Molecular Diagnosis of Infantile Mitochondrial Disease with Targeted Next-Generation Sequencing

Mitochondrion-targeted RNA therapies as a potential treatment strategy for mitochondrial diseases

Detection of FLT3 Internal Tandem Duplication in Targeted, Short-Read-Length, Next-Generation Sequencing Data

Background: Primary immunodeficiencies (PIDs) are a diverse group of disorders caused by multiple genetic defects. Obtaining a molecular diagnosis for PID patients using a phenotype-based approach is often complex, expensive, and not always successful. Next-generation sequencing (NGS) methods offer an unbiased genotype-based approach, which can facilitate molecular diagnostics.Objective: To develop an efficient NGS method to identify variants in PID-related genes.Methods: We performed HaloPlex custom target enrichment and NGS using the Ion Torrent PGM to screen 173 genes in 11 healthy controls, 13 PID patients previously evaluated with either an identified mutation or SNP, and 120 patients with undiagnosed PIDs. Sensitivity and specificity were determined by comparing NGS and Sanger sequencing results for 33 patients. Run metrics and coverage analyses were done to identify systematic deficiencies.Results: A molecular diagnosis was identified for 18 of 120 patients who previously lacked a genetic diagnosis, including 9 who had atypical presentations and extensive previous genetic and functional studies. Our NGS method detected variants with 98.1% sensitivity and >99.9% specificity. Uniformity was variable (72–89%), and we were not able to reliably sequence 45 regions (45/2455 or 1.8% of total regions) due to low (<20) average read depth or <90% region coverage; thus, we optimized probe hybridization conditions to improve read-depth and coverage for future analyses, and established criteria to help identify true positives.Conclusion: While NGS methods are not as sensitive as Sanger sequencing for individual genes, targeted NGS is a cost-effective, first-line genetic test for the evaluation of patients with PIDs. This approach decreases time to diagnosis, increases diagnostic rate, and provides insight into the genotype–phenotype correlation of PIDs in a cost-effective way.

Background: Infantile nystagmus syndrome (INS) is a genetically heterogeneous disorder. Identifying genetic causes of INS would help clinicians to facilitate clinical diagnosis and provide appropriate treatment. The aim of this study was to determine the diagnostic utility of targeted next-generation sequencing (NGS) for INS. Materials and methods: We recruited 37 patients who were referred to the Neuro-ophthalmology clinics for evaluations of INS. NGS was performed using a targeted panel that included 98 candidate genes associated with INS. We identified pathogenic variants according to guidelines of the American College of Medical Genetics and Genomics. We also calculated the sensitivity and specificity of each clinical sign to assess the diagnostic yield of our gene panel. Results: After variant filtering, annotation, and interpretation, the potential pathogenic variants were detected in 13 of the 37 patients, achieving a molecular diagnostic rate of 35%. The identified genes were PAX6 (n = 4), FRMD7 (n = 4), GPR143 (n = 2), CACNA1F (n = 1), CNGA3 (n = 1) and GUCY2D (n = 1). In approximately 30% (n = 4) of the patients, the initial clinical diagnosis was revised after a molecular diagnosis was performed. The presence of a family history had the highest predictive power for a molecular diagnosis (sensitivity = 61.5%, specificity = 91.7%), and the sensitivity increased when the family history was considered together with one of two clinical signs such as pendular nystagmus waveforms or anterior segment dysgenesis. Conclusions: Our study shows that targeted NGS can be useful to determine a molecular diagnosis for patients with INS. Targeted NGS also helps to confirm a clinical diagnosis in atypical phenotypes or unresolved cases.

Mitochondrial disease was a clinically and genetically heterogeneous group of diseases, thus the diagnosis was very difficult to clinicians. Our objective was to analyze clinical and genetic characteristics of children with mitochondrial disease in China. We tested 141 candidate patients who have been suspected of mitochondrial disorders by using targeted next-generation sequencing (NGS), and summarized the clinical and genetic data of gene confirmed cases from Neurology Department, Beijing Children's Hospital, Capital Medical University from October 2012 to January 2015. In our study, 40 cases of gene confirmed mitochondrial disease including eight kinds of mitochondrial disease, among which Leigh syndrome was identified to be the most common type, followed by mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS). The age-of-onset varies among mitochondrial disease, but early onset was common. All of 40 cases were gene confirmed, among which 25 cases (62.5%) with mitochondrial DNA (mtDNA) mutation, and 15 cases (37.5%) with nuclear DNA (nDNA) mutation. M.3243A>G (n=7) accounts for a large proportion of mtDNA mutation. The nDNA mutations include SURF1 (n=7), PDHA1 (n=2), and NDUFV1, NDUFAF6, SUCLA2, SUCLG1, RRM2B, and C12orf65, respectively.

