Next-generation Sequencing Gene Panel Research Articles

Clinical Impact of Genetic Diagnosis of Sensorineural Hearing Loss in Adults.

Adult genetic sensorineural hearing loss (SNHL) may be underestimated. The diagnosis of genetic hearing loss is challenging, given its extreme genetic and phenotypic heterogeneity, particularly in adulthood. This study evaluated the utility of next-generation sequencing (NGS) in the etiological diagnosis of adult-onset SNHL. Adults (>16 yr old) with SNHL were recruited at the Otolaryngology Department at Marqués de Valdecilla University Hospital (Spain). Environmental factors, acoustic trauma, endolymphatic hydrops, and age-related hearing loss were excluding criteria. An NGS gene panel was used, including 196 genes (OTOgenics v3) or 229 genes (OTOgenics v4) related to syndromic and nonsyndromic hearing loss. Sixty-five patients were included in the study (average age at the onset of SNHL, 41 yr). Fifteen pathogenic/likely pathogenic variants considered to be causative were found in 15 patients (23% diagnostic yield) in TECTA (4), KCNQ4 (3), GJB2 (2), ACTG1 (1), COL2A1 (1), COCH (1), COCH/COL2A1 (1), STRC (1), and ABHD12 (1). Three patients had syndromic associations (20% of patients with genetic diagnosis) that had not been previously diagnosed (two Stickler type I and one polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, cataract syndrome). Seven variants of unknown significance were found in COL11A1 (1), GSMDE (2), DNTM1 (1), SOX10 (1), EYA4 (1), and TECTA (1). NGS gene panels can provide diagnostic yields greater than 20% for adult SNHL, with a significant proportion of variant of unknown significance that could potentially contribute to increasing diagnostic output. Identifying a genetic cause enables genetic counseling, provides prognostic information and can reveal unrecognized syndromes contributing to an accurate management of their associated manifestations.

Next-Generation Sequencing Gene Panels in Inheritable Cardiomyopathies and Channelopathies: Prevalence of Pathogenic Variants and Variants of Unknown Significance in Uncommon Genes.

The diffusion of next-generation sequencing (NGS)-based approaches allows for the identification of pathogenic mutations of cardiomyopathies and channelopathies in more than 200 different genes. Since genes considered uncommon for a clinical phenotype are also now included in molecular testing, the detection rate of disease-causing variants has increased. Here, we report the prevalence of genetic variants detected by using a NGS custom panel in a cohort of 133 patients with inherited cardiomyopathies (n = 77) or channelopathies (n = 56). We identified 82 variants, of which 50 (61%) were identified in genes without a strong or definitive evidence of disease association according to the NIH-funded Clinical Genome Resource (ClinGen; “uncommon genes”). Among these, 35 (70%) were variants of unknown significance (VUSs), 13 (26%) were pathogenic (P) or likely pathogenic (LP) mutations, and 2 (4%) benign (B) or likely benign (LB) variants according to American College of Medical Genetics (ACMG) classifications. These data reinforce the need for the screening of uncommon genes in order to increase the diagnostic sensitivity of the genetic testing of inherited cardiomyopathies and channelopathies by allowing for the identification of mutations in genes that are not usually explored due to a currently poor association with the clinical phenotype.

P.76 Gene variant and neuromuscular findings from a long-chain fatty acid oxidation disorder gene panel program

