Heterozygous Variant Of Unknown Significance Research Articles

Expanding the Phenotype: Neurodevelopmental Disorder, Mitochondrial, With Abnormal Movements and Lactic Acidosis, With or Without Seizures (NEMMLAS) due to WARS2 Biallelic Variants, Encoding Mitochondrial Tryptophanyl-tRNA Synthase

WARS2 encodes a tryptophanyl tRNA synthetase, which is involved in mitochondrial protein synthesis. Biallelic mutations in WARS2 are rare and have been associated with a spectrum of clinical presentations, including neurodevelopmental disorder with abnormal movements, lactic acidosis with or without seizures (NEMMLAS). Here we present the case of an 8-year-old girl with ataxia and parkinsonism with periventricular white matter abnormalities on magnetic resonance imaging (MRI) and global developmental delay. The initial investigations revealed an elevated lactate level. Extensive metabolic testing, including a muscle biopsy, was inconclusive. Cerebrospinal fluid (CSF) neurotransmitter levels were low; however, a trial of levodopa was unremarkable. The chromosomal microarray and initial ataxia gene panel was normal. Zinc supplementation for a heterozygous variant of unknown significance in the CP gene on the ataxia exome panel was not effective in treating her symptoms. Reanalysis of the ataxia exome panel highlighted biallelic mutations in WARS2, which lead to the diagnosis of neurodevelopmental disorder, mitochondrial, with abnormal movements and lactic acidosis, with or without seizures (NEMMLAS). This lead to parental genetic testing, redirected therapy, and helped to expand the symptomology of this rare condition. Here we emphasize the importance of imminent and repeat expanded genetic testing to ensure early diagnosis and treatment for rare pediatric disorders. The patient is being trialed on a mitochondrial cocktail in an attempt to compensate for defects in mitochondrial protein synthesis associated with this variant. Longitudinal monitoring of disease manifestation will help establish the currently unknown natural history of this condition.

Spurious elevations of total vitamin B12 identified with polyethylene glycol precipitation procedure

Our laboratory has classified patients with methylmalonic aciduria using somatic cell studies for over four decades. We have accumulated 127 fibroblast lines from patients with persistent elevated methylmalonic acid (MMA) levels in which no genetic cause could be identified. Cultured fibroblasts from 26 of these patients had low [14C]propionate incorporation into macromolecules, possibly reflecting decreased methylmalonyl-CoA mutase function.Genome sequencing (GS), copy-number variation (CNV) analysis, and RNA sequencing were performed on genomic DNA and complementary DNA (cDNA) from these 26 patients.No patient had two pathogenic variants in any gene associated with cobalamin metabolism. Nine patients had heterozygous variants of unknown significance previously identified by a next-generation sequencing (NGS) panel targeting cobalamin metabolic genes. Three patients had pathogenic changes in genes not associated with cobalamin metabolism (PCCA, EPCAM, and a 17q12 duplication) that explain parts of their phenotypes other than elevated MMA.Genome and RNA sequencing did not detect any additional putative causal genetic defects in known cobalamin genes following somatic cell studies and the use of a targeted NGS panel. They did detect pathogenic variants in other genes in three patients that explained some aspects of their clinical presentation.

MON-103 Chromosome 15q13.3 Microdeletion And MAGEL2 Gene Mutation In A Pediatric Patient With Extreme Obesity And Tall Stature: A Case Report

