Homoplasmic Variants Research Articles

The interplay between mitochondrial DNA genotypes, female infertility, ovarian response, and mutagenesis in oocytes.

Is there an association between different mitochondrial DNA (mtDNA) genotypes and female infertility or ovarian response, and is the appearance of variants in the oocytes favored by medically assisted reproduction (MAR) techniques? Ovarian response was negatively associated with global non-synonymous protein-coding homoplasmic variants but positively associated with haplogroup K; the number of oocytes retrieved in a cycle correlates with the number of heteroplasmic variants in the oocytes, principally with variants located in the hypervariable (HV) region and rRNA loci, as well as non-synonymous protein-coding variants. Several genes have been shown to be positively associated with infertility, and there is growing concern that MAR may facilitate the transmission of these harmful variants to offspring, thereby passing on infertility. The potential role of mtDNA variants in these two perspectives remains poorly understood. This cohort study included 261 oocytes from 132 women (mean age: 32 ± 4 years) undergoing ovarian stimulation between 2019 and 2020 at an academic center. The oocyte mtDNA genotypes were examined for associations with the women's fertility characteristics. The mtDNA of the oocytes underwent deep sequencing, and the mtDNA genotypes were compared between infertile and fertile groups using Fisher's exact test. The impact of the mtDNA genotype on anti-Müllerian hormone (AMH) levels and the number of (mature) oocytes retrieved was assessed using the Mann-Whitney U test for univariate analysis and logistic regression for multivariate analysis. Additionally, we examined the associations of oocyte maturation stage, infertility status, number of ovarian stimulation units, and number of oocytes retrieved with the type and load of heteroplasmic variants using univariate analysis and Poisson or linear regression analysis. Neither homoplasmic mtDNA variants nor haplogroups in the oocytes were associated with infertility status or with AMH levels. Conversely, when the relationship between the number of oocytes retrieved and different mtDNA genotypes was examined, a positive association was observed between the number of metaphase (MII) oocytes (P = 0.005) and haplogroup K. Furthermore, the presence of global non-synonymous homoplasmic variants in the protein-coding region was significantly associated with a reduced number of total oocytes and MII oocytes retrieved (P < 0.001 for both). Regarding the type and load of heteroplasmic variants in the different regions, there were no significant associations according to maturation stage of the oocyte or to fertility status; however, the number of oocytes retrieved correlated positively with the total number of heteroplasmic variants, and specifically with non-synonymous protein-coding, HV and rRNA variants (P < 0.001 for all). The current work is constrained by its retrospective design and single-center approach, potentially limiting the generalizability of our findings. The small sample size for specific types of infertility restricts this aspect of the findings. This work suggests that mitochondrial genetics may have an impact on ovarian response and corroborates previous findings indicating that the size of the oocyte cohort after stimulation correlates with the presence of potentially deleterious variants in the oocyte. Future epidemiological and functional studies based on the results of the current study will provide valuable insights to address gaps in knowledge to assess any prospective risks for MAR-conceived offspring. This work was supported by the Research Foundation Flanders (FWO, Grant numbers 1506617N and 1506717N to C.S.), by the Fonds Wetenschappelijk Fonds, Willy Gepts Research Foundation of Universitair Ziekenhuis Brussel (Grant numbers WFWG14-15, WFWG16-43, and WFWG19-19 to C.S.), and by the Methusalem Grant of the Vrije Universiteit Brussel (to K.S.). M.R. and E.C.d.D. were supported predoctoral fellowships by the FWO, Grant numbers 1133622N and 1S73521N, respectively. The authors declare no conflict of interests. N/A.

Pathogenic mitochondrial DNA variants are associated with response to anti-VEGF therapy in ovarian cancer PDX models

BackgroundMitochondrial DNA (mtDNA) pathogenic variants have been reported in several solid tumors including ovarian cancer (OC), the most lethal gynecologic malignancy, and raised interest as they potentially induce mitochondrial dysfunction and rewiring of cellular metabolism. Despite advances in recent years, functional characterization of mtDNA variants in cancer and their possible modulation of drug response remain largely uncharted.MethodsHere, we characterized mtDNA variants in OC patient derived xenografts (PDX) and investigated their impact on cancer cells at multiple levels.ResultsGenetic analysis revealed that mtDNA variants predicted as pathogenic, mainly involving complex I and IV genes, were present in all but one PDX (n = 20) at different levels of heteroplasmy, including 7 PDXs with homoplasmic variants. Functional analyses demonstrated that pathogenic mtDNA variants impacted on respiratory complexes activity and subunits abundance as well as on mitochondrial morphology. Moreover, PDX cells bearing homoplasmic mtDNA variants behaved as glucose-addicted and could barely survive glucose starvation in vitro. RNA-seq analysis indicated that mtDNA mutated (heteroplasmy > 50%) PDXs were endowed with upregulated glycolysis and other pathways connected with cancer metabolism. These findings led us to investigate whether pathogenic mtDNA variants correlated with response to anti-VEGF therapy, since the latter was shown to reduce glucose availability in tumors. Strikingly, PDXs bearing homoplasmic pathogenic mtDNA variants associated with improved survival upon anti-VEGF treatment in mice, compared with mtDNA wild type or low heteroplasmy PDXs.ConclusionsThese results hint at mtDNA variants as potential biomarkers of response to antiangiogenic drugs.

