Concordant Variants Research Articles

Genome-wide meta-analyses of cross substance use disorders in European, African, and Latino ancestry populations.

Genetic risks for substance use disorders (SUDs) are due to both SUD-specific and SUD-shared genes. We performed the largest multivariate analyses to date to search for SUD-shared genes using samples of European (EA), African (AA), and Latino (LA) ancestries. By focusing on variants having cross-SUD and cross-ancestry concordant effects, we identified 45 loci. Through gene-based analyses, gene mapping, and gene prioritization, we identified 250 SUD-shared genes. These genes are highly expressed in amygdala, cortex, hippocampus, hypothalamus, and thalamus, primarily in neuronal cells. Cross-SUD concordant variants explained ~ 50% of the heritability of each SUD in EA. The top 5% individuals having the highest polygenic scores were approximately twice as likely to have SUDs as others in EA and LA. Polygenic scores had higher predictability in females than in males in EA. Using real-world data, we identified five drugs targeting identified SUD-shared genes that may be repurposed to treat SUDs.

Multisite Validation of a Functional Assay to Adjudicate SCN5A Brugada Syndrome-Associated Variants.

Brugada syndrome is an inheritable arrhythmia condition that is associated with rare, loss-of-function variants in SCN5A. Interpreting the pathogenicity of SCN5A missense variants is challenging, and ≈79% of SCN5A missense variants in ClinVar are currently classified as variants of uncertain significance. Automated patch clamp technology enables high-throughput functional studies of ion channel variants and can provide evidence for variant reclassification. An in vitro SCN5A-Brugada syndrome automated patch clamp assay was independently performed at Vanderbilt University Medical Center and Victor Chang Cardiac Research Institute. The assay was calibrated according to ClinGen Sequence Variant Interpretation recommendations using high-confidence variant controls (n=49). Normal and abnormal ranges of function were established based on the distribution of benign variant assay results. Odds of pathogenicity values were derived from the experimental results according to ClinGen Sequence Variant Interpretation recommendations. The calibrated assay was then used to study SCN5A variants of uncertain significance observed in 4 families with Brugada syndrome and other arrhythmia phenotypes associated with SCN5A loss-of-function. Variant channel parameters generated independently at the 2 research sites showed strong correlations, including peak INa density (R2=0.86). The assay accurately distinguished benign controls (24/25 concordant variants) from pathogenic controls (23/24 concordant variants). Odds of pathogenicity values were 0.042 for normal function and 24.0 for abnormal function, corresponding to strong evidence for both American College of Medical Genetics and Genomics/Association for Molecular Pathology benign and pathogenic functional criteria (BS3 and PS3, respectively). Application of the assay to 4 clinical SCN5A variants of uncertain significance revealed loss-of-function for 3/4 variants, enabling reclassification to likely pathogenic. This validated high-throughput assay provides clinical-grade functional evidence to aid the classification of current and future SCN5A-Brugada syndrome variants of uncertain significance.

A PCR-independent approach for mtDNA enrichment and next-generation sequencing: comprehensive evaluation and clinical application

BackgroundSequencing the mitochondrial genome has been increasingly important for the investigation of primary mitochondrial diseases (PMD) and mitochondrial genetics. To overcome the limitations originating from PCR-based mtDNA enrichment, we set out to develop and evaluate a PCR-independent approach in this study, named Pime-Seq (PCR-independent mtDNA enrichment and next generation Sequencing).ResultsBy using the optimized mtDNA enrichment procedure, the mtDNA reads ratio reached 88.0 ± 7.9% in the sequencing library when applied on human PBMC samples. We found the variants called by Pime-Seq were highly consistent among technical repeats. To evaluate the accuracy and reliability of this method, we compared Pime-Seq with lrPCR based NGS by performing both methods simultaneously on 45 samples, yielding 1677 concordant variants, as well as 146 discordant variants with low-level heteroplasmic fraction, in which Pime-Seq showed higher reliability. Furthermore, we applied Pime-Seq on 4 samples of PMD patients retrospectively, and successfully detected all the pathogenic mtDNA variants. In addition, we performed a prospective study on 192 apparently healthy pregnant women during prenatal screening, in which Pime-Seq identified pathogenic mtDNA variants in 4 samples, providing extra information for better health monitoring in these cases.ConclusionsPime-Seq can obtain highly enriched mtDNA in a PCR-independent manner for high quality and reliable mtDNA deep-sequencing, which provides us an effective and promising tool for detecting mtDNA variants for both clinical and research purposes.

