Custom Next-generation Sequencing Research Articles

Post-zygotic mosaicism of SMARCB1 variants in patients with rhabdoid tumors: a not so rare condition exposing to successive tumors.

Rhabdoid tumors (RT) are aggressive, rare tumors predominantly affecting young children, characterized by bi-allelic SMARCB1 gene inactivation. While most SMARCB1 alterations are acquired de novo, a third of cases exhibit germline alterations, defining Rhabdoid Tumors Predisposition Syndrome (RTPS1). With increased sensitivity of next-generation sequencing (NGS), mosaicisms in genes linked to genetic diseases are more detectable. This study focuses on exploring SMARCB1 germline alterations, notably mosaicism in blood samples of children with RT and in parents, using a custom NGS panel. A cohort of 280 children and 140 parents with germline analysis was studied. Germline DNA from 111 children with RT and 32 parents were re-analyzed with a custom NGS panel with 1,500X average depth targeting the SMARCB1 gene to identify intragenic variants not detected with conventional low-sensitivity methods. Follow-up data was obtained for 77 patients. Nine previously undetected mosaicism cases were identified, totaling 17/280 patients with a mosaic variant (6.1%) in the cohort, with variant allele frequencies between 0.9% and 33%, thus highlighting the prior underestimation of its prevalence. Follow-up data showed that 4 out of 7 survivors with mosaic variants developed distinct novel tumors, two sharing SMARCB1 alterations with the initial tumor, emphasizing the potential clinical impact of SMARCB1 mosaicism. The hitherto underestimated rate of SMARCB1 mosaicism in RT underscores the need for optimized genetic counseling and oncological monitoring. The findings have significant medical implications, considering the dire prognosis of RT.

Discovery and Validation of Survival-Specific Genes in Papillary Renal Cell Carcinoma Using a Customized Next-Generation Sequencing Gene Panel.

Papillary renal cell carcinoma (PRCC), the second most common kidney cancer, is morphologically, genetically, and molecularly heterogeneous with diverse clinical manifestations. Genetic variations of PRCC and their association with survival are not yet well-understood. This study aimed to identify and validate survival-specific genes in PRCC and explore their clinical utility. Using machine learning, 293 patients from the Cancer Genome Atlas-Kidney Renal Papillary Cell Carcinoma (TCGA-KIRP) database were analyzed to derive genes associated with survival. To validate these genes, DNAs were extracted from the tissues of 60 Korean PRCC patients. Next generation sequencing was conducted using a customized PRCC gene panel of 202 genes, including 171 survival-specific genes. Kaplan-Meier and Log-rank tests were used for survival analysis. Fisher's exact test was performed to assess the clinical utility of variant genes. A total of 40 survival-specific genes were identified in the TCGA-KIRP database through machine learning and statistical analysis. Of them, 10 (BAP1, BRAF, CFDP1, EGFR, ITM2B, JAK1, NODAL, PCSK2, SPATA13, and SYT5) were validated in the Korean-KIRP database. Among these survival gene signatures, three genes (BAP1, PCSK2, and SPATA13) showed survival specificity in both overall survival (OS) (p = 0.00004, p = 1.38 × 10-7, and p = 0.026, respectively) and disease-free survival (DFS) (p = 0.00002, p = 1.21 × 10-7, and p = 0.036, respectively). Notably, the PCSK2 mutation demonstrated survival specificity uniquely in both the TCGA-KIRP (OS: p = 0.010 and DFS: p = 0.301) and Korean-KIRP (OS: p = 1.38 × 10-7 and DFS: p = 1.21 × 10-7) databases. We discovered and verified genes specific for the survival of PRCC patients in the TCGA-KIRP and Korean-KIRP databases. The survival gene signature, including PCSK2 commonly obtained from the 40 gene signature of TCGA and the 10 gene signature of the Korean database, is expected to provide insight into predicting the survival of PRCC patients and developing new treatment.

