Somatic mtDNA mutations cause aging phenotypes without affecting reactive oxygen species production

The mitochondrial theory of aging proposes that reactive oxygen species (ROS) generated inside the cell will lead, with time, to increasing amounts of oxidative damage to various cell components. The main site for ROS production is the respiratory chain inside the mitochondria and accumulation of mtDNA mutations, and impaired respiratory chain function have been associated with degenerative diseases and aging. The theory predicts that impaired respiratory chain function will augment ROS production and thereby increase the rate of mtDNA mutation accumulation, which, in turn, will further compromise respiratory chain function. Previously, we reported that mice expressing an error-prone version of the catalytic subunit of mtDNA polymerase accumulate a substantial burden of somatic mtDNA mutations, associated with premature aging phenotypes and reduced lifespan. Here we show that these mtDNA mutator mice accumulate mtDNA mutations in an approximately linear manner. The amount of ROS produced was normal, and no increased sensitivity to oxidative stress-induced cell death was observed in mouse embryonic fibroblasts from mtDNA mutator mice, despite the presence of a severe respiratory chain dysfunction. Expression levels of antioxidant defense enzymes, protein carbonylation levels, and aconitase enzyme activity measurements indicated no or only minor oxidative stress in tissues from mtDNA mutator mice. The premature aging phenotypes in mtDNA mutator mice are thus not generated by a vicious cycle of massively increased oxidative stress accompanied by exponential accumulation of mtDNA mutations. We propose instead that respiratory chain dysfunction per se is the primary inducer of premature aging in mtDNA mutator mice.     

Somatic mutations and immune checkpoint biomarkers

The development of molecular testing for identifying somatic mutations and immune checkpoint biomarkers has directed treatment towards personalized medicine for patients with non‐small cell lung cancer. The choice of molecular testing in a clinical setting is influenced by cost, expertise in the technology, instrumentation setup and sample type availability. The molecular techniques described in this review include immunohistochemistry (IHC), fluorescent in situ hybridization, direct sequencing, real‐time polymerase chain reaction (PCR), denaturing high‐performance liquid chromatography, matrix‐assisted laser desorption/ionization time of flight mass spectrometry and next‐generation sequencing (NGS). IHC is routinely used in clinical practice for the classification, differentiation, histology and identification of targetable alterations of epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK) and programmed death ligand‐1 (PD‐L1). Recently, the PD‐L1 pathway was identified as being exploited by tumour cells, allowing immune resistance and tumour evasion. The development of immune checkpoint inhibitors as treatment for tumours expressing checkpoints has highlighted the need for standardized IHC assays to inform treatment decisions for patients. Direct sequencing was historically the gold standard for mutation testing for EGFR, KRAS (Kirsten rat sarcoma viral oncogene homologue) and BRAF (v‐Raf murine sarcoma viral oncogene homologue B1) requiring a high ratio of tumour to normal cells, but this has been superseded by more sensitive methods. NGS is a new emerging technique, which allows high‐throughput coverage of frequently mutated genes, including less common BRAF and MET mutations and alterations in tumour suppressor genes. When an NGS platform is unavailable, PCR‐based technologies offer an efficient and cost‐effective single gene test to guide patient treatment. This article will review these techniques and discuss the future of molecular platforms underpinning clinical management decisions.     

mtDNA mutations increase tumorigenicity in prostate cancer

Mutations in the mtDNA have been found to fulfill all of the criteria expected for pathogenic mutations causing prostate cancer. Focusing on the cytochrome oxidase subunit I (COI) gene, we found that 11-12% of all prostate cancer patients harbored COI mutations that altered conserved amino acids (mean conservation index=83%), whereas <2% of no-cancer controls and 7.8% of the general population had COI mutations, the latter altering less conserved amino acids (conservation index=71%). Four conserved prostate cancer COI mutations were found in multiple independent patients on different mtDNA backgrounds. Three other tumors contained heteroplasmic COI mutations, one of which created a stop codon. This latter tumor also contained a germ-line ATP6 mutation. Thus, both germ-line and somatic mtDNA mutations contribute to prostate cancer. Many tumors have been found to produce increased reactive oxygen species (ROS), and mtDNA mutations that inhibit oxidative phosphorylation can increase ROS production and thus contribute to tumorigenicity. To determine whether mutant tumors had increased ROS and tumor growth rates, we introduced the pathogenic mtDNA ATP6 T8993G mutation into the PC3 prostate cancer cell line through cybrid transfer and tested for tumor growth in nude mice. The resulting mutant (T8993G) cybrids were found to generate tumors that were 7 times larger than the wild-type (T8993T) cybrids, whereas the wild-type cybrids barely grew in the mice. The mutant tumors also generated significantly more ROS. Therefore, mtDNA mutations do play an important role in the etiology of prostate cancer.     