Circulating Tumor DNA in Head and Neck Squamous Cell Carcinoma: Association with Metabolic Tumor Burden Determined with FDG-PET/CT.

The detection of circulating tumor DNA (ctDNA) with next-generation sequencing (NGS) in venous blood is a promising tool for the genomic profiling of head and neck squamous cell carcinoma (HNSCC). The association between ctDNA findings and metabolic tumor burden detected with FDG-PET/CT imaging is of particular interest for developing prognostic and predictive algorithms in HNSCC. Twenty-six prospectively enrolled HNSCC patients were eligible for further analysis. All patients underwent tumor tissue and venous liquid biopsy sampling and FDG-PET/CT before definitive oncologic treatment. An NGS-based commercial panel was used for a genomic analysis of the samples. Maximum variant allele frequency (VAF) in blood correlated positively with whole-body (WB) metabolic tumor volume (MTV) and total lesion glycolysis (TLG) (r = 0.510, p = 0.008 and r = 0.584, p = 0.002, respectively). A positive liquid biopsy was associated with high WB-TLG using VAF ≥ 1.00% or ≥5.00% as a cut-off value (p = 0.006 or p = 0.003, respectively). Additionally, ctDNA detection was associated with WB-TLG when only concordant variants detected in both ctDNA and tissue samples were considered. A high metabolic tumor burden based on FDG imaging is associated with a positive liquid biopsy and high maximum VAF. Our findings suggest a complementary role of metabolic and genomic signatures in the pre-treatment evaluation of HNSCC.

MP14-08 CHARACTERIZING VARIABILITY IN GENOMIC ALTERATIONS BETWEEN INDEX AND REPEAT TRANSURETHRAL RESECTION LESIONS IN NON-MUSCLE INVASIVE BLADDER CANCER

You have accessJournal of UrologyCME1 Apr 2023MP14-08 CHARACTERIZING VARIABILITY IN GENOMIC ALTERATIONS BETWEEN INDEX AND REPEAT TRANSURETHRAL RESECTION LESIONS IN NON-MUSCLE INVASIVE BLADDER CANCER Prithvi Murthy, Kyle Rose, Billie Gould, Facundo Davaro, Pan Du, Lucia Camperlengo, Philippe Spiess, Wade Sexton, Scott Gilbert, Daniel Grass, Aram Vosoughi, and Roger Li Prithvi MurthyPrithvi Murthy More articles by this author , Kyle RoseKyle Rose More articles by this author , Billie GouldBillie Gould More articles by this author , Facundo DavaroFacundo Davaro More articles by this author , Pan DuPan Du More articles by this author , Lucia CamperlengoLucia Camperlengo More articles by this author , Philippe SpiessPhilippe Spiess More articles by this author , Wade SextonWade Sexton More articles by this author , Scott GilbertScott Gilbert More articles by this author , Daniel GrassDaniel Grass More articles by this author , Aram VosoughiAram Vosoughi More articles by this author , and Roger LiRoger Li More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003234.08AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Repeat transurethral resection (re-TUR) in non-muscle invasive bladder cancer (NMIBC) is recommended for patients with high-risk Ta and T1 lesions, and often more extensively samples the tumor base and detrusor musculature. In this hypothesis-generating analysis, we seek to characterize the variability in genomic alterations between index and re-TUR specimens as they may be implicated in muscle-invasion as well as choice of and response to intravesical therapies. METHODS: Baseline mutational profiling of six index NMIBC tumors and paired repeat resections was performed via PredicineWES+™ whole-exome sequencing across 20,000 genes with boosted sequencing of 600 cancer-related genes in patients with high-risk Ta and T1 lesions on index resection. Non-synonymous (NS) mutations observed in index and re-TUR specimens identified in a minimum of two samples are reported. RESULTS: Five and one patients had T1HG and TaHG index TURBT, respectively and two patients were upstaged to pT2 on re-TUR. As shown in the OncoPrint (Figure 1), 46 genes with NS mutations in at least two samples were identified including TP53, PIK3CA, and ARID1A. A median of 140 (range 48-420) and 90 NS (range 0-313) mutations were identified in the index and re-TUR specimens, respectively. On average, in patients with malignancy on re-TUR, lesions were 83% concordant with the index (range 33%-97%). One patient was pT0 on re-TUR for which no concordant variants were identified. Of non-concordant mutations, cell-cycle (CCND3, CCNE1, CCNE2) and PD-L1 (CD274) gene amplifications were uniquely expressed on the index lesion. CONCLUSIONS: Genomic alterations between index and re-TUR lesions are highly concordant even when pathologic upstaging occurs. However, index lesions harbored unique mutations in cell cycle and immune checkpoint genes. Surface tumor cells may behave differently than base tumor cells, with varying therapeutic targets. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e184 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Prithvi Murthy More articles by this author Kyle Rose More articles by this author Billie Gould More articles by this author Facundo Davaro More articles by this author Pan Du More articles by this author Lucia Camperlengo More articles by this author Philippe Spiess More articles by this author Wade Sexton More articles by this author Scott Gilbert More articles by this author Daniel Grass More articles by this author Aram Vosoughi More articles by this author Roger Li More articles by this author Expand All Advertisement PDF downloadLoading ...