With advances in sequencing technologies, high-throughput next-generation sequencing (NGS) has triggered increased attention on its application in clinical laboratories and facilitates the molecular diagnosis and treatment of infectious diseases. Compared with conventional microbiology laboratory methods, NGS has greatly increased the sensitivity and accuracy of diagnosis, and reduced detection time for infectious pathogens, especially for diagnosis of complex and mixed infections. However, there are still some problems that hamper the NGS application in infections diagnosis, including lack of standardization, cost, and variation in data interpretation, etc. In recent years, with the developing of policies and legislation, and guidance and supports from Chinese government, the sequencing industry has gained continuously healthy development and sequencing application market gradually becomes mature. Meanwhile, worldwide microbiology experts are striving to develop the standards and reach the consensus, there are more and more clinical laboratories equipped with sequencing instruments and personnel with expertise. All of these measures would certainly promote the clinical application of NGS, and making full use of high-throughput NGS could contribute to the accurate clinical diagnosis and appropriate treatment. The current article describes the application of high-throughput next-generation sequencing technology in the laboratory diagnosis of clinical microbial infectious diseases, as well as the policy system support and development direction.

Introduction Pheochromocytomas and paragangliomas (PPGLs) are highly heritable tumours, with up to 40% of cases carrying germline variants. Current guidelines recommend genetic testing for all patients with PPGLs. Next-generation sequencing (NGS) enables accurate, fast, and inexpensive genetic testing. This study aimed to compare the costs related to PPGL genetic testing between the sequential testing using the decisional algorithm proposed in the 2014 Endocrine Society guidelines and targeted NGS gene panels. Methods Patients with proven PPGLs were enrolled. A gene list covering 17 susceptibility genes related to hereditary PPGLs was developed for targeted sequencing. Validation was carried out by Sanger sequencing. We simulated the diagnostic workflow to examine the anticipated costs based on each strategy for genetic testing. Results Twenty-nine patients were included, among whom a germline variant was identified in 34.5%. A total of 22.7% with apparently sporadic PPGL carried a variant. Five genes were involved (RET, n = 3; SDHB, n = 3; SDHD, n = 2; EGLN1, n = 1; and NF1, n = 1). According to the diagnostic workflow, the average cost of the targeted NGS (534.7 US dollars per patient) is lower than that of the sequential testing (734.5 US dollars per patient). The targeted NGS can also reduce the number of hospital visits from 4.1 to 1 per person. The cost can be further reduced to 496.24 US dollars per person (32% reduction) if we apply a new syndromic-driven diagnostic algorithm to establish priorities for specific genetic testing for syndromic and selected cases, and targeted NGS for non-syndromic patients. Conclusions Targeted NGS can reduce both the cost of PPGL genetic testing and the number of hospital visits, compared with the conventional approach. Our proposed algorithm is the preferred approach due to its significant reduction of the cost of genetic testing. Key message Pheochromocytomas and paragangliomas are highly heritable neoplasms. The targeted next-generation sequencing (NGS) gene panels have proven to be fast, accurate, and inexpensive for the genetic analysis. According to this cost analysis, it is economically reasonable to use targeted NGS gene panels for genetic screening.

Targeted Next Generation Sequencing (TNGS) is an efficient and economical Next Generation Sequencing (NGS) platform and the preferred choice when specific genomic regions are of interest. So far, only institutions located in middle and high-income countries have developed and implemented the technology, however, the efficiency and cost savings, as opposed to more traditional sequencing methodologies (e.g. Sanger sequencing) make the approach potentially well suited for resource-constrained regions as well. In April 2018, scientists from the Plasmodium Diversity Network Africa (PDNA) and collaborators met during the 7th Pan African Multilateral Initiative of Malaria (MIM) conference held in Dakar, Senegal to explore the feasibility of applying TNGS to genetic studies and malaria surveillance in Africa. The group of scientists reviewed the current experience with TNGS platforms in sub-Saharan Africa (SSA) and identified potential roles the technology might play to accelerate malaria research, scientific discoveries and improved public health in SSA. Research funding, infrastructure and human resources were highlighted as challenges that will have to be mitigated to enable African scientists to drive the implementation of TNGS in SSA. Current roles of important stakeholders and strategies to strengthen existing networks to effectively harness this powerful technology for malaria research of public health importance were discussed.

