Pathogenic Mechanisms of Somatic Mutation and Genome Mosaicism in Aging.

Age-related accumulation of postzygotic DNA mutations results in tissue genetic heterogeneity known as somatic mosaicism. Although implicated in aging as early as the 1950s, somatic mutations in normal tissue have been difficult to study because of their low allele fractions. With the recent emergence of cost-effective high-throughput sequencing down to the single-cell level, enormous progress has been made in our capability to quantitatively analyze somatic mutations in human tissue in relation to aging and disease. Here we first review how recent technological progress has opened up this field, providing the first broad sets of quantitative information on somatic mutations in vivo necessary to gain insight into their possible causal role in human aging and disease. We then propose three major mechanisms that can lead from accumulated de novo mutations across tissues to cell functional loss and human disease.

Comparing clinical and genomic features based on the tumor location in patients with resected pancreatic cancer.

BACKGROUND: Pancreatic cancer is anatomically divided into pancreatic head and body/tail cancers, and some studies have reported differences in prognosis. However, whether this discrepancy is induced from the difference of tumor biology is hotly debated. Therefore, we aimed to evaluate the differences in clinical outcomes and tumor biology depending on the tumor location. METHODS: In this retrospective cohort study, we identified 800 patients with pancreatic ductal adenocarcinoma who had undergone upfront curative-intent surgery. Cox regression analysis was performed to explore the prognostic impact of the tumor location. Among them, 153 patients with sufficient tumor tissue and blood samples who provided informed consent for next-generation sequencing were selected as the cohort for genomic analysis. RESULTS: Out of the 800 patients, 500 (62.5%) had pancreatic head cancer, and 300 (37.5%) had body/tail cancer. Tumor location in the body/tail of the pancreas was not identified as a significant predictor of survival outcomes compared to that in the head in multivariate analysis (hazard ratio, 0.94; 95% confidence interval, 0.77-1.14; P = 0.511). Additionally, in the genomic analyses of 153 patients, there were no significant differences in mutational landscapes, distribution of subtypes based on transcriptomic profiling, and estimated infiltration levels of various immune cells between pancreatic head and body/tail cancers. CONCLUSIONS: We could not find differences in prognosis and tumor biology depending on tumor location in pancreatic ductal adenocarcinoma. Discrepancies in prognosis may represent a combination of lead time, selection bias, and clinical differences, including the surgical burden between tumor sites.

Prevalence of FRAC Group 11 Fungicide Resistance in Stemphylium vesicarium Isolates, but Not S. beticola Isolates, Causing Stemphylium Leaf Spot of Spinach (Spinacia oleracea).

Stemphylium leaf spot of spinach, caused by Stemphylium beticola and S. vesicarium, is a disease of economic importance in fresh market, processing, and seed production. There have been increasing reports of difficulty managing the disease in the southern United States using fungicides in Fungicide Resistance Action Committee (FRAC) group 11. Isolates of S. beticola and S. vesicarium obtained from spinach leaves and seed from 2001 to 2020 were screened for resistance to azoxystrobin and pyraclostrobin in vitro, in vivo, and using PCR assays to detect mutations in cytochrome b associated with resistance in other fungi (F129L, G137R, and G143A). EC50 values for mycelial growth and conidial germination of S. vesicarium isolates in vitro were significantly less (mean of 0.35 µg/ml) than that of S. vesicarium (mean of 14.17 µg/ml) with both fungicides. All isolates were slightly more sensitive to pyraclostrobin than azoxystrobin in both assays. In vivo assays of plants inoculated with the isolates of S. vesicarium demonstrated poor efficacy of fungicides with each of the two active ingredients. Only the G143A mutation was detected in all spinach isolates of S. vesicarium, including an isolate of S. vesicarium collected in 2003 and 82.9% of isolates from spinach seed lots harvested from crops grown in or after 2017 in Europe, New Zealand, and the United States. The FRAC 11 mutations were not detected in any isolates of S. beticola. The in vitro, in vivo, and DNA mutation assays suggest FRAC group 11 fungicide resistance is widespread in spinach isolates of S. vesicarium but not S. beticola.