Long-chain fatty acid oxidation disorders (LC-FAOD) are rare, life-threatening, autosomal recessive conditions that can be diagnosed clinically with plasma acylcarnitine analysis and molecular testing. Undiagnosed LC-FAOD may present with hypoglycemia, cardiomyopathy, cardiac arrhythmias, and neuromuscular symptoms. Patients with a clinical diagnosis or suspicion of LC-FAOD are eligible for this no-charge, next-generation sequencing gene panel which includes 6 genes associated with LC-FAOD plus 18 genes associated with disorders that cause abnormal acylcarnitine profiles. As of 28 October 2021, LC-FAOD gene variants were identified in 153 (37%) of 417 patients tested, including 83 variants of uncertain significance (VUS), 8 likely pathogenic (LP), and 102 pathogenic (P) variants. Twenty-three patients had positive (2 P/LP) LC-FAOD results and 19 had potential positive (2 variants, at least 1 VUS) results. VUS resolution analysis led to the reclassification of 6 variants from VUS to LP or P. Five patients had variants in two or more LC-FAOD genes and 22 had variants in one LC-FAOD gene and one or more non-LC-FAOD genes. Fifty-one patients had only one LC-FAOD gene variant identified. The most common neuromuscular symptoms among patients ages ≥13 (76 reported) were myopathy (42), elevated creatine kinase (36), and rhabdomyolysis (30), and among patients <13 years (83 reported) were elevated creatine kinase (22), and myopathy (12). Program results demonstrate the diverse composition of gene variants in patients referred for LC-FAOD genetic testing. Approaches to resolve VUS and identify previously undetected variants in patients with suspected LC-FAOD are important and necessary.

Tumor Mutational Burden by Whole-Genome Sequencing in Resected NSCLC of Never Smokers.

Data are scarce about tumor mutational burden (TMB) as a biomarker in never smokers with non-small cell lung cancer (NSCLC). TMB was assessed by whole-genome sequencing (WGS) and compared with in silico reduced whole-exome sequencing (WES) and targeted commercial next-generation sequencing (NGS) gene panels in 92 paired tumor-normal samples from never smokers who underwent NSCLC resection with curative intent. Analyses were performed to test for association with survival after surgery and to identify the optimal prognostic TMB cutoff. Tumors of never smokers with NSCLC had low TMB scores (median 1.57 mutations/Mb; range, 0.13-17.94). A TMB cutoff of 1.70 mutations/Mb was associated with a 5-year overall survival of 58% in the high-TMB (42% of cases) compared with 86% in low-TMB patients (Wald P = 0.0029). TMB scores from WGS and WES were highly correlated (Spearman ρ = 0.93, P < 2.2e-16). TMB scores from NGS panels demonstrated high intraindividual fluctuations and identified high-TMB patients with 65% concordance in average compared with WGS. In resected NSCLC of never smokers, high TMB was associated with worse prognosis. WES provided a good estimate of TMB while targeted NGS panels seem to lack adequate depth and resolution in the setting of low mutation burden. TMB is a prognostic indicator of survival in resected NSCLC from individuals who never smoked. In this setting of low mutation counts, TMB can be accurately measured by WGS or WES, but not NGS panels.

The Usefulness of a Targeted Next Generation Sequencing Gene Panel in Providing Molecular Diagnosis to Patients With a Broad Spectrum of Neurodevelopmental Disorders.

Background: Neurodevelopmental disorders comprise a clinically and genetically heterogeneous group of conditions that affect 2%–5% of children and represents a public health challenge due to complexity of the etiology. Only few patients with unexplained syndromic and non-syndromic NDDs receive a diagnosis through first-tier genetic tests as array-CGH and the search for FMR1 CGG expansion. The aim of this study was to evaluate the clinical performance of a targeted next-generation sequencing (NGS) gene panel as a second-tier test in a group of undiagnosed patients with NDDs. Method: A 221-gene next-generation sequencing custom panel was designed and used to analyze a cohort of 338 patients with a broad spectrum of NDDs (202 males and 136 females) including Intellectual Disability (ID), Autism Spectrum Disorders (ASD), Epilepsy, language and motor disorders. Results: A molecular diagnosis was established in 71 patients (21%) and a de novo origin was present in 38 (64.4%) of the available trios. The diagnostic yield was significantly higher in females than in males (29.4% vs. 15.3%; p = 0.0019) in particular in ASD (36.8% vs. 7.6%; p = 0.0026) and Epilepsy (38.9% vs. 14.4% p = 0.001). The most involved genes were SLC2A1, SCN1A, ANKRD11, ATP1A2, CACNA1A, FOXP1, and GNAS altered in more than two patients and accounting for the 19.7% of the diagnosis. Conclusion: Our findings showed that this NGS panel represents a powerful and affordable clinical tool, significantly increasing the diagnostic yield in patients with different form of NDDs in a cost- and time-effective manner without the need of large investments in data storage and bioinformatic analysis.