Somatic mutations and clonal expansions in paroxysmal nocturnal hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematopoietic stem cell disorder caused by a mutation of the X-linked PIGA gene, resulting in a deficient expression of glycosylphosphatidylinositol (GPI)-anchored proteins. While large clonal expansions of GPI(?) cells cause hemolytic symptoms, tiny GPI(?) cell populations can be found in healthy individuals and remain miniscule throughout life. The slight expansion of PNH clones often occurs in patients with acquired aplastic anemia (AA), an autoimmune bone marrow (BM) failure caused by autoreactive cytotoxic T lymphocyte attack on hematopoietic stem and progenitor cells (HSPCs). The presence of PNH clones is thought to represent the immune pathophysiology of BM failure and be derived from GPI(?) HSPCs that evaded immune attack against HSPCs. However, which mechanisms underlie the selection of GPI(?) HSPCs as well as their overwhelming clonal expansion remains unclear. Ancestral or secondary somatic mutations in GPI(?) HSPCs contribute to the clonal expansion of the aberrant HSPCs in certain patients with PNH; however, it remains unclear whether such driver mutations are responsible for clonal expansion of all patients. Increased sensitivity to TGF-β in GPI(?) HSPCs partly explains the predominance of GPI(?) erythrocytes in immune-mediated BM failure. CD4⁺ T cells specific to antigens presented by HLA-DR15 on HSPCs also contribute to the immune escape of GPI(–) HSPCs. Studying the evolution of HSPCs in AA and PNH will yield further information for understanding human autoimmunity and stem cell biology.     

Somatic second-hit mutations leads to polycystic liver diseases

Polycystic liver diseases(PCLDs) are a heterogeneous group of genetic disorders characterized by the development of multiple fluid-filled cysts in the liver,which derive from cholangiocytes,the epithelial cells lining the bile ducts.When these cysts grow,symptoms such as abdominal distension,nausea,and abdominal pain may occur.PCLDs may exist isolated(i.e.,autosomal dominant polycystic liver disease,ADPLD) or in combination with renal cystogenesis(i.e.,autosomal dominant polycystic kidney disease and autosomal recessive polycystic liver disease).The exact prevalence of PCLDs is unknown,but is estimated to occur in approximately 1:1000 persons.Although the pathogenesis of each form of PCLD appears to be different,increasing evidences indicate that hepatic cystogenesis is a phenomenon that may involve somatic loss of heterozygosity(LOH) in those pathological conditions inherited in a dominant form.A recent report,using highly sophisticated methodology,demonstrated that ADPLD patients with a germline mutation in the protein kinase C substrate 80K-H(PRKCSH) gene mostly develop hepatic cystogenesis through a second somatic mutation.While hepatocystin,the PRKCSH-encoding protein,was absent in the hepatic cysts with LOH,it was still expressed in the heterozygous cysts.On the other hand,no additional trans-heterozygous mutations on the SEC63 homolog(S.cerevisiae /SEC63) gene(also involved in the development of PCLDs) were observed.These data indicate that PCLD is recessive at the cellular level,and point out the important role of hepatocystin loss in cystogenesis.In this commentary,we discuss the knowledge regarding the role of somatic second-hit mutations in the development of PCLDs,and the most relevant findings have been highlighted.     

Somatic mutations in acquired pure red cell aplasia

Acquired pure red cell aplasia (PRCA) is a syndrome characterized by anemia and a marked reduction of erythroid progenitor cells with various etiologies. The 3 major subtypes of PRCA are idiopathic PRCA, large granular lymphocytic leukemia-associated PRCA and thymoma-associated PRCA, which are thought to be caused by a T-cell-mediated mechanism. In these 3 subtypes, an expansion of clonal cytotoxic T cells is often detected. In addition, those T cells recurrently harbor somatic mutations of STAT3, a gene coding one of the important signal transducers in the JAK/STAT system. Somatic mutations of clonal hematopoiesis (CH)-related genes, including epigenetic modifying genes, have also been reported, however, the data are still not mature enough upon which to draw conclusion, Somatic mutations of STAT3 and CH-related genes may be unique characteristics of acquired PRCA. However, their involvement in dyserythropoiesis or clinical relevance to the clinical course of those somatic mutations. Mutational landscapes, their involvements in dyserythropoiesis and clinical relevance in acquired PRCA remains unclear, and further investigation is needed.     

