POLG mutations in sporadic mitochondrial disorders with multiple mtDNA deletions

The accumulation of multiple mitochondrial DNA (mtDNA) deletions in stable tissues is a distinctive feature of several autosomal disorders, characterized by Progressive External Ophthalmoplegia (PEO), ptosis, and proximal myopathy. At least three nuclear genes are responsible for these disorders: ANT1 and C10orf2 cause autosomal dominant PEO, while mutations of DNA polymerase gammaA (POLG1 or POLG) gene on chromosome 15q25 causes both autosomal dominant and recessive forms of PEO. To investigate the contribution of these genes to the sporadic cases of PEO with multiple mtDNA deletions, we studied 31 mitochondrial myopathy patients without any family history for the disorder: 23 had PEO with myopathy, with or without the additional features of pigmentary retinopathy, ataxia, neurosensorial hypoacusia and diabetes mellitus, 7 presented isolated myopathy and one a peripheral neuropathy with ptosis. In all patients Southern blot of muscle DNA showed multiple mtDNA deletions; screening for ANT1 and C10ORF2 genes was negative. POLG analysis revealed mutations in eight patients; in six of them the mutations were allelic, while two patients were heterozygous. Five mutations were new, namely one stop codon (c.2407C>T/p.R709X) and four missense mutations (c.1085G>C/p.G268A; c.1967G>A/p.R562Q; c.2702G>C/p.R807P; c.3076C>T/p.H932W). A high degree of conservation was observed for all the new missense mutations. Only patients presenting PEO as part of their clinical phenotype had POLG mutations, in seven of them together with myopathic signs and in one with a sensori-motor peripheral neuropathy.     

Pathogenic role of mtDNA duplications in mitochondrial diseases associated with mtDNA deletions

We estimated the frequency of multiple mtDNA rearrangements by Southern blot in 32 patients affected by mitochondrial disorders associated with single deletions in order to assess genotype-phenotype correlations and elucidate the pathogenic significance of mtDNA duplications. Muscle in situ hybridization studies were performed in patients showing mtDNA duplications at Southern blot. We found multiple rearrangements in 12/32 (37.5%) patients; in particular, mtDNA duplications were detected in 4/4 Kearns-Sayre syndrome (KSS), in 1 Pearson's syndrome, in 1/3 encephalomyopathies with progressive external ophthalmoplegia (PEO), and in 2/23 PEO. In situ studies documented an exclusive accumulation of deleted mtDNAs in cytochrome c oxidase negative fibers of patients with mtDNA duplications. The presence of mtDNA duplications significantly correlated with onset of symptoms before age 15 and occurrence of clinical multisystem involvement. Analysis of biochemical data documented a predominant reduction of complex III in patients without duplications compared to patients with mtDNA duplications. Our data indicate that multiple mtDNA rearrangements are detectable in a considerable proportion of patients with single deletions and that mtDNA duplications do not cause any oxidative impairment. They more likely play a pathogenic role in the determination of clinical expression of mitochondrial diseases associated with single mtDNA deletions, possibly generating deleted mtDNAs in embryonic tissues by homologous recombination.     

HoxD cluster scanning deletions identify multiple defects leading to paralysis in the mouse mutant Ironside

A spontaneous semidominant mutation (Ironside, Irn) was isolated in mice, leading to severe hindlimb paralysis following multiple deletions in cis at the HoxD locus. To understand its cellular and molecular etiology, we embarked on a comparative analysis using systematic HoxD cluster deletions, produced via targeted meiotic recombination (TAMERE). Different lines of mice were classified according to the severity of their paralyses, and subsequent analyses revealed that multiple causative factors were involved, alone or in combination, in the occurrence of this pathology. Among them are the loss of Hoxd10 function, the sum of remaining Hoxd gene activity, and the ectopic gain of function of the neighboring gene Evx2, all contributing to the mispositioning, the absence, or misidentification of specific lumbo-sacral pools of motoneurons, nerve root homeosis, and hindlimb innervation defects. These results highlight the importance of a systematic approach when studying such clustered gene families, and give insights into the function and regulation of Hox and Evx2 genes during early spinal cord development.     

mtDNA lineage analysis of mouse L-cell lines reveals the accumulation of multiple mtDNA mutants and intermolecular recombination

