Heteroplasmy Levels Research Articles

The clinical spectrum of MELAS and associated disorders across ages: a retrospective cohort study.

Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) is a severe multisystemic disease, although some have a milder phenotype. We aimed to evaluate the clinical spectrum of this disease from MELAS patients to asymptomatic carriers and identify predictors of severity. We reviewed 81 patients, who had MELAS or had positive genetics without meeting clinical criteria. Patients who met criteria including lactic acidosis, encephalomyopathy, and stroke-like episodes (SLE) were categorized as MELAS, symptomatic non-MELAS, and asymptomatic. MELAS was further categorized as "standard-onset" if the first stroke-like episode (SLE) occurred before age 40 or "late-onset." Eighty-one patients were included: 42 MELAS (13 late-onset), 30 symptomatic non-MELAS, and 9 asymptomatic. MELAS patients had lower BMI at onset (mean 18.6 vs. 25.1 asymptomatic and 22.0 symptomatic non-MELAS, p < 0.05). There was a trend toward higher serum heteroplasmy in MELAS compared to symptomatic non-MELAS and asymptomatic (means 39.3, 29.3, and 21.8% p = 0.09). Symptomatic non-MELAS had more sensorineural hearing loss as first presenting symptom (51.6% vs. 24.4%, p < 0.05). MELAS had higher prevalence of seizures (88.1% vs. 16.7%, p < 0.05) and shorter survival from onset to death (50% mortality at 25 years vs. 10%, p < 0.05). Late-onset MELAS had longer disease duration from first symptom to first SLE (mean 16.6 vs. 9.3 yrs) and also lived longer (mean age at death 62 vs. 30). Standard-onset MELAS had more neurologic involvement at onset than late-onset (51.7% vs. 15.4%). Late-onset patients had more prevalent diabetes (69.2% vs. 13.8%) and nephropathy (53.8% vs. 10.3%). Patients with late-onset MELAS also had more organ systems involved (mean 4.1 vs. 2.7, p < 0.05). There was a trend toward higher heteroplasmy levels in standard-onset (mean 44.8% vs. 25.3%, p = 0.18). Our study highlights the spectrum of MELAS. The lower BMI in MELAS at presentation as well as higher rates of sensorineural hearing loss as initial symptom in symptomatic non-MELAS may be useful clinical markers. While many patients present before age 40 with SLE, some can present with SLE later in life. Standard onset MELAS is more likely to present with neurologic symptoms. Late-onset is more likely to suffer diabetes or nephropathy and have more organ systems involved.

Whole mitogenome sequencing uncovers a relation between mitochondrial heteroplasmy and leprosy severity

BackgroundIn recent years, the mitochondria/immune system interaction has been proposed, so that variants of mitochondrial genome and levels of heteroplasmy might deregulate important metabolic processes in fighting infections, such as leprosy.MethodsWe sequenced the whole mitochondrial genome to investigate variants and heteroplasmy levels, considering patients with different clinical forms of leprosy and household contacts. After sequencing, a specific pipeline was used for preparation and bioinformatics analysis to select heteroplasmic variants.ResultsWe found 116 variants in at least two of the subtypes of the case group (Borderline Tuberculoid, Borderline Lepromatous, Lepromatous), suggesting a possible clinical significance to these variants. Notably, 15 variants were exclusively found in these three clinical forms, of which five variants stand out for being missense (m.3791T > C in MT-ND1, m.5317C > A in MT-ND2, m.8545G > A in MT-ATP8, m.9044T > C in MT-ATP6 and m.15837T > C in MT-CYB). In addition, we found 26 variants shared only by leprosy poles, of which two are characterized as missense (m.4248T > C in MT-ND1 and m.8027G > A in MT-CO2).ConclusionWe found a significant number of variants and heteroplasmy levels in the leprosy patients from our cohort, as well as six genes that may influence leprosy susceptibility, suggesting for the first time that the mitogenome might be involved with the leprosy process, distinction of clinical forms and severity. Thus, future studies are needed to help understand the genetic consequences of these variants.

Abstract 12838: Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-Like Episodes (MELAS) and m.3243A&gt;G Mutations Have Higher Prevalence of Wolff-Parkinson-White Syndrome

