Customized Gene Panel Research Articles

279O ctDNA detection in cerebrospinal fluid and concordance with the primary tumor in a multicenter prospective study of patients with glioma

Gliomas are the most common primary brain tumors, but there are currently many limitations for the implementation of liquid biopsy in this disease. Cerebrospinal fluid (CSF) is emerging as the most reliable reservoir for ctDNA analysis, although evidence is still limited. Prospective study of patients with gliomas diagnosed at Hospital Clinico Universitario San Carlos and Hospital Universitario La Paz (Madrid, Spain). High throughput next generation sequencing (NGS) using a customized gene panel (Illumina, Inc.) of 8 genes (IDH1, IDH2, ATRX, TP53, PTEN, PIK3CA, EGFR, BRAF) was used in paired CSF and FFPE and/or fresh tumor samples, and run in an Illumina Miseq instrument (Illumina, Inc.). Only confirmed pathogenic mutations were considered as valid and are reported here. Mutation concordance occurred when the same pathogenic gene variants were detected in tumor and CSF. Between February 2017 and March 2020, 31 glioma patients (pts) were enrolled in which 2-5 ml of CSF were collected intraoperatively prior to surgical manipulation of the tumor. M:F ratio: 22:9. Median age 51 (Min-Max: 20-78). CSF collected at new diagnosis (n=22); relapse (n=9). WHO 5th Ed: IDHMUT astrocytoma (n=9), IDHMUT oligodendroglioma (n=6), IDHWT glioblastoma (n=16). CSF-ctDNA-positive: 18/31 (58%). CSF-ctDNA-negative: 13/31 (42%). No. of mutations in CSF: 1 (9/18), 2 (7/18), 3 (2/18). Frequency of CSF-ctDNA mutated genes: EGFR (8/18: 44%), PTEN (7/18: 39%), TP53 (6/18: 33%), IDH1 (4/18: 22%), PIK3CA (4/18: 22%). Tumor-CSF mutation concordance: 15/18 (83%). After a median follow-up since CSF collection of 20 months (m) (Min-Max: 0-57), median OS was not reached (NR) (95%CI NR-NR). No correlation was found between detection of ctDNA in CSF and distance from closest CSF reservoir, tumor size or IDH status. CSF is a reliable reservoir for ctDNA analyses in patients with gliomas. Larger, prospective studies should be conducted in order to refine the potential role of liquid biopsy in this disease.

360P Epigenetic engineering on CDX2 allows to determine the interplaying consequences with other genetic and transcriptomic events in colorectal cancer

The loss of the caudal homeobox 2 protein (Cdx2) is associated with poor prognosis in colorectal cancer (CRC) patients. However, the mechanisms that generate the loss of expression and the interplay with other genetic and transcriptomic aberrations remain unknown. Understanding the regulatory mechanism and interaction processes of CDX2, through a multi-omic approach, would help to change the genomic/transcriptomic tumor pattern, favoring sensitivity to standard treatment. CDX2 promoter methylation and mutations were studied by Miseq in both 7 CRC cell lines and 50 CRC patients. Azaytidine was used as demethylation treatment. A parallel study using CRISPR-dCAS9 fused with the catalytic TET and DNMT domains was required for the expression reversal studies. Determination of CDX2 status has been performed using a new approach developed in-house. A custom gene panel was used to study other genomic aberrations. Transcriptomic analysis by RNAseq was assessed through differential expression by DESeq2. Among the analyzed CG positions, a promoter island with an increased methylation rate tumor/normal and inverse correlation with expression was identified. Azacytidine treatment showed a dose-dependent correlation between methylation and expression (p<0.001). We also demostrated a specific reversion of CDX2 expression using CRISPR-dCAS9, subsequently changes in FOLFOX sensitivity was observed (p<0.05). Patients with CDX2- had higher levels of methylation in the studied CpG island than those with CDX2+. NGS showed no functional mutations or copy-number changes on CDX2-. Upstream mutations displayed an interaction between CDX2- and mutations in FLNA. Transcriptomic analysis revealed changes in INFa response pathway and MAPK proliferation genes. CDX2 promoter hypermethylation is the main regulatory mechanism of CDX2 expression. Loss of CDX2 interacts with immune and proliferation pathways at genomic and transcriptomic levels, conferring an agresive phenotype. Epigenetic engineering modifications of CDX2 could favor increase the sensitivity to standar therapy in this setting.