Concurrent genotyping of mitochondrial DNA and nuclear DNA in rootless hair shafts and blood samples for enhanced analysis

Hair is an important type of biological evidence at crime scenes. However, the highly degraded nature of DNA fragments in hair shafts poses challenges for the detection of nuclear DNA (nuDNA) through capillary electrophoresis-based short tandem repeat (STR) genotyping. In this study, an all-in-one multiplex system named MGIEasy Signature Identification Library Prep Kit (MGI Tech, China) was employed to the simultaneous genotyping of both mitochondrial DNA (mtDNA) and nuDNA in hair shafts. This system is based on massively parallel sequencing (MPS) technology and encompasses Amelogenin, STRs, single nucleotide polymorphisms (SNPs) and mtDNA hypervariable regions (HVRs) in a single reaction. A total of 370 hair shafts, together with 180 blood samples as the references, were examined. The mtDNA analysis of 110 unrelated blood samples unveiled a total of 150 homoplasmic variants and 105 distinct haplotypes, revealing population polymorphisms in the Guangdong Han Chinese. The study also delved into the detection of mtDNA heteroplasmy, revealing 8.18 % and 16.36 % of individuals with point heteroplasmies (PHPs) in blood and hair shaft samples, respectively. Additionally, hair shafts with DNA extracted using the Investigator method yielded higher average depth of coverage (DoC), lower drop-out rate for SNP genotyping, higher nuDNA genotyped rates and success rates, than those using the MinElute method. In the longitudinal research, a gradual decrease in the total DoC of mtDNA fragments was observed along the length of the hair shaft, from the proximal root to the distal end. In contrast, the DoC of nuDNA exhibited a relatively stable pattern along the length of the hair shafts. The study contributes valuable insights into the simultaneous detection of nuDNA and mtDNA in hair shafts, emphasizing the need for optimized DNA extraction and detection methods for these highly degraded samples.

Neonatal Hemochromatosis Secondary to an Inborn Error of Metabolism (Mitochondrial Disorder): Post- mortem Diagnosis in the Fetus of a Young Primipara

Abstract Introduction/Objective Neonatal hemochromatosis (NH), an extremely rare congenital phenotype of acute hepatic failure with hepatic and extrahepatic siderosis, is primarily (98%) caused by gestational alloimmune liver disease (GALD) and, rarely (2%), by infection, metabolic disorders, bile acid defects, and mitochondrial hepatopathy. This report presents an extremely rare case of NH caused by an inborn error of metabolism (suspected mitochondrial dysfunction). This low-birth-weight female was born with acute liver failure, metabolic acidosis, multisystem failure, and progressive lactate acidosis, and expired on day 7. On autopsy, liver was less than 50% of normal size and without nodularity. Microscopic architecture was preserved with erythropoiesis, canalicular bile plugs, microvesicular steatosis (oil red O stain) and abundant coarsely granular siderosis (iron stain). GALD was unlikely because of the absence of the typical hepatocytic injury, fibrosis or biliary destruction, and the negative immunohistochemistry for MAC (C5b-C9). Extrahepatic siderosis was demonstrated in thyroid follicles, thymic Hassall corpuscles, myocardium, and adenohypophysis. Sanger sequencing and deletion testing of mitochondrial genome on a maternal buccal biopsy identified a homoplasmic variant (m.15482 T&amp;gt;C p.(S246P) in maternal MT-CYB gene but without molecular diagnosis of a mitochondrial DNA disorder. Infant plasma had increased C6DC, C5, C12-OH acylcarnitine, alanine, and proline. Urine contained elevated organic acids (lactate/pyruvate, 2-ketoacids, 3-methylglutarate, 3-methylglutaconate), concerning acute mitochondrial respiratory chain dysfunction. Methods/Case Report: Case Study Results (if a Case Study enter NA) NA Conclusion Diagnosis of NH warrants exclusion of the most common etiology, GALD. Mitochondrial genetic mutations are the most difficult to diagnosis because of heterogeneity and typically undiagnosed maternal defects. This is the first report of NH with a homoplasmic maternal variant of uncertain significance. Neonatal lactic acidosis is the strongest clinical clue for mitochondrial dysfunction and should trigger phenotyping, maternal mitochondrial genetic variants (mtDNA, nuDNA), functional, biochemical, and genetic studies.