Background: Clinical practice guidelines call for consideration of genetic syndromes in evaluation of pediatric patients with extreme early onset obesity with clinical features of genetic obesity syndromes or strong family history of obesity. We describe a case of two genetic variations discovered in an obese pediatric patient. Clinical Case: 10-year-old female of African American descent who presented for evaluation of early onset extreme obesity. Patient noted to have history of hyperphagia with behavioral problems and concern for abnormal coordination. Family history was limited by fact that patient was adopted in infancy without sustained contact with biological family. On physical exam, her height was in the 98th percentile, weight >99th percentile, and BMI 194% of the 95th percentile (Z+3.01). She was noted to have acanthosis nigricans without dysmorphic features. She underwent laboratory evaluation notable for HbA1c in pre-diabetes range at 5.8% (<5.7%) and dyslipidemia with triglycerides elevated at 148mg/dL (35-110mg/dL). Given her early onset of extreme obesity, she underwent genetic testing for obesity syndromes. She was found to have a heterozygous variant of unknown significance of MAGEL2 gene (c. 2572A>T). Although this particular variant has not yet been reported in the literature, a single pathogenic variant in the paternally inherited MAGEL2 gene causes disease. Pathogenic variants in the MAGEL2 gene have been associated with Schaaf-Yang or Prader-Willi-like syndrome. Schaaf-Yang syndrome has clinical overlap with Prader-Willi syndrome with a wide phenotypic spectrum and can include short stature, hypogonadism, intellectual disability, hypotonia, feeding difficulties. Our patient’s clinical phenotype is unique as she has no reported history of hypotonia or feeding difficulties, no intellectual disability, tall stature, and no dysmorphic features on exam. She was noted to have a 1.5MB microdeletion on chromosome 15q13.3 on chromosomal microarray. 15q13.3 microdeletion has been associated with a highly variable phenotype which has been reported to include obesity, intellectual disability, epilepsy, and dysmorphism. Finally, she was found to have a heterozygous variant of unknown significance of TTC8 gene. Homozygous mutations in the TTC8 gene are found in Bardet-Biedl syndrome. Conclusion: This case describes a pediatric patient with extreme obesity found to have both a chromosome 15q13.3 microdeletion and variant in MAGEL2 gene with a unique phenotype. Genetic obesity syndromes should be considered in patients who present with early onset extreme obesity, particularly in the setting of a strong family history, physical exam findings, or neurodevelopmental abnormalities. Reference: Fountain MD, Aten E, Cho MT, et al. The phenotypic spectrum of Schaaf-Yang syndrome: 18 new affected individuals from 14 families. Genet Med. 2016;19(1):45-52.

Thyroglobulin gene mutations in Chinese patients with congenital hypothyroidism

Mutations in Thyroglobulin (TG) are common genetic causes of congenital hypothyroidism (CH). But the TG mutation spectrum and its frequency in Chinese CH patients have not been investigated. Here we conducted a genetic screening of TG gene in a cohort of 382 Chinese CH patients. We identified 22 rare non-polymorphic variants including six truncating variants and 16 missense variants of unknown significance (VUS). Seven patients carried homozygous pathogenic variants, and three patients carried homozygous or compound heterozygous VUS. 48 out of 382 patients carried one of 18 heterozygous VUS which is significantly more often than their occurrences in control cohort (P < 0.0001). Unique to Asian population, the c.274+2T>G variant is the most common pathogenic variant with an allele frequency of 0.021. The prevalence of CH due to TG gene defect in Chinese population was estimated to be approximately 1/101,000. Our study uncovered ethnicity specific TG mutation spectrum and frequency.