Determining the genetic contribution in patients with non-syndromic ascending thoracic aortic aneurysms: Correlation with findings from computational pathology

ObjectivesThis study aims to identify the clinical utility of targeted-genetic sequencing in a cohort of patients with TAA and establish a new method for regional histological characterisation of TAA disease. MethodsFifty-four patients undergoing surgery for proximal TAA were recruited. Exclusions: connective tissue disease, bicuspid aortic valves, redo surgery. All patients underwent next generation sequencing (NGS) using a custom gene panel containing 63 genes previously associated with TAA on Illumina MiSeqor NextSeq550 platforms. Explanted TAA tissue was obtained en-bloc from 34/54 patients, and complete circumferential strips of TAA tissue processed into whole slides which were subsequently digitalised. Computational pathology methods were employed to quantify elastin, cellularity and collagen in six equally divided regions across the whole aneurysm circumference. ResultsOf 54 patients, clearly pathogenic or potentially pathogenic variants were found in 7.4%: namely LOX, PRKG1, TGFBR1 and SMAD3 genes. 55% had at least one variant of unknown significance (VUS) and seven of the VUSs were in genes with a strong disease association (category A) genes, whilst 15 were from moderate risk (category B) genes. Elastin and collagen abundance displayed high regional variation throughout the aneurysm circumference. In patients with <60% total elastin, the loss of elastin was more significant on the outer curve (38.0% vs 47.4%, p = 0.0094). The presence of VUS, higher pulse wave velocity and advancing age were predictors of elastin loss (regression analysis: p < 0.05). ConclusionsThese findings demonstrate the heterogeneity of TAA disease microstructure and the potential link between histological appearance and clinical factors, including genetic variation.

TWNK in Parkinson's Disease: A Movement Disorder and Mitochondrial Disease Center Perspective Study.

BackgroundParkinsonian features have been described in patients harboring variants in nuclear genes encoding for proteins involved in mitochondrial DNA maintenance, such as TWNK.ObjectivesThe aim was to screen for TWNK variants in an Italian cohort of Parkinson's disease (PD) patients and to assess the occurrence of parkinsonism in patients presenting with TWNK‐related autosomal dominant progressive external ophthalmoplegia (TWNK‐adPEO).MethodsGenomic DNA of 263 consecutively collected PD patients who underwent diagnostic genetic testing was analyzed with a targeted custom gene panel including TWNK, as well as genes causative of monogenic PD. Genetic and clinical data of 18 TWNK‐adPEO patients with parkinsonism were retrospectively analyzed.ResultsSix of 263 PD patients (2%), presenting either with isolated PD (n = 4) or in combination with bilateral ptosis (n = 2), carried TWNK likely pathogenic variants. Among 18 TWNK‐adPEO patients, 5 (28%) had parkinsonism.ConclusionsWe show candidate TWNK variants occurring in PD without PEO. This finding will require further confirmatory studies. © 2022 Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson Movement Disorder Society.

Utility of targeted next generation sequencing for inborn errors of immunity at a tertiary care centre in North India

Inborn errors of immunity (IEI) are a heterogeneous group of monogenic disorders that include primary immunodeficiency’s and other disorders affecting different aspects of the immune system. Next-Generation Sequencing (NGS) is an essential tool to diagnose IEI. We report our 3-year experience in setting up facilities for NGS for diagnosis of IEI in Chandigarh, North India. We used a targeted, customized gene panel of 44 genes known to result in IEI. Variant analysis was done using Ion Reporter software. The in-house NGS has enabled us to offer genetic diagnoses to patients with IEI at minimal costs. Of 121 patients who were included pathogenic variants were identified in 77 patients. These included patients with Chronic Granulomatous Disease, Severe Combined Immune Deficiency, leukocyte adhesion defect, X-linked agammaglobulinemia, Ataxia Telangiectasia, Hyper-IgE syndrome, Wiskott Aldrich syndrome, Mendelian susceptibility to mycobacterial diseases, Hyper-IgM syndrome, autoimmune lymphoproliferative syndrome, and GATA-2 deficiency. This manuscript discusses the challenges encountered while setting up and running targeted NGS for IEI in our unit. Genetic diagnosis has helped our patients with IEI in genetic counselling, prenatal diagnosis, and accessing appropriate therapeutic options.