Next-generation sequencing of host genetics risk factors associated with COVID-19 severity and long-COVID in Colombian population

Coronavirus disease 2019 (COVID-19) was considered a major public health burden worldwide. Multiple studies have shown that susceptibility to severe infections and the development of long-term symptoms is significantly influenced by viral and host factors. These findings have highlighted the potential of host genetic markers to identify high-risk individuals and develop target interventions to reduce morbimortality. Despite its importance, genetic host factors remain largely understudied in Latin-American populations. Using a case–control design and a custom next-generation sequencing (NGS) panel encompassing 81 genetic variants and 74 genes previously associated with COVID-19 severity and long-COVID, we analyzed 56 individuals with asymptomatic or mild COVID-19 and 56 severe and critical cases. In agreement with previous studies, our results support the association between several clinical variables, including male sex, obesity and common symptoms like cough and dyspnea, and severe COVID-19. Remarkably, thirteen genetic variants showed an association with COVID-19 severity. Among these variants, rs11385942 (p < 0.01; OR = 10.88; 95% CI = 1.36–86.51) located in the LZTFL1 gene, and rs35775079 (p = 0.02; OR = 8.53; 95% CI = 1.05–69.45) located in CCR3 showed the strongest associations. Various respiratory and systemic symptoms, along with the rs8178521 variant (p < 0.01; OR = 2.51; 95% CI = 1.27–4.94) in the IL10RB gene, were significantly associated with the presence of long-COVID. The results of the predictive model comparison showed that the mixed model, which incorporates genetic and non-genetic variables, outperforms clinical and genetic models. To our knowledge, this is the first study in Colombia and Latin-America proposing a predictive model for COVID-19 severity and long-COVID based on genomic analysis. Our study highlights the usefulness of genomic approaches to studying host genetic risk factors in specific populations. The methodology used allowed us to validate several genetic variants previously associated with COVID-19 severity and long-COVID. Finally, the integrated model illustrates the importance of considering genetic factors in precision medicine of infectious diseases.

A custom next-generation sequencing panel for 1p/19q codeletion and mutational analysis in gliomas.

The World Health Organization has updated their classification system for the diagnosis of gliomas, combining histological features with molecular data including isocitrate dehydrogenase 1 and codeletion of chromosomal arms 1p and 19q. 1p/19q codeletion analysis is commonly performed by fluorescence in situ hybridization (FISH). In this study, we developed a 57-gene targeted next-generation sequencing (NGS) panel including 1p/19q codeletion detection mainly to assess diagnosis and potential treatment response in melanoma, gastrointestinal stromal tumor, and glioma patients. Loss of heterozygosity analysis was performed using the NGS method on 37 formalin-fixed paraffin-embedded glioma tissues that showed 1p and/or 19q loss determined by FISH. Conventional methods were applied for the validation of some glioma-related gene mutations. In 81.1% (30 of 37) and 94.6% (35 of 37) of cases, 1p and 19q were found to be in agreement whereas concordance for 1p/19q codeletion and no 1p/19q codeletion was found in 94.7% (18 of 19) and 94.4% (17 of 18) of cases, respectively. Overall, comparing NGS results with those of conventional methods showed high concordance. In conclusion, the NGS panel allows reliable analysis of 1p/19q codeletion and mutation at the same time.

Tumor mutational burden assessment and standardized bioinformatics approach using custom NGS panels in clinical routine

BackgroundHigh tumor mutational burden (TMB) was reported to predict the efficacy of immune checkpoint inhibitors (ICIs). Pembrolizumab, an anti-PD-1, received FDA-approval for the treatment of unresectable/metastatic tumors with high TMB as determined by the FoundationOne®CDx test. It remains to be determined how TMB can also be calculated using other tests.ResultsFFPE/frozen tumor samples from various origins were sequenced in the frame of the Institut Curie (IC) Molecular Tumor Board using an in-house next-generation sequencing (NGS) panel. A TMB calculation method was developed at IC (IC algorithm) and compared to the FoundationOne® (FO) algorithm.Using IC algorithm, an optimal 10% variant allele frequency (VAF) cut-off was established for TMB evaluation on FFPE samples, compared to 5% on frozen samples. The median TMB score for MSS/POLE WT tumors was 8.8 mut/Mb versus 45 mut/Mb for MSI/POLE-mutated tumors. When focusing on MSS/POLE WT tumor samples, the highest median TMB scores were observed in lymphoma, lung, endometrial, and cervical cancers. After biological manual curation of these cases, 21% of them could be reclassified as MSI/POLE tumors and considered as “true TMB high.” Higher TMB values were obtained using FO algorithm on FFPE samples compared to IC algorithm (40 mut/Mb [10–3927] versus 8.2 mut/Mb [2.5–897], p < 0.001).ConclusionsWe herein propose a TMB calculation method and a bioinformatics tool that is customizable to different NGS panels and sample types. We were not able to retrieve TMB values from FO algorithm using our own algorithm and NGS panel.