Analysis of chronic kidney disease patients by targeted next-generation sequencing identifies novel variants in kidney-related genes.

Despite the enormous economic and societal burden of chronic kidney disease (CKD), its pathogenesis remains elusive, impeding specific diagnosis and targeted therapy. Herein, we sought to elucidate the genetic causes of end-stage renal disease (ESRD) and identify genetic variants associated with CKD and related traits in Saudi kidney disease patients. We applied a genetic testing approach using a targeted next-generation sequencing gene panel including 102 genes causative or associated with CKD. A total of 1,098 Saudi participants were recruited for the study, including 534 patients with ESRD and 564 healthy controls. The pre-validated NGS panel was utilized to screen for genetic variants, and then, statistical analysis was conducted to test for associations. The NGS panel revealed 7,225 variants in 102 sequenced genes. Cases had a significantly higher number of confirmed pathogenic variants as classified by the ClinVar database than controls (i.e., individuals with at least one allele of a confirmed pathogenic variant that is associated with CKD; 279 (0.52) vs. 258 (0.45); p-value = 0.03). A total of 13 genetic variants were found to be significantly associated with ESRD in PLCE1, CLCN5, ATP6V1B1, LAMB2, INVS, FRAS1, C5orf42, SLC12A3, COL4A6, SLC3A1, RET, WNK1, and BICC1, including four novel variants that were not previously reported in any other population. Furthermore, studies are necessary to validate these associations in a larger sample size and among individuals of different ethnic groups.

Clinical-genetic findings in a group of subjects with macular dystrophies due to mutations in rare inherited retinopathy genes.

To describe the results of clinical and molecular analyses in a group of patients suffering from inherited macular dystrophies, in which next-generation sequencing (NGS) efficiently detected rare causative mutations. A total of eight unrelated Mexican subjects with a clinical and multimodal imaging diagnosis of macular dystrophy were included. Visual assessment methods included best corrected visual acuity, color fundus photography, Goldmann visual field tests, kinetic perimetry, dark/light adapted chromatic perimetry, full-field electroretinography, autofluorescence imaging, and spectral domain-optical coherence tomography imaging. Genetic screening was performed by means of whole exome sequencing with subsequent Sanger sequencing validation of causal variants. All patients exhibited a predominantly macular or cone-dominant disease. Patients' ages ranged from 12 to 60years. Three cases had mutations in genes associated with autosomal dominant inheritance (UNC119 and PRPH2) while the remaining five cases had mutations in genes associated with autosomal recessive inheritance (CNGA3, POC1B, BEST1, CYP2U1, and PROM1). Of the total of 11 different pathogenic alleles identified, three were previously unreported disease-causing variants. Macular dystrophies can be caused by defects in genes that are not routinely analyzed or not included in NGS gene panels. In this group of patients, whole exome sequencing efficiently detected rare genetic causes of hereditary maculopathies, and our findings contribute to expanding the current knowledge of the clinical and mutational spectrum associated with these disorders.

Whole Genome Sequencing Applied in Familial Hamartomatous Polyposis Identifies Novel Structural Variations.

Hamartomatous polyposis syndromes (HPS) are rare cancer-predisposing disorders including Juvenile polyposis (JPS), Peutz–Jeghers (PJS) and PTEN hamartomatous syndromes (PHS). Penetrant mutations in corresponding genes (SMAD4, BMPR1A, STK11, PTEN and AKT1), are usually diagnosed via a next-generation-sequencing gene panel (NGS-GP) for tailored surveillance and preimplantation testing for monogenic disorders (PGT-M). Five probands with HPS phenotype, with no genetic diagnosis per genetic workup, underwent whole-genome sequencing (WGS) that identified structural genetic alterations: two novel inversions in BMPRA1 and STK11, two BMPR1A-deletions, known as founders among Bukharan Jews, and BMPR1A microdeletion. BMPR1A inversion was validated by “junction fragment” amplification and direct testing. PGT-M was performed via multiplex-PCR and enabled successful birth of a non-carrier baby. WGS may be considered for HPS patients with no NGS-GP findings to exclude structural alterations.

MTPpilot: An Interactive Software for Visualization of Next-Generation Sequencing Results in Molecular Tumor Boards.