To characterize the mutations in mitochondrial DNA (mtDNA) and mitochondrion-related nuclear genes (nDNA), and clinical features in Chinese patients with mitochondrial ataxia. Targeted next-generation sequencing (NGS) technology was performed to screen the whole mtDNA sequence and nDNA genes in a cohort of 33 unrelated ataxia patients. A total of 5 pedigrees were finally genetically diagnosed as mitochondrial ataxia, with 3 pathogenic mutations (m.8344A>G, m.9176T>C, and m.9185T>C), one likely pathogenic mutation (m.3995A>G) in mtDNA, and one pathogenic mutation (c.1159_1162dupAAGT, p.Ser388Terfs) in PDHA1. The prevalence of mitochondrial ataxia in our patient cohort is 15.2%. In addition, all 4 patients with mtDNA mutations experienced symptoms of ataxia with age at onset ranging from 12 to 39years (21±12.2) and developed extrapyramidal symptoms during the disease course. One male patient with pyruvate dehydrogenase deficiency showed an acute intermittent ataxia phenotype. Our results implicate that mitochondrial ataxia might not be as rare in Chinese as previously assumed. This study firstly defines the mutations of mitochondrial ataxia in a Chinese population by targeted NGS, which broadens the clinical spectrum of mtDNA mutations and highlights the importance of screening mtDNA and nDNA mutations among undefined ataxia patients.

Introduction Currently, genetic testing of mitochondrial DNA mutations includes screening for single-nucleotide variants, several base pair insertions or deletions, large-scale deletions, or relative depletion of total mitochondrial DNA content. Within the last decade, next-generation sequencing (NGS) has resulted in remarkable advances in the field of mitochondrial diseases (MD) and has become a routine step of the diagnostic workup. Areas covered We aimed to present an overview of current technologies employed in molecular diagnosis of mitochondrial DNA diseases. We report on the recent contributions of NGS testing to the diagnosis and understanding of MD Expert opinion The progress of NGS technologies allows the simultaneous detection of mutations and quantification of the heteroplasmy level, ensuring sensitivity and specificity requested for the detection of mitochondrial DNA point mutations. NGS protocols enabling the simultaneous analysis of mitochondrial and nuclear DNA are now efficient and cost-saving approaches, and have become the gold-standard technique in diagnostic laboratories.

To assess the value of next-generation sequencing (NGS) technology in the diagnosis of osteoarticular brucellosis pathogenesis. Fifty eight patients admitted to the Department of Orthopaedics, Hebei Provincial Chest Hospital from January 2021 to January 2023 were retrospectively analyzed, and the patients were classified into 48 cases in the osteoarticular brucellosis group and 10 cases in the nonosteoarticular brucellosis group according to the final clinical diagnosis. All patients underwent serum agglutination test (SAT), CT-guided puncture or surgical sampling of lesions for bacteriological culture and NGS after admission. The diagnostic efficacy of these three methods for osteoarticular brucellosis was compared using the final clinical diagnosis as the reference standard. Among the 58 patients with suspected osteoarticular brucellosis, 40 cases (68.97%) were positive by NGS, 33 cases (56.89%) by SATand 10 cases (17.24%) by culture, and the differences were statistically significant (p < 0.05). Using the final clinical diagnosis as a criterion, the sensitivity of NGS, SAT, and culture for the detection of osteoarticular brucellosis was 83.33%, 62.50%, and 20.83%, respectively, the specificity was 100.00%, 70.00%, and 100.00%, the diagnostic accuracy was 86.20%, 63.79%, and 34.49%, and the κ values were 0.799, 0.590, and 0.504, respectively. NGS has a high pathogen detection rate and sensitivity in the pathogenetic diagnosis of patients with osteoarticular brucellosis and can provide clinical guidance for the diagnosis and management of patients with osteoarticular brucellosis.