Exploring the evidence for mitochondrial dysfunction and genetic abnormalities in the etiopathogenesis of tropical ataxic neuropathy

Tropical ataxic neuropathy (TAN) is characterised by ataxic polyneuropathy, degeneration of the posterior columns and pyramidal tracts, optic atrophy, and sensorineural hearing loss. It has been attributed to nutritional/toxic etiologies, but evidence for the same has been equivocal. TAN shares common clinical features with inherited neuropathies and mitochondrial disorders, it may be hypothesised that genetic abnormalities may underlie the pathophysiology of TAN. This study aimed to establish evidence for mitochondrial dysfunction by adopting an integrated biochemical and multipronged genetic analysis. Patients (n = 65) with chronic progressive ataxic neuropathy with involvement of visual and/or auditory pathways underwent deep phenotyping, genetic studies including mitochondrial DNA (mtDNA) deletion analysis, mtDNA and clinical exome sequencing (CES), and respiratory chain complex (RCC) assay. The phenotypic characteristics included dysfunction of visual (n = 14), auditory (n = 12) and visual + auditory pathways (n = 29). Reduced RCC activity was present in 13 patients. Mitochondrial DNA deletions were noted in five patients. Sequencing of mtDNA (n = 45) identified a homoplasmic variant (MT-ND6) and a heteroplasmic variant (MT-COI) in one patient each. CES (n = 45) revealed 55 variants in nuclear genes that are associated with neuropathy (n = 27), deafness (n = 7), ataxia (n = 4), and mitochondrial phenotypes (n = 5) in 36 patients. This study provides preliminary evidence that TAN is associated with a spectrum of genetic abnormalities, including those associated with mitochondrial dysfunction, which is in contradistinction from the prevailing hypothesis that TAN is related to dietary toxins. Analysing the functional relevance of these genetic variants may improve the understanding of the pathogenesis of TAN.

Complete mitochondrial genomes of patients from Thailand with cardiovascular diseases.

Several previous studies have reported that both variation and haplogroups of mitochondrial (mt) DNA were associated with various kinds of diseases, including cardiovascular diseases, in different populations, but such studies have not been carried out in Thailand. Here, we sequenced complete mtDNA genomes from 82 patients diagnosed with three types of cardiovascular disease, i.e., Hypertrophic Cardiomyopathy (HCM) (n = 26), Long Q-T Syndrome (LQTS) (n = 7) and Brugada Syndrome (BrS) (n = 49) and compared these with 750 previously published mitogenome sequences from interviewed normal individuals as a control group. Both patient and control groups are from the same geographic region of northeastern Thailand. We found 9, 2, and 5 novel mutations that were not both damaging and deleterious in HCM, LQTS, and BrS patients, respectively. Haplogroup R9c was significantly associated with HCM (P = 0.0032; OR = 62.42; 95%CI = 6.892-903.4) while haplogroup M12b was significantly associated with LQTS (P = 0.0039; OR = 32.93; 95% CI = 5.784-199.6). None of the haplogroups was found to be significantly associated with BrS. A significantly higher density of mtDNA variants in the rRNA genes was found in patients with HCM and BrS (P < 0.001) than in those with LQTS or the control group. Effects of detected SNPs in either protein coding or tRNA genes of all the mitogenome sequences were also predicted. Interestingly, three SNPs in two tRNA genes (MT-TA m.5618T>C and m.5631G>A heteroplasmic variants in two BrS patients and MT-TQ m.4392C>T novel homoplasmic variant in a HCM patient) were predicted to alter tRNA secondary structure, possibly leading to abnormal tRNA function.

Penetrance and expressivity of mitochondrial variants in a large clinically unselected population.

Whole genome sequencing (WGS) from large clinically unselected cohorts provides a unique opportunity to assess the penetrance and expressivity of rare and/or known pathogenic mitochondrial variants in population. Using WGS from 179 862 clinically unselected individuals from the UK Biobank, we performed extensive single and rare variant aggregation association analyses of 15 881 mtDNA variants and 73 known pathogenic variants with 15 mitochondrial disease-relevant phenotypes. We identified 12 homoplasmic and one heteroplasmic variant (m.3243A>G) with genome-wide significant associations in our clinically unselected cohort. Heteroplasmic m.3243A>G (MAF = 0.0002, a known pathogenic variant) was associated with diabetes, deafness and heart failure and 12 homoplasmic variants increased aspartate aminotransferase levels including three low-frequency variants (MAF ~0.002 and beta~0.3 SD). Most pathogenic mitochondrial disease variants (n = 66/74) were rare in the population (<1:9000). Aggregated or single variant analysis of pathogenic variants showed low penetrance in unselected settings for the relevant phenotypes, except m.3243A>G. Multi-system disease risk and penetrance of diabetes, deafness and heart failure greatly increased with m.3243A>G level ≥ 10%. The odds ratio of these traits increased from 5.61, 12.3 and 10.1 to 25.1, 55.0 and 39.5, respectively. Diabetes risk with m.3243A>G was further influenced by type 2 diabetes genetic risk. Our study of mitochondrial variation in a large-unselected population identified novel associations and demonstrated that pathogenic mitochondrial variants have lower penetrance in clinically unselected settings. m.3243A>G was an exception at higher heteroplasmy showing a significant impact on health making it a good candidate for incidental reporting.