Error-Corrected Deep Targeted Sequencing of Circulating Cell-Free DNA from Colorectal Cancer Patients for Sensitive Detection of Circulating Tumor DNA.

Circulating tumor DNA (ctDNA) is a promising biomarker, reflecting the presence of tumor cells. Sequencing-based detection of ctDNA at low tumor fractions is challenging due to the crude error rate of sequencing. To mitigate this challenge, we developed ultra-deep mutation-integrated sequencing (UMIseq), a fixed-panel deep targeted sequencing approach, which is universally applicable to all colorectal cancer (CRC) patients. UMIseq features UMI-mediated error correction, the exclusion of mutations related to clonal hematopoiesis, a panel of normal samples for error modeling, and signal integration from single-nucleotide variations, insertions, deletions, and phased mutations. UMIseq was trained and independently validated on pre-operative (pre-OP) plasma from CRC patients (n = 364) and healthy individuals (n = 61). UMIseq displayed an area under the curve surpassing 0.95 for allele frequencies (AFs) down to 0.05%. In the training cohort, the pre-OP detection rate reached 80% at 95% specificity, while it was 70% in the validation cohort. UMIseq enabled the detection of AFs down to 0.004%. To assess the potential for detection of residual disease, 26 post-operative plasma samples from stage III CRC patients were analyzed. From this we found that the detection of ctDNA was associated with recurrence. In conclusion, UMIseq demonstrated robust performance with high sensitivity and specificity, enabling the detection of ctDNA at low allele frequencies.

Genetic and histological analysis intraplacental choriocarcinoma: a case report.

We report on single case of intraplacental choriocarcinoma (IC) coexisting with feto-maternal hemorrhage from our hospital, a rare malignant tumor that occurs in the chorionic villous trophoblast. To investigate genetic and epigenetic changes to the carcinogenesis of IC, we employed cancer gene panel analysis and whole methylation analysis from a recent case of IC. By Short Tandem Repeats analysis, we confirmed that the tumor of present IC was derived from concurrent normal chorionic villous trophoblast cells. No mutation was found in 145 cancer-related genes. Meanwhile, amplification in MDM2 gene was observed. Furthermore, we observed deferentially methylated CpG sites between tumor and surrounding normal placenta in present IC case. These observations suggest that IC might be arisen as a result of aberrations of methylation rather than of DNA mutations. Further studies are needed to clarify association between aberrant methylation and choriocarcinogenesis.

Whole exome sequencing of 28 families of Danish descent reveals novel candidate genes and pathways in developmental dysplasia of the hip.

Developmental dysplasia of the hip (DDH) is a common condition involving instability of the hip with multifactorial etiology. Early diagnosis and treatment are critical as undetected DDH is an important cause of long-term hip complications. Better diagnostics may be achieved through genetic methods, especially for patients with positive family history. Several candidate genes have been reported but the exact molecular etiology of the disease is yet unknown. In the present study, we performed whole exome sequencing of DDH patients from 28 families with at least two affected first-degree relatives. Four genes previously not associated with DDH (METTL21B, DIS3L2, PPP6R2, and TM4SF19) were identified with the same variants shared among affected family members, in more than two families. Among known association genes, we found damaging variants in DACH1, MYH10, NOTCH2, TBX4, EVC2, OTOG, and SHC3. Mutational burden analysis across the families identified 322 candidate genes, and enriched pathways include the extracellular matrix, cytoskeleton, ion-binding, and detection of mechanical stimulus. Taken altogether, our data suggest a polygenic mode of inheritance for DDH, and we propose that an impaired transduction of the mechanical stimulus is involved in the etiopathological mechanism. Our findings refine our current understanding of candidate causal genes in DDH, and provide a foundation for downstream functional studies.

Optimized allotopic expression of mitochondrial ND6 transgene restored complex I and apoptosis deficiencies caused by LHON-linked ND6 14484T > C mutation.