Genetic and clinical profile of patients with hypophosphatemic rickets

Nutritional vitamin D deficiency is the most frequent cause of rickets followed by genetic causes, that include entities like classic hypophosphatemic rickets (FGF23 related), Dent disease, Fanconi syndrome, renal tubular acidosis, and vitamin D dependent rickets. Hypophosphatemia is a feature in all these forms. The diagnosis relies on a combination of clinical, biochemical and radiological features, but genetic testing is required to confirm the diagnosis. We screened 66 patients with hypophosphatemic rickets referred to this center between May 2015 and July 2019 using whole exome sequencing (WES) in addition to the measurement of their intact serum fibroblast growth factor 23 (FGF23) levels. WES revealed 36 pathogenic and 28 likely pathogenic variants in 16 different genes (PHEX, FGF23, DMP1, ENPP1, CLCN5, CTNS, SLC2A2, GATM, SLC34A1, EHHADH, SLC4A1, ATP6V1B1, ATP6V0A4, CYP27B1, VDR and FGFR1) in 63 patients which helped differentiate between the various forms of hypophosphatemic rickets. Intact serum FGF23 levels were significantly higher in patients with variations in PHEX, FGF23, DMP1 or ENPP1 genes. The major genetic causes of rickets were classic hypophosphatemic rickets with elevated FGF23 levels, distal renal tubular acidosis, and vitamin D dependent rickets. Based on the present results, we propose a customized gene panel for targeted exome sequencing, which will be useful for confirming the diagnosis in most patients with hypophosphatemic rickets.

Stratification of Oligometastatic Prostate Cancer Patients by Liquid Biopsy: Clinical Insights from a Pilot Study.

We propose a pilot, prospective, translational study with the aim of identifying possible molecular markers underlying metastatic prostate cancer (PC) evolution with the use of liquid biopsy. Twenty-eight castrate sensitive, oligometastatic PC patients undergoing bone and/or nodal stereotactic body radiotherapy (SBRT) were recruited. Peripheral blood samples were collected before the commencement of SBRT, then they were processed for circulating cell free DNA (cfDNA) extraction. Deep targeted sequencing was performed using a custom gene panel. The primary endpoint was to identify differences in the molecular contribution between the oligometastatic and polymetastatic evolution of PC to same-first oligo-recurrent disease presentation. Seventy-seven mutations were detected in 25/28 cfDNA samples: ATM in 14 (50%) cases, BRCA2 11 (39%), BRCA1 6 (21%), AR 13 (46%), ETV4, and ETV6 2 (7%). SBRT failure was associated with an increased risk of harboring the BRCA1 mutation (OR 10.5) (p = 0.043). The median cfDNA concentration was 24.02 ng/mL for ATM mutation carriers vs. 40.04 ng/mL for non-carriers (p = 0.039). Real-time molecular characterization of oligometastatic PC may allow for the identification of a true oligometastatic phenotype, with a stable disease over a long time being more likely to benefit from local, curative treatments or the achievement of long-term disease control. A prospective validation of our promising findings is desirable for a better understanding of the real impact of liquid biopsy in detecting tumor aggressiveness and clonal evolution.

Genetic Analysis of the LOXHD1 Gene in Chinese Patients With Non-Syndromic Hearing Loss.

Non-syndromic hearing loss (NSHL) is a common neurosensory disease with an extreme genetic heterogeneity which has been linked to variants in over 120 genes. The LOXHD1 gene (DFNB77), encoding lipoxygenase homology domain 1, is a rare hearing loss gene found in several populations. To evaluate the importance of LOXHD1 variants in Chinese patients with NSHL, we performed genetic analysis on LOXHD1 in 2,901 sporadic Chinese patients to identify the aspect and frequency of LOXHD1 causative variants. Next-generation sequencing using a custom gene panel of HL was conducted on 2,641 unrelated patients and whole-exome sequencing on the remaining 260 patients. A total of 33 likely causative variants were identified in 21 patients, including 20 novel variants and 13 previously reported pathogenic variants. Each of the 20 novel variants was evaluated according to ACMG criteria. These findings showed that causative variants in LOXHD1 were found in about 0.72% (21/2,901) of Chinese NSHL patients. This study is by far the largest number of novel variants identified in this gene expanding the range of pathogenic variants in LOXHD1, and suggests that variants in this gene occur relatively commonly in Chinese NSHL patients. This extensive investigation of LOXHD1 in Chinese NSHL patients proposed six recurrent LOXHD1 variants. These findings may assist in both molecular diagnosis and genetic counseling.

Rapid Identification of Drug-Resistant Tuberculosis Genes Using Direct PCR Amplification and Oxford Nanopore Technology Sequencing.