A new family with a case of severe early-onset muscle fatigue and a peculiar maternally inherited painful swelling in chewing muscles associated with homoplasmic m.15992A>T mutation in mitochondrial tRNAPro

A 16-year-old boy was evaluated for a history of exercise-induced fatigability associated with nausea even after minimal effort, lower limbs muscle hypotrophy, and swelling of the masseter muscles after chewing. Laboratory tests were remarkable for hyperlactatemia and metabolic acidosis after short physical activity. The muscle biopsy showed non-specific mitochondrial alterations and an increase in intrafibral lipids. Biochemical analysis showed reduced activity of the respiratory chain complexes. Mitochondrial DNA sequencing revealed the presence of a homoplasmic variant m.15992A>T in the MT-TP gene, coding for the mt-tRNAPro in the patient, in his mother and in his brother. Pathogenic or likely pathogenic variants in MT-TP gene are rare. They are responsible for different clinical presentation, almost ever involving the muscle tissue. We report the first family with exercise-induced muscle weakness and swelling of the chewing muscles due to m.15992A>T variant in absence of J1c10 haplogroup, confirming its pathogenicity.

Mitochondrial DNA heteroplasmy analysis in keratoconus patients from China.

Background: Mitochondrial DNA (mtDNA) variants have been implicated in keratoconus (KC). The present study aimed to characterize the mtDNA heteroplasmy profile in KC and explore the association of mitochondrial heteroplasmic levels with KC. Methods: Mitochondrial sequencing of peripheral blood samples and corneal tomography were conducted in 300 KC cases and 300 matched controls. The number of heteroplasmic and homoplasmic variants was calculated across the mitochondrial genome. Spearman's correlation was used to analyze the correlation between the number of heteroplasmic variants and age. The association of mtDNA heteroplasmic level with KC was analyzed by logistic regression analysis. Moreover, the relationship between mitochondrial heteroplasmic levels and clinical parameters was determined by linear regression analysis. Results: The distribution of mtDNA heteroplasmic variants showed the highest number of heteroplasmic variants in the non-coding region, while the COX3 gene exhibited the highest number in protein-coding genes. Comparisons of the number of heteroplasmic and homoplasmic non-synonymous variants in protein-coding genes revealed no significant differences between KC cases and controls (all p > 0.05). In addition, the number of heteroplasmic variants was positively associated with age in all subjects (r = 0.085, p = 0.037). The logistic regression analyses indicated that the heteroplasmic levels of m.16180_16181delAA was associated with KC (p < 0.005). Linear regression analyses demonstrated that the heteroplasmic levels of m.16180_16181delAA and m.302A>C were not correlated with thinnest corneal thickness (TCT), steep keratometry (Ks), and flat keratometry (Kf) (all p > 0.05) in KC cases and controls separately. Conclusion: The current study characterized the mtDNA heteroplasmy profile in KC, and revealed that the heteroplasmic levels of m.16180_16181delAA were associated with KC.

Whole Mitochondrial Genome Detection and Analysis of Two- to Four-Generation Maternal Pedigrees Using a New Massively Parallel Sequencing Panel.

Mitochondrial DNA (mtDNA) is an effective genetic marker in forensic practice, especially for aged bones and hair shafts. Detection of the whole mitochondrial genome (mtGenome) using traditional Sanger-type sequencing is laborious and time-consuming. Additionally, its ability to distinguish point heteroplasmy (PHP) and length heteroplasmy (LHP) is limited. The application of massively parallel sequencing in mtDNA detection helps researchers to study the mtGenome in-depth. The ForenSeq mtDNA Whole Genome Kit, which contains a total of 245 short amplicons, is one of the multiplex library preparation kits for the mtGenome. We used this system to detect the mtGenome in the blood samples and hair shafts of thirty-three individuals from eight two-generation pedigrees, one three-generation pedigree, and one four-generation pedigree. High-quality sequencing results were obtained. Ten unique mtGenome haplotypes were observed in the mothers from the ten pedigrees. A total of 26 PHPs were observed using the interpretation threshold of 6%. Eleven types of LHPs in six regions were evaluated in detail. When considering homoplasmic variants only, consistent mtGenome haplotypes were observed between the twice-sequenced libraries and between the blood and hair shafts from the same individual and among maternal relatives in the pedigrees. Four inherited PHPs were observed, and the remainder were de novo/disappearing PHPs in the pedigrees. Our results demonstrate the effective capability of the ForenSeq mtDNA Whole Genome Kit to generate the complete mtGenome in blood and hair shafts, as well as the complexity of mtDNA haplotype comparisons between different types of maternal relatives when heteroplasmy is considered.

Leber hereditary optic neuropathy presenting as bilateral visual loss and white matter disease.

Leber hereditary optic neuropathy (LHON) is a rare maternally inherited mitochondrial disorder that typically affects young male adults in their second and third decades of life. It usually manifests as painless, subacute, progressive, bilateral vision loss, with more than 90% of affected individuals losing their vision before age 50. Compared with other diseases that cause optic neuritis (multiple sclerosis or neuromyelitis optica spectrum disorders), LHON has worsening visual function in the first 6-12 months of disease progression, is predominantly male, the optic nerve is affected bilaterally from onset, there is no gadolinium enhancement on MRI, no response to disease-modifying therapy, and there is a family history of mutation in mitochondrial DNA. In this article, we describe an interesting and challenging case of LHON due to a homoplasmic variant in the MT -CO3 gene that was initially misdiagnosed as a monophasic demyelinating disorder (clinically isolated syndrome vs acute disseminated encephalomyelitis vs neuromyelitis optica spectrum disorders).