The development of a next-generation sequencing panel targeting cannabinoid synthase genes to distinguish between marijuana and hemp.

Hemp and marijuana, both derived from Cannabis sativa L. (C. sativa), are subject to divergent legal regulations due to their different Δ9-tetrahydrocannabinol (Δ9-THC) contents. Cannabinoid synthase genes are considered the key enzymes that determine the chemical composition or chemotype of a particular cultivar. However, existing methods for crop type differentiation based on previous synthase gene theories have limitations in terms of precision and specificity, and a wider range of cannabis varieties must be considered when examining cannabis-based genetic markers. A custom next-generation sequencing (NGS) panel was developed targeting all synthase genes, including Δ9-THC acid synthase, cannabidiolic acid synthase, and cannabichromenic acid synthase, as well as the pseudogenes across diverse C. sativa samples, spanning reference hemp and marijuana, commercial hemp derivatives, and seized marijuana extracts. Interpretation of NGS data revealed a relationship between genotypes and underlying chemotypes, with the principal component analysis indicating a clear distinction between hemp and marijuana clusters. This differentiation was attributed to variations in both synthase genes and pseudogene variants. Finally, this study proposes a genetic cannabis classification method using a differentiation flow chart with novel synthase markers. The flow chart successfully differentiated hemp from marijuana with a 1.3% error rate (n=147).

Application of Newly Customized Myeloid NGS Panel in the Diagnosis of Myeloid Malignancies.

Genetic mutations are major factors in the diagnosis and prognosis of leukemia, and it is difficult to assess these variants using single-gene analysis. Therefore, this study aimed to develop a fast and cost-effective method for genetic screening of myeloid malignancies using a customized next-generation sequencing (NGS) panel. A customized myeloid panel was designed and investigated in 15 acute myeloid leukemia patients. The panel included 11 genes that were most commonly mutated in myeloid malignancies. This panel was designed to sequence the complete genome of CALR, IDH1, IDH2, JAK2, FLT3, NPM1, MPL, TET2, SF3B1, TP53, and MLL. Among the 15 patients, 14 actual pathogenic variants were identified in nine samples, and negative results were found in six samples. Positive findings were observed for JAK2, FLT3, SF3B1, and TET2. Interestingly, non-classical FLT3 mutations (c.1715A>C, c.2513delG, and c.2507dupT) were detected in patients who were negative for FLT3-ITD and TKD by routine molecular results. All identified variants were pathogenic, and the high coverage of the assay allowed us to predict variants at a low frequency (1%) with 1000x coverage. Utilizing a custom panel allowed us to identify variants that were not detected by routine tests or those that were not routinely investigated. Using the costuming panel will enable us to sequence all genes and discover new potential pathogenic variants that are not possible with other commercially available panels that focus only on hotspot regions. This study's strength in utilizing NGS and implanting a customized panel to identify new pathogenic variants that might be common in our population and important in routine diagnosis for providing optimal healthcare for personalized medicine.

The polygenic implication of clopidogrel responsiveness: Insights from platelet reactivity analysis and next-generation sequencing.