Background: Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) is one of the most common mitochondrial disorders. Cardiovascular involvement has been reported in up to 30% of MELAS patients with varying clinical presentations from non-specific cardiogenic abnormalities to conduction abnormalities, cardiomyopathies, heart failure, fatal arrhythmias and cardiac death. Although conduction defects are a known complication, the frequency of Wolff-Parkinson-White (WPW) Syndrome among MELAS patients and mutations associated with MELAS is uncertain, and their association with cardiomyopathy and treatment outcomes have rarely been reported. Methods: A retrospective chart review of 50 MELAS patients from January 2007 to December 2022 from the Neuroscience Institute at University of Texas Houston was conducted. Medical histories, genetic testing, electrocardiograms, echocardiograms, and electrophysiology studies were reviewed. Results: 43 patients were included (mean age 23.8, median age 18.5, range 2-58). Eight of 43 patients with MELAS (20 %), inclusive of one of 3 patients with m.4317A&gt;G (33.3%) and seven of 40 patients with m.3243A&gt;G variants had electrocardiographic findings consistent with WPW. Other conduction abnormalities were noted in ten of 20 patients (50%) with m.3243A&gt;G with neurological involvement and six of 20 patients (30%) with m.3243A&gt;G mutation without strokes. Four patients required electrophysiology studies with ablation (mean age 8.5, range 7-10), one for inappropriate sinus tachycardia resistant to several medications, and three patients with WPW syndrome all of whom required repeat ablations. Conduction abnormalities had a positive correlation with higher heteroplasmy levels (mean 35 % vs 51%, 95% CI -30.2 to -2.88, p=0.019). Six patients with MELAS had cardiomyopathy of varying severity which were all associated with conduction abnormalities, including two patients with WPW syndrome (p=0.014). Conclusion: The prevalence of WPW in patients with MELAS syndrome and the m.3243A&gt;G variant appears much higher than in the normal population and may require multiple electrophysiology studies ablations to treat. Routine cardiology screening is recommended for early detection.

High-throughput single-cell analysis reveals progressive mitochondrial DNA mosaicism throughout life.

Heteroplasmic mitochondrial DNA (mtDNA) mutations are a major cause of inherited disease and contribute to common late-onset human disorders. The late onset and clinical progression of mtDNA-associated disease is thought to be due to changing heteroplasmy levels, but it is not known how and when this occurs. Performing high-throughput single-cell genotyping in two mouse models of human mtDNA disease, we saw unanticipated cell-to-cell differences in mtDNA heteroplasmy levels that emerged prenatally and progressively increased throughout life. Proliferating spleen cells and nondividing brain cells had a similar single-cell heteroplasmy variance, implicating mtDNA or organelle turnover as the major force determining cell heteroplasmy levels. The two different mtDNA mutations segregated at different rates with no evidence of selection, consistent with different rates of random genetic drift in vivo, leading to the accumulation of cells with a very high mutation burden at different rates. This provides an explanation for differences in severity seen in human diseases caused by similar mtDNA mutations.

Mitochondrial heteroplasmic shifts reveal a positive selection of breast cancer

BackgroundBreast cancer is, despite screening, not always detected early enough and is together with other tumor types known to shed genetic information in circulation. Unlike single-copy nuclear DNA, mitochondrial DNA (mtDNA) copies range from 100s to 10,000s per cell, thus providing a potentially alternative to identify potential missing cancer information in circulation at an early stage.MethodsTo characterize mitochondrial mutation landscapes in breast cancer, whole mtDNA sequencing and bioinformatics analyses were performed on 86 breast cancer biopsies and 50 available matched baseline cancer-free whole blood samples from the same individuals, selected from a cohort of middle-aged women in Sweden. To determine whether the mutations can be detected in blood plasma prior to cancer diagnosis, we further designed a nested case-control study (n = 663) and validated the shortlisted mutations using droplet digital PCR.ResultsWe detected different mutation landscapes between biopsies and matched whole blood samples. Compared to whole blood samples, mtDNA from biopsies had higher heteroplasmic mutations in the D-loop region (P = 0.02), RNR2 (P = 0.005), COX1 (P = 0.037) and CYTB (P = 0.006). Furthermore, the germline mtDNA mutations had higher heteroplasmy level than the lost (P = 0.002) and de novo mutations (P = 0.04). The nonsynonymous to synonymous substitution ratio (dN/dS) was higher for the heteroplasmic mutations (P = 7.25 × 10−12) than that for the homoplasmic mutations, but the de novo (P = 0.06) and lost mutations (P = 0.03) had lower dN/dS than the germline mutations. Interestingly, we found that the critical regions for mitochondrial transcription: MT-HSP1 (odds ratio [OR]: 21.41), MT-TFH (OR: 7.70) and MT-TAS2 (OR: 3.62), had significantly higher heteroplasmic mutations than the rest of the D-loop sub-regions. Finally, we found that the presence of mt.16093T > C mutation increases 67% risk of developing breast cancer.ConclusionsOur findings show that mitochondrial genetic landscape changes during cancer pathogenesis and positive selection of mtDNA heteroplasmic mutations in breast cancer. Most importantly, the mitochondrial mutations identified in biopsies can be traced back in matched plasma samples and could potentially be used as early breast cancer diagnostic biomarkers.