Mycobacterium tuberculosis antimicrobial resistance has been continually reported and is a major public health issue worldwide. Rapid prediction of drug resistance is important for selecting appropriate antibiotic treatments, which significantly increases cure rates. Gene sequencing technology has proven to be a powerful strategy for identifying relevant drug resistance information. This study established a sequencing method and bioinformatics pipeline for resistance gene analysis using an Oxford Nanopore Technologies sequencer. The pipeline was validated by Sanger sequencing and exhibited 100% concordance with the identified variants. Turnaround time for the nanopore sequencing workflow was approximately 12 h, facilitating drug resistance prediction several weeks earlier than that of traditional phenotype drug susceptibility testing. This study produced a customized gene panel assay for rapid bacterial identification via nanopore sequencing, which improves the timeliness of tuberculosis diagnoses and provides a reliable method that may have clinical application.

Multigene Panel Sequencing Reveals Cancer-Specific and Common Somatic Mutations in Colorectal Cancer Patients: An Egyptian Experience.

This study aims at identifying common pathogenic somatic mutations at different stages of colorectal carcinogenesis in Egyptian patients. Our cohort included colonoscopic biopsies collected from 120 patients: 20 biopsies from patients with inflammatory bowel disease, 38 from colonic polyp patients, and 62 from patients with colorectal cancer. On top of this, the cohort included 20 biopsies from patients with non-specific mild to moderated colitis. Targeted DNA sequencing using a customized gene panel of 96 colorectal related genes running on the Ion Torrent NGS technology was used to process the samples. Our results revealed that 69% of all cases harbored at least one somatic mutation. Fifty-seven genes were found to carry 232 somatic non-synonymous variants. The most frequently pathogenic somatic mutations were localized in TP53, APC, KRAS, and PIK3CA. In total, 16 somatic mutations were detected in the CRC group and in either the IBD or CP group. In addition, our data showed that 51% of total somatic variants were CRC-specific variants. The average number of CRC-specific variants per sample is 2.4. The top genes carrying CRC-specific mutations are APC, TP53, PIK3CA, FBXW7, ATM, and SMAD4. It seems obvious that TP53 and APC genes were the most affected genes with somatic mutations in all groups. Of interest, 85% and 28% of the APC and TP53 deleterious somatic mutations were located in Exon 14 and Exon 3, respectively. Besides, 37% and 28% of the total somatic mutations identified in APC and TP53 were CRC-specific variants, respectively. Moreover, we identified that, in 29 somatic mutations in 21 genes, their association with CRC patients was unprecedented. Ten detected variants were likely to be novel: six in PIK3CA and four variants in FBXW7. The detected P53, Wnt/βcatenin, Angiogenesis, EGFR, TGF-β and Interleukin signaling pathways were the most altered pathways in 22%, 16%, 12%, 10%, 9% and 9% of the CRC patients, respectively. These results would contribute to a better understanding of the colorectal cancer and in introducing personalized therapies for Egyptian CRC patients.

820 New Insights into the Genomic Landscape of Skull Base Meningiomas

INTRODUCTION: The management of skull base meningiomas is challenging due to their complexity and proximity to crucial nearby structures. The identification of oncogenic mutations has provided further insights into the tumorigenesis of meningioma and the possibility of targeted therapy. METHODS: Tissue samples were collected from 71 patients diagnosed with meningioma from 2008 to 2016. Tissues were treated and archived as formalin-fixed paraffin-embedded (FFPE). Samples were subjected to targeted sequencing using a customized gene panel. RESULTS: The study cohort comprised 71 patients with histologically proven meningioma (grades I, II, and III) .The median patient age was 54.8 years (range 27-96 years). Most of the patients were female (51/71, 72.82%), while 19/71 (27.14%) were male. 56/71 patients had grade I (80.0%), 13/71 (18.57%) had grade II and 1/71 (1.428%) had grade III tumors. A total of 51/71 of the tumors were found in the skull base (72.82%). 22/71 (31.42%) of the tumors were meningothelial, 6/71 (8.57%) were atypical, 6/71 (8.57%) were transitional, 3/71 (4.28) were secretory, and 2/71 (2.86%) were chordoid. We detected an average of 1.56 ± 1.07 genomic alterations (GAs) per patient. the most common mutations were in NF2 (52/71), PIK3CA (22/71), FGFR3 (13/71), SMO (11/71) and AKT1 (10/71), with Tertp (1/71) mutations being the least frequent. The NF2-positive tumors were predominantly of grade I 43/52 (82.69%) with lower rates of recurrence (7/52, 13.41%) in tumors harboring NF2 mutations compared to tumor harboring non-NF2 mutations (8/18, 44.44%). Single FGFR3 mutations reported in three patients, all had WHO grade I tumors, with no recurrence in our cohort. CONCLUSIONS: This cohort focusing skull base meningiomas, highlights a range of mutations outside the known cancer driver NF2 that may be linked to meningioma prognosis. Among our findings were the identification of a rare TERTp mutation and the first report of FGFR3 mutations that may represent biomarkers for the identification of skull base meningioma patients with a favorable prognosis. Taken together, these findings highlight how genetic profiling can guide optimal treatment strategies, prognostic prediction, and patient management for skull meningioma.

