disease characterized by recurrent attacks of edema affecting the subcutaneous and/or submucosal layers of tissue, face, lips, neck, extremities of the body, oral cavity, intestine and/or larynx. In the latter case, the disease becomes life-threatening. The majority of HAE cases are associated with decreased levels of C1 (C1-esterase inhibitor), there are also descriptions of HAE with dysfunctional C1 inhibitor and HAE with normal C1 inhibitor. In the first and second variants, mutations in the C1NH gene are the cause of the disease. HAE with normal quantitative and functional levels of C1-inhibitor has the same clinical manifestations but with mutations in other genes, including F12, PLG, ANGPT1, KNG1, MYOF, and HS3ST6. Currently, mutations in the HS3ST6 gene remain poorly understood; only one missense mutation (p.Thr144Ser, rs746467957) associated with the development of HAE has been described.The aim of our work was to study new mutations in the HS3ST6 gene and analyze in silico their prognostic nature and clinical significance for the development of hereditary angioedema.The material was whole blood samples obtained from 13 patients with symptoms of hereditary angioedema without reduced levels and function of C1-INH.Whole exome sequencing of patients, bioinformatic analysis of HS3ST6 gene mutations using a number of databases and Web resources to predict the effect of mutations on the protein and assess the conservatism of the positions of the mutations detected was involved in study methods.Mutations in the HS3ST6 gene were identified in four patients, including two cases with two mutations simultaneously. Application of bioinformatic analysis allowed us to obtain new data on four missense mutations in the studied gene. Potential pathogenetic significance was determined for three of them. The mutation NC_000016.9:g.1962132G&gt;A (p.A163V) is most likely to be involved in pathogenesis of HAE by indirect disruption of heparan sulfate O-sulfation directly within the protein. The NC_000016.9:g.1962024G&gt;A mutation (p.P199L) appears to lead to the development of the disease through disruption of docking with SDC2 heparan sulfate. In the NC_000016.9:g.1962046C&gt;T (p.A192T) mutation, destabilization of the 192 amino acid position next to PAPS, may contribute to disruption of heparan sulfate O-sulfation through disruption of protein functional activity and, therefore, catalysis transfer of sulfo group to heparan sulfate syndecan-2. Thus, in all three cases, the formation of HAE appears to be possible due to disruption of the O-sulfation steps of heparan sulfate syndecan-2.Considering that in silico methods offer new opportunities to assess the pathogenetic significance of mutations, the application of bioinformatic analysis can contribute to a detailed investigation of the causes of hereditary angioedema. The present work convincingly demonstrates that rare mutations in the HS3ST6 gene may be involved in the pathogenesis of HAE and provoke edema due to increased bradykinin release.

Association of genetic variants and survival in patients with acute myeloid leukemia in rural Appalachia.

Previous population health studies examining adults with acute myeloid leukemia (AML); however many of these, such as the Cancer Genome Atlas, are derived from databases collected by large urban centers. Due to its unique industry and environmental exposures, we hypothesized the West Virginia Appalachian population may have different mutational trends and clinical outcomes. To address the concern of under-representation of rural minorities in cancer genomic databases, we performed exploratory whole exome sequencing in patients with newly diagnosed AML in rural Appalachia. Correlations between genetic variants and clinical outcome variables were examined via retrospective chart review. A total of 26 patients were identified and whole exome sequencing was performed. Median age was 68 years old. Twenty-one patients had de novo AML (84%). As per European LeukemiaNet (ELN) criteria, 8 patients were favorable (32%), 12 were intermediate (48%), and 5 were adverse risk (20%). Eight patients proceeded to transplant. The median progression-free survival and overall survival were 16.5months and 26.6months, respectively. We noted an increased tumor mutation burden and a higher frequency of specific known driver mutations when compared to The Cancer Genome Atlas database; we also found novel mutations in MUC3A, MUC5AC, HCAR3, ORT2B, and PABPC. Survival outcomes were slightly lower than national average and BCOR mutation correlated with inferior outcomes. Our findings provide novel insight into detrimental mutations in AML in a rural, underrepresented population. We discovered several novel mutations and higher frequency of some known driver mutations, which will help us identify therapeutic targets to improve patient outcomes.