Somatic PTPN11 mutations in childhood acute myeloid leukaemia

Somatic mutations in PTPN11, the gene encoding the transducer SHP-2, have emerged as a novel class of lesions that upregulate RAS signalling and contribute to leukaemogenesis. In a recent study of 69 children and adolescents with de novo acute myeloid leukaemia (AML), we documented a non-random distribution of PTPN11 mutations among French-American-British (FAB) subtypes. Lesions were restricted to FAB-M5 cases, where they were relatively common (four of 12 cases). Here, we report on the results of a molecular screening performed on 181 additional unselected patients, enrolled in participating institutions of the Associazione Italiana Ematologia Oncologia Pediatrica-AML Study Group, to provide a more accurate picture of the prevalence, spectrum and distribution of PTPN11 mutations in childhood AML and to investigate their clinical relevance. We concluded that PTPN11 defects do not represent a frequent event in this heterogeneous group of malignancies (4.4%), although they recur in a considerable percentage of patients with FAB-M5 (18%). PTPN11 lesions rarely occur in other subtypes. Within the FAB-M5 group no clear association of PTPN11 mutations with any clinical variable was evident. Nearly two third of the patients with this subtype were found to harbour an activating mutation in PTPN11, NRAS, KRAS2 or FLT3.     

Somatic mutations of mitochondrial DNA in digestive tract cancers

BACKGROUND: Somatic mutations of mitochondrial DNA (mtDNA) have been reported to play an important role in the carcinogenesis of several human cancers. However, there are few reports on mtDNA mutations in digestive tract cancers, including esophageal, gastric and colorectal cancers. The present study examined somatic mtDNA mutations in these cancers. METHODS: Samples of 82 esophageal cancers, 96 gastric cancers and 138 colorectal cancers were collected. Mutations in the D310 mononucleotide repeat of mtDNA were examined by microsatellite assay. RESULTS: Frequencies of mtDNA mutations were similar in each digestive tract cancer: 14% (7/51) in esophageal cancers, 15% (14/94) in gastric cancers and 8% (11/133) in colorectal cancers. There were no significant relationships between mtDNA mutations and clinicopathological features, such as patient age or sex, tumor location, depth of tumor invasion and lymph node metastasis in each digestive tract cancer. CONCLUSIONS: The results suggest that mtDNA mutations play a role in the development but not progression in each digestive tract cancer, and that the role of mtDNA mutations might be similar among the digestive tract cancers.     

Extensive tissue-related and allele-related mtDNA heteroplasmy suggests positive selection for somatic mutations