Implications of mitochondrial DNA variants in pediatric B-cell acute lymphoblastic leukemia

BackgroundResearch on the role of variations in the mitochondrial genome in pathogenesis of acute lymphoblastic leukemia (ALL) has been unfolding at a rapid rate. Our laboratory has previously described higher number of copies of the mitochondrial genomes per cell in pediatric ALL patients as compared to the healthy controls. In the current study, we evaluated the pattern of mitochondrial genome variations in 20 de-novo pediatric B-ALL cases and seven controls. Quantitative real-time Polymerase Chain Reaction was used for estimation of mitochondrial genomes’ copy number in bone marrow samples of each ALL patient and peripheral blood samples of controls. The complete mitochondrial genomes of all samples were sequenced using the Illumina platform.ResultsSequencing data analysis using multiple mitochondrial genome databases revealed 325 variants in all 27 samples, out of which 221 variants were previously known while 104 were unassigned, new variants. The 325 variants consisted of 7 loss-of-function variants, 131 synonymous variants, 75 missense variants, and 112 non-coding variants. New, missense variants (n = 21) were identified in genes encoding the electron transport chain complexes with most of them encoding ND4, ND5 of complex I. Missense and loss-of-function variants were found to be deleterious by many predictor databases of pathogenicity. MuTect2 identified true somatic variants present only in tumors between patient-sibling pairs and showed overlap with missense and loss-of-function variants. Online MtDNA-server showed heteroplasmic and homoplasmic variants in mitochondrial genome.ConclusionsThe data suggest that some of these variations might have a deleterious impact on the expression of mitochondrial encoded genes with a possible functional relevance in leukemia.

Benchmarking the Effectiveness and Accuracy of Multiple Mitochondrial DNA Variant Callers: Practical Implications for Clinical Application.

Mitochondrial DNA (mtDNA) mutations contribute to human disease across a range of severity, from rare, highly penetrant mutations causal for monogenic disorders to mutations with milder contributions to phenotypes. mtDNA variation can exist in all copies of mtDNA or in a percentage of mtDNA copies and can be detected with levels as low as 1%. The large number of copies of mtDNA and the possibility of multiple alternative alleles at the same DNA nucleotide position make the task of identifying allelic variation in mtDNA very challenging. In recent years, specialized variant calling algorithms have been developed that are tailored to identify mtDNA variation from whole-genome sequencing (WGS) data. However, very few studies have systematically evaluated and compared these methods for the detection of both homoplasmy and heteroplasmy. A publicly available synthetic gold standard dataset was used to assess four mtDNA variant callers (Mutserve, mitoCaller, MitoSeek, and MToolBox), and the commonly used Genome Analysis Toolkit “best practices” pipeline, which is included in most current WGS pipelines. We also used WGS data from 126 trios and calculated the percentage of maternally inherited variants as a metric of calling accuracy, especially for homoplasmic variants. We additionally compared multiple pathogenicity prediction resources for mtDNA variants. Although the accuracy of homoplasmic variant detection was high for the majority of the callers with high concordance across callers, we found a very low concordance rate between mtDNA variant callers for heteroplasmic variants ranging from 2.8% to 3.6%, for heteroplasmy thresholds of 5% and 1%. Overall, Mutserve showed the best performance using the synthetic benchmark dataset. The analysis of mtDNA pathogenicity resources also showed low concordance in prediction results. We have shown that while homoplasmic variant calling is consistent between callers, there remains a significant discrepancy in heteroplasmic variant calling. We found that resources like population frequency databases and pathogenicity predictors are now available for variant annotation but still need refinement and improvement. With its peculiarities, the mitochondria require special considerations, and we advocate that caution needs to be taken when analyzing mtDNA data from WGS data.

Comparison of mitochondrial DNA sequences from whole blood and lymphoblastoid cell lines

Lymphoblastoid cell lines (LCLs) provide an unlimited source of genomic DNA for genetic studies. Here, we compared mtDNA sequence variants, heteroplasmic or homplasmic, between LCL (sequenced by mitoRCA-seq method) and whole blood samples (sequenced through whole genome sequencing approach) of the same 130 participants in the Framingham Heart Study. We applied harmonization of sequence coverages and consistent quality control to mtDNA sequences. We identified 866 variation sites in the 130 LCL samples and 666 sites in the 130 blood samples. More than 94% of the identified homoplasmies were present in both LCL and blood samples while more than 70% of heteroplasmic sites were uniquely present either in LCL or in blood samples. The LCL and whole blood samples carried a similar number of homoplasmic variants (p = 0.45) per sample while the LCL carried a greater number of heteroplasmic variants than whole blood per sample (p < 2.2e−16). Furthermore, the LCL samples tended to accumulate low level heteroplasmies (heteroplasmy level in 3–25%) than their paired blood samples (p = 0.001). These results suggest that cautions should be taken in the interpretation and comparison of findings when different tissues/cell types or different sequencing technologies are applied to obtain mtDNA sequences.