Genetic Evidence That Dimerization of Glycophorin a Is Critical for Red Cell Invasion By Plasmodium Falciparum but Not for the Binding of EBA-175

Introduction: Genome-wide association studies showed that resistance to malaria may be linked to the GYPA/B/E locus (Leffler et al, Science, 2017) and it is assumed that GYPA is the receptor for P. falciparum EBA-175 ligand (Sim et al, Science, 1994). The crystal structure of the erythrocyte binding domain of EBA-175 (RII) suggests that its binding to GYPA induces dimerization of RII by assembling GYPA extracellular domain (Tolia et al, Cell, 2005), a model consistent with studies pointing out the importance of the dimer status of GYPA in this interaction (Durainsingh et al, PNAS, 2003). Thanks to the description of a new GYPA variant, we investigated the role of GYPA dimerization in RBC invasion by P. falciparum. Results: Whole exome sequencing in patient with an alloantibody to a high-prevalence antigen of unknown specificity led to the finding of eight candidate variants including a homozygous 284T>C GYPA mutation resulting in ILE95THR substitution and previously reported only at heterozygous state (frequency 0.0038 % in the Genome Aggregation Database). Others RBCs with rare blood type including Dantu and En(a-) were as reactive as the standard panel cells and the antibody was claimed to be of unknown specificity. Regarding the importance of Ile95 for GYPA dimerization (Trenker et al, JACS, 2015), we analyzed GYPA organization in RBC membranes from the proband and his homozygous brother. Indeed, immunoblotting with a specific anti-GYPA antibody showed the absence of the dimeric form of GYPA in the Ile95Thr and Dantu RBCs. Flow cytometry showed a slight decrease of GYPA but normal expression of Band3 and Wrb antigen in both variants. However, a second anti-GYPA antibody recognizing another epitope of this protein showed a strong decrease of GYPA in these variants. These results indicate a structural alteration of the GYPA in both Ile95Thr and Dantu RBCs. Interestingly, flow cytometry revealed that recombinant PfEBA-175 is able to bind the monomeric form of GYPA in Ile95Thr but not in Dantu RBCs. Finally, ex vivo invasion assays showed a drastic decreased invasion for all Ile95Thr, Dantu, En(a-) and Mk/Mk RBCs, suggesting a crucial role of GYPA dimerization for RBC invasion by P.falciparum. Conclusion: We reported a new Ile95Thrh variant of GYPA organized in a monomeric form at the RBC membrane. Although EBA-175 binding is normal, this variant seems to be resistant to P. falciparum invasion, as it is the case for Dantu and En(a-) variants. Our findings provide genetic evidence that dimerization of GYPA is critical for RBC invasion by Plasmodium falciparum but not for the binding of EBA-175. This outcome could explain the poorly elucidated mechanism of Dantu RBC resistance to P. falciparum invasion.

Associations of Tissue Tumor Mutational Burden and Mutational Status With Clinical Outcomes With Pembrolizumab Plus Chemotherapy Versus Chemotherapy For Metastatic NSCLC