Targeting of CRISPR-Cas12a crRNAs into human mitochondria

Mitochondrial gene editing holds great promise as a therapeutic approach for mitochondrial diseases caused by mutations in the mitochondrial DNA (mtDNA). Current strategies focus on reducing mutant mtDNA heteroplasmy levels through targeted cleavage or base editing. However, the delivery of editing components into mitochondria remains a challenge. Here we investigate the import of CRISPR-Cas12a system guide RNAs (crRNAs) into human mitochondria and study the structural requirements for this process by northern blot analysis of RNA isolated from nucleases-treated mitoplasts. To investigate whether the fusion of crRNA with known RNA import determinants (MLS) improve its mitochondrial targeting, we added MLS hairpin structures at 3′-end of crRNA and demonstrated that this did not impact crRNA ability to program specific cleavage of DNA in lysate of human cells expressing AsCas12a nuclease. Surprisingly, mitochondrial localization of the fused crRNA molecules was not improved compared to non-modified version, indicating that structured scaffold domain of crRNA can probably function as MLS, assuring crRNA mitochondrial import. Then, we designed a series of crRNAs targeting different regions of mtDNA and demonstrated their ability to program specific cleavage of mtDNA fragments in cell lysate and their partial localization in mitochondrial matrix in human cells transfected with these RNA molecules. We hypothesize that mitochondrial import of crRNAs may depend on their secondary structure/sequence. We presume that imported crRNA allow reconstituting the active crRNA/Cas12a system in human mitochondria, which can contribute to the development of effective strategies for mitochondrial gene editing and potential future treatment of mitochondrial diseases.

Leber’s hereditary optic neuropathy: clinical and genetic analysis of Bulgarian patients

Leber’s hereditary optic neuropathy (LHON) is a rare maternally inherited disease caused by mutations in mitochondrial DNA (mtDNA) genes encoding subunits of complex I in the mitochondrial respiratory chain. The most common mutations causing LHON are G11778A, G3460A and T14484C, but there are also several less common mutations. LHON presents as acute or subacute bilateral visual loss, usually affecting young males. The aim of this study was to assess the clinical symptomatology and genetic analysis of Bulgarian patients with LHON. Twenty-two patients were diagnosed with LHON based on clinical evaluation and genetic examination (17 males and 5 females); 12 of them were previously reported, while 8 males and 2 females are newly diagnosed. A full neuroophthalmologic and genetic examination was performed. Eight patients had a family history of LHON, while 14 were isolated cases. The age at onset ranged from 3 to 43 years, and visual acuity ranged from counting fingers to 0.9. Genetic testing revealed various mutations, including a rare mutation G3635A in MT-ND1 in five affected members of one pedigree and digenic inheritance of G11778A and T14484C in three individuals from a different family. A variant m.15988A > G in the mitochondrial gene MT-TP with a high level of heteroplasmy was found in one patient. In addition to the most common mutations causing LHON, our patients also had rare mutations. These results suggest that genetic analysis of the entire mtDNA sequence is recommended in cases with strong clinical suspicion of LHON, since new rare mtDNA pathogenic variants are being identified.

Myopathy and Ophthalmologic Abnormalities in Association With Multiple Skeletal Muscle Mitochondrial DNA Deletions.

Establishing a molecular diagnosis of mitochondrial diseases due to pathogenic mitochondrial DNA (mtDNA) variants can be difficult because of varying levels of tissue heteroplasmy, and identifying these variants is important for clinical management. Here, we present clinical and molecular findings in 8 adult patients with classical features of mitochondrial ophthalmologic and/or muscle disease and multiple mtDNA deletions isolated to muscle. The patients were identified via a retrospective review of patients seen in both a tertiary ophthalmology center and a genetics clinic with a clinical diagnosis of chronic progressive external ophthalmoplegia, optic nerve abnormalities, and/or mitochondrial myopathy. Age at onset of symptoms ranged from 18 to 61 years. Ocular manifestations included bilateral optic neuropathy in one patient, bilateral optic disc cupping without optic neuropathy in 2 patients, ptosis in 4 patients, and ocular motility deficits in 2 patients. Five patients had generalized weakness. Pathogenic variants in mtDNA were not found in the blood or buccal sample from any patient, but 7 of 8 patients had multiple mtDNA deletions identified in muscle tissue. One patient had a single mtDNA deletion identified in the muscle. Heteroplasmy was less than 15% for all of the identified deletions, with the exception of one deletion that had a heteroplasmy of 50%-60%. None of the patients were found to have a nuclear gene variant known to be associated with mitochondrial DNA maintenance. mtDNA deletions were identified in adult patients with ophthalmologic and/or musle abnormalities and may underlie their clinical presentations.