PURPOSEComprehensive targeted next-generation sequencing (NGS) panels are routinely used in modern molecular cancer diagnostics. In molecular tumor boards, the detected genomic alterations are often discussed to decide the next treatment options for patients with cancer. With the increasing size and complexity of NGS panels, the discussion of these results becomes increasingly complex, especially if they are reported in a text-based form, as it is the standard in current molecular pathology.METHODSWe have developed the Molecular Tumor Profiling pilot (MTPpilot) webservice using HTML, PHP, JavaScript, and MySQL to support the clinical discussion of NGS results at molecular tumor boards.RESULTSMTPpilot integrates various public genome, network, and cancer mutation databases with interactive visualization tools to assess the functional impact of mutations and support clinical decision making at tumor boards.CONCLUSIONMTPpilot is tailored for discussion of NGS gene panel results at molecular tumor boards. It is freely available as a webservice at MTPpilot.

Genetic Profile of Left Ventricular Noncompaction Cardiomyopathy in Children-A Single Reference Center Experience.

Background: Left ventricular noncompaction cardiomyopathy (LVNC) is a rare cardiac disorder characterised by the presence of a two-layer myocardium with prominent ventricular trabeculation, intertrabecular deep depressions and an increased risk of heart failure, atrial and ventricular arrhythmias and systemic thromboembolic events in affected patients. The heterogeneous molecular aetiology solved in 10%–50% of patients more frequently involves sarcomeric, cytoskeletal or ion channel protein dysfunction—mainly related to causative MYH7, TTN or MYBPC3 variants. The aim of the study was to determine the molecular spectrum of isolated LVNC in a group of children examined in a single paediatric reference centre. Methods: Thirty-one paediatric patients prospectively diagnosed with LVNC by echocardiography and cardiovascular magnetic resonance examination were recruited into the study group. The molecular analysis included next-generation sequencing (gene panel or whole exome) and classic Sanger sequencing. All selected variants with high priority were co-segregated in the available parents. Results: We identified 16 distinct variants in 11 genes in 16 patients (52%), including 10 novel alterations. The most frequent defects in our cohort were found in the genes HCN4 (n = 4), MYH7 (n = 2) and PRDM16 (n = 2). Other likely disease-causing variants were detected in ACTC1, ACTN2, HCCS, LAMA4, MYH6, RBM20, TAFFAZIN and TTN. Patients with established molecular defects more often presented with arrhythmia, thromboembolic events and death, whereas the predominant symptoms in patients with no identified molecular defects were heart failure and the presence of late gadolinium enhancement. Conclusion: This study expands the genetic and clinical spectrum of childhood LVNC. Although the molecular aetiology of LVNC varies widely, the comprehensive testing of a wide panel of cardiomyopathy-related genes helped to identify underlying molecular defects in more than half of the children in the study group. The molecular spectrum in our cohort correlated with the occurrence of arrhythmia, death and a family history of cardiomyopathy. We confirmed that genetic testing is an integral part of the work-up and management LVNC in children.

PB1854: NEXT GENERATION SEQUENCING APPROACHES IN CHRONIC LYMPHOCYTIC LEUKAEMIA: FINDINGS FROM EXTERNAL QUALITY ASSESSMENT