Spectrum of Mutations Associated with Hereditary Erythrocytosis

▪Background: Mechanisms of hereditary erythrocytosis have been elucidated recently. These include high oxygen affinity (HOA) hemoglobin (Hb) variants, bisphosphoglycerate mutase (BPGM) deficiency, abnormalities in the erythropoietin receptor (EPOR) and oxygen-sensing pathway (OSP) proteins prolyl hydroxylase domain-2 (PHD2), hypoxia-inducible factor-2 alpha subunit (HIF2A), and von Hippel-Lindau (VHL). We present our experience with these disorders.Methods: Evaluation of erythrocytosis patients' blood samples for HOA Hb variants by protein or DNA sequencing methods has been performed in our laboratory for over 30 years. Testing has included alkaline/acid electrophoresis, isoelectric focusing, capillary electrophoresis, high performance liquid chromatography, mass spectrometry and Sanger sequencing. BPGM deficiency was determined by enzyme activity assay and/or genetic testing. Since 2012 we have evaluated the EPOR, EGLN1 (PHD2), EPAS1(HIF2A), and VHL genes. Reflexive evaluations, which include testing for p50 and pertinent Sanger sequencing of HBB, HBA1, HBA2, EPOR (exon 8), EGLN1 (exons 1-5), EPAS1 (exon 12) and VHL, have been performed on a subset of cases. Genetic results were correlated with clinical and phenotypic data.Results: Retrospective database review identified 62 confirmed HOA Hb variants (48β, 14α) with clinical erythrocytosis (Table 1). Multiple Hb variants were silent on at least one protein method but showed decreased p50 values. One homozygous BPGM mutation case was identified. Of the 394 cases evaluated for EPOR and OSP abnormalities, 39 cases (10%) contained genetic alterations. Of these, 11 were known pathogenic mutations and 28 were novel alterations including 4 pathogenic, 12 likely pathogenic and 12 variants of unknown significance (VUS). Eighteen EGLN1, 10 EPAS1, and 7 EPOR alterations were detected, all heterozygous. Four VHL mutation cases were identified: 3 homozygous/compound heterozygous known mutations and one novel heterozygous VUS (p.R200Q). All EPOR mutations resulted in a premature stop codon. Most of the EPAS1 alterations were located near proline 531, although two predicted splice site disturbances were found. The EGLN1 alterations were novel, variable, and affect the enzyme's catalytic domain. All 4 VHL cases involved the R200 amino acid position in at least one allele. Serum Epo levels varied: decreased in EPOR (5/5); normal in PHD2 (11/11) and most HIF2A (6/7) and VHL (3/4) cases; increased in 1 HIF2A and 1 VHL case. p50 was normal in EPOR and OSP cases (31/31 tested).Conclusion: A spectrum of abnormalities was identified. Salient points include: 1) Diagnostically, a high index of suspicion for HOA Hb variants is required as they can give false negative results by routine protein detection methods. p50 and Epo levels were useful for triage. 2) Phenotypically, serum Epo levels were low in EPOR and normal in PHD2 and HIF2A cases. Unexpectedly, the two classic Chuvash polycythemia VHL cases were associated with normal Epo levels. 3) Mechanistically, the alterations were truncating in EPOR, variable and scattered in EGLN1 and clustered near proline 531 in EPAS1. VHL mutations were rare and consistently affected the R200 amino acid position in at least one allele. Novel alterations were frequent, especially in EGLN1. 4) Clinically, many patients were asymptomatic but a subset demonstrated recurrent headaches and one had chest pain. Clotting complications included cerebrovascular accident in one EPAS1 and EGLN1 case each and portal vein thrombosis in one EGLN1 case.Table 1Clinically significant high oxygen affinity Hb variants at Mayo ClinicnBetaAlpha>30MalmoTarrant10 - 20Bethesda, San Diego, Andrew-Minneapolis, M-Saskatoon, Olympia, Syracuse, AbruzzoDallas5 - 10Ty Gard, Ypsilanti, Alberta, Coimbra, North ChicagoJ-Cape Town2 - 4Brigham, Kempsey, Little Rock, Puttelange, Wood, Brisbane, Cowtown, Creteil, Linkoping, Osler, Potomac, Providence, Ranier, Sparta, Vanderbilt, Heathrow, Helsinki, Hiroshima, Homozygous HPFH, McKees-Rocks, Pierre Benite, ReginaChesapeake, Ethiopia, Chiapas, Columbia Missouri, Burlington, Legnano1Alcorn County*, Bologna-St. Orsola, Bunbury, Cambridge-MA, Cardarelli, Chandigarh, Johnstown, Nantes, Nebraska, Olomouc, Palmerston North, Pitie-Salpetriere, Saint-Jacques, South MilwaukeeLinwood, Longview*, Milledgeville, Sarasota Springs, Voorhees*novel DisclosuresPardanani:Stemline: Research Funding.

Modified Ham Test Distinguishes aHUS from TTP and Predicts Response to Eculizumab