Mitochondrial DNA variation across 56,434 individuals in gnomAD.

Genomic databases of allele frequency are extremely helpful for evaluating clinical variants of unknown significance; however, until now, databases such as the Genome Aggregation Database (gnomAD) have focused on nuclear DNA and have ignored the mitochondrial genome (mtDNA). Here, we present a pipeline to call mtDNA variants that addresses three technical challenges: (1) detecting homoplasmic and heteroplasmic variants, present, respectively, in all or a fraction of mtDNA molecules; (2) circular mtDNA genome; and (3) misalignment of nuclear sequences of mitochondrial origin (NUMTs). We observed that mtDNA copy number per cell varied across gnomAD cohorts and influenced the fraction of NUMT-derived false-positive variant calls, which can account for the majority of putative heteroplasmies. To avoid false positives, we excluded contaminated samples, cell lines, and samples prone to NUMT misalignment due to few mtDNA copies. Furthermore, we report variants with heteroplasmy ≥10%. We applied this pipeline to 56,434 whole-genome sequences in the gnomAD v3.1 database that includes individuals of European (58%), African (25%), Latino (10%), and Asian (5%) ancestry. Our gnomAD v3.1 release contains population frequencies for 10,850 unique mtDNA variants at more than half of all mtDNA bases. Importantly, we report frequencies within each nuclear ancestral population and mitochondrial haplogroup. Homoplasmic variants account for most variant calls (98%) and unique variants (85%). We observed that 1/250 individuals carry a pathogenic mtDNA variant with heteroplasmy above 10%. These mtDNA population allele frequencies are freely accessible and will aid in diagnostic interpretation and research studies.

Expression pattern of mitochondrial respiratory chain enzymes in skeletal muscle of patients with mitochondrial myopathy associated with the homoplasmic m.14674T>C variant.

Two homoplasmic variants in tRNAGlu (m.14674T>C/G) are associated with reversible infantile respiratory chain deficiency. This study sought to further characterize the expression of the individual mitochondrial respiratory chain complexes and to describe the natural history of the disease. Seven patients from four families with mitochondrial myopathy associated with the homoplasmic m.14674T>C variant were investigated. All patients underwent skeletal muscle biopsy and mtDNA sequencing. Whole‐genome sequencing was performed in one family. Western blot and immunohistochemical analyses were used to characterize the expression of the individual respiratory chain complexes. Patients presented with hypotonia and feeding difficulties within the first weeks or months of life, except for one patient who first showed symptoms at 4 years of age. Histopathological findings in muscle included lipid accumulation, numerous COX‐deficient fibers, and mitochondrial proliferation. Ultrastructural abnormalities included enlarged mitochondria with concentric cristae and dense mitochondrial matrix. The m.14674T>C variant in MT‐TE was identified in all patients. Immunohistochemistry and immunoblotting demonstrated pronounced deficiency of the complex I subunit NDUFB8. The expression of MTCO1, a complex IV subunit, was also decreased, but not to the same extent as NDUFB8. Longitudinal follow‐up data demonstrated that not all features of the disorder are entirely transient, that the disease may be progressive, and that signs and symptoms of myopathy may develop during childhood. This study sheds new light on the involvement of complex I in reversible infantile respiratory chain deficiency, it shows that the disorder may be progressive, and that myopathy can develop without an infantile episode.

MitoScape: A big-data, machine-learning platform for obtaining mitochondrial DNA from next-generation sequencing data.

The growing number of next-generation sequencing (NGS) data presents a unique opportunity to study the combined impact of mitochondrial and nuclear-encoded genetic variation in complex disease. Mitochondrial DNA variants and in particular, heteroplasmic variants, are critical for determining human disease severity. While there are approaches for obtaining mitochondrial DNA variants from NGS data, these software do not account for the unique characteristics of mitochondrial genetics and can be inaccurate even for homoplasmic variants. We introduce MitoScape, a novel, big-data, software for extracting mitochondrial DNA sequences from NGS. MitoScape adopts a novel departure from other algorithms by using machine learning to model the unique characteristics of mitochondrial genetics. We also employ a novel approach of using rho-zero (mitochondrial DNA-depleted) data to model nuclear-encoded mitochondrial sequences. We showed that MitoScape produces accurate heteroplasmy estimates using gold-standard mitochondrial DNA data. We provide a comprehensive comparison of the most common tools for obtaining mtDNA variants from NGS and showed that MitoScape had superior performance to compared tools in every statistically category we compared, including false positives and false negatives. By applying MitoScape to common disease examples, we illustrate how MitoScape facilitates important heteroplasmy-disease association discoveries by expanding upon a reported association between hypertrophic cardiomyopathy and mitochondrial haplogroup T in men (adjusted p-value = 0.003). The improved accuracy of mitochondrial DNA variants produced by MitoScape will be instrumental in diagnosing disease in the context of personalized medicine and clinical diagnostics.