Background: Chronic lymphocytic leukaemia (CLL) represents a clinically heterogeneous, lymphoproliferative neoplasm resulting in chronic B-lymphocyte proliferation. Historically, large cytogenetic aberrations detected by fluorescent in situ hybridisation were identifiable in patients, enabling prognostic assessment. With the development of next generation sequencing (NGS), variants have been identified in several genes that are recurrently mutated in CLL, including TP53, ATM, BIRC3, NOTCH1 and SF3B1, with the prognostic implications of variants in these genes being reported. However, only TP53 variant status has been incorporated into routine clinical diagnostics to guide prognosis, risk stratification and appropriate therapeutic interventions based on findings indicating decreased survival and resistance to immunochemotherapy. Aims: The aims of this study were to assess NGS gene panel content and design approaches in CLL to determine levels of harmonisation amongst laboratories. Methods: The United Kingdom National External Quality Assessment Service for Leucocyte Immunophenotyping has provided external quality assessment for use of NGS technologies in CLL since 2018. The programme routinely requests NGS panel information (gene content and exon coverage) and other methodological data from participating laboratories. Data returns from 2018-2021 relating to NGS gene content and panel analysis were evaluated. Results: In total, 127 genes were analysed across 34 laboratories, ranging from 1-82 genes analysed in the context of CLL per laboratory (median of 12 genes). Of the 34 laboratories evaluated, five analysed TP53 only, with four of these utilising a non-disease specific myeloid NGS panel. The most commonly analysed genes were TP53 (100% laboratories), SF3B1 (73.5%) and NOTCH1 (70.6%). For these genes, there was a high-level of standardisation in sequencing approaches; with 91.2% of laboratories sequencing coding exons 2-11 in TP53 and 100% laboratories sequencing ‘hotspot’ exon 34 of NOTCH1, accounting for >80% variants in NOTCH1 associated with CLL. For SF3B1, 87% laboratories sequenced exons 14-16, which are hotspots for variants. Other frequently analysed genes included MYD88 (61.8% laboratories), BTK (58.8%), ATM and PLCG2 (52.9%). Summary/Conclusion: The number of genes analysed in the context of CLL amongst laboratories was highly variable. Use of NGS genetic testing approaches in haemato-oncology aids in accurate diagnosis and prognosis, however, the cost of developing these panels is not insubstantial and as such, laboratories often design panels to encompass multiple lymphoid/haematological neoplasms. Whilst recommendations outline the need for TP53 testing in CLL, the lack of technical and clinical guidelines on minimal NGS panel content and bioinformatic analysis that provide additional prognostic information has resulted in a wide variation in bioinformatic analysis approaches. This inevitably results in wide disparity of data generated and subsequent clinical interpretation. Guidelines are urgently needed for the identification of minimal NGS panel content in CLL and in other mature lymphoid neoplasms, outlining key genes directly involved in the diagnostic, prognostic and theranostic outputs in order to standardise patient testing and care.

A Novel Somatic Mutation of CACNA1H p.V1937M in Unilateral Primary Hyperaldosteronism.

BackgroundSomatic mutations for excess aldosterone production have been frequently identified as important roles in the pathogenesis of unilateral primary hyperaldosteronism (uPA). Although CACNA1H mutation represents a minor etiology in primary aldosteronism, it plays a significant role in causing uPAs in sporadic cases.ObjectiveTo identify novel somatic CACNA1H mutation in patients with uPA and investigate the pathophysiological, immunohistological, and clinical characteristics of the variant.MethodsWe applied a customized and targeted gene panel next-generation sequencing approach to detect mutations from the uPA cohort in Taiwan Primary Aldosteronism Investigation study group. Information from pre-diagnostic to postoperative data was collected, including past history, medications, blood pressure readings, biochemical data, and image studies. The functional role of the variant was confirmed by in vitro studies, demonstrating aldosterone production in variant-transfected human adrenal cell lines.ResultsWe identified a novel somatic CACNA1H mutation c.5809G>A (p.Val1937Met) in a uPA case. The CACNA1H gene encodes the pore-forming alpha-1H subunit of the voltage-dependent T-type calcium channel Cav3.2. This somatic CACNA1H p.V1937M variant showed excellent clinical and biochemical outcomes after ipsilateral adrenalectomy. The functional effect of somatic CACNA1H p.V1937M variant results in increased CYP11B2 expression and aldosterone biosynthesis in HAC15 cells. A distinct heterogeneous foamy pattern of CYP11B2 and CYP17A1 expression was identified in immunohistological staining, supporting the pathological evidence of aldosterone synthesis.ConclusionsThe somatic mutation of CACNA1H p.V1937M might be a pathogenic driver in aldosterone overproduction. This study provides new insight into the molecular mechanism and disease outcomes of uPA.

Application of a custom NGS gene panel revealed a high diagnostic utility for molecular testing of hereditary ataxias.