Negative correlation between organ heteroplasmy, particularly hepatic heteroplasmy, and age at death revealed by post-mortem studies of m.3243A > G cases

Mitochondrial DNA m.3243A > G mutation causes mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) and its associated multi-organ disorders, including diabetes. To clarify associations between m.3243A > G organ heteroplasmy and clinical phenotypes, including the age at death, we combined genetic and pathological examinations from seven unreported and 36 literature cases of autopsied subjects. Clinical characteristics of subjects were as follows: male, 13; female, 28; unknown, 2; the age at death, 36.9 ± 20.2 [4–82] years; BMI, 16.0 ± 2.9 [13.0–22.3]; diabetes, N = 21 (49%), diabetes onset age 38.6 ± 14.2 years; deafness, N = 27 (63%); stroke-like episodes (StLEp), N = 25 (58%); congestive heart failure (CHF), N = 15 (35%); CHF onset age, 51.3 ± 14.5 years. Causes of death (N = 32) were as follows: cardiac, N = 13 (41%); infection, N = 8 (25%); StLEp, N = 4 (13%); gastrointestinal, N = 4 (13%); renal, N = 2 (6%); hepatic, N = 1 (2%). High and low heteroplasmies were confirmed in non-regenerative and regenerative organs, respectively. Heteroplasmy of the liver, spleen, leukocytes, and kidney for all subjects was significantly associated with the age at death. Furthermore, the age at death was related to juvenile-onset (any m.3243A > G-related symptoms appeared before 20) and stroke-like episodes. Multiple linear regression analysis with the age at death as an objective variable showed the significant contribution of liver heteroplasty and juvenile-onset to the age at death. m.3243A > G organ heteroplasmy levels, particularly hepatic heteroplasmy, are significantly associated with the age at death in deceased cases.

Increases in computationally predicted deleterious variants of unknown significance and sperm mtDNA copy numbers may be associated with semen quality.

Mitochondria are essential for sperm motility because they provide the energy required for the movement. Changes in sperm mtDNA, such as point mutations, large-scale deletions, or copy number variations, may interfere with ATP production and reduce sperm motility. However, it is not clear if changes in mtDNA are linked to semen quality. To explore the association between sperm mitochondrial DNA (mtDNA) changes and semen quality. Sixty-five oligo and/or astheno and/or terato patients (O/A/T) patients and 41 controls were recruited from couples undergoing assisted reproduction. Semen and blood samples were collected from the same individual on the day of oocyte retrieval to extract, isolate and purify mtDNA for next-generation sequencing. mtDNA copy numbers were assessed in 64 patient and 39 control sperm DNA samples using quantitative real-time PCR. The 4977bp deletion was assessed in 20 patient and 20 control sperm DNA samples using polymerase chain reaction. The mtDNA of patients was more likely to carry pathogenic variants or variants of unknown significance (VUSs) (P=0.091) with higher heteroplasmy levels (P<0.05) than that of controls. Interestingly, 33.85% of O/A/T patients (22 out of 65) lacked unique variants in their spermatozoa. but presented an exceptionally high mtDNA copy number (P<0.0001). Moreover, we observed a decrease in the heteroplasmy level of common mtDNA variants shared by somatic and gamete cells (P<0.0001) and the emergence of a very large number of de novo mtDNA variants with low-level heteroplasmy in spermatozoa. The increases in the number of computationally predicted deleterious VUS and mtDNA copies in spermatozoa may be associated with semen quality. Exposure to environmental mutation pressure that causes novel mtDNA variants with low-level heteroplasmy may occur during spermatogenesis. Furthermore, when a certain harmful threshold is reached, male germ cells may degrade mtDNA with mutations and replicate the correct mtDNA sequence to maintain the mitochondrial function in spermatozoa.

CmVCall: An automated and adjustable nanopore analysis pipeline for heteroplasmy detection of the control region in human mitochondrial genome