BACKGROUND: Leber's hereditary optic neuropathy (LHON) is a maternally inherited eye disease due to mutations in mitochondrial DNA. However, there is no effective treatment for this disease. LHON-linked ND6 14484T > C (p.M64V) mutation caused complex I deficiency, diminished ATP production, increased production of reactive oxygen species (ROS), elevated apoptosis, and impaired mitophagy. Here, we investigated if the allotopic expression of human mitochondrial ND6 transgene corrected the mitochondrial dysfunctions due to LHON-associated m.14484T > C mutation. METHODS: Nucleus-versions of ND6 was generated by changing 6 non-universal codons with universal codons and added to mitochondrial targeting sequence of COX8. Stable transfectants were generated by transferring human ND6 cDNA expressed in a pCDH-puro vector into mutant cybrids carrying the m.14484T > C mutation and control cybrids. The effect of allotopic expression of ND6 on oxidative phosphorylation (OXPHOS) was evaluated using Blue Native gel electrophoresis and extracellular flux analyzer. Assessment of ROS production in cell lines was performed by flow cytometry with MitoSOX Red reagent. Analyses for apoptosis and mitophagy were undertaken via flow cytometry, TUNEL and immunofluorescence assays. RESULTS: The transfer of human ND6 into the cybrids carrying the m.14484T > C mutation raised the levels of ND6, ND1 and ND4L but did not change the levels of other mitochondrial proteins. The overexpression of ND6 led to 20~23% increases in the assembly and activity of complex I, and ~ 53% and ~ 33% increases in the levels of mitochondrial ATP and ΔΨm in the mutant cybrids bearing m.14484T > C mutation. Furthermore, mutant cybrids with overexpression of ND6 exhibited marked reductions in the levels of mitochondrial ROS. Strikingly, ND6 overexpression markedly inhibited the apoptosis process and restored impaired mitophagy in the cells carrying m.14484T > C mutation. However, overexpression of ND6 did not affect the ND6 level and mitochondrial functions in the wild-type cybrids, indicating that this ND6 level appeared to be the maximum threshold level to maintain the normal cell function. CONCLUSION: We demonstrated that allotopic expression of nucleus-versions of ND6 restored complex I, apoptosis and mitophagy deficiencies caused by the m.14484T > C mutation. The restoration of m.14484T > C mutation-induced mitochondrial dysfunctions by overexpression of ND6 is a step toward therapeutic interventions for LHON and mitochondrial diseases.

DNA Deamination Is Required for Human APOBEC3A-Driven Hepatocellular Carcinoma In Vivo.

Although the APOBEC3 family of single-stranded DNA cytosine deaminases is well-known for its antiviral factors, these enzymes are rapidly gaining attention as prominent sources of mutation in cancer. APOBEC3's signature single-base substitutions, C-to-T and C-to-G in TCA and TCT motifs, are evident in over 70% of human malignancies and dominate the mutational landscape of numerous individual tumors. Recent murine studies have established cause-and-effect relationships, with both human APOBEC3A and APOBEC3B proving capable of promoting tumor formation in vivo. Here, we investigate the molecular mechanism of APOBEC3A-driven tumor development using the murine Fah liver complementation and regeneration system. First, we show that APOBEC3A alone is capable of driving tumor development (without Tp53 knockdown as utilized in prior studies). Second, we show that the catalytic glutamic acid residue of APOBEC3A (E72) is required for tumor formation. Third, we show that an APOBEC3A separation-of-function mutant with compromised DNA deamination activity and wildtype RNA-editing activity is defective in promoting tumor formation. Collectively, these results demonstrate that APOBEC3A is a "master driver" that fuels tumor formation through a DNA deamination-dependent mechanism.

Systematic review and meta-analysis: Diagnostic performance of DNA alterations in pancreatic juice for the detection of pancreatic cancer.