Hereditary ataxias (HA) are a rare group of heterogeneous disorders. Here, we present the results of molecular testing of a group of ataxia patients using a custom-designed next-generation sequencing (NGS) panel. Due to the genetic and clinical overlapping of hereditary ataxias and spastic paraplegias (HSP), the panel encompasses together HA and HSP genes. The NGS libraries, comprising coding sequences for 152 genes, were performed using KAPA HyperPlus and HyperCap Target Enrichment Kit, sequenced on the MiSeq instrument. The results were analyzed using the BaseSpace Variant Interpreter and Integrative Genomics Viewer. All pathogenic and likely pathogenic variants were confirmed using Sanger sequencing. A total of 29 patients with hereditary ataxias were enrolled in the NGS testing, and 16 patients had a confirmed molecular diagnosis with diagnostic accuracy rate of 55.2%. Pathogenic or likely pathogenic mutations were identified in 10 different genes: POLG (PEOA1, n = 3; SCAE, n = 2), CACNA1A (EA2, n = 2), SACS (ARSACS, n = 2), SLC33A1 (SPG42, n = 2), STUB1 (SCA48, n = 1), SPTBN2 (SCA5, n = 1), TGM6 (SCA35, n = 1), SETX (AOA2, n = 1), ANO10 (SCAR10, n = 1), and SPAST (SPG4, n = 1). We demonstrated that an approach based on the targeted use of the NGS panel can be highly effective and a useful tool in the molecular diagnosis of ataxia patients. Furthermore, we highlight the fact that a sequencing panel targeting both ataxias and HSP genes increases the diagnostic success level.

A Novel Targeted Amplicon Next-Generation Sequencing Gene Panel for the Diagnosis of Common Variable Immunodeficiency Has a High Diagnostic Yield: Results from the Perth CVID Cohort Study

With the advent of next-generation sequencing (NGS), monogenic forms of common variable immunodeficiency (CVID) have been increasingly described. Our study aimed to identify disease-causing variants in a Western Australian CVID cohort using a novel targeted NGS panel. Targeted amplicon NGS was performed on 22 unrelated subjects who met the formal European Society for Immunodeficiencies-Pan-American Group for Immunodeficiency diagnostic criteria for CVID and had at least one of the following additional criteria: disease onset at age <18 years, autoimmunity, low memory B lymphocytes, family history, and/or history of lymphoproliferation. Candidate variants were assessed by in silico predictions of deleteriousness, comparison to the literature, and classified according to the American College of Medical Genetics and Genomics-Association for Molecular Pathology criteria. All detected genetic variants were verified independently by an external laboratory, and additional functional studies were performed if required. Pathogenic or likely pathogenic variants were detected in 6 of 22 (27%) patients. Monoallelic variants of uncertain significance were also identified in a further 4 of 22 patients (18%). Pathogenic variants, likely pathogenic variants, or variants of uncertain significance were found in TNFRSF13B, TNFRSF13C, ICOS, AICDA, IL21R, NFKB2, and CD40LG, including novel variants and variants with unexpected inheritance pattern. Targeted amplicon NGS is an effective tool to identify monogenic disease-causing variants in CVID, and is comparable or superior to other NGS methods. Moreover, targeted amplicon NGS identified patients who may benefit from targeted therapeutic strategies and had important implications for family members.

Wide Next-Generation Sequencing Characterization of Young Adults Non-Small-Cell Lung Cancer Patients.