Abstract P5-05-08: Dual ctDNA and tissue sequencing improves detection of actionable variants in breast cancer patients

Abstract Background: Next-generation sequencing of circulating tumor DNA (ctDNA) and solid-tissue can identify clinically actionable genomic variants that may be used for both treatment selection and disease surveillance. Due to differences in tumor biology and assay design, ctDNA and solid biopsies may identify unique variants. Here, we investigate a real-world dataset of breast cancer patients to determine whether clinically actionable variant detection is enhanced by dual ctDNA and solid tissue testing. Methods: We used the deidentified Tempus Lens database to retrospectively analyze stage IV breast cancer patients with known hormonal subtype. Each patient had dual testing defined as Tempus xF (ctDNA) and Tempus xT (tumor tissue)—which resulted in clinical reports for both tests. Patients were further stratified according to the timing of ctDNA biopsy relative to tissue biopsy. Concurrent dual testing was defined as samples collected ≤30 days apart and longitudinal dual testing was defined as liquid >30 days after solid. Variants were included in analyses if they met the limit of detection criteria of both assays. Clinical actionability was defined by indication-matched OncoKB Level 1-3. Fisher exact test was used to calculate significance. Results: Of the 1,341 breast cancer patients with dual ctDNA and tissue sequencing, at least one actionable variant was identified in 61% (n=823) of patients. In the subset of concurrent tested patients (n=782), 60% (n=473) had one or more actionable findings: 54% (n=257/473) of patients with actionable variants had perfectly concordant variants, 29% (n=136/473) had at least one unique variant detected only by solid tumor testing, and 20% (n=93/473) had at least one unique variant detected only by ctDNA testing. Similarly, in the longitudinal set (n=559), 63% (n=350) had one or more actionable findings: 34% (n=118/350) were concordant, 43% (n=150/350) were unique to solid, and 27% (n=96/350) were unique to ctDNA. When stratifying concurrent patients by OncoKB levels of evidence, 72% (n=98/136) of patients with variants unique in solid had at least one level 1-2 variant, while 39% (n=53/136) contained unique level 3 variants. Level 1-2 variants in PIK3CA were the most frequent variants seen uniquely in solid tumors, occurring in 54% (n=73/136) of patients. In contrast, in patients with unique ctDNA variants, 37% (n=34/93) of patients had at least one level 1-2 variants and 72% (n=67/93) had level 3 variants. Level 3 variants in ESR1 were the most frequent variants seen uniquely in ctDNA, occurring in 57% (n=53/93) of patients. The proportion of concurrent patients with actionable variants found exclusively in ctDNA significantly differed by subtype (p=0.04): Luminal A (22%) and Luminal B (23%) contained the most patients with unique ctDNA variants. This ability to detect additional variants in ctDNA remained true even if profiling occurred over time. Indeed, in patients with ESR1 variants tested with ctDNA > 1 year after tissue, 78% (n=43/55) had ESR1 variants only detected in blood. Conclusions: We show that dual testing in breast cancer patients improves the identification of clinically actionable variants which may be missed by either ctDNA or solid tissue biopsy alone. Adoption of dual testing should be considered as standard practice to provide a comprehensive view of actionable molecular alterations. Citation Format: Matthew Mackay, Kabir Manghnani, Adam Hockenberry, Joshua Drews, James Chen, Rotem Ben-Shachar, Justin Guinney. Dual ctDNA and tissue sequencing improves detection of actionable variants in breast cancer patients [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-05-08.