Clopidogrel is widely used worldwide as an antiplatelet therapy in patients with acute coronary disease. Genetic factors influence interindividual variability in response. Some studies have explored the polygenic contributions in the drug response, generating pharmacogenomic risk scores (PgxPRS). Importantly, these factors are less explored in underrepresented populations, such as Latin-American countries. Identifying patients at risk of high-on-treatment platelet reactivity (HTPR) is highly valuable in translational medicine. In this study we used a custom next-generation sequencing (NGS) panel composed of 91 single nucleotide polymorphisms (SNPs) and 28 genes related to clopidogrel metabolism, to analyze 70 patients with platelet reactivity values, assessed through closure time (CT). Our results demonstrated the association of SNPs with HTPR and non-HTPR, revealing the strongest associations with rs2286823 (OR: 5,0; 95% CI: 1,02-24,48; p: 0,03), rs2032582 (OR: 4,41; 95% CI: 1,20-16,12; p: 0,019), and rs1045642 (OR: 3,38; 95% CI: 0,96-11,9; p: 0,05). Bivariate regression analysis demonstrated the significant association of several SNPs with the CT value, a "surrogate" biomarker of clopidogrel response. Exploratory results from the LASSO regression model showed a high discriminatory capacity between HTPR and non-HTPR patients (AUC: 0,955), and the generated PgxPRS demonstrated a significant negative association between the risk score, CT value, and the condition of HTPR and non-HTPR. To our knowledge, our study addresses for the first time the analysis of the polygenic contribution in platelet reactivity using NGS and establishes PgxPRS derived from the LASSO model. Our results demonstrate the polygenic implication of clopidogrel response and offer insights applicable to the translational medicine of antiplatelet therapy in an understudied population.

Development and validation of next-generation sequencing panel for personalized Helicobacter pylori eradication treatment targeting multiple species.

The decreasing Helicobacter pylori eradication rate is primarily attributed to antibiotic resistance, and further exacerbated by uniform drug administration disregarding a host's metabolic capability. Consequently, applying personalized treatment based on antibiotic resistance-associated variants and the host's metabolic phenotype can potentially increase the eradication rate. A custom next-generation sequencing panel for personalized H. pylori eradication treatment (NGS-PHET) was designed which targeted the regions for amoxicillin, clarithromycin, metronidazole, tetracycline, and levofloxacin-resistance in H. pylori and human proton-pump inhibitor (PPI) metabolism. The libraries were constructed following customized methods and sequenced simultaneously. The customized framework criteria, grounded in previously reported antibiotic resistance associated variants and the host's PPI metabolism, was applied to the NGS-PHET results and suggested a personalized treatment for each subject, which was validated through each subject's actual eradication outcome. Both previously reported and novel variants were identified from H. pylori sequencing results. Concurrently, five CYP2C19 homozygous extensive metabolizers and three CYP3A4 intermediate metabolizers were identified. Among the total of 12 subjects, clarithromycin triple therapy was suggested for five subjects, bismuth quadruple therapy was suggested for six subjects, and rifabutin triple therapy was suggested for one subject by following the customized framework criteria. The treatment suggestion for nine of the 12 subjects was consistent with the treatment that each subject achieved eradication with. Applying the methodology using the NGS-PHET and customized framework helps to perform eradication treatment quickly and effectively in most patients with antibiotic-resistant H. pylori strains, and is also useful in research to find novel antibiotic-resistance candidates.

Perspectives for next generation sequencing in patients with follicular lymphoma

Aim. To study the prognostic significance of gene mutations and intracellular signaling pathways involved in lymphomagenesis in patients with follicular lymphoma using next generation sequencing (NGS).Materials and methods. The prospective study included 26 patients with a median age of 51.5 years. Mutational screening was performed for cohort using custom NGS Panel of 118 genes. Gene set enrichment analysis (GSEA) was performed using Metascape. The data was analyzed in SPSS Statistics 26 and R 4.2.2.Results. The highest mutation frequency was noted in the genes: KMT2C – 50 %, KMT2D – 50 %, CREBBP – 31 %, NOTCH2 – 31 %, GNAS – 23 %. Missense mutations occurred with a frequency of 84.3 %. ARID1A gene mutation is an unfavorable prognostic factor according to progressive-free (p = 0.014) and event-free (p = 0.029) survival analysis. Tumor mutation burden (TMB) was defined as the number of mutations per megabase (Mb) of the coding sequence, the median TMB was 5.0 (3.3–8.3) mutations/Mb. The TMB threshold of 6 mutations/Mb divided patients into groups with high (44 %) and low (56 %) TMB. In the high TMB group, 2-year event-free survival was 27.3 % (95 % confidence interval 6.0–61.0), which was significantly lower than in low TMB group – 72.7 % (95 % confidence interval 41.9–91.6; p = 0.037). The most enriched cellular pathways according to GSEA results were regulation of cell activation (–log10(q-value) = 6.357), chromatin remodeling (–log10(q-value) = 5.707), histone modification (–log10(q-value) = 4.569). We have also demonstrated other possibilities of GSEA using follicular lymphoma as an example.Conclusion. TMB is a significant prognostic factor in patients with follicular lymphoma. We have shown that mutations in the MYC, CREBBP, EZH2, KMT2D genes lead to dysregulation in several intracellular processes, mediating complex molecular changes. The most enriched intracellular pathways in follicular lymphoma are those of chromatin remodeling, regulation of cell activation and histone modification.