Individuals with Sickle Cell Disease Have a Higher Burden of Mitochondrial DNA Heteroplasmy

Background: The simple point mutation that causes sickle cell disease (SCD) belies the extensive systemic damage it can cause. While the sickle pathology is initiated by polymerization of HbS, the multiple end-organ damage is inflicted by years of on-going inflammation and vasculopathy. An emerging marker of inflammation is the accumulation of acquired heteroplasmy mutations in mitochondrial DNA (mtDNA). Given the underlying chronic inflammation in SCD, we hypothesized that SCD patients display increased rates of mtDNA mutations, and previously confirmed (1). Here, we further performed indepth analyses in an ethnically matched normal (HbAA) as well as sickle trait (HbAS) subjects from another independent cohort, the Jackson Heart Study (JHS).Methods: We analyzed and compared whole genome sequencing (WGS) data from the from NIH cohort of 676 SCD patients of African ancestry with that of 621 ethnic-matched indviduals from the 1000 Genomes Project (1KG), and 3,580 individuals from the JHS cohort. The NIH SCD cohort included 561 HbSS & HbSβ 0thalassemia (combined), 90 HbSC, and 25 HbSβ + thalassemia genotypes, the 1KG cohort - 516 HbAA and 105 HbAS and JHS cohort - 3,200 HbAA, 89 HbAC (hemoglobin C trait), and 291 HbAS. Additionally, to further understand any potential sequencing depth bias, as well as to compare between two patient cohorts (NIH SCD & JHS cohorts) with underlying conditons that may influence the heteroplasmy bias, we downsampled 300 NIH cohort HbSS samples to a sequencing depth similar to JHS cohort, and compared their heteroplasmy burden. Mitochondrial sequences extracted from the cleaned WGS data of these 3 cohorts were analyzed for heteroplasmic and homoplasmic variants using mitoCaller from the package mitoAnalyzer.Results: The average depth per locus was ~6,671X for the NIH SCD cohort , ~2,879X for the 1KG cohort, and ~2169X for JHS cohort. We compared the quantity of heteroplasmic variants across the different NIH SCD genotype with 1KG (HbAA & HbAS), and JHS (HbAA, HbAC and HbAS) genotypic groups. The median number of heteroplasmic variants per individual increased progressively from HbAA, HbAS, HbSβ +thalassemia, and HbSC with the highest median number of 118 in HbSS & HbSβ 0 (Fig 1A) in NIH SCD cohort. It is noteworthy that the median mtDNA heteroplasmy in HbAA individuals in 1KG cohort was significantly lower than those in JHS cohort (Table insert in Fig 1A) which may be related to the underlying cardiovascular disease in the JHS cohort; whereas similar heteroplasmy burden in HbAS individuals between these 2 cohorts may underscore the genotype (HbAS) as the driver of heteroplasmy in these cohorts. We compared the heteroplasmy burden of a downsampled subset (n=300) NIH HbSS with that of JHS HbAA, HbAC and HbAS genotypes (Fig 1B). Although, the 70% reduction in sequencing depth resulted in the slight reduction in heteroplasmy burden, we noticed higher heteroplasmic variability (standard deviation) in this subset of NIH HbSS patients. This variability may be attributable to extreme variation in SCD phenotypic severity. We then applied cumulative distribution function to this downsampled subset and compared with JHS genotypes. We found the NIH HbSS patients have disproportionately higher proportion of heteroplasmy variants (Fig 1D) when compared to the JHS genotypes (HbAA, HbAC, and HbAS).Conclusion: We conclude that there is an increased prevalence of heteroplasmic mtDNA variants in SCD compared to ethnic-matched normal (HbAA) populations. Normal individuals with HbAA in JHS cohort have significantly higher heteroplasmic burden compared to those in 1KG cohort, suggesting an underlying cardiovascular disease in JHS cohort as a driving factor. Within each 1KG and JHS cohorts, individuals with sickle cell trait (HbAS) have similar heteroplasmy burden and also higher than those with HbAA, highlighting the potential significance of this genotype. Reducing the sequencing depth by > 70% (downsampling) led to the filtering out of heteroplasmy variants that would have been discovered with the original deeper sequencing depth of ~7300X. Nonetheless, downsampled HbSS samples still retained disproportionately higher heteroplasmy burden compared to non-SCD subjects. We are currently investigating if there is any correlation between mtDNA heteroplasmy burden and severity of clinical phenotypes among the SCD patients.1. Ahmad, MM et al, Blood 136 (1):11-11 (2020) [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Homoplasmy of the m. 8993 T>G variant in a patient without MRI findings of Leigh syndrome, ataxia or retinal abnormalities

Leigh syndrome is a progressive neurodegenerative syndrome caused by multiple mitochondrial DNA and nuclear DNA pathological variants. Patients with Leigh syndrome consistently have distinct brain lesions found on MRI scanning involving abnormal signal in the basal ganglia, brainstem and/or cerebellum. Other clinical findings vary depending on the genetic etiology and epigenetic factors. Mitochondrial DNA-derived Leigh syndrome phenotype is thought to be modulated by heteroplasmy level. The classic example is the clinical expression of the pathological variant, m. 8993 T>G. At heteroplasmy levels above 90%, the resulting phenotype is Leigh syndrome, but at levels 70–90% patients present with a syndrome of neuropathy, ataxia and retinitis pigmentosa. We describe a 15-year old girl with homoplasmic variant in m.8993 T>G and clinical and biochemical findings consistent with Leigh syndrome but with normal brain MRI findings and without retinal abnormalities or ataxia.