Genetic associations between human mitochondrial DNA (mtDNA) heteroplasmy and mitochondrial diseases, aging, and cancer have been elaborated, contributing a lot to the further understanding of mtDNA polymorphic spectrum in anthropology, population, and forensic genetics. In the past decade, heteroplasmy detection using Sanger sequencing and next generation sequencing (NGS) was hampered by the former’s inefficiency and the latter’s inherent bias due to amplification and mapping of short reads, respectively. Nanopore sequencing stands out for its ability to yield long contiguous segments of DNA, providing a new insight into heterogeneity authentication. In addition to MinION from Oxford Nanopore Technologies, an alternative nanopore sequencer QNome (Qitan Technology) has also been applied to various biological research and the forensic applicability of this platform has been proved recently. In this study, we evaluated the performance of four commonly used variant callers in the heterogeneity authentication of the control region of human mtDNA based on simulations of different ratios generated by mixing QNome nanopore sequencing reads of two synthetic sequences. Then, an open-source and python-based nanopore analytics pipeline, CmVCall was developed and incorporated multiple programs including reads filtering, removal of nuclear mitochondrial sequences (NUMTs), alignment, optional ‘Correction’ mode, and heterogeneity identification. CmVCall can achieve high precision, accuracy, and recall of 100%, 99.9%, and 92.3% with a 5% heteroplasmy level in ‘Correction’ mode. Moreover, blood, saliva, and hair shaft samples from monozygotic (MZ) twins were used for heterogeneity evaluation and comparison with the NGS data. Results of MZ twin samples showed that CmVCall could identify more point heteroplasmy sites, revealing significant levels of inter- and intra-individual mtDNA polymorphism. In conclusion, we believe that this analysis pipeline will lay a solid foundation for the development of a comprehensive nanopore analysis pipeline targeting the whole mitochondrial genome.

Neuropathological hallmarks in autopsied cases with mitochondrial diseases caused by the mitochondrial 3243A>G mutation.

The mitochondrial (m.) 3243A>G mutation is known to be associated with various mitochondrial diseases including mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). Their clinical symptoms have been estimated to occur with an increased mitochondrial DNA (mtDNA) heteroplasmy and reduced activity of oxidative phosphorylation (OXPHOS) complexes, but their trends in the central nervous system remain unknown. Six autopsied mutant cases and three disease control cases without the mutation were enrolled in this study. The mutant cases had a disease duration of 1-27 years. Five of six mutant cases were compatible with MELAS. In the mutant cases, cortical lesions including a laminar necrosis were frequently observed in the parietal, lateral temporal, and occipital lobes; less frequently in the frontal lobe including precentral gyrus; and not at all in the medial temporal lobe. The mtDNA heteroplasmy in brain tissue samples of the mutant cases was strikingly high, ranging from 53.8% to 85.2%. The medial temporal lobe was preserved despite an inhospitable environment having high levels of mtDNA heteroplasmy and lactic acid. OXPHOS complex I was widely decreased in the mutant cases. The swelling of smooth muscle cells in the vessels on the leptomeninges, with immunoreactivity (IR) against mitochondria antibody, and a decreased nuclear/cytoplasmic ratio of choroidal epithelial cells were observed in all mutant cases but in none without the mutation. Common neuropathological findings such as cortical laminar necrosis and basal ganglia calcification were not always observed in the mutant cases. A high level of mtDNA heteroplasmy was observed throughout the brain in spite of heterogeneous cortical lesions. A lack of medial temporal lesion, mitochondrial vasculopathy in vessels on the leptomeninges, and an increased cytoplasmic size of epithelial cells in the choroid plexus could be neuropathological hallmarks helpful in the diagnosis of mitochondrial diseases.

The effects of heteroplasmy level of atherosclerosis-associated mitochondrial DNA mutations on growth rate of cells

The effects of heteroplasmy level of atherosclerosis-associated mitochondrial DNA mutations on growth rate of cells

Novel economical, accurate, sensitive, single-cell analytical method for mitochondrial DNA quantification in mtDNA mutation carriers.

Although a variety of analytical methods have been developed to detect mitochondrial DNA (mtDNA) heteroplasmy, there are special requirements of mtDNA heteroplasmy quantification for women carrying mtDNA mutations receiving the preimplantation genetic diagnosis (PGD) and prenatal diagnosis (PD) in clinic. These special requirements include various sample types, large samplenumber, long-term follow-up, and the need for detection of single-cell from biopsied embryos. Therefore, developing an economical, accurate, high-sensitive, and single-cell analytical method for mtDNA heteroplasmy is necessary. In this study, we developed the Sanger sequencing combined droplet digital polymerase chain reaction (ddPCR) method for mtDNA quantification and compared the results to next-generation sequencing (NGS). A total of seventeen families with twelve mtDNA mutations were recruited in this study. The results showed that both Sanger sequencing and ddPCR could be used to analyze the mtDNA heteroplasmy in single-cell samples. There was no statistically significant difference in heteroplasmy levels in common samples with high heteroplasmy (≥ 5%), low heteroplasmy (< 5%), and single-cell samples, either between Sanger sequencing and NGS methods, or between ddPCR and NGS methods (P > 0.05). However, Sanger sequencing was unable to detect extremely low heteroplasmy accurately. But even in samples with extremely low heteroplasmy (0.40% and 0.92%), ddPCR was always able to quantify them. Compared to NGS, Sanger sequencing combined ddPCR analytical methods greatly reduced the cost of sequencing. In conclusion, this study successfully established an economical, accurate, sensitive, single-cell analytical method based on the Sanger sequencing combined ddPCR methods for mtDNA heteroplasmy quantification in aclinical setting.