Abstract P2-01-15: Developing highly sensitive high NGS data efficient ctDNA detection assays for breast cancer surveillance

Abstract Introduction: Growing data established the importance of monitoring dynamic changes in circulating tumor DNA (ctDNA) to identify early signs of therapeutic responses, allowing for timely management of treatment to achieve more effective personalized therapy. Higher assay accuracy and consistency, and lower assay cost will support more clinical validation trials and benefit more cancer patients with non-invasive ctDNA NGS tests that can simultaneously map multiple genomic alterations at an affordable price. Method: The NVIGEN X - Precision Cancer Profiling test is a next generation sequencing (NGS) based circulating tumor DNA detection assay using the hybridization capture approach with customized gene panels. Our ctDNA NGS assay was developed with the use of high performance magnetic nanobeads, which enhances assay workflow at key steps including cfDNA extraction, NGS library preparation, and target enrichment. Experiments with individual plasma samples and DNA mutant fragments spiked in plasma samples were carried out to establish the assay performance such as sensitivity, specificity, consistency and data efficiency. NGS data QC metrics of the NVIGEN assay were compared with other assays in peer reviewed publications. Results: We developed a focus 32 gene panel that covers 144 kb of gene regions of clinical significance for breast cancer treatment monitoring and guidance, such as AKT1, ERBB2, PIK3CA, EGFR, ESR1, BRCA1/2, and CD274. Our results demonstrated the capability of NVIGEN X ctDNA NGS assay to detect rare copies (8 cp) of gene mutation at 0.07% MAF from DNA mutant fragments spiked into plasma samples. The NVIGEN X ctDNA NGS assays consistently presented 2-5% duplication rate, &amp;gt;80% on-target rate, &amp;lt;10% CV for key NGS data metrics, and on average required 1.36X paired reads per 1X unique coverage. Compared with the Roche Avenio assays (targeted, expanded and surveillance panels) as published in 2020 which on average required 9.36X paired reads per 1X unique coverage, the NVIGEN X -precision cancer profiling assays demonstrated 85% reduction in NGS data need to generate each unique coverage. Compared with the original Capp-seq data as published in the 2014 Nature Medicine paper which required in average 13.78 or 27.56 paired reads per unique coverage, the NVIGEN X assay demonstrated &amp;gt;90% reduction in NGS data need per unique coverage. Conclusion: The NVIGEN X - Precision Cancer Profiling assay provided high NGS assay performance with high sensitivity, specificity, and consistency, and significantly improved NGS data efficiency. This allows for dramatically reduced assay cost and will help support routine applications of ctDNA NGS tests to improve cancer patient treatment. Experiments of applying NVIGEN X assays for clinical research with patient samples are ongoing and will be presented. Citation Format: Aihua Fu, Wenwu Cui, Minh V. Ton, Kevan Wang, Weiwei Gu, Tianhong Li, Heather A. Parsons, Minetta C. Liu, George W. Sledge. Developing highly sensitive high NGS data efficient ctDNA detection assays for breast cancer surveillance [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-01-15.

Epidemiology and Molecular Profile of Mucosal Melanoma: A Population-Based Study in Southern Europe.

Simple SummaryThere are few population-based studies focused on the epidemiology of mucosal melanoma, a rare neoplasm. Its poor prognosis, the different etiology from cutaneous melanoma and the lack of effective treatment beyond corrective surgery, make the knowledge of the mutational profile of this type of cancer a useful tool in understanding its natural history and also for the investigation of new target therapies. The aim of our population-based study is to analyze the incidence and survival of mucosal melanoma, which mainly arises from the head and neck sphere, genitourinary tract and rectal area, and to carry out the mutational analysis of selected cases. We used the Girona Cancer Registry database, which registered all cancer cases in Girona, a province of Spain in southern Europe, during the period of 1994–2018.Background: Mucosal melanoma is a rare neoplasm on which few epidemiological population-based studies have been published. A good surgical approach is the standard treatment, but the prognosis is worse than that of skin melanoma. The analysis of mucosal melanoma’s mutational profile can help to develop target therapies in advanced disease or adjuvant settings. Methods: We analyzed the database of the Cancer Registry of Girona, a region located in the north-east of Spain, in the period of 1994–2018. We selected cases of primary invasive melanoma, excluding those located in the skin, eye, central nervous system and an unknown primary site. Epidemiological analysis included incidence and survival. Mutational profile analysis was performed with a custom gene panel. Results: Forty-two patients were identified: 14 (33%) had vulvar-vaginal melanoma, 15 (35.7%) had rectal melanoma, 12 (28.6%) had melanoma located in the head and neck sphere and 1 male patient had a urethral melanoma. European age-standardized incidence rates for vulvar-vaginal, rectal and head and neck melanoma were 0.09, 0.1 and 0.09 cases/100,000 inhabitant-years, respectively. Five-year observed survival rates were 37.5%, 20% and 25% for these types of cancers. NRAS Q61 was the most frequent mutation found. Conclusion: Our study confirms the steady incidence and low survival of mucosal melanomas in a region of southern Europe. NRAS and NF1 play a role in the molecular landscape of mucosal melanoma. MEK and PI3K/mTOR inhibitors could be reasonable treatment options and are being studied in clinical trials.