NGS Custom Panel Implementation in Patients with Non-Syndromic Autism Spectrum Disorders in the Clinical Routine of a Tertiary Hospital.

Autism spectrum disorder (ASD) is a set of neurodevelopmental disorders characterized by deficiencies in communication, social interaction, and repetitive and restrictive behaviors. The discovery of genetic involvement in the etiology of ASD has made this condition a strong candidate for genome-based diagnostic tests. Next-generation sequencing (NGS) is useful for the detection of variants in the sequence of different genes in ASD patients. Herein, we present the implementation of a personalized NGS panel for autism (AutismSeq) for patients with essential ASD over a prospective period of four years in the clinical routine of a tertiary hospital. The cohort is composed of 48 individuals, older than 3 years, who met the DSM-5 (The Diagnostic and Statistical Manual of Mental Disorders) diagnostic criteria for ASD. The NGS customized panel (AutismSeq) turned out to be a tool with good diagnostic efficacy in routine clinical care, where we detected 12 "pathogenic" (including pathogenic, likely pathogenic, and VUS (variant of uncertain significance) possibly pathogenic variations) in 11 individuals, and 11 VUS in 10 individuals, which had previously been negative for chromosomal microarray analysis and other previous genetic studies, such as karyotype, fragile-X, or MLPA/FISH (Multiplex Ligation dependent Probe Amplification/Fluorescence in situ hybridization) analysis. Our results demonstrate the high genetic and clinical heterogeneity of individuals with ASD and the current difficulty of molecular diagnosis. Our study also shows that an NGS-customized panel might be useful for diagnosing patients with essential/primary autism and that it is cost-effective for most genetic laboratories.

1237P Design and validation of a custom next-generation sequencing panel in melanoma, glioma and gastrointestinal stromal tumor

1237P Design and validation of a custom next-generation sequencing panel in melanoma, glioma and gastrointestinal stromal tumor

Next-generation sequencing improves precision medicine in hearing loss.

Background: An early etiological diagnosis of hearing loss positively impacts children's quality of life including language and cognitive development. Even though hearing loss associates with extremely high genetic and allelic heterogeneity, several studies have proven that Next-Generation Sequencing (NGS)-based gene panel testing significantly reduces the time between onset and diagnosis. Methods: In order to assess the clinical utility of our custom NGS GHELP panel, the prevalence of pathogenic single nucleotide variants, indels or copy number variants was assessed by sequencing 171 nuclear and 8 mitochondrial genes in 155 Spanish individuals with hearing loss. Results: A genetic diagnosis of hearing loss was achieved in 34% (52/155) of the individuals (5 out of 52 were syndromic). Among the diagnosed cases, 87% (45/52) and 12% (6/52) associated with autosomal recessive and dominant inheritance patterns respectively; remarkably, 2% (1/52) associated with mitochondrial inheritance pattern. Although the most frequently mutated genes in this cohort were consistent with those described in the literature (GJB2, OTOF or MYO7A), causative variants in less frequent genes such as TMC1, FGF3 or mitCOX1 were also identified. Moreover, 5% of the diagnosed cases (3/52) were associated with pathogenic copy number variants. Conclusion: The clinical utility of NGS panels that allows identification of different types of pathogenic variants-not only single nucleotide variants/indels in both nuclear and mitochondrial genes but also copy number variants-has been demonstrated to reduce the clinical diagnostic odyssey in hearing loss. Thus, clinical implementation of genomic strategies within the regular clinical practice, and, more significantly, within the newborn screening protocols, is warranted.