Next-generation sequencing reveals mitogenome diversity in plasma extracellular vesicles from colorectal cancer patients

BackgroundRecent reports have demonstrated that the entire mitochondrial genome can be secreted in extracellular vesicles (EVs), but the biological attributes of this cell-free mitochondrial DNA (mtDNA) remain insufficiently understood. We used next-generation sequencing to compare plasma EV-derived mtDNA to that of whole blood (WB), peripheral blood mononuclear cells (PBMCs), and formalin-fixed paraffin-embedded (FFPE) tumor tissue from eight rectal cancer patients and WB and fresh-frozen (FF) tumor tissue from eight colon cancer patients.MethodsTotal DNA was isolated before the mtDNA was enriched by PCR with either two primer sets generating two long products or multiple primer sets (for the FFPE tumors), prior to the sequencing. mtDNA diversity was assessed as the total variant number, level of heteroplasmy (mutant mtDNA copies mixed with wild-type copies), variant distribution within the protein-coding genes, and the predicted functional effect of the variants in the different sample types. Differences between groups were compared by paired Student’s t-test or ANOVA with Dunnett’s multiple comparison tests when comparing matched samples from patients. Mann–Whitney U test was used when comparing differences between the cancer types and patient groups. Pearson correlation analysis was performed.ResultsIn both cancer types, EV mtDNA presented twice as many variants and had significantly more low-level heteroplasmy than WB mtDNA. The EV mtDNA variants were clustered in the coding regions, and the proportion of EV mtDNA variants that were missense mutations (i.e., estimated to moderately affect the mitochondrial protein function) was significantly higher than in WB and tumor tissues. Nonsense mutations (i.e., estimated to highly affect the mitochondrial protein function) were only observed in the tumor tissues and EVs.ConclusionTaken together, plasma EV mtDNA in CRC patients exhibits a high degree of diversity.Trial registrationClinicalTrials.gov: NCT01816607. Registered 22 March 2013.

Maternally inherited diabetes mellitus and deafness (MIDD): A case report and review of literature

Abstract Maternally inherited diabetes and deafness (MIDD) is a rare subtype of diabetes with a prevalence of up to 1% globally but often missed if not suspected. We present a case of MIDD and a thorough review of the literature related to it. The phenotypic presentation of diabetes depends on heteroplasmy levels of m.3243A&gt;G mutation of the individual patient. Patients with MIDD may have involvement of other organs such as eye, muscles, kidney, and heart. In our patient, along with diabetes and deafness, there was also an associated vision loss with a strong maternal inheritance of diabetes. A young male who presented with diabetic ketoacidosis (DKA) and hearing and vision impairment and on further diagnostic work up turned out to be a case of genetically confirmed (m.3243A&gt;G mutation) MIDD. To the best of our knowledge, this should be the first case of MIDD presenting as DKA being reported from India.

P-228 Reliability of blastomere versus trophectoderm biopsy in preimplantation genetic testing for mitochondrial DNA disorders