The role of mitochondrial DNA (mtDNA) mutations and mtDNA recombination in cancer cell proliferation and developmental biology remains controversial. While analyzing the mtDNAs of several mouse L cell lines, we discovered that every cell line harbored multiple mtDNA mutants. These included four missense mutations, two frameshift mutations, and one tRNA homopolymer expansion. The LA9 cell lines lacked wild-type mtDNAs but harbored a heteroplasmic mixture of mtDNAs, each with a different combination of these variants. We isolated each of the mtDNAs in a separate cybrid cell line. This permitted determination of the linkage phase of each mtDNA and its physiological characteristics. All of the polypeptide mutations inhibited their oxidative phosphorylation (OXPHOS) complexes. However, they also increased mitochondrial reactive oxygen species (ROS) production, and the level of ROS production was proportional to the cellular proliferation rate. By comparing the mtDNA haplotypes of the different cell lines, we were able to reconstruct the mtDNA mutational history of the L-L929 cell line. This revealed that every heteroplasmic L-cell line harbored a mtDNA that had been generated by intracellular mtDNA homologous recombination. Therefore, deleterious mtDNA mutations that increase ROS production can provide a proliferative advantage to cancer or stem cells, and optimal combinations of mutant loci can be generated through recombination.     

Increased mitochondrial biogenesis in muscle improves aging phenotypes in the mtDNA mutator mouse

Aging is an intricate process that increases susceptibility to sarcopenia and cardiovascular diseases. The accumulation of mitochondrial DNA (mtDNA) mutations is believed to contribute to mitochondrial dysfunction, potentially shortening lifespan. The mtDNA mutator mouse, a mouse model with a proofreading-deficient mtDNA polymerase γ, was shown to develop a premature aging phenotype, including sarcopenia, cardiomyopathy and decreased lifespan. This phenotype was associated with an accumulation of mtDNA mutations and mitochondrial dysfunction. We found that increased expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), a crucial regulator of mitochondrial biogenesis and function, in the muscle of mutator mice increased mitochondrial biogenesis and function and also improved the skeletal muscle and heart phenotypes of the mice. Deep sequencing analysis of their mtDNA showed that the increased mitochondrial biogenesis did not reduce the accumulation of mtDNA mutations but rather caused a small increase. These results indicate that increased muscle PGC-1α expression is able to improve some premature aging phenotypes in the mutator mice without reverting the accumulation of mtDNA mutations.     

Homozygous mutations in C1QBP as cause of progressive external ophthalmoplegia (PEO) and mitochondrial myopathy with multiple mtDNA deletions

Biallelic mutations in the C1QBP gene have been associated with mitochondrial cardiomyopathy and combined respiratory‐chain deficiencies, with variable onset (including intrauterine or neonatal forms), phenotypes, and severity. We studied two unrelated adult patients from consanguineous families, presenting with progressive external ophthalmoplegia (PEO), mitochondrial myopathy, and without any heart involvement. Muscle biopsies from both patients showed typical mitochondrial alterations and the presence of multiple mitochondrial DNA deletions, whereas biochemical defects of the respiratory chain were present only in one subject. Using next‐generation sequencing approaches, we identified homozygous mutations in C1QBP. Immunoblot analyses in patients' muscle samples revealed a strong reduction in the amount of the C1QBP protein and varied impairment of respiratory chain complexes, correlating with disease severity. Despite the original study indicated C1QBP mutations as causative for mitochondrial cardiomyopathy, our data indicate that mutations in C1QBP have to be considered in subjects with PEO phenotype or primary mitochondrial myopathy and without cardiomyopathy.     

Families of mtDNA re-arrangements can be detected in patients with mtDNA deletions: duplications may be a transient intermediate form

In three patients with mitochondrial DNA duplications, thereare two additional re-arranged molecules derived from mitochondrialDNA. Two forms of closed circular deletions of mitochondrialDNA have been characterised in all three patients, one beinga monomer, and the other a dimer. The junction fragments appearto be the same in the deletion and the duplication, suggestingthat both re-arrangements arose from the same initial recombinationevent, followed by homologous recombination. Sequential musclebiopsy and cell culture studies suggest that the duplicationis present only transiently in muscle and cloned fibroblastlines. The duplicated molecule could thus be an intermediatein the formation of the deletion. Evidence is presented forthe presence of duplicated mtDNA in 6/11 patients known to havedeletions of mitochondrial DNA in muscle, suggesting that thiscould be a general mechanism for major re-arrangements of mitochondrialDNA. There may be parallels between the families of re-arrangementsfound in plant mitochondrial DNA, and the three distinct re-arrangedmolecules described here.     

Interstitial deletions of the short arm of chromosome 4 in patients with a similar combination of multiple minor anomalies and mental retardation

Interstitial deletions of chromosome 4 have been described rarely and have had variable presentations. We describe the phenotypic characteristics associated with interstitial deletion of the p14–16 region of chromosome 4 in 7 patients with multiple minor anomalies in common, and with mental retardation. A review of published cases of interstitial deletions of the short arm of chromosome 4 is provided. These deletions present a distinct phenotype which is different from that of Wolf-Hirschhorn syndrome. © 1995 Wiley-Liss, Inc.     

