Mitochondrial and nuclear disease panel (Mito‐aND‐Panel): Combined sequencing of mitochondrial and nuclear DNA by a cost‐effective and sensitive NGS‐based method

Schöser, Benedikt; Scharf, Florentine; Kleinle, Stephanie; Schön, Ulrike; Holinski‐Feder, Elke; Horvath, Rita; Benet‐Pagès, Anna; Diebold, Isabel

doi:10.1002/mgg3.500

Cited by 25 publications

(18 citation statements)

References 39 publications

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“…For forensic and diagnostic applications, coverage of the mitochondrial genome is not strictly specified and is more stringent on the concomitant replication of identification results via Sanger sequencing (Scientific Working Group on DNA Analysis Methods [SWGDAM]/National Association of Testing Authorities [NATA] guidelines). Multiple studies examining the use of NGS for medical diagnostic testing have specified that a coverage depth of >1000X is sufficient to reliably call mtDNA variants at low heteroplasmy [32–34]. Further optimisation of total coverage should be performed when considering heteroplasmic associations.…”

Section: Discussionmentioning

confidence: 99%

Ion torrent high throughput mitochondrial genome sequencing (HTMGS)

et al. 2019

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The implementation and popularity of next generation sequencing (NGS) has led to the development of various rapid whole mitochondrial genome sequencing techniques. We summarise an efficient and cost-effective NGS approach for mitochondrial genomic DNA in humans using the Ion Torrent platform, and further discuss our bioinformatics pipeline for streamlined variant calling. Ion 316 chips were utilised with the Ion Torrent semi-conductor platform Personal Genome Machine (PGM) to perform tandem sequencing of mitochondrial genomes from the core pedigree (n = 315) of the Norfolk Island Health Study. Key improvements from commercial methods focus on the initial PCR step, which currently requires extensive optimisation to ensure the accurate and reproducible elongation of each section of the complete mitochondrial genome. Dual-platform barcodes were incorporated into our protocol thereby extending its potential application onto Illumina-based systems. Our bioinformatics pipeline consists of a modified version of GATK best practices tailored for mitochondrial genomic data. When compared with current commercial methods, our method, termed high throughput mitochondrial genome sequencing (HTMGS), allows high multiplexing of samples and the use of alternate library preparation reagents at a lower cost per sample (~1.7 times) when compared to current commercial methodologies. Our HTMGS methodology also provides robust mitochondrial sequencing data (>450X average coverage) that can be applied and modified to suit various study designs. On average, we were able to identify ~30 variants per sample with 572 variants observed across 315 samples. We have developed a high throughput sequencing and analysis method targeting complete mitochondrial genomes; with the potential to be platform agnostic with analysis options that adhere to current best practices.

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Section: Discussionmentioning

confidence: 99%

Ion torrent high throughput mitochondrial genome sequencing (HTMGS)

et al. 2019

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“…Here, we report one of the largest cohorts of pediatric patients with a molecular diagnosis of a MD. While most of the previous works have reported on the diagnostic yield of NGS techniques in MD patients [9,10,11,12,13,14,15,16,17,18,19,20,21], in this study we focused our attention on muscular features of pediatric MD patients with a definitive molecular diagnosis, as the ultimate biomarker (Table S1).…”

Section: Discussionmentioning

confidence: 99%

“…NGS has facilitated diagnosis, and several recently reported patient series associated more than 290 genes as causative of MD [19]. Different genetic approaches have been applied, including targeted mitochondrial gene panels, whole exome (WES) and whole genome sequencing (WGS) and, recently, the combined genetic analysis of both mtDNA and nDNA [20,21]. As a result, not only is diagnostic efficacy enhanced, but new pathomechanisms are being unraveled, and unexpected findings are being recognized [17].…”

Section: Introductionmentioning

confidence: 99%

Muscle Involvement in a Large Cohort of Pediatric Patients with Genetic Diagnosis of Mitochondrial Disease

Jou

Ortigoza‐Escobar

O’Callaghan

et al. 2019

JCM

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Mitochondrial diseases (MD) are a group of genetic and acquired disorders which present significant diagnostic challenges. Here we report the disease characteristics of a large cohort of pediatric MD patients (n = 95) with a definitive genetic diagnosis, giving special emphasis on clinical muscle involvement, biochemical and histopathological features. Of the whole cohort, 51 patients harbored mutations in nuclear DNA (nDNA) genes and 44 patients had mutations in mitochondrial DNA (mtDNA) genes. The nDNA patients were more likely to have a reduction in muscle fiber succinate dehydrogenase (SDH) stains and in SDH-positive blood vessels, while a higher frequency of mtDNA patients had ragged red (RRF) and blue fibers. The presence of positive histopathological features was associated with ophthalmoplegia, myopathic facies, weakness and exercise intolerance. In 17 patients younger than two years of age, RRF and blue fibers were observed only in one case, six cases presented cytochrome c oxidase (COX) reduction/COX-fibers, SDH reduction was observed in five and all except one presented SDH-positive blood vessels. In conclusion, muscle involvement was a frequent finding in our series of MD patients, especially in those harboring mutations in mtDNA genes.

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“…This technology reduces the cost of genetic testing for inherited diseases. [15,16] Since renal biopsy is invasive, NGS could be a more economical alternative for the diagnosis of AS. In particular, this could be the first option for juveniles or children with proteinuria or hematuria, with a family history of kidney disease.…”

Section: Discussionmentioning

confidence: 99%

Novel mutations in patients with X-linked Alport syndrome

Oh¹,

Kim²,

Kim³

et al. 2019

Medicine

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Rationale: A genotype-phenotype correlation is known to be associated with Alport syndrome (AS). Identifying novel mutations can expand the knowledge about the natural course of AS. Patient concerns: The first patient was a-15-year-old boy detected with proteinuria during the school health check-up. The second case was a-29-year-old woman, who visited the outpatient clinic for edema. Diagnosis: We performed targeted next-generation sequencing to identify the mutations associated with AS. Results were confirmed by Sanger sequencing and multiplex ligation-dependent probe amplification. Missense mutation (c.2332G>C, p.Gly778Arg) was identified in the first case and an exon 16 deletion was also identified in the second case. Intervention: We treated both cases with angiotensin receptor blocker (ARB). Outcomes: The amount of proteinuria in the first case did not change after ARB therapy, during the follow-up period (1 year). Proteinuria in the woman decreased to half of the baseline level, 1 year after treatment. Glomerular filtration rate was also maintained during the follow-up. Conclusion: We identified novel mutations in Koreans with an X-linked AS mutation in the COL4A5 gene and an individual phenotype. This is the first report of AS patients with a novel missense mutation and copy number variation.

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Mitochondrial and nuclear disease panel (Mito‐aND‐Panel): Combined sequencing of mitochondrial and nuclear DNA by a cost‐effective and sensitive NGS‐based method

Cited by 25 publications

References 39 publications

Ion torrent high throughput mitochondrial genome sequencing (HTMGS)

Ion torrent high throughput mitochondrial genome sequencing (HTMGS)

Muscle Involvement in a Large Cohort of Pediatric Patients with Genetic Diagnosis of Mitochondrial Disease

Novel mutations in patients with X-linked Alport syndrome

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