Abstract Study question Can a single blastomere or trophectoderm (TE) biopsy accurately reflect the heteroplasmy levels of the whole embryo for mitochondrial DNA (mtDNA) disorders? Summary answer Heteroplasmy levels of single blastomere and TE are comparable to those of the rest of the embryo, including the inner cell mass (ICM). What is known already Oocytes of women carrying mtDNA mutations can display varying mutation loads – percentage of mutated mtDNA copies. Preimplantation genetic testing (PGT) aims at reducing the risk of having affected children by screening for embryos with minimal mtDNA heteroplasmy. The technique involves either a blastomere biopsy on cleavage stage embryos or TE biopsy from blastocysts, followed by genetic analysis to select embryos suitable for transfer. However, it remains unclear whether there is a uniform segregation of heteroplasmic mtDNA during the early stages of embryogenesis, and therefore whether the mutation load from blastomere or TE biopsies are representative of the whole embryo. Study design, size, duration We investigated the suitability of PGT for mtDNA disorders by comparing the mutation load between a single blastomere recovered at the eight-cell stage, TE cells and rest of the same blastocyst (n = 15) in a mouse model heteroplasmic for the m.5024C&amp;gt;T mutation. We also compared the levels of heteroplasmy between TE and ICM of heteroplasmic human blastocysts (n = 5) and between blastomeres of arrested cleavage stage embryos (n = 4) donated by two mtDNA disease patients. Participants/materials, setting, methods To obtain heteroplasmic mouse embryos, we mated female mice carrying a mitochondrial tRNAAla mutation (m.5024C&amp;gt;T) with wild-type males. Whereas in human experiments, we analysed preimplantation embryos from two different carriers of the m.3243A&amp;gt;G mutation, associated with MELAS syndrome, who showed 16% and 32% mutation load in their peripheral blood. After embryo biopsy, NGS was performed in three independent technical replicates and profiles were compared between samples from the same embryo to assess heteroplasmy levels. Main results and the role of chance Analysis of heteroplasmic mouse embryos showed a strong correlation in mtDNA heteroplasmy between blastomere and the corresponding embryo (r2 = 0.76), and between TE and the rest of the blastocyst (r2 = 0.94) (n = 15). Moreover, a strong correlation was observed between the biopsied blastomere and TE from the same embryo (r2 = 0.90). Heteroplasmy levels in the embryos analysed ranged from 55.3% to 88.3%, and the mean overall mutation load was 73.7 ± 7.9%. Development of biopsied embryos was comparable to those of non-manipulated mutant (n = 10) and wild-type embryos (n = 10) (blastocyst rate = 85.7%). For human blastocysts (n = 5), statistical analysis showed a strong correlation in heteroplasmy levels between ICM and TE portions (r2 = 0.98). Moreover, a modest correlation was observed among blastomeres of cleavage stage human embryos (r2 = 0.60) (n = 24 from 4 embryos). The mutation load in the embryos analysed ranged from 10.3% to 57.4%, and the mean overall heteroplasmy was 42.8 ± 12.2% in cleavage stage embryos and 38.6 ± 17.3% in blastocysts. Overall, concordance in heteroplasmy levels between single blastomeres, TE portions and corresponding blastocyst was established in human and mouse embryos. Limitations, reasons for caution These results should be further validated by increasing the sample size and by the inclusion of different mtDNA mutations. Furthermore, current data are suggestive, but not definitive, to guarantee that the mtDNA mutation load in the embryo will remain constant throughout life. Wider implications of the findings Taken together, results suggest that TE biopsy may be better than blastomere biopsy for mtDNA disorders. Moreover, biopsy at the blastocyst stage is likely less harmful for embryo development. Following, we will also investigate how heteroplasmic mtDNA segregates at the peri-implantation stages after the resumption of mtDNA replication. Trial registration number Not applicable

P-706 Non-invasive analysis of mitochondrial DNA mutation for embryo evaluation.

Abstract Study question Is it possible to amplify whole mitochondrial DNA (mtDNA) and detect all mtDNA mutation in human embryo from spent media after embryo culture? Summary answer We were able to amplify whole mtDNA from 43% of spent medium after 24-hour culture. The most of mtDNA mutation in embryo could be detected. What is known already Preimplantation genetic testing for aneuploidy (PGT-A) with embryo biopsy is now commonly used to improve pregnancy rate. However, invasive embryo biopsy requires highly trained embryologist and induced higher risk of pre-eclampsia. Therefore, non-invasive methods with cell-free DNA in spent medium have been studied. Our group previously showed euploid or viable embryos at post-implantation stage have lower number of non-synonymous mtDNA mutations and this may help to select the best blastocyst to transfer. Although cell-free DNA is reported to contain mtDNA, it remains unclear whether mtDNA mutations number can be assessed from cell-free DNA. Study design, size, duration Donated low grade blastocysts were cultured for additional 24 hours with new media, cell-free mtDNA were obtained from the spent culture medium (non-invasive samples). For comparison, trophectoderm cells were biopsied from each embryo (invasive sample). The mtDNA selective amplifications were done to both samples. We performed mtDNA sequencing by next generation sequencing (NGS) and compared mtDNA mutations between non-invasive and invasive samples to clarify the capability of non-invasive assessment for mtDNA mutation. Participants/materials, setting, methods Under the ethical review of Yokohama City University and informed consent with patients, we collected human blastocysts which were discarded because of slow development or morphologically low-grade. We extracted whole DNA from biopsy and medium samples and selectively amplified mtDNA by long-range PCR for two overlapping amplicons. The mtDNA sequencing with NGS was performed to successfully amplified mtDNA. The 90% or more heteroplasmy level non-synonymous mutations were investigated. Main results and the role of chance We collected 34 discarded embryos from 13 patients. All embryos were cultured individually for additional 24 hours with 20 μl drop of newly prepared media. Amplified mtDNA were visualized by the presence of 8 kb band following agarose gel-apheresis for two fragments PCR. Both fragments were detected in 41% (14/34) of non-invasive samples and all invasive samples. Only one fragment was detected in 14% (5/34) of non-invasive samples. The completely amplified non-invasive samples and corresponding invasive samples were performed mtDNA sequencing with NGS. In results, 13 non-synonymous mutations were identified in invasive samples. All of these mutations were also detected in non-invasive samples. Although the number and location of mutations completely matched between the invasive and non-invasive samples in 78.5% (11/14) embryos, in other 3 embryos, mtDNA mutation numbers were higher in non-invasive samples even though no mutation existed in invasive samples. This means, if selective mtDNA amplification is completed, mtDNA mutations can be evaluated from the non-invasive samples with a probability of about 80% but include 20% false positive mtDNA mutations. To realize this, we need to improve the mtDNA amplification method. Limitations, reasons for caution We can used only clinically discarded embryos because ethical restriction. And it was necessary to additional 24-hours culture to obtain individually cultured spent media because embryos are co-cultured until they are donated. Because of small number of samples available, we keep collecting more samples for more accurate evaluation. Wider implications of the findings If the mtDNA mutations can be analyzed from spent medium, high-quality embryos can be selected without invasive embryo biopsy and complicated procedure. If this non-invasive mtDNA analysis can be performed to clinical embryo for embryo transfer, it may contribute to further improvement of pregnancy rate without any invasion to embryo. Trial registration number the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant-in-Aid for Scientific Research C (Grant Number JP21K09474) and Early-Career Scientists (Grant Number JP20K18169)