Whole-exome sequencing of BRCA-negative breast cancer patients and case–control analyses identify variants associated with breast cancer susceptibility

BackgroundFor the majority of individuals with early-onset or familial breast cancer referred for genetic testing, the genetic basis of their familial breast cancer remains unexplained. To identify novel germline variants associated with breast cancer predisposition, whole-exome sequencing (WES) was performed.MethodsWES on 290 BRCA1/BRCA2-negative Singaporeans with early-onset breast cancer and/or a family history of breast cancer was done. Case–control analysis against the East-Asian subpopulation (EAS) from the Genome Aggregation Database (gnomAD) identified variants enriched in cases, which were further selected by occurrence in cancer gene databases. Variants were further evaluated in repeated case–control analyses using a second case cohort from the database of Genotypes and Phenotypes (dbGaP) comprising 466 early-onset breast cancer patients from the United States, and a Singapore SG10K_Health control cohort.ResultsForty-nine breast cancer-associated germline pathogenic variants in 37 genes were identified in Singapore cases versus gnomAD (EAS). Compared against SG10K_Health controls, 13 of 49 variants remain significantly enriched (False Discovery Rate (FDR)-adjusted p < 0.05). Comparing these 49 variants in dbGaP cases against gnomAD (EAS) and SG10K_Health controls revealed 23 concordant variants that were significantly enriched (FDR-adjusted p < 0.05). Fourteen variants were consistently enriched in breast cancer cases across all comparisons (FDR-adjusted p < 0.05). Seven variants in GPRIN2, NRG1, MYO5A, CLIP1, CUX1, GNAS and MGA were confirmed by Sanger sequencing.ConclusionsIn conclusion, we have identified pathogenic variants in genes associated with breast cancer predisposition. Importantly, many of these variants were significant in a second case cohort from dbGaP, suggesting that the strategy of using case–control analysis to select variants could potentially be utilized for identifying variants associated with cancer susceptibility.

Longitudinal Shifts of Solid Tumor and Liquid Biopsy Sequencing Concordance in Metastatic Breast Cancer.

PURPOSETissue-based next-generation sequencing (NGS) in metastatic breast cancer (mBC) is limited by the inability to noninvasively track tumor evolution. Cell-free DNA (cfDNA) NGS has made sequential testing feasible; however, the relationship between cfDNA and tissue-based testing in mBC is not well understood. Here, we evaluate concordance between tissue and cfDNA NGS relative to cfDNA sampling frequency in a large, clinically annotated mBC data set.METHODSTempus LENS was used to analyze deidentified records of mBC cases with Tempus xT (tissue) and xF (cfDNA) sequencing results. Then, various metrics of concordance were assessed within overlapping probe regions of the tissue and cfDNA assays (104 genes), focusing on pathogenic variants. Variant allele frequencies of discordant and concordant pathogenic variants were also compared. Analyses were stratified by mBC subtype and time between tests.RESULTSRecords from 300 paired tissue and liquid biopsies were analyzed. Median time between tissue and blood collection was 78.5 days (standard deviation = 642.99). The median number of pathogenic variants/patient was one for cfDNA and two for tissue. Across the cohort, 77.8% of pathogenic tissue variants were found in cfDNA and 75.7% of pathogenic cfDNA variants were found in tissue when tests were ≤ 7 days apart, which decreased to 50.3% and 51.8%, respectively, for > 365 days. Furthermore, the median patient-level variant concordance was 67% when tests were ≤7 days apart and 30%-37% when > 30 days. The median variant allele frequencies of discordant variants were generally lower than those of concordant variants within the same time frame.CONCLUSIONWe observed high concordances between tissue and cfDNA results that generally decreased with longer durations between tests. Thus, cfDNA NGS reliably measures tissue genomics and is likely beneficial for longitudinal monitoring of molecular changes in mBC.