Simple SummaryMolecular characterization of advanced non-small-cell lung cancer (NSCLC) is mandatory before any treatment decision making. Next-generation sequencing (NGS) approaches represent the best strategy in this context. In our study, we analyzed a case series of young (under 65 years old) NSCLC patients with a wide NGS gene panel assay. The most frequent altered genes were TP53 (64.55%), followed by KRAS (44.1%), STK11 (26.9%), CDKN2A (21.5%), CDKN2B (14.0%), EGFR (16.1%), and RB1 (10.8%). Tumor mutational burden (TMB) was also evaluated considering different cut-offs, and we found a significant association between TMB and STK11 and KRAS mutations. Conversely, EGFR and EML4-ALK alterations were more frequently found in tumors with low TMB. We compared results obtained from this approach with those obtained from a single or few genes approach, observing perfect concordance of the results.Molecular characterization of advanced non-small-cell lung cancer (NSCLC) is mandatory before any treatment decision making. Next-generation sequencing (NGS) approaches represent the best strategy in this context. The turnaround time for NGS methodologies and the related costs are becoming more and more adaptable for their use in clinical practice. In our study, we analyzed a case series of young (under 65 years old) NSCLC patients with a wide NGS gene panel assay. The most frequent altered genes were TP53 (64.55%), followed by KRAS (44.1%), STK11 (26.9%), CDKN2A (21.5%), CDKN2B (14.0%), EGFR (16.1%), and RB1 (10.8%). Tumor mutational burden (TMB) was also evaluated. Considering the cut-off of 10 mut/Mb, 62 (68.9%) patients showed a TMB < 10 mut/Mb, whereas 28 (31.1%) showed a TMB ≥ 10 mut/Mb. STK11 and KRAS mutations were significantly associated with a higher TMB (p = 0.019 and p = 0.004, respectively). Conversely, EGFR and EML4-ALK alterations were more frequently found in tumors with low TMB (p = 0.019 and p < 0.001, respectively). We compared results obtained from this approach with those obtained from a single or few genes approach, observing perfect concordance of the results.

Preliminary Results of NGS Gene Panel Test Using NSCLC Sputum Cytology and Therapeutic Effect Using Corresponding Molecular-Targeted Drugs.

As more molecular-targeted drugs for advanced non-small cell lung cancer are brought to market, batch tests for the identification of gene mutations are needed at initial diagnosis. However, since current gene panel tests require a sufficient amount of tissue sample, there are many instances where panel tests cannot be performed. Therefore, we have developed a highly sensitive next generation sequencing (NGS) panel test to facilitate cytological specimens. Herein, we describe three cases positive for epidermal growth factor receptor (EGFR) exon 19 deletion, MET exon 14 skipping, and KRAS G12A using NGS analysis from sputum. In each case, genetic information was consistent with companion diagnostic analysis obtained from tissue samples collected under bronchoscopy. In cases of EGFR and MET mutations, the corresponding tyrosine kinase inhibitors were highly effective. This is the first report to demonstrate that a novel panel test could detect gene mutations in sputum samples in clinical practice and compare the gene allele ratio with the sample directly collected from the lesion.

Unraveling Structural Rearrangements of the CFH Gene Cluster in Atypical Hemolytic Uremic Syndrome Patients Using Molecular Combing and Long-Fragment Targeted Sequencing

Complement factor H (CFH) and its related proteins have an essential role in regulating the alternative pathway of the complement system. Mutations and structural variants (SVs) of the CFH gene cluster, consisting of CFH and its five related genes (CFHR1-5), have been reported in renal pathologies as well as in complex immune diseases like age-related macular degeneration and systemic lupus erythematosus. SV analysis of this cluster is challenging because of its high degree of sequence homology. Following first-line next-generation sequencing gene panel sequencing, we applied Genomic Vision's Molecular Combing Technology to detect and visualize SVs within the CFH gene cluster and resolve its structural haplotypes completely. This approach was tested in three patients with atypical hemolytic uremic syndrome and known SVs and 18 patients with atypical hemolytic uremic syndrome or complement factor 3 glomerulopathy with unknown CFH gene cluster haplotypes. Three SVs, a CFH/CFHR1 hybrid gene in two patients and a rare heterozygous CFHR4/CFHR1 deletion in trans with the common CFHR3/CFHR1 deletion in a third patient, were newly identified. For the latter, the breakpoints were determined using a targeted enrichment approach for long DNA fragments (Samplix Xdrop) in combination with Oxford Nanopore sequencing. Molecular combing in addition to next-generation sequencing was able to improve the molecular genetic yield in this pilot study. This (cost-)effective approach warrants validation in larger cohorts with CFH/CFHR-associated disease.