▪Introduction: Atypical hemolytic uremic syndrome (aHUS) is a disease of excessive alternative pathway of complement (APC) activation that can be treated with eculizumab. Clinicians have been hesitant to administer eculizumab because of the lack of a diagnostic assay for aHUS and the expense of the drug. Mutations in genes that regulate the APC (factor H/CFH, I/CFI, CD46, etc.) underlie up to 50% of aHUS patients, but the clinical relevance of these mutations is often unclear. We developed the modified Ham test to distinguish aHUS from thrombotic thrombocytopenic purpura (TTP). This functional assay measures cell viability (WST-1) of a PIGA mutant cell line following exposure to patient serum. The cells are more sensitive to APC killing because complement regulators CD55 and CD59 are absent from the cell surface. CD46 is a transmembrane APC regulator that remains on the surface of the PIGA- null TF1 cells. Here, we show that the modified Ham test predicts response to eculizumab and can be adapted to assess the contribution of APC mutations to clinical phenotype.Method: We studied adult patients with thrombotic microangiopathies (TMA) who presented to our hospital from July 2014 through June 2015 and two patients from outside institutions. aHUS was defined by the presence of a TMA with platelet count <100 x 109/L, serum creatinine >2.25 mg/dL, and ADAMTS13 activity >10%. TTP was defined as TMA in association with an ADAMTS13 ≤ 10%. Response to eculizumab was defined as TMA event-free status for at least 12 weeks and normalization of hematologic values. The modified Ham test was performed utilizing PIGA-null myeloid TF1 (PIGA only knockout/KO) as previously described. We additionally knocked-out CD46 / membrane cofactor protein (MCP) gene in the PIGA-null TF-1 cell line (PIGA/CD46 double KO), and wild-type TF1 cells (CD46 only KO) using CRISPR/Cas9 technology.Results: The modified Ham test was performed using PIGA-null TF1 reagent cells with 37 serum samples from 29 patients with TMAs. 13 patients were diagnosed with aHUS, 15 with TTP, and 1 with TMA of unknown etiology. APC-mediated cell killing was significantly increased in patients with aHUS compared to TTP (Figure 1A, grey shapes symbolize patients on eculizumab). In the ROC analysis, the cut-off value of 20.5% killing suggests an aHUS diagnosis with 100% specificity and 94.1% sensitivity. One patient presented with a TMA of unknown etiology (normal ADAMTS13 levels, and did not satisfy criteria for aHUS due to a creatinine of <1.3; no known complement-related mutations or autoantibodies were detected). The patient showed no increased killing with the modified Ham test and had a transient response to plasma exchange and eculizumab but relapsed after 8 weeks of eculizumab treatment.12 aHUS patients received eculizumab and all responded to treatment. Eculizumab was discontinued in 6 patients after a median 11 doses (range 5-37) and all remain in remission for 8 months (range 4-15). The modified Ham test was positive in all aHUS patients that responded to eculizumab (positive predictive value 100%) and negative in 1 responder with a heterozygous variant of unknown significance in DGKE. Among 12 patients with aHUS, 7 patients had mutations in genes previously associated with aHUS (3 CFH, 1 DGKE, 1 CFH and CD46, 1 CFH and ADAMTS13, and 1 CFH and thrombomodulin). Six of those patients had additional heterozygous deletions in the CFHR3-CFHR1 gene region.To study the impact of CD46 insufficiency on complement regulation we exposed PIGA/CD46 double KO TF1, and CD46 only KO lines to aHUS serum and measured cell viability in the modified Ham test. The CD46 KO line was no more susceptible to killing than wild-type TF1 cells and the PIGA/CD46 double KO cells were no more sensitive to killing than the PIGAonly KO cells, suggesting that CD46 is a weak complement regulator compared to the combination of CD55 and CD59 (Figure 1B).Conclusions: The modified Ham test reliably distinguishes most forms of aHUS from TTP and predicts response to eculizumab. Mutations in CD46 do not impact susceptibility to APC killing in the modified Ham test. The modified Ham test may be adapted to inform investigators and clinicians on the correlation between genotype and phenotype. [Display omitted] DisclosuresMoliterno:Incyte: Membership on an entity's Board of Directors or advisory committees. Sperati:Alexion Pharmaceuticals: Other: Principal investigator for the international aHUS registry. Brodsky:Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Whole exome sequencing is an efficient, sensitive and specific method for determining the genetic cause of short-rib thoracic dystrophies.

Short-rib thoracic dystrophies (SRTDs) are congenital disorders due to defects in primary cilium function. SRTDs are recessively inherited with mutations identified in 14 genes to date (comprising 398 exons). Conventional mutation detection (usually by iterative Sanger sequencing) is inefficient and expensive, and often not undertaken. Whole exome massive parallel sequencing has been used to identify new genes for SRTD (WDR34, WDR60 and IFT172); however, the clinical utility of whole exome sequencing (WES) has not been established. WES was performed in 11 individuals with SRTDs. Compound heterozygous or homozygous mutations were identified in six confirmed SRTD genes in 10 individuals (IFT172, DYNC2H1, TTC21B, WDR60, WDR34 and NEK1), giving overall sensitivity of 90.9%. WES data from 993 unaffected individuals sequenced using similar technology showed two individuals with rare (minor allele frequency <0.005) compound heterozygous variants of unknown significance in SRTD genes (specificity >99%). Costs for consumables, laboratory processing and bioinformatic analysis were <AU$850 per sample. WES is sensitive, specific, efficient and cost-effective for mutation screening as well as gene discovery in SRTDs and can be considered a first-line methodology for mutation identification in affected individuals.

