Pathology-related mutation A7526G (A9G) helps in the understanding of the 3D structural core of human mitochondrial tRNAAsp

More than 130 mutations in human mitochondrial tRNA (mt-tRNA) genes have been correlated with a variety of neurodegenerative and neuromuscular disorders. Their molecular impacts are of mosaic type, affecting various stages of tRNA biogenesis, structure, and/or functions in mt-translation. Knowledge of mammalian mt-tRNA structures per se remains scarce however. Primary and secondary structures deviate from classical tRNAs, while rules for three-dimensional (3D) folding are almost unknown. Here, we take advantage of a myopathy-related mutation A7526G (A9G) in mt-tRNA^Asp to investigate both the primary molecular impact underlying the pathology and the role of nucleotide 9 in the network of 3D tertiary interactions. Experimental evidence is presented for existence of a 9-12-23 triple in human mt-tRNA^Asp with a strongly conserved interaction scheme in mammalian mt-tRNAs. Mutation A7526G disrupts the triple interaction and in turn reduces aspartylation efficiency.     

Mitochondrial myopathy in a child with a muscle-restricted mutation in the mitochondrial transfer RNAAsn gene

We report an 11-year-old boy with exercise-related myopathy, and a novel mutation m.5669G>A in the mitochondrial tRNA Asparagine gene (mt-tRNA(Asn), MTTN). Muscle biopsy studies showed COX-negative, SDH-positive fibers at histochemistry and biochemical defects of oxidative metabolism. The m.5669G>A mutation was present only in patient's muscle resulting in the first muscle-specific MTTN mutation. Mt-tRNA(Asn) steady-state levels and in silico predictions supported the pathogenicity of this mutation. A mitochondrial myopathy should be considered in the differential diagnosis of exercise intolerance in children.     

Effect of endogenous carotenoids on "adaptive" mutation in Escherichia coli FC40

The appearance over many days of Lac(+) frameshift mutations in Escherichia coli strain FC40 incubated on lactose selection plates is a classic example of apparent "adaptive" mutation in an episomal gene. We show that endogenously overproduced carotenoids reduce adaptive mutation under selective conditions by a factor of around two. Carotenoids are known to scavenge singlet oxygen suggesting that the accumulation of oxidative base damage may be an integral part of the adaptive mutation phenomenon. If so, the lesion cannot be 7,8-dihydro-8-oxoguanine since adaptive mutation in FC40 is unaffected by mutM and mutY mutations. If active oxygen species such as singlet oxygen are involved in adaptive mutation then they should also induce frameshift mutations in FC40 under non-selective conditions. We show that such mutations can be induced under non-selective conditions by protoporphyrin photosensitisation and that this photodynamic induction is reduced by a factor of just over two when endogenous carotenoids are present. We argue that the involvement of oxidative damage would in no way be inconsistent with current understanding of the mechanism of adaptive mutation and the role of DNA polymerases.     

The impact of mitochondrial tRNA mutations on the amount of ATP synthase differs in the brain compared to other tissues

The impact of point mutations in mitochondrial tRNA genes on the amount and stability of respiratory chain complexes and ATP synthase (OXPHOS) has been broadly characterized in cultured skin fibroblasts, skeletal muscle samples, and mitochondrial cybrids. However, less is known about how these mutations affect other tissues, especially the brain. We have compared OXPHOS protein deficiency patterns in skeletal muscle mitochondria of patients with Leigh (8363G>A), MERRF (8344A>G), and MELAS (3243A>G) syndromes. Both mutations that affect mt-tRNA(Lys) (8363G>A, 8344A>G) resulted in severe combined deficiency of complexes I and IV, compared to an isolated severe defect of complex I in the 3243A>G sample (mt-tRNA(LeuUUR). Furthermore, we compared obtained patterns with those found in the heart, frontal cortex, and liver of 8363G>A and 3243A>G patients. In the frontal cortex mitochondria of both patients, the patterns of OXPHOS deficiencies differed substantially from those observed in other tissues, and this difference was particularly striking for ATP synthase. Surprisingly, in the frontal cortex of the 3243A>G patient, whose ATP synthase level was below the detection limit, the assembly of complex IV, as inferred from 2D-PAGE immunoblotting, appeared to be hindered by some factor other than the availability of mtDNA-encoded subunits.     