Good performance of the criteria of American College of Medical Genetics and Genomics/Association for Molecular Pathology in prediction of pathogenicity of genetic variants causing thoracic aortic aneurysms and dissections

BackgroundThe identification of pathogenic variant in patients with thoracic aortic aneurysms and dissections (TAAD) was previously found to be a significant indicator pointing to earlier need for surgical intervention. In order to evaluate available methods for classifying identified genetic variants we have compared the event-free survival in a cohort of TAAD patients classified as genotype-positive versus genotype-negative by the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG-AMP) criteria or by ClinVar database.MethodsWe analyzed previously unreported cohort of 132 patients tested in the routine clinical setting for genetic variants in a custom panel of 30 genes associated with TAAD or the TruSight Cardio commercial panel of 174 genes associated with cardiac disease. The identified variants were classified using VarSome platform. Kaplan–Meier survival curves were constructed to compare the event-free survival between probands defined as ‘genotype-positive’ and ‘genotype-negative’ using different classifications in order to compare their performance.ResultsOut of 107 rare variants found, 12 were classified as pathogenic/likely pathogenic by ClinVar, 38 were predicted to be pathogenic/likely pathogenic by ACMG. Variant pathogenicity as assessed by ACMG criteria was a strong predictor of event free survival (event free survival at 50 years 83% vs. 50%, for genotype positive patients vs. reference, respectively, p = 0.00096). The performance of ACMG criteria was similar to that of ClinVar (event free survival at 50 years 87% vs. 50%, for genotype positive patients vs. reference, respectively p = 0.023) but independent from it as shown by analysing variants with no ClinVar record (event free survival at 50 years 80% vs. 50%, p = 0.0039). Variants classified as VUS by ACMG criteria or ClinVar did not affect event-free survival. TAAD specific custom gene panel performed similar to the larger universal cardiac panel.ConclusionsIn our cohort of unrelated TAAD patients ACMG classification tool available at VarSome was useful in assessing pathogenicity of novel genetic variants. Gene panel containing the established genes associated with the highest risk of hereditary TAAD (ACTA1, COL3A1, FBN1, MYH11, SMAD3, TGFB2, TGFBR1, TGFBR2, MYLK) was sufficient to identify prevailing majority of variants most likely to be causative of the disease.

Identification of Survival-Specific Genes in Clear Cell Renal Cell Carcinoma Using a Customized Next-Generation Sequencing Gene Panel.

Purpose: Although mutations are associated with carcinogenesis, little is known about survival-specific genes in clear cell renal cell carcinoma (ccRCC). We developed a customized next-generation sequencing (NGS) gene panel with 156 genes. The purpose of this study was to investigate whether the survival-specific genes we found were present in Korean ccRCC patients, and their association with clinicopathological findings. Materials and Methods: DNA was extracted from the formalin-fixed, paraffin-embedded tissue of 22 ccRCC patients. NGS was performed using our survival-specific gene panel with an Illumina MiSeq. We analyzed NGS data and the correlations between mutations and clinicopathological findings and also compared them with data from the Cancer Genome Atlas-Kidney Renal Clear Cell Carcinoma (TCGA-KIRC) and Renal Cell Cancer-European Union (RECA-EU). Results: We found a total of 100 mutations in 37 of the 156 genes (23.7%) in 22 ccRCC patients. Of the 37 mutated genes, 11 were identified as clinicopathologically significant. Six were novel survival-specific genes (ADAMTS10, CARD6, NLRP2, OBSCN, SECISBP2L, and USP40), and five were top-ranked mutated genes (AKAP9, ARID1A, BAP1, KDM5C, and SETD2). Only CARD6 was validated as an overall survival-specific gene in this Korean study (p = 0.04, r = −0.441), TCGA-KIRC cohort (p = 0.0003), RECA-EU (p = 0.0005). The 10 remaining gene mutations were associated with clinicopathological findings; disease-free survival, mortality, nuclear grade, sarcomatoid component, N-stage, sex, and tumor size. Conclusions: We discovered 11 survival-specific genes in ccRCC using data from TCGA-KIRC, RECA-EU, and Korean patients. We are the first to find a correlation between CARD6 and overall survival in ccRCC. The 11 genes, including CARD6, NLRP2, OBSCN, and USP40, could be useful diagnostic, prognostic, and therapeutic markers in ccRCC.