High-throughput sequencing, also known as the high-throughput next generation sequencing (NGS), can sequence thousands of genes at one time, greatly improve the efficiency of DNA sequencing and reduce the cost. Recently, NGS has been widely used in medical research and clinical diagnosis. It has extensive applications in non-invasive prenatal screening, tumor screening, diagnosis and treatment, microbial pathogens detection, genetic diseases screening and diagnosis as well as organ transplantation rejection screening. However, as a new technology, NGS also has many issues in related clinical application. The quality management system is still imperfect and faces many challenges. Thus the joint efforts of medical laboratories and related departments are needed to promote the standardization of NGS in clinical diagnosis. Key words: Next generation sequencing technology; Molecular diagnosis; Quality management

Understanding the Implications of Mitochondrial DNA Variation in the Health of Black Southern African Populations: The 2014 Workshop.

To study the clinical features and gene mutation spectrum of children with sideroblastic anemia (SA) and the clinical value of targeted next-generation sequencing in the molecular diagnosis of children with SA. Clinical data were collected from 36 children with SA. Targeted next-generation sequencing was used to detect mutations in SA-related pathogenic genes and genes associated with heme synthesis and mitochondrial iron metabolism. The association between genotype and clinical phenotype was analyzed. Of the 36 patients, 32 had congenital sideroblastic anemia (CSA) and 4 had myelodysplastic syndrome with ring sideroblasts (MDS-RS). Mutations in CSA-related genes were detected in 19 children (19/36, 53%), among whom 9 (47%) had ALAS2 mutation, 4 (21%) had SLC25A38 mutation, and 6 (32%) had mitochondrial fragment deletion. No pathogenic gene mutation was detected in 4 children with MDS-RS. Among the 19 mutations, 89% (17/19) were known mutations and 11% (2/19) were novel mutations. The novel mutation of the ALAS2 gene c.1153A>T(p.I385F) was rated as "possibly pathogenic" and the novel mutation of the SLC25A38 gene c.175C>T(p.Q59X) was rated as "pathogenic". ALAS2 and SLC25A38 gene mutations are commonly seen in children with CSA, but mitochondrial gene fragment deletion also accounts for a relatively high proportion. For children with hypoplastic anemia occurring in infancy, mitochondrial disease should be considered.

Diagnostic process of mitochondrial disorders (MD) is challenging because of the clinical variability and genetic heterogeneity of these conditions. Next-Generation Sequencing (NGS) technology offers a high-throughput platform for nuclear MD. We included 59 of 72 patients that undergone WES and targeted exome sequencing panel suspected to have potential PMDs. Patients who were included in the analysis considering the possible PMD were reviewed retrospectively and scored according to the Mitochondrial Disease Criteria Scale. Sixty-one percent of the patients were diagnosed with whole-exome sequencing (WES) (36/59) and 15% with targeted exome sequencing (TES) (9/59). Patients with MD-related gene defects were included in the mito group, patients without MD-related gene defects were included in the nonmito group, and patients in whom no etiological cause could be identified were included in the unknown etiology group. In 11 out of 36 patients diagnosed with WES, a TES panel was applied prior to WES. In 47 probands in 39 genes (SURF1, SDHAF1, MTO1, FBXL4, SLC25A12, GLRX5, C19oRF12, NDUFAF6, DARS2, BOLA3, SLC19A3, SCO1, HIBCH, PDHA1, PDHAX, PC, ETFA, TRMU, TUFM, NDUFS6, WWOX, UBCD TREX1, ATL1, VAC14, GFAP, PLA2G6, TPRKB, ATP8A2, PEX13, IGHMBP2, LAMB2, LPIN1, GFPT1, CLN5, DOLK) (20 mito group, 19 nonmito group) 59 variants (31 mito group, 18 nonmito group) were detected. Seven novel variants in the mito group (SLC25A12, GLRX5, DARS2, SCO1, PC, ETFA, NDUFS6), nine novel variants in the nonmito group (IVD, GCDH, COG4, VAC14, GFAP, PLA2G6, ATP8A2, PEX13, LPIN1) were detected. We explored the feasibility of identifying pathogenic alleles using WES and TES in MD. Our results show that WES is the primary method of choice in the diagnosis of MD until at least all genes responsible for PMD are found and are highly effective in facilitating the diagnosis process.