De NovoRPS20 Mutations in Diamond Blackfan Anemia

Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by pure red blood cell aplasia, variable congenital anomalies and increased risk of malignancy. Approximately 50-60% of cases are due to germline, heterozygous mutations in 1 of 13 genes that encode components of either the small (RPS7, RPS10, RPS17, RPS19, RPS24, RPS26 and RPS29) or large (RPL5, RPL11, RPL15, RPL26, RPL31 and RPL35A) ribosomal subunits. The mutations may be either inherited or de novo. Consistent with DBA being a ribosomopathy, defects in ribosome assembly, altered ribosomal RNA processing and nucleolar stress are observed in cells of patients with DBA and ribosomal protein gene mutations. Mutations in GATA1, a hematopoietic transcription factor, have also been reported in rare X-linked recessive cases.Whole exome sequencing was independently carried out on two white/Hispanic probands with genetically uncharacterized DBA (BMF92 and NCI-62-1). Both presented with transfusion-dependent anemia during the first week of life. Bone marrow evaluations revealed marked erythroid hypoplasia characteristic of DBA. One of the probands (BMF92) experienced a spontaneous remission of his anemia toward the end of the first year of life, possibly related to corticosteroids administered for bronchiolitis. Subsequently, his erythrocyte adenosine deaminase(eADA) level was measured and elevated [10.5 units (mol/min/gm hg); reference range 0.42-3.5 units]. BMF92’s hypoplastic anemia later recurred and was found to be steroid refractory. NCI-62-1 is a male, now 12 years of age and red cell transfusion dependent. BMF92 and NCI-62-1 are of normal stature and have no documented congenital anomalies, although both have experienced chronic colitis. In both cases, family history for DBA was negative and parental eADA levels were within normal limits.Whole exome sequencing of peripheral blood DNA, validated by Sanger or Ion Torrent targeted sequencing, identified heterozygous variants of unknown significance in the ribosomal protein gene RPS20. Parental studies revealed both variants to be de novo. The variants were mutated at the same genomic position, but resulted in different amino acid substitutions in RPS20 [hg19 chr8 56985758, c.251A>T, p.I84N (BMF92) and 56985758, c.251A>C, p.I84S (NCI-62-1)]. Buccal swab analysis performed in BMF92 confirmed the presence of the variant. Both variants were novel, based on inspection of several databases, including dbSNP, 1000 Genomes, NHLBI Exome Sequencing Project, clinical WES data at the Whole Genome Laboratory at Baylor College of Medicine, Kaviar, Human Genome Mutation Database and ClinVar, totally well over 10,000 individuals as well as the COSMIC database, which reports somatic mutations in cancer. In silicoanalyses were consistent with the variants being damaging and disease causing (PolyPhen-2, SIFT and Mutation Taster), affecting highly evolutionarily conserved residues (GERP, PhyloP and Sitewise likelihood-ratio score) and decreasing the stability of the protein structure (MUpro and I-Mutant2.0).The above data strongly suggest that the RPS20 variants in these cases are disease causing. A recent report implicated a germline RPS20 truncating mutation in a four-generation pedigree with familial nonpolyposis colorectal carcinoma; however, the mutation carriers were reported to not manifest features of DBA (Nieminen et al., Gastroenterology 2014). This raises the likelihood of allele-specific effects. Functional assays of our probands’ mutations, including quantification of RPS20 protein and ribosomal RNA precursor steady state levels in patient versus control lymphoblastoid cell lines as well as analysis of the impact of mutant RPS20 protein expression on p53 and downstream targets are underway to determine their contribution to the DBA phenotype. DisclosuresNo relevant conflicts of interest to declare.

Mutation analysis of 18 nephronophthisis associated ciliopathy disease genes using a DNA pooling and next generation sequencing strategy

BackgroundNephronophthisis associated ciliopathies (NPHP-AC) comprise a group of autosomal recessive cystic kidney diseases that includes nephronophthisis (NPHP), Senior-Loken syndrome (SLS), Joubert syndrome (JBTS), and Meckel-Gruber syndrome (MKS). To date, causative...