Prediction of fetal blood group antigens from maternal plasma using Ion AmpliSeq HD technology.

Fetal blood group (BG) and platelet (HPA) antigens may trigger maternal immunization, causing a fetal disease. Noninvasive prenatal diagnostics (NIPT) predicts fetal genotype, identifying pregnancies with no risk. All current techniques detect fetal antigen alleles with unspecific background and without estimation of fetal fraction, thus new protocols for detection of fetal BG/HPA alleles with ultrahigh sensitivity still need to be tested to improve NIPT. To design NIPT of clinically important antigens using Ion AmpliSeq HD technology. Plasma DNA from 36 pregnant women (9-33 week of gestation, 24 immunized with anti-HPA-1a,-3b,-15a, -K, or -D+C+S), with known BG/HPA genotypes of their neonates/partners, was tested on Ion S5 System using the Ion AmpliSeq HD designer custom gene panel. NGS contained 25 rs-targets encoding relevant BG/HPA antigens and 10 markers. Using the NGS protocol, 76 out of 85 differences in fetal/maternal BG/HPA genotypes were determined in concentration above 2% fetal paternally inherited allele chimerism. The level of unspecific reads for BG/HPA alleles was below 0.87%. In 24 immunized women NGS revealed feto-maternal incompatibility in 11 cases (from 2.44% to 7.41%) and excluded in 10 (<0.05%), three cases had inconclusive results (1.79%, 0.19%, 0.11%). The presence of fetal DNA was confirmed in each case by detecting markers with at least 2% chimerism. The use of Ion AmpliSeq HD technology improves the prediction of feto-maternal incompatibility, increasing the sensitivity of BG/HPA NIPT and serving confirmation of the fetal DNA at the same workflow.

Multifaceted and Age-Dependent Phenotypes Associated With Biallelic PNPLA6 Gene Variants: Eight Novel Cases and Review of the Literature.

A wide spectrum of neurodegenerative diseases has been associated with pathogenic variants in the PNPLA6 (patatin-like phospholipase domain-containing protein 6) gene, including spastic paraplegia type 39, Gordon—Holmes, Boucher—Neuhauser, Oliver—Mc Farlane, and Laurence—Moon syndromes. These syndromes present variable and overlapping clinical symptoms, encompassing cerebellar ataxia, hypogonadotropic hypogonadism, chorioretinal dystrophy, spastic paraplegia, muscle wasting, peripheral neuropathy, and cognitive impairment. In the present study, we performed a wide genetic screening in 292 patients presenting with ataxia or spastic paraplegia using a probe-based customized gene panel, covering >200 genes associated with spinocerebellar diseases. We identified six novel and four recurrent PNPLA6 gene variants in eight patients (2.7%). Six patients presented an infantile or juvenile onset (age <18), and two patients had an adult onset. Cerebellar ataxia was observed in seven patients and spastic paraplegia in one patient. Progression of cerebellar symptoms was slow in all patients, who retained ambulation even after a mean disease duration of 15 years. Brain MRI showed cerebellar atrophy in 6/8 patients, more pronounced in superior and dorsal vermis lobules (I to VII). Additional clinical features included hypogonadotropic hypogonadism (5/8), growth hormone deficiency (2/8), peripheral axonal neuropathy (4/8), cognitive impairment (3/8), chorioretinal dystrophy (2/8), and bilateral vestibular areflexia with a reduced visual vestibule-ocular reflex (1/8). In accordance with previous studies, chorioretinal dystrophy was the most frequent presenting symptom in early onset patients, hypogonadotropic hypogonadism in juvenile onset cases, and cerebellar ataxia in adult patients. One patient had an initial clinical presentation compatible with Cerebellar Ataxia with Neuropathy and Vestibular Areflexia Syndrome (CANVAS), but no pathological expansions in the RFC1 gene. In conclusion, patients with PNPLA6 variants present a variable age of onset spanning from infancy to adulthood, and each clinical symptom has an age-dependent manifestation thus requiring a multi-systemic diagnostic approach. The description of patients presenting very late-onset cerebellar ataxia suggests that PNPLA6 genetic screening should also be considered in the diagnostic workout of adult cerebellar ataxia.