Design and Validation of a Custom Next-Generation Sequencing Panel in Pediatric Acute Lymphoblastic Leukemia.

The molecular landscape of acute lymphoblastic leukemia (ALL) is highly heterogeneous, and genetic lesions are clinically relevant for diagnosis, risk stratification, and treatment guidance. Next-generation sequencing (NGS) has become an essential tool for clinical laboratories, where disease-targeted panels are able to capture the most relevant alterations in a cost-effective and fast way. However, comprehensive ALL panels assessing all relevant alterations are scarce. Here, we design and validate an NGS panel including single-nucleotide variants (SNVs), insertion-deletions (indels), copy number variations (CNVs), fusions, and gene expression (ALLseq). ALLseq sequencing metrics were acceptable for clinical use and showed 100% sensitivity and specificity for virtually all types of alterations. The limit of detection was established at a 2% variant allele frequency for SNVs and indels, and at a 0.5 copy number ratio for CNVs. Overall, ALLseq is able to provide clinically relevant information to more than 83% of pediatric patients, making it an attractive tool for the molecular characterization of ALL in clinical settings.

Outcome prediction in resectable esophageal adenocarcinoma based on clinical variables, radiomics, and ctDNA.

423 Background: Despite the advent of precision medicine, prediction of survival outcome of esophageal cancer patients remains a challenge. Here we aim to investigate the value of prediction models integrating multi-signal data including radiomics and circulating tumor DNA (ctDNA) data in addition to clinical data for the prediction of resectable esophageal adenocarcinoma (rEAC) related outcomes. Methods: In total n=111 rEAC patients treated with neoadjuvant chemoradiotherapy (nCRT; n=71) +/- anti-PD-L1 (n=40) were included. Baseline clinical variables (n=9) were based on the SOURCE survival prediction model (van den Boorn et al. JNCCN. 2021). The baseline ctDNA data from plasma was derived from fragmentomic and copy number aberrations (ichorCNA) from shallow whole genome sequencing (&lt;5-fold coverage) and a custom next-generation sequencing panel (n=23 genes). Baseline radiomic original features were extracted by the pyradiomics package from CT-image delineated tumor volumes. An initial redundancy filtering was performed to remove correlating variables (r&gt;0.6). We evaluated the predictive performance of baseline ctDNA and radiomics features on overall survival (OS), progression free survival (PFS), and time to progression (TTP), through fitting Cox-regression models. Four ctDNA features were included in the models: P20-150, ichorCNA, fragment end score and mutation detection. For the radiomics features we performed an additional back- and forward variable selection based on Akaike’s Information Criterion. Using the likelihood ratio test we tested if the model fit changed after adding ctDNA and radiomics features to a model with clinical variables. Results: The addition of radiomics to clinical variables improved model fit for OS (p=0.017). Baseline prediction of OS resulted in a C-index of 0.65 using clinical variables only, 0.65 with ctDNA, 0.68 with radiomics and 0.68 with ctDNA and radiomics combined. For PFS model fit improved after adding radiomics (p=0.020) and ctDNA and radiomics combined (p=0.017). Baseline prediction of PFS resulted in a C-index of 0.64 using clinical variables, 0.65 with ctDNA, 0.67 with radiomics, and 0.68 with ctDNA and radiomics combined. For TTP model fit improved after adding radiomics (p=0.008) and radiomics and ctDNA combined (p=0.002). Baseline prediction of TTP resulted in a C-index of 0.64 with clinical variables, 0.65 with ctDNA, 0.71 with radiomics, and 0.72 with ctDNA and radiomics combined. Based on the cox-regression models using clinical variables and radiomics, risk stratification by splitting the cohort in a high and low risk group was possible for OS, PFS and TTP (p&lt;0.001). Conclusions: Combining clinical variables from SOURCE with radiomics data improved predictions of OS, PFS, and TTP among patients with rEAC. Multi-signal integration of clinical and radiomics variables could potentially be used to identify risk groups.