Heteroplasmy in human mtDNA may play a role in cancer, other diseases, and aging, but patterns of heteroplasmy variation across different tissues have not been thoroughly investigated. Here, we analyzed complete mtDNA genome sequences at ∼3,500× average coverage from each of 12 tissues obtained at autopsy from each of 152 individuals. We identified 4,577 heteroplasmies (with an alternative allele frequency of at least 0.5%) at 393 positions across the mtDNA genome. Surprisingly, different nucleotide positions (nps) exhibit high frequencies of heteroplasmy in different tissues, and, moreover, heteroplasmy is strongly dependent on the specific consensus allele at an np. All of these tissue-related and allele-related heteroplasmies show a significant age-related accumulation, suggesting positive selection for specific alleles at specific positions in specific tissues. We also find a highly significant excess of liver-specific heteroplasmies involving nonsynonymous changes, most of which are predicted to have an impact on protein function. This apparent positive selection for reduced mitochondrial function in the liver may reflect selection to decrease damaging byproducts of liver mitochondrial metabolism (i.e., “survival of the slowest”). Overall, our results provide compelling evidence for positive selection acting on some somatic mtDNA mutations.Although mtDNA heteroplasmy (intraindividual variability in mtDNA sequences) was initially thought to be rare in humans, studies using next-generation sequencing platforms have documented extensive heteroplasmy at low levels (<2%) (1–4). Heteroplasmy is thought to represent an intermediate stage in the fixation of mtDNA mutations within an individual, and heteroplasmic mtDNA mutations have been implicated in various diseases, cancer, and aging (5–8). Heteroplasmies occur preferentially at positions with a high mutation rate (2, 9), suggesting that mutation and drift are the primary forces influencing heteroplasmy. In addition, elevated levels of nonsynonymous (NS) heteroplasmies within individuals relative to NS polymorphisms among individuals (2) suggest that there is negative selection against heteroplasmies that involve deleterious amino acid changes. In other words, deleterious amino acid changes can be observed as heteroplasmies as long as the allele frequency stays below a certain threshold and “normal” mitochondrial function is maintained; exceeding this threshold results in impaired mitochondrial function, as is commonly observed for disease-associated mtDNA mutations (10).However, fundamental aspects about the nature of heteroplasmy remain unknown. For example, although certain nucleotide positions (nps) in the mtDNA genome are prone to heteroplasmy (2, 4, 11), it is not clear to what extent these positions simply have a high mutation rate, and thus are more prone to heteroplasmy across all tissues, vs. a role for tissue-specific processes in heteroplasmy (i.e., certain tissues may be more prone to heteroplasmy due to their metabolic requirements, rate of cellular turnover, etc.). Tissue-specific patterns of heteroplasmy are commonly observed with mtDNA mutations associated with disease and are thought to reflect the differing bioenergetic requirements of different tissues (10). Moreover, mice constructed to be heteroplasmic for different haplotypes show tissue-specific patterns of segregation over time (12, 13), suggesting positive selection related to mtDNA function in different tissues. However, these results may be specific to disease-associated mutations and to the artificial mouse constructs. A recent study of heteroplasmy in 10 tissues from two individuals found some tissue-specific patterns that suggested positive selection (11), but, currently, there is little evidence to support a significant role for positive selection as a force promoting heteroplasmy (5, 14).Previous studies of variation in heteroplasmy across different tissues have been limited in terms of number of individuals and/or tissues studied (1, 2, 11, 15–19). Here, we present the results of the most comprehensive study to date (to our knowledge) of patterns of heteroplasmy in different tissues in humans. Our results suggest a surprisingly significant role for positive selection as a force influencing some somatic heteroplasmic mutations.     

Somatic mutations, acetylator status, and prognosis in colorectal cancer

Background—Somatic mutations inK-ras and TP53 may be associated with bothacetylator status and prognosis in colorectal cancer.
Aims—To determine whether cancers withsomatic mutations are more frequent in fast acetylators and whethermutations or acetylator status influence prognosis after colorectal surgery.
Patients—One hundred consecutive subjectsundergoing elective surgery for colorectal cancer.
Methods—Acetylator status was determined bypolymerase chain reaction (PCR) genotyping for polymorphism in theN-acetyltransferase 2 (NAT2) gene. Mutations in K-ras(codon 12) and TP53 were determined by PCR analysisusing restriction enzyme digestion and single strand conformationpolymorphism respectively. Survival from colorectal cancer for up tofive years after diagnosis was analysed using the Kaplan-Meier productlimit estimator. Cox proportional hazards regression was used tocompare survival rates after adjusting for tumour stage.
Results—Mutations in K-ras andTP53 were independent of acetylator status. By log ranktest, survival was significantly reduced in subjects with TP53mutations (p=0.003) but was not significantly related toacetylator status or the presence of K-ras mutations. Afteradjustment for tumour stage, subjects with both TP53 and K-ras mutations had a 4.2-fold case fatality (95%confidence interval 1.5 to 11.6) when compared with that of aTP53 negative reference group.
Conclusion—The presence of both TP53and K-ras mutations in colorectal tumours is an adverseprognostic marker which is independent of tumour stage.

Keywords:colorectal cancer; TP53 andK-ras mutations; acetylator status; prognosis

     

Somatic mosaicism for oncogenic NRAS mutations in juvenile myelomonocytic leukemia

Juvenile myelomonocytic leukemia (JMML) is a rare pediatric myeloid neoplasm characterized by excessive proliferation of myelomonocytic cells. Somatic mutations in genes involved in GM-CSF signal transduction, such as NRAS, KRAS, PTPN11, NF1, and CBL, have been identified in more than 70% of children with JMML. In the present study, we report 2 patients with somatic mosaicism for oncogenic NRAS mutations (G12D and G12S) associated with the development of JMML. The mutated allele frequencies quantified by pyrosequencing were various and ranged from 3%-50% in BM and other somatic cells (ie, buccal smear cells, hair bulbs, or nails). Both patients experienced spontaneous improvement of clinical symptoms and leukocytosis due to JMML without hematopoietic stem cell transplantation. These patients are the first reported to have somatic mosaicism for oncogenic NRAS mutations. The clinical course of these patients suggests that NRAS mosaicism may be associated with a mild disease phenotype in JMML.