BACKGROUND AND AIMS: Pancreatic cancer has a dismal prognosis. So far, imaging has been proven incapable of establishing an early enough diagnosis. Thus, biomarkers are urgently needed for early detection and improved survival. Our aim was to evaluate the pooled diagnostic performance of DNA alterations in pancreatic juice. METHODS: A systematic literature search was performed in EMBASE, MEDLINE Ovid, Cochrane CENTRAL and Web of Science for studies concerning the diagnostic performance of DNA alterations in pancreatic juice to differentiate patients with high-grade dysplasia or pancreatic cancer from controls. Study quality was assessed using QUADAS-2. The pooled prevalence, sensitivity, specificity and diagnostic odds ratio were calculated. RESULTS: Studies mostly concerned cell-free DNA mutations (32 studies: 939 cases, 1678 controls) and methylation patterns (14 studies: 579 cases, 467 controls). KRAS, TP53, CDKN2A, GNAS and SMAD4 mutations were evaluated most. Of these, TP53 had the highest diagnostic performance with a pooled sensitivity of 42% (95% CI: 31-54%), specificity of 98% (95%-CI: 92%-100%) and diagnostic odds ratio of 36 (95% CI: 9-133). Of DNA methylation patterns, hypermethylation of CDKN2A, NPTX2 and ppENK were studied most. Hypermethylation of NPTX2 performed best with a sensitivity of 39-70% and specificity of 94-100% for distinguishing pancreatic cancer from controls. CONCLUSIONS: This meta-analysis shows that, in pancreatic juice, the presence of distinct DNA mutations (TP53, SMAD4 or CDKN2A) and NPTX2 hypermethylation have a high specificity (close to 100%) for the presence of high-grade dysplasia or pancreatic cancer. However, the sensitivity of these DNA alterations is poor to moderate, yet may increase if they are combined in a panel.

Single-cell whole-genome sequencing reveals the functional landscape of somatic mutations in B lymphocytes across the human lifespan.

Accumulation of mutations in somatic cells has been implicated as a cause of aging since the 1950s. However, attempts to establish a causal relationship between somatic mutations and aging have been constrained by the lack of methods to directly identify mutational events in primary human tissues. Here we provide genome-wide mutation frequencies and spectra of human B lymphocytes from healthy individuals across the entire human lifespan using a highly accurate single-cell whole-genome sequencing method. We found that the number of somatic mutations increases from <500 per cell in newborns to >3,000 per cell in centenarians. We discovered mutational hotspot regions, some of which, as expected, were located at Ig genes associated with somatic hypermutation (SHM). B cell-specific mutation signatures associated with development, aging, or SHM were found. The SHM signature strongly correlated with the signature found in human B cell tumors, indicating that potential cancer-causing events are already present even in B cells of healthy individuals. We also identified multiple mutations in sequence features relevant to cellular function (i.e., transcribed genes and gene regulatory regions). Such mutations increased significantly during aging, but only at approximately one-half the rate of the genome average, indicating selection against mutations that impact B cell function. This full characterization of the landscape of somatic mutations in human B lymphocytes indicates that spontaneous somatic mutations accumulating with age can be deleterious and may contribute to both the increased risk for leukemia and the functional decline of B lymphocytes in the elderly.

Mitochondrial Dysfunction and Therapeutic Perspectives in Cardiovascular Diseases.

High mortality rates due to cardiovascular diseases (CVDs) have attracted worldwide attention. It has been reported that mitochondrial dysfunction is one of the most important mechanisms affecting the pathogenesis of CVDs. Mitochondrial DNA (mtDNA) mutations may result in impaired oxidative phosphorylation (OXPHOS), abnormal respiratory chains, and ATP production. In dysfunctional mitochondria, the electron transport chain (ETC) is uncoupled and the energy supply is reduced, while reactive oxygen species (ROS) production is increased. Here, we discussed and analyzed the relationship between mtDNA mutations, impaired mitophagy, decreased OXPHOS, elevated ROS, and CVDs from the perspective of mitochondrial dysfunction. Furthermore, we explored current potential therapeutic strategies for CVDs by eliminating mtDNA mutations (e.g., mtDNA editing and mitochondrial replacement), enhancing mitophagy, improving OXPHOS capacity (e.g., supplement with NAD+, nicotinamide riboside (NR), nicotinamide mononucleotide (NMN), and nano-drug delivery), and reducing ROS (e.g., supplement with Coenzyme Q10 and other antioxidants), and dissected their respective advantages and limitations. In fact, some therapeutic strategies are still a long way from achieving safe and effective clinical treatment. Although establishing effective and safe therapeutic strategies for CVDs remains challenging, starting from a mitochondrial perspective holds bright prospects.