Investigation of the Genetic Etiology in Idiopathic Generalized Epileptic Disorders by Targeted Next-generation Sequencing Technique

Idiopathic generalized epilepsy is the most common group of epilepsy disorders in children and adolescents. Various types of genetic abnormality were identified among the hereditary factors that explain epilepsy. To determine the variations in the etiopathogenesis, treatment protocol planning, and prognosis of idiopathic generalized epilepsy using the next-generation sequencing method. A cross-sectional study. This study included 32 patients with idiopathic generalized epilepsy. Genomic DNA was obtained from peripheral venous blood samples taken from the patients. A total of 18 genes encoding ion channel subunits that are involved in monogenic disorders and are associated with idiopathic generalized epilepsy were included. The targeted custom next-generation sequencing panel was designed to cover all coding exons and all exon/intron splice site regions of 18 genes. We detected 9 (28%) variations, including 1 likely pathogenic (a variant in the SCN1A gene) and 8 of unknown clinical significance (2 in the CLCN2 genes, GABBR2, SCN1B, SLC2A1, SLC4A10 genes, and 2 in the TBC1D24 gene). Study results should be supported by functional advanced studies, with increased existing knowledge in the relevant variations.

A Novel Mutation Associated with Neonatal Lethal Cardiomyopathy Leads to an Alternative Transcript Expression in the X-Linked Complex I NDUFB11 Gene.

We report a neonatal patient with hypertrophic cardiomyopathy (HCM), lactic acidosis and isolated complex I deficiency. Using a customized next-generation sequencing panel, we identified a novel hemizygous variant c.338G>A in the X-linked NDUFB11 gene that encodes the NADH: ubiquinone oxidoreductase subunit B11 of the mitochondrial respiratory chain (MRC) complex I (CI). Molecular and functional assays performed in the proband’s target tissues—skeletal and heart muscle—showed biochemical disturbances of the MRC, suggesting a pathogenic role for this variant. In silico analyses initially predicted an amino acid missense change p.(Arg113Lys) in the NDUFB11 CI subunit. However, we showed that the molecular effect of the c.338G>A variant, which is located at the last nucleotide of exon 2 of the NDUFB11 gene in the canonical ‘short’ transcript (sized 462 bp), instead causes a splicing defect triggering the up-regulation of the expression of an alternative ‘long’ transcript (sized 492 bp) that can also be detected in the control individuals. Our results support the hypothesis that the canonical ‘short’ transcript is required for the proper NDUFB11 protein synthesis, which is essential for optimal CI assembly and activity, whereas the longer alternative transcript seems to represent a non-functional, unprocessed splicing intermediate. Our results highlight the importance of characterizing the molecular effect of new variants in the affected patient’s tissues to demonstrate their pathogenicity and association with the clinical phenotypes.

Favorable prognostic impact of phosphatase and tensin homolog alterations in wild-type isocitrate dehydrogenase and telomerase reverse transcriptase promoter glioblastoma.

Telomerase reverse transcriptase promoter (TERTp) mutations are a biological marker of glioblastoma; however, the prognostic significance of TERTp mutational status is controversial. We evaluated this impact by retrospectively analyzing the outcomes of patients with isocitrate dehydrogenase (IDH)- and TERTp-wild-type glioblastomas. Using custom next-generation sequencing, we analyzed 208 glioblastoma samples harboring wild-type IDH. TERTp mutations were detected in 143 samples (68.8%). The remaining 65 (31.2%) were TERTp-wild-type. Among the TERTp-wild-type glioblastoma samples, we observed a significant difference in median progression-free survival (18.6 and 11.4 months, respectively) and overall survival (not reached and 15.7 months, respectively) in patients with and without phosphatase and tensin homolog (PTEN) loss and/or mutation. Patients with TERTp-wild-type glioblastomas with PTEN loss and/or mutation were younger and had higher Karnofsky Performance Status scores than those without PTEN loss and/or mutation. We divided the patients with TERTp-wild-type into 3 clusters using unsupervised hierarchical clustering: Good (PTEN and TP53 alterations; lack of CDKN2A/B homozygous deletion and platelet-derived growth factor receptor alpha (PDGFRA) alterations), intermediate (PTEN alterations, CDKN2A/B homozygous deletion, lack of PDGFRA, and TP53 alterations), and poor (PDGFRA and TP53 alterations, CDKN2A/B homozygous deletion, and lack of PTEN alterations) outcomes. Kaplan-Meier survival analysis indicated that these clusters significantly correlated with the overall survival of TERTp-wild-type glioblastoma patients. Here, we report that PTEN loss and/or mutation is the most useful marker for predicting favorable outcomes in patients with IDH- and TERTp-wild-type glioblastomas. The combination of 4 genes, PTEN, TP53, CDKN2A/B, and PDGFRA, is important for the molecular classification and individual prognosis of patients with IDH- and TERTp-wild-type glioblastomas.