Identification of a PROM1 mutation in a Spanish family with inherited retinal dystrophies

Background: We report a Spanish family, comprising an affected mother and daughter, respectively diagnosed with retinitis pigmentosa and Stargardt-like macular dystrophy, in whom we identified a PROM1 mutation. Methods: A custom gene panel consisting of 119 inherited retinal dystrophies (IRD)-genes was applied in the two affected individuals of this family and sequenced using the Illumina´s NextSeq500 platform. Results: The analysis of the resulting data allowed us to identify the pathogenic PROM1 mutation c.1117C&gt;T (p.Arg373Cys) as the primary cause of the disease in both patients. No additional variants contributing to the extent of retinal dysfunction were detected. Conclusion: The variable expressivity of the detected PROM1 mutation is the most likely responsible for the intrafamilial phenotypic variability observed in this family. Screening of this mutation should be considered in patients with compatible clinical manifestations, especially when accompanied by an autosomal dominant family history.

Alterations in SLC4A2, SLC26A7 and SLC26A9 Drive Acid-Base Imbalance in Gastric Neuroendocrine Tumors and Uncover a Novel Mechanism for a Co-Occurring Polyautoimmune Scenario.

Autoimmune polyendocrine syndrome (APS) is assumed to involve an immune system malfunction and entails several autoimmune diseases co-occurring in different tissues of the same patient; however, they are orphans of its accurate diagnosis, as its genetic basis and pathogenic mechanism are not understood. Our previous studies uncovered alterations in the ATPase H+/K+ Transporting Subunit Alpha (ATP4A) proton pump that triggered an internal cell acid–base imbalance, offering an autoimmune scenario for atrophic gastritis and gastric neuroendocrine tumors with secondary autoimmune pathologies. Here, we propose the genetic exploration of APS involving gastric disease to understand the underlying pathogenic mechanism of the polyautoimmune scenario. The whole exome sequencing (WES) study of five autoimmune thyrogastric families uncovered different pathogenic variants in SLC4A2, SLC26A7 and SLC26A9, which cotransport together with ATP4A. Exploratory in vitro studies suggested that the uncovered genes were involved in a pathogenic mechanism based on the alteration of the acid–base balance. Thus, we built a custom gene panel with 12 genes based on the suggested mechanism to evaluate a new series of 69 APS patients. In total, 64 filtered putatively damaging variants in the 12 genes of the panel were found in 54.17% of the studied patients and none of the healthy controls. Our studies reveal a constellation of solute carriers that co-express in the tissues affected with different autoimmune diseases, proposing a unique genetic origin for co-occurring pathologies. These results settle a new-fangled genetics-based mechanism for polyautoimmunity that explains not only gastric disease, but also thyrogastric pathology and disease co-occurrence in APS that are different from clinical incidental findings. This opens a new window leading to the prediction and diagnosis of co-occurring autoimmune diseases and clinical management of patients.

Identification of Nephrin gene variants in Indian children associated with Steroid sensitive and Steroid resistant nephrotic syndrome

Nephrotic syndrome (NS) remains the most frequent indication of glomerular disease in childhood. The NPHS1 gene encoding nephrin protein is one of the common mutant genes in SRNS (Steroid Resistant Nephrotic syndrome) patients. These gene mutations are more likely to progress into ESKD (end-stage kidney disease). In the present study, we investigated the pathogenesis of the NPHS1 gene in Indian patients with SSNS and SRNS. Ninety-six children have been involved in the present study in which 37 belong to Steroid Sensitive Nephrotic syndrome (SSNS), 27 belong to Steroid Resistant Nephrotic syndrome (SRNS) 32 are healthy individuals. We designed a panel of NPHS1 gene (CDS- Coding sequence) and performed Illumina sequencing (MiSeq). We found a total of 45 variants in the NPHS1 gene. In addition, we found novel pathogenic c.2441A > G and two likely pathogenic missense variants, p.Gly395Ser, p.Ser400Pro in SSNS and SRNS patients by ACMG criteria. Therefore, early diagnosis of steroid resistant nephrotic syndrome in the Indian population can be made by identifying genetic variants using a customized gene panel.