Overexpression of human mitochondrial valyl tRNA synthetase can partially restore levels of cognate mt-tRNAVal carrying the pathogenic C25U mutation

Phenotypic diversity associated with pathogenic mutations of the human mitochondrial genome (mtDNA) has often been explained by unequal segregation of the mutated and wild-type genomes (heteroplasmy). However, this simple hypothesis cannot explain the tissue specificity of disorders caused by homoplasmic mtDNA mutations. We have previously associated a homoplasmic point mutation (1624C>T) in MTTV with a profound metabolic disorder that resulted in the neonatal deaths of numerous siblings. Affected tissues harboured a marked biochemical defect in components of the mitochondrial respiratory chain, presumably due to the extremely low (<1%) steady-state levels of mt-tRNA^Val. In primary myoblasts and transmitochondrial cybrids established from the proband (index case) and offspring, the marked respiratory deficiency was lost and steady-state levels of the mutated mt-tRNA^Val were greater than in the biopsy material, but were still an order of magnitude lower than in control myoblasts. We present evidence that the generalized decrease in steady-state mt-tRNA^Val observed in the homoplasmic 1624C>T-cell lines is caused by a rapid degradation of the deacylated form of the abnormal mt-tRNA^Val. By both establishing the identity of the human mitochondrial valyl-tRNA synthetase then inducing its overexpression in transmitochondrial cell lines, we have been able to partially restore steady-state levels of the mutated mt-tRNA^Val, consistent with an increased stability of the charged mt-tRNA. These data indicate that variations in the levels of VARS2L between tissue types and patients could underlie the difference in clinical presentation between individuals homoplasmic for the 1624C>T mutation.     

A "de novo" mutation of the LDL-receptor gene as the cause of familial hypercholesterolemia

Familial hypercholesterolemia (FH) is a common genetic disorder caused by mutations of the LDL-receptor gene and transmitted as a co-dominant trait. However, there are some forms of hypercholesterolemia which have a recessive type of transmission. We have identified a subject with the clinical phenotype of heterozygous FH whose parents had normal plasma lipid values, suggesting a recessive type of transmission. The analysis of the LDL-receptor gene revealed that the patient was heterozygous for a G>C transversion in exon 4, which results in a serine for cysteine substitution at position 88 (C88S) of the receptor protein. Since this novel mutation was not found in the proband's parents and non-paternity was excluded, we concluded that the patient was a carrier of a "de novo" mutation. Haplotype analysis of LDL-receptor locus indicated that this "de novo" mutation occurred in the paternal germ line. The C88S mutation is the likely cause of LDL-receptor defect as it was present in the proband's hypercholesterolemic son and was not found in 200 chromosomes of control subjects.     

A proposed consensus panel of organisms for determining evolutionary conservation of mt-tRNA point mutations

Assigning pathogenicity to mt-tRNA variants requires multiple strands of evidence. Evolutionary conservation is often considered mandatory, but lack of a standard panel of organisms to assess conservation complicates comparison between reports and undermines the value of conservation-based evidence. We demonstrate that intra-species MTT sequence variation is sufficiently low for sequence data from a single organism to adequately represent a species. On this basis, we propose a standardised panel of organisms for conservation assessment and describe integration of this conservation panel into a pathogenicity scoring system designed to assess mt-tRNA variation associated with mitochondrial disease.     

Leber's hereditary optic neuropathy: no significant evidence for primary or secondary pathogenicity of the 15257 mutation

Leber's hereditary optic neuropathy (LHON) is a maternally inherited disease of the optic nerves associated with various mitochondrial DNA (mtDNA) mutations. Four of these mutations, at nucleotide positions (np) 3460, 11778, 14484 and 15257, have been postulated to be of primary pathogenetical importance. Previously, we described the molecular and clinical findings in patients with the 11778 and 14484 mutations. Here we describe the molecular and clinical findings of patients in eight pedigrees with the 3460 mutation and in three pedigrees with the 15 257 mutation. In all three 15257 positive pedigrees the 3460, the 11778 or the 14484 mutation was also found. The first combination has not been reported before. We compared the clinical findings in these pedigrees with those of the 3460, 11778 and 14484 positive pedigrees that lack the 15257 mutation. No significant differences were found with respect to the age of onset, visual outcome or the probability of developing LHON. We conclude that there is no evidence that the 15257 mutation, which has been reported in normal controls, has primary causal significance, because it may coincide with the 3460, 11778 and 14484 mutations. We presume that the 15257 mutation has no secondary pathogenic importance, since it has no clear contribution to the degree or the probability of phenotypic expression.     