A SEC61A1 variant is associated with autosomal dominant polycystic liver disease.

Autosomal dominant polycystic liver and kidney disease is a spectrum of hereditary diseases, which display disturbed function of primary cilia leading to cyst formation. In autosomal dominant polycystic kidney disease a genetic cause can be determined in almost all cases. However, in isolated polycystic liver disease (PLD) about half of all cases remain genetically unsolved, suggesting more, so far unidentified genes to be implicated in this disease. Customized next-generation sequencing was used to identify the underlying pathogenesis in two related patients with PLD. A variant identified in SEC61A1 was further analysed in immortalized patients' urine sediment cells and in an epithelial cell model. In both patients, a heterozygous missense change (c.706C>T/p.Arg236Cys) was found in SEC61A1, which encodes for a subunit of the translocation machinery of protein biosynthesis at the endoplasmic reticulum (ER). While kidney disease is absent in the proposita, her mother displays an atypical polycystic kidney phenotype with severe renal failure. In immortalized urine sediment cells, mutant SEC61A1 is expressed at reduced levels, resulting in decreased levels of polycystin-2 (PC2). In an epithelial cell culture model, we found the proteasomal degradation of mutant SEC61A1 to be increased, whereas its localization to the ER is not affected. Our data expand the allelic and clinical spectrum for SEC61A1, adding PLD as a new and the major phenotypic trait in the family described. We further demonstrate that mutant SEC61A1 results in enhanced proteasomal degradation and impaired biosynthesis of PC2.

An effective preselection criterion for MODY with an increasingly positive genetic testing rate by NGS: results from two cohorts of Chinese children.

The purpose of this study was to determine the frequency of maturity-onset diabetes of the young (MODY) in two selected cohorts of Chinese children with diabetes and clinically suspected MODY, using next-generation sequencing (NGS). Ninety-three children who met the comprehensive criteria of suspected MODY were enrolled in two cohorts. A custom NGS panel or a whole exon group was used for sequencing. We identified 55/93 (59.1%) children with pathogenic and likely pathogenic MODY variants. Forty-two (76.3%) were confirmed to have the GCK (MODY2) mutation. Additionally, five had the HNF1A (MODY3), two the HNF1B (MODY5), one the 17q12 microdeletion (MODY5), two the HNF4A (MODY1), two the ABCC8 (MODY12), and one the PDX1 mutation (MODY4). Of these, 13 novel variants were detected in different genes. By comparing the gene-positive with gene-negative children, we found that discriminatory factors for MODY at diagnosis included lower HbA1c [7.4% vs. 10.2% (53 vs. 86 mmol/mol); P = 0.002], lower body mass index z score (0.2 vs. 1.0; P = 0.01), lower onset age (8.1 vs. 11.2 years; P = 0.001), and lower C-peptide (1.4 vs. 2.5 ng/mL; P = 0.02). In conclusion, the criteria used in this study for screening MODY are effective, and MODY2 is the most common subtype (76%), followed by MODY3 and MODY5. Some rare MODY subtypes have been reported in Chinese children.NEW & NOTEWORTHY We proved the clinical suspicion of maturity-onset diabetes of the young (MODY) according to the comprehensive criterion for next-generation sequencing testing, which helps to identify both common and rare MODYs, leading to accurate diagnosis and personalized treatment.