Expansion of the mutation and phenotypic spectrum of hereditary spastic paraplegia

Hereditary spastic paraplegias (HSPs) are a heterogeneous group of rare neurodegenerative disorders affecting the corticospinal tracts, and more than 80 HSP loci have been mapped to cause HSP. In this study, we aim to perform a genetic and clinical study of ten (6 male, 4 female) sporadic Chinese HSP patients. Next-generation sequencing (NGS) gene panels combined with multiplex ligation-dependent probe amplification assay (MLPA) analysis and the trinucleotide repeat dynamic mutation detection are available for the ten patients. Among the 10 patients, one SPG7 patient, one SPG11 patient, and one pure SPG31 patient were detected. Two variants (deletion of exon 3-9 of SPG7 gene and the heterozygous mutation c.1861C > T/p.Q621* of SPG11 gene) were novel and three (c.1150_1150 + 1insCTAC/p.G384Afs*13 in SPG7 gene, c.3075dupA/p.E1026Rfs*4 in SPG11 gene, and c.478delA/p.R160Gfs*63 of REEP1 gene/SPG31) were previously reported. The SPG11 patient presented mild intellectual with peripheral neuropathy and thin corpus callosum (TCC) with no white matter abnormalities (WMA). The SPG7 patient detected in this study is the third SPG7 family reported in China; he manifested peripheral neuropathy, scoliosis, and polydactyly which expand the phenotype spectrum of SPG7. The AAO overlapped among each HSP subtype, which limited the ability to predict the subtype of HSP from AAO. Compared with non-Asian patients, the mutation frequency of SPG7 is relatively low in Asian populations. Considering the varieties of mutation types of HSP, we suggested targeted sequencing gene panels should be combined with MLPA for diagnosis of HSP.

Genetic care in geographically isolated small island communities: 8years of experience in the Dutch Caribbean.

Worldwide, there are large inequalities in genetic service delivery. In 2011, we established a bi‐annual joint pediatric‐genetics clinic with a visiting clinical geneticist in the Dutch Caribbean. This retrospective study evaluates the yield of diagnostic testing and the clinical utility of a diagnosis for patients with rare diseases on these relatively isolated, resource‐limited islands. A total of 331 patients that were referred to the clinical geneticist between November 2011 and November 2019 and had genetic testing were included in this study. A total of 508 genetic tests were performed on these patients. Microarray, next‐generation sequencing gene panels, and single‐gene analyses were the most frequently performed genetic tests. A molecularly confirmed diagnosis was established in 33% of patients (n = 108). Most diagnosed patients had single nucleotide variants or small insertions and/or deletions (48%) or copy number variants (34%). Molecular diagnostic yield was highest in patients referred for seizures and developmental delay/intellectual disability. The genetic diagnosis had an impact on clinical management in 52% of patients. Referrals to other health professionals and changes in therapy were the most frequently reported clinical consequences. In conclusion, despite limited financial resources, our genetics service resulted in a reasonably high molecular diagnostic yield. Even in this resource‐limited setting, a genetic diagnosis had an impact on clinical management for the majority of patients. Our approach with a visiting clinical geneticist may be an example for others who are developing genetic services in similar settings.

A Multi-Gene Panel to Identify Lipedema-Predisposing Genetic Variants by a Next-Generation Sequencing Strategy.

Lipedema is a disabling disease characterized by symmetric enlargement of the lower and/or upper limbs due to deposits of subcutaneous fat, that is easily misdiagnosed. Lipedema can be primary or syndromic, and can be the main feature of phenotypically overlapping disorders. The aim of this study was to design a next-generation sequencing (NGS) panel to help in the diagnosis of lipedema by identifying genes specific for lipedema but also genes for overlapping diseases, and targets for tailored treatments. We developed an NGS gene panel consisting of 305 genes potentially associated with lipedema and putative overlapping diseases relevant to lipedema. The genomes of 162 Italian and American patients with lipedema were sequenced. Twenty-one deleterious variants, according to 3 out of 5 predictors, were detected in PLIN1, LIPE, ALDH18A1, PPARG, GHR, INSR, RYR1, NPC1, POMC, NR0B2, GCKR, PPARA in 17 patients. This extended NGS-based approach has identified a number of gene variants that may be important in the diagnosis of lipedema, that may affect the phenotypic presentation of lipedema or that may cause disorders that could be confused with lipedema. This tool may be important for the diagnosis and treatment of people with pathologic subcutaneous fat tissue accumulation.