"Wide-open" 1.3 A structure of a multidrug-resistant HIV-1 protease as a drug target

This report examines structural changes in a highly mutated, clinical multidrug-resistant HIV-1 protease, and the crystal structure has been solved to 1.3 A resolution in the absence of any inhibitor. This protease variant contains codon mutations at positions 10, 36, 46, 54, 62, 63, 71, 82, 84, and 90 that confer resistance to protease inhibitors. Major differences between the wild-type and the variant include a structural change initiated by the M36V mutation and amplified by additional mutations in the flaps of the protease, resulting in a "wide-open" structure that represents an opening that is 8 A wider than the "open" structure of the wild-type protease. A second structural change is triggered by the L90M mutation that results in reshaping the 23-32 segment. A third key structural change of the protease is due to the mutations from longer to shorter amino acid side chains at positions 82 and 84.     

X-linked Charcot-Marie-Tooth disease and connexin32

We studied 29 families with X-linked dominant CMT (CMTX1) neuropathy. Twenty-five families showed mutations in the coding region of the connexin32 (Cx32) gene. The mutations included five nonsense mutations, 17 missense mutations, two medium size deletions and one insertion. Most missense mutations showed a mild clinical phenotype and slowing of motor nerve conduction velocities. All five nonsense mutations, the larger deletion and the insertion showed severe clinical phenotype. Four CMTX1 families with mild clinical phenotype showed no point mutations of the Cx32 gene coding region. Two mutations of the non-coding region were identified. The first mutation was located in the nerve specific Cx32 promoter, the second mutation was located in the 5' untranslated region of the mRNA.     

Suppression of a nuclear frameshift mutation by a mitochondrial tRNA in the yeast Kluyveromyces lactis

A fragment of mitochondrial DNA containing the Kluyveromyces lactis gene for valine-tRNA (tRNAVAL) was isolated as a multicopy suppressor of a respiratory-deficient mutant of this yeast. The mutant produced a truncated Cox14p because of a +1 frameshift mutation in COX14, a nuclear gene encoding a protein imported into mitochondria which is necessary for respiration (Fiori et al. 2000 Yeast 16: 307-314). We report here that the mitochondrial tRNAVAL gene, when transformed into K. lactis cells, is transcribed outside mitochondria and suppresses the frameshift mutation in COX14 restoring the correct reading frame during translation of its mRNA. In fact, using histidine tagging, the existence of a suppressed Cox14p of normal length was demonstrated in mutants expressing the mt-tRNAVAL from the nucleus. Suppression could occur through a non-canonical four base pairing between the tRNAVAL and the mutated mRNA or through slippage of ribosomes during translation. This is a new case of informational suppression in that the suppression of a chromosomal mutation is not caused by a second mutation but to a mislocalization/expression of a mt-tRNA.     

Hypertrophic cardiomyopathy: two homozygous cases with "typical" hypertrophic cardiomyopathy and three new mutations in cases with progression to dilated cardiomyopathy

About 10% of cases of hypertrophic cardiomyopathy (HCM) evolve into dilated cardiomyopathy (DCM) with unknown causes. We studied 11 unrelated patients (pts) with HCM who progressed to DCM (group A) and 11 who showed "typical" HCM (group B). Mutational analysis of the beta-myosin heavy chain (MYH7), myosin-binding protein C (MYBPC3), and cardiac troponin T (TNNT2) genes demonstrated eight mutations affecting MYH7 or MYBPC3 gene, five of which were new mutations. In group A-pts, the first new mutation occurred in the myosin head-rod junction and the second occurred in the light chain-binding site. The third new mutation leads to a MYBPC3 lacking titin and myosin binding sites. In group B, two pts with severe HCM carried two homozygous MYBPC3 mutations and one with moderate hypertrophy was a compound heterozygous for MYBPC3 gene. We identified five unreported mutations, potentially "malignant" defects as for the associated phenotypes, but no specific mutations of HCM/DCM.     