Mitochondrial DNA Variation in Individuals with Sickle Cell Disease

Background: Sickle cell disease (SCD) is a complex multi-system disorder that predominantly affects individuals of African heritage. While the sickle pathology is initiated by polymerization of HbS, multiple end-organ damage is inflicted by years of on-going inflammation and vasculopathy. An emerging marker of inflammation is the accumulation of mutations in mitochondrial DNA (mtDNA), the phenotypic effect of which will depend on the nature of the gene that harbors the mutation, the mutant allele fraction, and the pathogenicity of the mutant allele. A mutation in mtDNA is heteroplasmic when it is present in only a proportion of mtDNA, and homoplasmic, when it is present in all mtDNA molecules. Mitochondrial heteroplasmy can also occur at different tissue or cell levels, even within the same individual. Given the underlying chronic inflammation in SCD, we hypothesize that SCD patients display increased rates of mtDNA mutations. Methods: We analyzed and compared whole genome sequencing (WGS) data from the cohort of 683 SCD patients (SCD cohort) of African ancestry with that of 621 individuals of African ancestry from the 1000 Genomes Project (1KG). The SCD cohort included 561 HbSS &amp; HbSβ0 thalassemia (combined), 90 HbSC, and 25 HbSβ+ thalassemia. The 1KG cohort included 516 HbAA and 105 sickle carriers (HbAS). mtDNA sequences of SCD and 1KG cohorts were initially aligned to the revised Cambridge Reference Sequence (rCRS NC_012920), and subsequently base recalibrated and deduplicated. Mitochondrial sequences extracted from the cleaned WGS data of both cohorts were analyzed for heteroplasmic and homoplasmic variants using mitoCaller from the package mitoAnalyzer. Results: The average depth per locus is ~6,828X for the SCD cohort and ~2,879X for the 1KG cohort. We performed a locus by locus comparison between the mtDNA sequences of both cohorts. No homoplasmic variants unique to the SCD cohort were found when compared to the 1KG cohort. In contrast, there were several "hotspots" of heteroplasmic variants that were unique to the SCD cohort, and largely shared amongst the SCD patient population (Figure 1A). To identify these unique variants, we used MITOMASTER and Ensembl VEP to annotate the heteroplasmic variants that had above 40% population frequency within the SCD cohort and below 10% population frequency in the 1KG cohort. Several heteroplasmic variants were non-synonymous and the selected variants originated from the Control Region (D-loop), RNR1, RNR2, ND1, ND4, and ND5. One of the heteroplasmic variants, 2623 A&amp;gt;G, was found to be age linked for HbSS &amp; HbSβ0 (Figure 1B), with its minor allele frequency (MAF) increasing with age. Further analysis needs to be done in order to determine if more variants unique to the SCD cohort are age-linked. We then compared the quantity of heteroplasmic variants across the different SCD genotypic groups with the 1KG HbAA and the 1KG HbAS groups. The median number of heteroplasmic variants per individual increased progressively from HbAA, HbAS, HbSβ+ thalassemia, and HbSC with the highest median number of 119 in HbSS &amp; HbSβ0 (Figure 1C). Mitochondrial heteroplasmy for 1KG HbAA and 1KG HbAS were statistically significant when tested against each other and against every SCD sub-group; however, the difference was not statistically significant between the different SCD genotypes (Table insert in Fig 1C). It is important to note that we did not apply a MAF threshold, thus many of the heteroplasmic variants may be present at very low levels. Conclusion: Our findings suggest that there is an increased prevalence of heteroplasmic variants in SCD compared to ethnic-matched healthy populations. Within the SCD genotypes, the heteroplasmic burden increased progressively (HbAS &amp;lt; HbSβ+ thalassemia &amp;lt; HbSC &amp;lt; HbSS &amp; HbSβ0) with genotypic groups that are associated with increasing phenotypic severity. mtDNA heteroplasmic burden for one variant also increased with age in HbSS &amp; HbSβ0 individuals, but further studies are needed to explore if mtDNA heteroplasmic burden correlates with the degree of organ damage and disease severity within the same genotypic group. Although it is not clear if the variants are a cause or effect of the sickle inflammatory pathology, our data suggest that mtDNA heteroplasmic burden is a potential biomarker of SCD severity. Disclosures No relevant conflicts of interest to declare.