A linear five-ring pyrrole-imidazole polyamide-triphenylphosphonium conjugate targeting a mitochondrial DNA mutation efficiently induces apoptosis of HeLa cybrid cells carrying the mutation.

Somatic mutations in mitochondrial DNA may provide a new avenue for cancer therapy due to their associations to a number of cancers and a tendency of homoplasmicity. In consideration of mitochondrial features and its relatively small genome size, a nucleotide-based targeting approach is a considerably more promising option. To explore the efficacy of short linear N-methylpyrrole-N-methylimidazole polyamide (PI polyamide), we synthesized a five-ring short PI polyamide that provided sequence-specific homing for the A3243G mitochondrial mutation upon conjugation with triphenylphosphonium cation (TPP). This PI polyamide-TPP was able to induce cytotoxicity in HeLamtA3243G cybrid cells, while preserving preferential binding for oligonucleotides containing the A3243G motif from melting temperature assays. The PI polyamide-TPP also localized in the mitochondria in HeLamtA3243G cells and induced mitochondrial reactive oxygen species production, mitophagy and apoptosis in a mutation-specific fashion compared to the wild-type HeLamtHeLa cybrids; normal human dermal fibroblasts were also relatively unaffected to suggest discriminating selectivity for the mutant mitochondria, offering a novel outlook for cancer therapy via mitochondrial homing of short linear PIP-TPPs.

Preimplantation genetic diagnosis for a carrier with m.3697G > A mitochondrial DNA mutation.

OBJECTIVE: To explore inheritance of the m.3697G > A mitochondrial DNA (mtDNA) mutation and the effectiveness of preimplantation genetic diagnosis (PGD) for the carrier. METHODS: The study encompassed a pedigree of m.3697G > A mtDNA mutation, including one asymptomatic patient who pursued for PGD treatment. Twelve cumulus oocyte complexes (COCs) were collected in the first PGD cycle and 11 COCs in the second cycle. The efficiency of cumulus cells, polar bodies, and trophectoderm (TE) in predicting the m.3697G > A heteroplasmy of embryos was analyzed. RESULTS: From 23 COCs, 20 oocytes were fertilized successfully. On day 5 and 6 post-fertilization, 15 blastocysts were biopsied. The m.3697G > A mutation load of TE biopsies ranged from 15.2 to 100%. In the first cycle, a blastocyst with mutation load of 31.7% and chromosomal mosaicism was transferred, but failed to yield a clinical pregnancy. In the second cycle, a euploid blastocyst with mutation load of 53.9% was transferred, which gave rise to a clinical pregnancy. However, the pregnancy was terminated due to fetal cleft lip and palate. The mutation loads of different tissues (47.7 ± 1.8%) from the induced fetus were comparable to that of the biopsied TE and amniotic fluid cell (49.7%). The mutation load of neither cumulus cells (R2 = 0.02, p = 0.58) nor polar bodies (R2 = 0.33, p = 0.13) correlated with TE mutation load which was regarded as a gold standard. CONCLUSIONS: The m.3697G > A mutation showed a random pattern of inheritance. PGD could be used to reduce the risk of inheritance of a high mutation load. Cumulus cells are not a suitable predictor of blastocyst mutation load.

The Role of Altered Mitochondrial Metabolism in Thyroid Cancer Development and Mitochondria-Targeted Thyroid Cancer Treatment.