Germline and somatic mutation profile in Cancer patients revealed by a medium-sized pan-Cancer panel

Gene mutation detection and the resulted precision-medicine therapy is transforming clinical practice. Here, we report the use of a custom-developed, medium-sized, pan-cancer probe panel for the detection of somatic and germline mutations. We used a hybridization capture-based NGS assay for targeted deep sequencing of all exons and selected introns of 181 key cancer driver genes, covering both inherited risks and somatic mutations. We performed paired-variant calling on tumor samples and their matched normal samples. We processed clinical patient samples of formalin-fixed, paraffin embedded tumors (FFPE samples) and cell-free peripheral blood (cfDNA samples). We found germline mutations of inherited cancer risk at 9%; and discovered a novel germline mutation in BRCA1. Somatic mutation rate in driver genes is at 73.1%, much higher than previously reported. On recommending precision-medicine therapeutics, we achieved 91.6% for patients with FFPE samples.     

Mitochondrial tRNA 5′-Editing in Dictyostelium discoideum and Polysphondylium pallidum

Mitochondrial tRNA (mt-tRNA) 5′-editing was first described more than 20 years ago; however, the first candidates for 5′-editing enzymes were only recently identified in a eukaryotic microbe (protist), the slime mold Dictyostelium discoideum. In this organism, eight of 18 mt-tRNAs are predicted to be edited based on the presence of genomically encoded mismatched nucleotides in their aminoacyl-acceptor stem sequences. Here, we demonstrate that mt-tRNA 5′-editing occurs at all predicted sites in D. discoideum as evidenced by changes in the sequences of isolated mt-tRNAs compared with the expected sequences encoded by the mitochondrial genome. We also identify two previously unpredicted editing events in which G-U base pairs are edited in the absence of any other genomically encoded mismatches. A comparison of 5′-editing in D. discoideum with 5′-editing in another slime mold, Polysphondylium pallidum, suggests organism-specific idiosyncrasies in the treatment of U-G/G-U pairs. In vitro activities of putative D. discoideum editing enzymes are consistent with the observed editing reactions and suggest an overall lack of tRNA substrate specificity exhibited by the repair component of the editing enzyme. Although the presence of terminal mismatches in mt-tRNA sequences is highly predictive of the occurrence of mt-tRNA 5′-editing, the variability in treatment of U-G/G-U base pairs observed here indicates that direct experimental evidence of 5′-editing must be obtained to understand the complete spectrum of mt-tRNA editing events in any species.     

A novel G3337A mitochondrial ND1 mutation related to cardiomyopathy co-segregates with tRNALeu(CUN) A12308G and tRNAThr C15946T mutations

We describe a novel mutation in human mitochondrial NADH dehydrogenase 1 gene (ND1), a G to A transition at nucleotide position 3337, which is co-segregated with two known mutations in tRNALeu(CUN) A12308G and tRNAThr C15946T. These mutations were detected in two unrelated patients with different clinical phenotypes, exhibiting cardiomyopathy as the common symptom. The ND1 G3337A mutation that was detected was found almost homoplasmic in the two patients and it was absent in 150 individuals that were tested as control group. Mitochondrial respiratory chain complex I activity of the patients platelets was also tested and found decreased compared to those of controls. We suggest that the co-existence of mutations in tRNA and ND1 genes may act synergistically affecting the clinical phenotype. Our study highlights the enormous phenotypic diversity that exists among pathogenic mtDNA mutations and re-emphasizes the need for a more careful clinical approach.     

RNA mutations of prox1 detected in human esophageal cancer cells by the shifted termination assay

We have recently reported a novel finding that a candidate tumor suppressor gene prox1 suffered adenosine-to-inosine (A-to-I) RNA mutation without genomic mutation in a subset of human cancer cells and lost its function. Hence, screening of mutations in both cDNA and genomic DNA could be important in the analysis of causes for cancers. Here, we applied a sensitive, accurate, and simple method, called shifted termination assay (STA) for detection of an A-to-I RNA mutation (R334G) in prox1. We prepared PCR-amplified samples containing the target base of RNA mutation from cDNAs and genomic DNAs of various cell lines and clinical samples, to demonstrate that the STA method can be used to identify not only genomic mutations but also RNA mutations more effectively compared to sequencing. By means of STA, we found prox1 R334G RNA mutations but not genomic DNA mutations in 4 of 8 cases of esophageal cancers. This method can help us to detect RNA mutation effectively and progress research of a potential oncogenic principle.     