#6681 MT-TL1 M.3243A&amp;gt;G MITOCHONDRIAL DNA VARIANT DETECTED FROM WHOLE EXOME SEQUENCING CAN EXPLAIN CASES OF ADULT UNKNOWN NEPHROPATHIES

Abstract Background and Aims In a population-based study, mainly constituted of unknown nephropathies (cohort Sorbonne University, Paris, France N = 1197 patients), beyond reporting the diagnostic yield of nuclear genes, we also investigated the incidence of the mtDNA pathogenic variant MT-TL1 m.3243A&amp;gt;G. This variation was systematically searched from whole exome sequencing (WES) data. In retrospect, we described the renal phenotype and studied the heteroplasmy level of the mtDNA variation in urine or kidney tissues compared to its blood fraction. The prevalence of m.3243A&amp;gt;G has been estimated ranging from 0.08% to up to 0.25%. Method From September 2018, to February 2023, WES has been prospectively performed, as a first exploration of adult's nephropathies of unknown origin or when a genetic renal disease was clinically suggested. As off-target result, we retrieved m.3243A&amp;gt;G variation in blood DNA and then determined the mtDNA heteroplasmic level respective from urine sample or kidney tissue when available with an orthogonal method. Results We report a molecular diagnosis in 294 over 1197 adult patients sequenced (diagnostic yield: 24%, Age 43 y/o in average). Among these 294 patients with molecular diagnoses, 48 were distinct monogenic disorders, out of which 8 accounted for 52% of the genetic diagnoses. MT-TL1 m.3243A&amp;gt;G pathogenic variant was detected in 1,7% of the patients (20 patients over 1197 patients). An orthogonal method confirmed the presence of m.3243A&amp;gt;G variant in 10 patients supporting the possibility to diagnose from blood DNA analysed by WES. In all cases, the presence of m.3243A&amp;gt;G had major clinical implications for the patients and their families: living related donor aborted for two patients, a molecular diagnosis in three patients with unknown nephropathies and in three patients with histological diagnosis of focal segmental glomerulosclerosis. Other mitochondrial diseases appeared less likely involved in our adult cohort (only one patient with COQ2 pathogenic variants). Conclusion This population-based study reinforces the place of WES as first-tier exploration for adult patients with chronic kidney disease in whom phenotypes are often poor and/or atypical. The mitochondrial genome analysis with the same assay allowed the detection of yet unsuspected MT-TL1 m.3243A&amp;gt;G variant in 1,7% of patients suggesting enrichment compared to current prevalence observed in other population. Our data suggest that the entity “mitochondrial nephropathy” can represent a significant part of adult unknown nephropathies.

Discrimination of monozygotic twins using mtDNA heteroplasmy through probe capture enrichment and massively parallel sequencing.

Differentiating between monozygotic (MZ) twins remains difficult because they have the same genetic makeup. Applying the traditional STR genotyping approach cannot differentiate one from the other. Heteroplasmy refers to the presence of two or more different mtDNA copies within a single cell and this phenomenon is common in humans. The levels of heteroplasmy cannot change dramatically during transmission in the female germ line but increase or decrease during germ-line transmission and in somatic tissues during life. As massively parallel sequencing (MPS) technology has advanced, it has shown the extraordinary quantity of mtDNA heteroplasmy in humans. In this study, a probe hybridization technique was used to obtain mtDNA and then MPS was performed with an average sequencing depth of above 4000. The results showed us that all ten pairs of MZ twins were clearly differentiated with the minor heteroplasmy threshold at 1.0%, 0.5%, and 0.1%, respectively. Finally, we used a probe that targeted mtDNA to boost sequencing depth without interfering with nuclear DNA and this technique can be used in forensic genetics to differentiate the MZ twins.