Characterization of large deletions in the DHCR7 gene

Pathogenic variants in the DHCR7 gene cause Smith–Lemli–Opitz syndrome (SLOS), a defect of cholesterol biosynthesis resulting in an autosomal recessive congenital metabolic malformation disorder. In approximately 4% of patients, the second mutation remains unidentified. In this study, 12 SLOS patients diagnosed clinically and/or by elevated 7‐dehydrocholesterol (7‐DHC) have been investigated by customized multiplex ligation‐dependent probe amplification (MLPA) analysis, because only one DHCR7 sequence variant has been detected. Two unrelated patients of this cohort carry different large deletions in the DHCR7 gene. One patient showed a deletion of exons 3–6. The second patient has a deletion of exons 1 and 2 (non‐coding) and lacks the major part of the promoter. These two patients show typical clinical and biochemical phenotypes of SLOS. Second disease‐causing mutations are p.(Arg352Trp) and p.(Thr93Met), respectively. Deletion breakpoints were characterized successfully in both cases. Such large deletions are rare in the DHCR7 gene but will resolve some of the patients in whom a second mutation has not been detected.     

Mice with an aspartylglucosaminuria mutation similar to humans replicate the pathophysiology in patients

Aspartyglucosaminuria (AGU) is a lysosomal storage disease with autosomal recessive inheritance that is caused by deficient activity of aspartylglucosaminidase (AGA), a lysosomal enzyme belonging to the newly described enzyme family of N-terminal hydrolases. An AGU mouse model was generated by targeted disruption of the AGA gene designed to mimic closely one human disease mutation. These homozygous mutant mice have no detectable AGA activity and excrete aspartylglucosamine in their urine. Analogously to the human disease, the affected homozygous animals showed storage in lysosomes in all analyzed tissues, including the brain, liver, kidney and skin, and lysosomal storage was already detected in fetuses at 19 days gestation. Electron microscopic studies of brain tissue samples demonstrated lysosomal storage vacuoles in the neurons and glia of the neocortical and cortical regions. Magnetic resonance images (MRI) facilitating monitoring of the brains of living animals indicated cerebral atrophy and hypointensity of the deep gray matter structures of brain-findings similar to those observed in human patients. AGU mice are fertile, and up to 11 months of age their movement and behavior do not differ from their age-matched littermates. However, in the Morris water maze test, a slow worsening of performance could be seen with age. The phenotype mimics well AGU in humans, the patients characteristically showing only slowly progressive mental retardation and relatively mild skeletal abnormalities.      

The occurrence of mtDNA heteroplasmy in multiple cetacean species

In population genetics and phylogenetic studies, mitochondrial DNA (mtDNA) is commonly used for examining differences both between and within groups of individuals. For these studies, correct interpretation of every nucleotide position is crucial but can be complicated by the presence of ambiguous bases resulting from heteroplasmy. Particularly for non-model taxa, the presence of heteroplasmy in mtDNA is rarely reported, therefore, it is unclear how commonly it occurs and how it can affect phylogenetic relationships among taxa and the overall understanding of evolutionary processes. We examined the occurrence of both site and length heteroplasmy within the mtDNA of ten marine mammal species, for most of which mtDNA heteroplasmy has never been reported. After sequencing a portion of the mtDNA control region for 5,062 individuals, we found heteroplasmy in at least 2% of individuals from seven species, including Stenella frontalis where 58.9% were heteroplasmic. We verified the presence of true heteroplasmy, ruling out artifacts from amplification and sequencing methods and the presence of nuclear copies of mitochondrial genes. We found no evidence that mtDNA heteroplasmy influenced phylogenetic relationships, however, its occurrence does have the potential to increase the genetic diversity for all species in which it is found. This study stresses the importance of both detecting and reporting the occurrence of heteroplasmy in wild populations in order to enhance the knowledge of both the introduction and the persistence of mutant mitochondrial haplotypes in the evolutionary process.     