Thyroid cancer (TC) is the most common type of endocrine malignancy. Tumour formation, progression, and metastasis greatly depend on the efficacy of mitochondria-primarily, the regulation of mitochondria-mediated apoptosis, Ca2+ homeostasis, dynamics, energy production, and associated reactive oxygen species generation. Recent studies have successfully confirmed the mitochondrial aetiology of thyroid carcinogenesis. In this review, we focus on the recent progress in understanding the molecular mechanisms of thyroid cancer relating to altered mitochondrial metabolism. We also discuss the repurposing of known drugs and the induction of mitochondria-mediated apoptosis as a new trend in the development of anti-TC therapy.

Molecular Hydrogen as a Novel Antitumor Agent: Possible Mechanisms Underlying Gene Expression.

While many antitumor drugs have yielded unsatisfactory therapeutic results, drugs are one of the most prevalent therapeutic measures for the treatment of cancer. The development of cancer largely results from mutations in nuclear DNA, as well as from those in mitochondrial DNA (mtDNA). Molecular hydrogen (H2), an inert molecule, can scavenge hydroxyl radicals (·OH), which are known to be the strongest oxidizing reactive oxygen species (ROS) in the body that causes these DNA mutations. It has been reported that H2 has no side effects, unlike conventional antitumor drugs, and that it is effective against many diseases caused by oxidative stress and chronic inflammation. Recently, there has been an increasing number of papers on the efficacy of H2 against cancer and its effects in mitigating the side effects of cancer treatment. In this review, we demonstrate the efficacy and safety of H2 as a novel antitumor agent and show that its mechanisms may not only involve the direct scavenging of ·OH, but also other indirect biological defense mechanisms via the regulation of gene expression.

The APOBEC Protein Family: United by Structure, Divergent in Function.

The APOBEC (apolipoprotein B mRNA editing catalytic polypeptide-like) family of proteins have diverse and important functions in human health and disease. These proteins have an intrinsic ability to bind to both RNA and single-stranded (ss) DNA. Both function and tissue-specific expression varies widely for each APOBEC protein. We are beginning to understand that the activity of APOBEC proteins is regulated through genetic alterations, changes in their transcription and mRNA processing, and through their interactions with other macromolecules in the cell. Loss of cellular control of APOBEC activities leads to DNA hypermutation and promiscuous RNA editing associated with the development of cancer or viral drug resistance, underscoring the importance of understanding how APOBEC proteins are regulated.

Inheritance of mitochondrial DNA in humans: implications for rare and common diseases.

The first draft human mitochondrial DNA (mtDNA) sequence was published in 1981, paving the way for two decades of discovery linking mtDNA variation with human disease. Severe pathogenic mutations cause sporadic and inherited rare disorders that often involve the nervous system. However, some mutations cause mild organ-specific phenotypes that have a reduced clinical penetrance, and polymorphic variation of mtDNA is associated with an altered risk of developing several late-onset common human diseases including Parkinson's disease. mtDNA mutations also accumulate during human life and are enriched in affected organs in a number of age-related diseases. Thus, mtDNA contributes to a wide range of human pathologies. For many decades, it has generally been accepted that mtDNA is inherited exclusively down the maternal line in humans. Although recent evidence has challenged this dogma, whole-genome sequencing has identified nuclear-encoded mitochondrial sequences (NUMTs) that can give the false impression of paternally inherited mtDNA. This provides a more likely explanation for recent reports of 'bi-parental inheritance', where the paternal alleles are actually transmitted through the nuclear genome. The presence of both mutated and wild-type variant alleles within the same individual (heteroplasmy) and rapid shifts in allele frequency can lead to offspring with variable severity of disease. In addition, there is emerging evidence that selection can act for and against specific mtDNA variants within the developing germ line, and possibly within developing tissues. Thus, understanding how mtDNA is inherited has far-reaching implications across medicine. There is emerging evidence that this highly dynamic system is amenable to therapeutic manipulation, raising the possibility that we can harness new understanding to prevent and treat rare and common human diseases where mtDNA mutations play a key role.

Mitochondrial DNA pathogenic mutations in multiple symmetric lipomatosis.