IntroductionWe evaluated tissue tumor mutational burden (tTMB) and mutations in STK11, KEAP1, and KRAS as biomarkers for outcomes with pembrolizumab plus platinum-based chemotherapy (pembrolizumab-combination) for NSCLC among patients in the phase 3 KEYNOTE-189 (ClinicalTrials.gov, NCT02578680; nonsquamous) and KEYNOTE-407 (ClinicalTrials.gov, NCT02775435; squamous) trials. MethodsThis retrospective exploratory analysis evaluated prevalence of high tTMB and STK11, KEAP1, and KRAS mutations in patients enrolled in KEYNOTE-189 and KEYNOTE-407 and the relationship between these potential biomarkers and clinical outcomes. tTMB and STK11, KEAP1, and KRAS mutation status was assessed using whole-exome sequencing in patients with available tumor and matched normal DNA. The clinical utility of tTMB was assessed using a prespecified cutpoint of 175 mutations/exome. ResultsAmong patients with evaluable data from whole-exome sequencing for evaluation of tTMB (KEYNOTE-189, n = 293; KEYNOTE-407, n = 312) and matched normal DNA, no association was found between continuous tTMB score and overall survival (OS) or progression-free survival for pembrolizumab-combination (Wald test, one-sided p > 0.05) or placebo-combination (Wald test, two-sided p > 0.05) in patients with squamous or nonsquamous histology. Pembrolizumab-combination improved outcomes for patients with tTMB greater than or equal to 175 compared with tTMB less than 175 mutations/exome in KEYNOTE-189 (OS, hazard ratio = 0.64 [95% confidence interval (CI): 0.38‒1.07] and 0.64 [95% CI: 0.42‒0.97], respectively) and KEYNOTE-407 (OS, hazard ratio = 0.74 [95% CI: 0.50‒1.08 and 0.86 [95% CI: 0.57‒1.28], respectively) versus placebo-combination. Treatment outcomes were similar regardless of KEAP1, STK11, or KRAS mutation status. ConclusionsThese findings support pembrolizumab-combination as first-line treatment in patients with metastatic NSCLC and do not suggest the utility of tTMB, STK11, KEAP1, or KRAS mutation status as a biomarker for this regimen.

Trio-based whole exome sequencing in patients with suspected sporadic inborn errors of immunity: A retrospective cohort study.

<i>De novo</i> variants (DNVs) are currently not routinely evaluated as part of diagnostic whole exome sequencing (WES) analysis in patients with suspected inborn errors of immunity (IEI). This study explored the potential added value of systematic assessment of DNVs in a retrospective cohort of 123 patients with a suspected sporadic IEI that underwent patient-parent trio-based WES. A (likely) molecular diagnosis for (part) of the immunological phenotype was achieved in 12 patients with the diagnostic <i>in silico</i> IEI WES gene panel. Systematic evaluation of rare, non-synonymous DNVs in coding or splice site regions led to the identification of 14 candidate DNVs in genes with an annotated immune function. DNVs were found in IEI genes (<i>NLRP3</i> and <i>RELA</i>) and in potentially novel candidate genes, including <i>PSMB10</i>, <i>DDX1</i>, <i>KMT2C,</i> and <i>FBXW11</i>. The <i>FBXW11</i> canonical splice site DNV was shown to lead to defective RNA splicing, increased NF-κB p65 signalling, and elevated IL-1β production in primary immune cells extracted from the patient with autoinflammatory disease. Our findings in this retrospective cohort study advocate the implementation of trio-based sequencing in routine diagnostics of patients with sporadic IEI. Furthermore, we provide functional evidence supporting a causal role for <i>FBXW11</i> loss-of-function mutations in autoinflammatory disease. This research was supported by grants from the European Union, ZonMW and the Radboud Institute for Molecular Life Sciences.