Ice-COLD-PCR enables rapid amplification and robust enrichment for low-abundance unknown DNA mutations

Identifying low-abundance mutations within wild-type DNA is important in several fields of medicine, including cancer, prenatal diagnosis and infectious diseases. However, utilizing the clinical and diagnostic potential of rare mutations is limited by sensitivity of the molecular techniques employed, especially when the type and position of mutations are unknown. We have developed a novel platform that incorporates a synthetic reference sequence within a polymerase chain reaction (PCR) reaction, designed to enhance amplification of unknown mutant sequences during COLD-PCR (CO-amplification at Lower Denaturation temperature). This new platform enables an Improved and Complete Enrichment (ice-COLD-PCR) for all mutation types and eliminates shortcomings of previous formats of COLD-PCR. We evaluated ice-COLD-PCR enrichment in regions of TP53 in serially diluted mutant and wild-type DNA mixtures. Conventional-PCR, COLD-PCR and ice-COLD-PCR amplicons were run in parallel and sequenced to determine final mutation abundance for a range of mutations representing all possible single base changes. Amplification by ice-COLD-PCR enriched all mutation types and allowed identification of mutation abundances down to 1%, and 0.1% by Sanger sequencing or pyrosequencing, respectively, surpassing the capabilities of other forms of PCR. Ice-COLD-PCR will help elucidate the clinical significance of low-abundance mutations and our understanding of cancer origin, evolution, recurrence-risk and treatment diagnostics.     

Multiple gene mutations, not the type of mutation, are the modifier of left ventricle hypertrophy in patients with hypertrophic cardiomyopathy

Genotype-phenotype correlation of hypertrophic cardiomyopathy (HCM) has been challenging because of the genetic and clinical heterogeneity. To determine the mutation profile of Chinese patients with HCM and to correlate genotypes with phenotypes, we performed a systematic mutation screening of the eight most commonly mutated genes encoding sarcomere proteins in 200 unrelated Chinese adult patients using direct DNA sequencing. A total of 98 mutations were identified in 102 mutation carriers. The frequency of mutations in MYH7, MYBPC3, TNNT2 and TNNI3 was 26.0, 18.0, 4.0 and 3.5 % respectively. Among the 200 genotyped HCM patients, 83 harbored a single mutation, and 19 (9.5 %) harbored multiple mutations. The number of mutations was positively correlated with the maximum wall thickness. We found that neither particular gene nor specific mutation was correlated to clinical phenotype. In summary, the frequency of multiple mutations was greater in Chinese HCM patients than in the Caucasian population. Multiple mutations in sarcomere protein may be a risk factor for left ventricular wall thickness.     

<Emphasis Type="Italic">MEFV</Emphasis> mutations in patients with familial Mediterranean fever from the Aegean region of Turkey

Familial Mediterranean Fever (FMF) which is frequently present in Mediterranean populations is caused by mutations in the MEFV gene. According to recent data, MEFV mutations are not the only cause of FMF, but these are major genetic determinants which cause FMF. It has also been suggested that there may be a number of other genes causing FMF. The MEFV gene is located at 16p13.3 and encodes a protein, pyrin/marenostrin. More than 70 disease associated mutations and totally 186 mutations and polymorphisms have been defined in affected individuals. We have retrospectively evaluated the molecular test results of 1,201 patients identified as having FMF clinical symptoms referred to the Molecular Genetics Laboratory of the Department of Medical Genetics, Faculty of Medicine, Ege University, Izmir/Turkey over the last 4 years. Patients were tested for 12 common mutations in the MEFV gene using a strip assay method (Innogenetics, Belgium). Out of the 1,201 patients tested (2,402 chromosomes) in the Aegean region in Turkey, 654 (54.45%) did not carry any mutations, among the 547 (45.55%) patients with mutations 246 patients were either homozygous (101) or compound heterozygous (145), 296 carried only one detected mutation, and five patients had three mutations. Allelic frequencies for the four most common mutations in the mutation positive groups were 47.60% (M694V), 16.75% (E148Q), 12.95% (V726A), 11.94% (M680I G/C).The remaining alleles (10.76%) showed rare mutations which were R761H, P369S, A744S, K695R, F479L, M694I. When the frequencies of mutations detected in our group were compared to the frequencies reported in the other regions of Turkey, an increase in V726A mutation frequency was observed. No patient showed a I692del mutation which is sometimes evident in other Mediterranean populations.