Dual tissue and plasma testing to improve detection of actionable variants in patients with solid cancers.

3017 Background: Next generation sequencing (NGS) of tumor tissue and plasma (circulating tumor DNA [ctDNA]) are used clinically to identify actionable genomic alterations, with implications for treatment selection and disease surveillance. Early studies have observed that solid tumor tissue and ctDNA testing may capture both overlapping and complementary alterations. Using the Tempus database, we examined whether dual tissue and ctDNA testing, “dual testing”, improved identification of actionable variants compared with either modality alone. Methods: We used Tempus Lens to retrospectively analyze 3153 de-identified stage 4 patients (breast [N = 644], colorectal [N = 841], non-small cell lung cancer (NSCLC) [N = 1232], and prostate [N = 436]). Each patient had dual testing—Tempus xF (ctDNA, 105 panel genes) and Tempus xT (tumor tissue, 595-648 panel genes), representing 6306 total samples. Samples were defined as concurrent if biopsied ≤30 days apart and longitudinal if plasma was collected between 31–365 days after tissue biopsy. All analyses were limited to single nucleotide variants and insertions/deletions that met the limit of detection criteria for both assays (104 genes). Indication matched actionable variants were defined by OncoKB Level 1 and 2 evidence, or R1 within both xF and xT (13 genes). Results: Of the 3153 patients with dual testing, 37% (1168) had actionable variants identified by at least one test. 94% (1100/1168) of these patients had variants identified via solid tumor profiling alone, 73% (856) had variants identified via ctDNA profiling alone, and 64% (745) had perfectly concordant variants. Thus, dual testing identified additional variants in 36% (423/1168) of these patients compared to any singular test. Of the 423 patients who had additional actionable alterations discovered through dual testing, ctDNA revealed unique alterations—which were not found in solid tissue testing—in 22% (95/423) of patients. Of these patients, 72% (68/95) had all actionable variants identified solely from ctDNA. Of the 251 patients with additional alterations identified by concurrent dual testing, 24% (61/251) had unique alterations identified in plasma. Similarly, of the 172 patients with additional alterations identified by longitudinal dual testing, 20% (34/172) had unique alterations identified in ctDNA alone. Conclusions: In the largest study of its kind, we show that dual tumor tissue and ctDNA testing—with samples collected either concurrently or longitudinally—identified more patients with actionable alterations than single modality testing alone and therefore should be considered as part of routine NGS testing. Additional studies to explore the genetic and intra-patient tumor heterogeneity of these variants as well as the impact of time between tissue and plasma sampling assessments and implications for timing of therapeutic recommendations are underway.

A joint analysis using exome and transcriptome data identifies candidate polymorphisms and genes involved with umbilical hernia in pigs

BackgroundUmbilical Hernia (UH) is characterized by the passage of part of the intestine through the umbilical canal forming the herniary sac. There are several potential causes that can lead to the umbilical hernia such as bacterial infections, management conditions and genetic factors. Since the genetic components involved with UH are poorly understood, this study aimed to identify polymorphisms and genes associated with the manifestation of umbilical hernia in pigs using exome and transcriptome sequencing in a case and control design.ResultsIn the exome sequencing, 119 variants located in 58 genes were identified differing between normal and UH-affected pigs, and in the umbilical ring transcriptome, 46 variants were identified, located in 27 genes. Comparing the two methodologies, we obtained 34 concordant variants between the exome and transcriptome analyses, which were located in 17 genes, distributed in 64 biological processes (BP). Among the BP involved with UH it is possible to highlight cell adhesion, cell junction regulation, embryonic morphogenesis, ion transport, muscle contraction, within others.ConclusionsWe have generated the first exome sequencing related to normal and umbilical hernia-affected pigs, which allowed us to identify several variants possibly involved with this disorder. Many of those variants present in the DNA were confirmed with the RNA-Seq results. The combination of both exome and transcriptome sequencing approaches allowed us to better understand the complex molecular mechanisms underlying UH in pigs and possibly in other mammals, including humans. Some variants found in genes and other regulatory regions are highlighted as strong candidates to the development of UH in pigs and should be further investigated.