Whole-exome sequencing of patients with juvenile myoclonic epilepsy

Background. Juvenile myoclonic epilepsy (JME) is the most common type of idiopathic generalized epilepsy with onset in adolescence and adulthood. During medical genetic counseling in probands with JME, aggravated epilepsy-related heredity is often detected. However, specific genetic variants of JME predisposition remain inconclusive. The use of contemporary methods of genetic analysis, particularly whole-exome and whole-genome sequencing, allows to detect, confirm and strengthen an association of any certain pathological phenotype with one or another pathogenic variant in a number of genes.Objective: to analyze the results of whole exome sequencing in patients with JME and seek for JME associations.Material and methods. The study included 7 patients with established JME diagnosis and 1 proband child without clinical signs of epilepsy. Whole exome sequencing was carried out by using MiSeq (Illumina, USA), bioinformatics analysis was performed on the Genomenal platform (Novel Software Systems, Russia).Results. Heterozygous carriage of pathogenic variants in the genes of recessive diseases was revealed: SACS, AHI1, CEP164, ANO10, RMND1, POMGNT1, FLG, ACTB. The analysis of the identified genetic variants in the patients examined showed no association with the clinical picture of the disease. Heterozygous missense mutations in CLCN2, EFHC1, JRK, ME2 genes and frameshift mutation in the CACNB4 gene were detected. Conclusion. In recent years, significant efforts were made to identify genes which predispose to JME. During our study, monogenic and/or polygenic pathogenic variants in patients with JME and a child of proband with JME were not identified. The high genetic heterogeneity of JME can explain numerous unsuccessful attempts to find genes predisposing to JME. Further research is necessary to confirm variants associated with potential JME. Advances in genomic technology can expand our understanding of the genetics of this pathology.

A TRPM7 mutation linked to familial trigeminal neuralgia: Omega current and hyperexcitability of trigeminal ganglion neurons

Trigeminal neuralgia (TN) is a unique pain disorder characterized by intense paroxysmal facial pain within areas innervated by the trigeminal nerve. Although most cases of TN are sporadic, familial clusters of TN suggest that genetic factors may contribute to this disorder. Whole-exome sequencing in patients with TN reporting positive family history demonstrated a spectrum of variants of ion channels including TRP channels. Here, we used patch-clamp analysis and Ca2+ and Na+ imaging to assess a rare variant in the TRPM7 channel, p.Ala931Thr, within transmembrane domain 3, identified in a man suffering from unilateral TN. We showed that A931T produced an abnormal inward current carried by Na+ and insensitive to the pore blocker Gd3+. Hypothesizing that replacement of the hydrophobic alanine at position 931 with the more polar threonine destabilizes a hydrophobic ring, near the voltage sensor domain, we performed alanine substitutions of F971 and W972 and obtained results suggesting a role of A931-W972 hydrophobic interaction in S3-S4 hydrophobic cleft stability. Finally, we transfected trigeminal ganglion neurons with A931T channels and observed that expression of this TRPM7 variant lowers current threshold and resting membrane potential, and increases evoked firing activity in TG neurons. Our results support the notion that the TRPM7-A931T mutation located in the S3 segment at the interface with the transmembrane region S4, generates an omega current that carries Na+ influx in physiological conditions. A931T produces hyperexcitability and a sustained Na+ influx in trigeminal ganglion neurons that may underlie pain in this kindred with trigeminal neuralgia.

Whole Exome Sequencing in Patients With Ectopic Posterior Pituitary.

Ectopic posterior pituitary (EPP), a condition in which the posterior pituitary gland is displaced due to defective neuronal migration, is frequently associated with hypopituitarism. Genetic variants play a role, but many cases remain unexplained. A large EPP cohort was studied to explore the importance of genetic variants and how they correlate with clinical findings. Whole exome sequencing was performed on a discovery sample of 27 cases to identify rare variants. The variants that met the criteria for rarity and biological relevance, or that were previously associated with EPP (ROBO1 and HESX1), were then resequenced in the 27 cases plus a replication sample of 51 cases. We identified 16 different variants in 12 genes in 15 of the 78 cases (19.2%). Complete anterior pituitary deficiency was twice as common in cases with variants of interest compared to cases without variants (9/15 [60%] vs 19/63 [30.1%], respectively; Z test, P = 0.06). Breech presentation was more frequent in the variant positive group (5/15 vs 1/63; Z test, P = 0.003). Four cases had variants in ROBO1 and 1 in HESX1, genes previously associated with EPP. The ROBO1 p.S18* variant has not been reported previously; ROBO1 p.Q1227H has not been associated with EPP previously. EPP cases with variants of interest identified in this study were more likely to present with severe clinical disease. Several variants were identified in genes not previously associated with EPP. Our findings confirm that EPP is a multigenic disorder. Future studies are needed to identify additional genes.