The frequency of dermatological manifestations in diseases due to mitochondrial DNA mutations is not well known, although multiple symmetric lipomatosis has been repeatedly associated to mitochondrial DNA mutations. Here, we present a patient suffering from multiple symmetric lipomatosis and other skin signs. We found a new mitochondrial DNA mutation, m.8357T>C, in the tRNALys -coding gene and, using a cybrid approach, confirmed its pathogenicity. A meta-analysis of the dermatological signs of the patient shows that they are not common in patients with confirmed mitochondrial DNA mutations and suggests that, in these cases, lipomatosis is not related to the oxidative phosphorylation dysfunction, but to an alteration of an additional function associated to particular mitochondrial tRNAs.

Genotypic and phenotypic characteristics of Korean children with childhood-onset Leber's hereditary optic neuropathy.

PURPOSE: We sought to identify the phenotypic and genotypic characteristics of Korean children with genetically confirmed Leber's hereditary optic neuropathy (LHON). METHODS: The medical records of 64 genetically confirmed LHON patients were reviewed. Seventeen patients aged 13 years or younger with optic atrophy with positive mitochondrial DNA (mtDNA) mutations were considered to demonstrate childhood-onset LHON. The non-childhood-onset group included 47 patients with genetically confirmed LHON who experienced disease onset later than 13 years of age. The type of mtDNA mutation, visual acuity (VA), color vision, fundus photography, retinal nerve fiber layer (RNFL) thickness, and visual field were investigated. RESULTS: Sequence analysis of the mitochondrial genome revealed five different kinds of LHON-associated mtDNA mutations among our childhood-onset patients, including m.11778G>A (58.8%), m.3496G>T (11.8%), m.3497C>T (5.9%), m.11696G>A (5.9%), and m.14502T>C (5.9%). The mean final best-corrected VA in the childhood-onset group was better than that in the non-childhood-onset group with the value of logMAR 0.29 (0.09-0.75) vs. 0.55 (0.27-1.29) (expressed as median (interquartile range); p = 0.05). Spontaneous visual recovery was observed in 35.3% of the childhood-onset group but in only 12.8% of the non-childhood-onset group (p = 0.04). Eight patients (47.1%) showed interocular asymmetry of the disease, with two presenting true unilateral involvement of the optic nerve and the other six patients demonstrating unilateral subclinical manifestations with bilateral optic atrophy. CONCLUSION: Involvement of secondary mitochondrial mutations was confirmed in patients with childhood-onset LHON. Characteristic clinical features of childhood-onset LHON included a higher proportion of subacute or insidious onset of symptoms, better VA, higher spontaneous recovery, and asymmetrical ocular involvement.

Molecular Basis of the Pathogenic Mechanism Induced by the m.9191T>C Mutation in Mitochondrial ATP6 Gene.

Probing the pathogenicity and functional consequences of mitochondrial DNA (mtDNA) mutations from patient's cells and tissues is difficult due to genetic heteroplasmy (co-existence of wild type and mutated mtDNA in cells), occurrence of numerous mtDNA polymorphisms, and absence of methods for genetically transforming human mitochondria. Owing to its good fermenting capacity that enables survival to loss-of-function mtDNA mutations, its amenability to mitochondrial genome manipulation, and lack of heteroplasmy, Saccharomyces cerevisiae is an excellent model for studying and resolving the molecular bases of human diseases linked to mtDNA in a controlled genetic background. Using this model, we previously showed that a pathogenic mutation in mitochondrial ATP6 gene (m.9191T>C), that converts a highly conserved leucine residue into proline in human ATP synthase subunit a (aL222P), severely compromises the assembly of yeast ATP synthase and reduces by 90% the rate of mitochondrial ATP synthesis. Herein, we report the isolation of intragenic suppressors of this mutation. In light of recently described high resolution structures of ATP synthase, the results indicate that the m.9191T>C mutation disrupts a four α-helix bundle in subunit a and that the leucine residue it targets indirectly optimizes proton conduction through the membrane domain of ATP synthase.