Thibaut de Champagne, Les Chansons— Texts and Melodies. Bilingual edition prepared by Christopher Callahan, Marie-Geneviève Grossel, and Daniel E. O'Sullivan. Paris: Champion, 2018, pp. 848.

Counted among the most original and prolific medieval French poets, Thibaut IV, count of Champagne and Brie, King of Navarre, was also called Thibaut the poet entertainer (1201–1253). This great-grandson of Eleanor of Aquitaine and great-grandfather of Marie de Champagne, was celebrated by Dante for the purity of his lyrics and widely admired for his mastery of all genres of the time. Remarkable as well for his interpretations of antique mythology and bestiaries, this grand prince and Crusade hero forged poems devoted to the Virgin, transmuting the lyrical lady into a celestial figura. The generous and capacious volume to hand, prepared by young scholars, one French and two Americans, is completely devoted to the famous poet; it is, since the long-respected 1925 edition by Wallensköld (SATF; just a single scholar), the first really complete one. It offers not only all the poems (love songs, debate poems, pastourelles, Crusade, and religious poems) accompanied by their melody, but also robust notes, concordant variants, and isolated melodies. The modern French translations are complemented by additional comments in the glossary, thus offering the reader a very generous and useful reference tool, the fruit of years of accumulated philological and musicological research.

Candidate lethal haplotypes and causal mutations in Angus cattle

BackgroundIf unmanaged, high rates of inbreeding in livestock populations adversely impact their reproductive fitness. In beef cattle, historical selection strategies have increased the frequency of several segregating fatal autosomal recessive polymorphisms. Selective breeding has also decreased the extent of haplotypic diversity genome-wide. By identifying haplotypes for which homozygotes are not observed but would be expected based on their frequency, candidates for developmentally lethal recessive loci can be localized. This analysis comes without the need for observation of the loss-associated phenotype (e.g., failure to implant, first trimester abortion, deformity at birth). In this study, haplotypes were estimated for 3961 registered Angus individuals using 52,545 SNP loci using findhap v2, which exploited the complex pedigree among the individuals in this population.ResultsSeven loci were detected to possess haplotypes that were not observed in homozygous form despite a sufficiently high frequency and pedigree-based expectation of homozygote occurrence. These haplotypes were identified as candidates for harboring autosomal recessive lethal alleles. Of the genotyped individuals, 109 were resequenced to an average 27X depth of coverage to identify putative loss-of-function alleles genome-wide and had variants called using a custom in-house developed pipeline. For the candidate lethal-harboring haplotypes present in these bulls, sequence-called genotypes were used to identify concordant variants. In addition, whole-genome sequence imputation of variants was performed into the set of 3961 genotyped animals using the 109 resequenced animals to identify candidate lethal recessive variants at the seven loci. Following the imputation, no variants were identified that were fully concordant with the marker-based diplotypes.ConclusionsSelective breeding programs could utilize the predicted lethal haplotypes associated with SNP genotypes. Sequencing and other methods for identifying the causal variants underlying these haplotypes can allow for more efficient methods of management such as gene editing. These two methods in total will reduce the negative impacts of inbreeding on fertility and maximize overall genetic gains.

A pipeline combining multiple strategies for prioritizing heterozygous variants for the identification of candidate genes in exome datasets

BackgroundThe identification of disease-causing variants in autosomal dominant diseases using exome-sequencing data remains a difficult task in small pedigrees. We combined several strategies to improve filtering and prioritizing of heterozygous variants using exome-sequencing datasets in familial Meniere disease: an in-house Pathogenic Variant (PAVAR) score, the Variant Annotation Analysis and Search Tool (VAAST-Phevor), Exomiser-v2, CADD, and FATHMM. We also validated the method by a benchmarking procedure including causal mutations in synthetic exome datasets.ResultsPAVAR and VAAST were able to select the same sets of candidate variants independently of the studied disease. In contrast, Exomiser V2 and VAAST-Phevor had a variable correlation depending on the phenotypic information available for the disease on each family. Nevertheless, all the selected diseases ranked a limited number of concordant variants in the top 10 ranking, using the three systems or other combined algorithm such as CADD or FATHMM.Benchmarking analyses confirmed that the combination of systems with different approaches improves the prediction of candidate variants compared with the use of a single method. The overall efficiency of combined tools ranges between 68 and 71% in the top 10 ranked variants.ConclusionsOur pipeline prioritizes a short list of heterozygous variants in exome datasets based on the top 10 concordant variants combining multiple systems.

Precision Molecular Diagnosis Defines Specific Therapy in Combined Immunodeficiency with Megaloblastic Anemia Secondary to MTHFD1 Deficiency

Methylenetetrahydrofolate dehydrogenase (MTHFD1) deficiency has recently been reported to cause a folate-responsive syndrome displaying a phenotype that includes megaloblastic anemia and severe combined immunodeficiency. To describe our investigative approach to the molecular diagnosis and evaluation of immune dysfunction in a family with MTHFD1 deficiency. The methods used were exome sequencing and analysis of variants in genes involved in the folate metabolic pathway in a family with 2 affected siblings. Routine laboratory and research data were analyzed to gain an in-depth understanding of innate, humoral, and cell-mediated immune function before and after folinic acid supplementation. Interrogation of exome data for concordant variants between the siblings in the genes involved in folate metabolic pathway identified a heterozygous mutation in exon 3 of the MTHFD1 gene that was shared with their mother. In view of highly suggestive phenotype, we extended our bioinformatics interrogation for structural variants in the MTHFD1 gene by manual evaluation of the exome data for sequence depth coverage of all the exons. A deletion involving exon 13 that was shared with their father was identified. Routine laboratory data showed lymphopenia involving all subsets and poor response to vaccines. Invitro analysis of dendritic cell and lymphocyte function was comparable to that in healthy volunteers. Treatment with folinic acid led to immune reconstitution, enabling discontinuation of all prophylactic therapies. Exome sequencing demonstrated MTHFD1 deficiency as a novel cause of a combined immunodeficiency. Folinic acid was established as precision therapy to reverse the clinical and laboratory phenotype of this primary immunodeficiency.

Whole genome analyses reveal no pathogenetic single nucleotide or structural differences between monozygotic twins discordant for amyotrophic lateral sclerosis

The contribution of genetic and environmental factors to the pathogenesis of sporadic amyotrophic lateral sclerosis (ALS) remains unclear. To investigate the genetic component of the disease, we performed whole genome sequencing on ALS discordant monozygotic twins. Illumina whole genome sequencing on white blood cell DNA of five ALS-discordant monozygotic twin pairs (10 samples in total) yielded ∼30x coverage per individual. All single nucleotide variants, indels, and structural variants (copy number variants, inversions and translocations) were called and evaluated for functional consequence, evolutionary conservation, population frequency and overlap with known ALS associated variants and genes. Results showed that no validated discordant coding or regulatory single nucleotide variants or indels were found, and nor were any genome-wide discordant structural variants detected. Concordant variants of particular interest were: 1) two rare, highly-conserved heterozygous non-synonymous variants in SYT9 and EWSR1, genes previously associated with ALS (out of 2044 rare heterozygous variants detected); 2) three rare homozygous missense variants; and 3) three novel copy number deletions that overlapped genes. In conclusion, no convincing coding or regulatory nucleotide or genome-wide structural differences were found between ALS discordant monozygotic twins. The results suggest that more work is needed to elucidate possible environmental, epigenetic, oligogenic and somatic genetic factors that could underlie susceptibility to sporadic ALS.

Human papillomavirus type 16 in infants: use of DNA sequence analyses to determine the source of infection

Perinatal transmission of human papillomavirus type 16 (HPV-16) and persistence of virus DNA in infants until 6 months of age has been described. To confirm the origin of infant infections as maternal, we determined the nucleotide sequence of the upstream regulatory region (URR; bp 7540 to 157) of HPV-16 in samples from 13 HPV-16 DNA-positive mothers and their infants at 6 weeks and 2 years of age. Identical HPV-16 variant URR sequences were found in two mother/infant samples and similar variants were found in three sets. Four mothers with samples which contained prototypic HPV-16 sequences delivered infants who also had the prototypic sequence. Four mothers with variant URRs delivered infants who harboured either prototypic or different URR variants. Thus, concordant variants or prototypic sequences were detected in nine of 13 mother/infant samples, indicating that up to 69.2% of HPV-16-positive infants acquire virus from their mothers.