Antigenic marker of human erythrokaryocytes similar to mouse erythroblast antigen

Identical antigenic determinants were detected by immunofluorescence and the cytotoxic test on the surface of human erythrokaryocytes with the aid of antibodies against a specific antigen of mouse erythroblasts (EBAG) discovered previously during a study of Rauscher leukemia. The antigen is present on the membrane of most nucleated erythroid cells of the liver of human embryos in the early stages of development and in adult human bone marrow, but is not found in fetal thymocytes, neonatal kidney cells, adult human liver, or in peripheral blood erythrocytes. EBAG evidently possesses an interspecific determinant which is common for mammalian nucleated erythroid cells and which may serve as their specific marker.Laboratory of Immunochemistry and Diagnosis of Tumors, Oncologic Scientific Center, Academy of Medical Sciences of the USSR, Moscow. Department of Control of Virus Preparations, L. A. Tarasevich Institute of Standardization and Control of Medical Biological Preparations, Moscow. Department of Hematology, Central Postgraduate Medical Institute, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR L. M. Shabad.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 86, No. 9, pp. 330–332, September, 1978.     

The detection of large deletions or duplications in genomic DNA

While methods for the detection of point mutations and small insertions or deletions in genomic DNA are well established, the detection of larger (>100 bp) genomic duplications or deletions can be more difficult. Most mutation scanning methods use PCR as a first step, but the subsequent analyses are usually qualitative rather than quantitative. Gene dosage methods based on PCR need to be quantitative (i.e., they should report molar quantities of starting material) or semi-quantitative (i.e., they should report gene dosage relative to an internal standard). Without some sort of quantitation, heterozygous deletions and duplications may be overlooked and therefore be under-ascertained. Gene dosage methods provide the additional benefit of reporting allele drop-out in the PCR. This could impact on SNP surveys, where large-scale genotyping may miss null alleles. Here we review recent developments in techniques for the detection of this type of mutation and compare their relative strengths and weaknesses. We emphasize that comprehensive mutation analysis should include scanning for large insertions and deletions and duplications.     

Localization of cloned mouse chromosome 7-specific DNA to lethal albino deletions

Mouse chromosome X- and 7-specific DNA fragments have been isolated from a recombinant DNA library enriched for X⁽⁷⁾ chromosome sequences. The library was enriched by flow sorting the X⁽⁷⁾ chromosome, a derivation of the Cattanach translocation, prior to library construction. A DNA fragment was found to be located in a region deleted in newborn mice doubly heterozygous for the two albino deletions c^3H and c^6H in chromosome 7. These chromosome-specific DNA fragments will be useful for studying X inactivation spreading in the X-autosome translocation (T(X;7)1Ct) and for investigating the developmental effects of the lethal albino deletions.     

High-throughput discovery of rare insertions and deletions in large cohorts

Pooled-DNA sequencing strategies enable fast, accurate, and cost-effect detection of rare variants, but current approaches are not able to accurately identify short insertions and deletions (indels), despite their pivotal role in genetic disease. Furthermore, the sensitivity and specificity of these methods depend on arbitrary, user-selected significance thresholds, whose optimal values change from experiment to experiment. Here, we present a combined experimental and computational strategy that combines a synthetically engineered DNA library inserted in each run and a new computational approach named SPLINTER that detects and quantifies short indels and substitutions in large pools. SPLINTER integrates information from the synthetic library to select the optimal significance thresholds for every experiment. We show that SPLINTER detects indels (up to 4 bp) and substitutions in large pools with high sensitivity and specificity, accurately quantifies variant frequency (r = 0.999), and compares favorably with existing algorithms for the analysis of pooled sequencing data. We applied our approach to analyze a cohort of 1152 individuals, identifying 48 variants and validating 14 of 14 (100%) predictions by individual genotyping. Thus, our strategy provides a novel and sensitive method that will speed the discovery of novel disease-causing rare variants.     

Analysis of multiple mitochondrial DNA deletions in inclusion body myositis

Inclusion body myositis (IBM) is a sporadic progressive myopathy, which is morphologically characterized by inflammatory cell infiltrates and rimmed vacuoles in muscle fibers. Mitochondrial changes are regularly present with ragged-red fibers showing deficiency of cytochrome c oxidase. In these muscle fiber segments, there is accumulation of mitochondria with mitochondrial DNA (mtDNA) deletions. There are different deletions in different muscle fibers. In this study, we have sequenced for the first time the multiple mtDNA deletions in muscle from four patients with IBM. The deletion breakpoints were sequenced from cloned polymerase chain reaction (PCR)-amplified mtDNA fragments. The sequencing was performed directly from the bacterial colonies used for cloning. Of 122 analyzed clones, 33 different deletions were identified. The majority of these have not previously been described. There was a marked predominance of deletion breakpoints in certain regions of mtDNA. These predominant breakpoint regions are similar to those described in other conditions with multiple deletions, such as autosomal dominant progressive external ophthalmoplegia (adPEO) and normal aging, but different from those described in diseases due to single deletions such as Kearns-Sayre syndrome and sporadic PEO. These findings indicate that common factors are involved in the development of multiple mtDNA deletions in IBM, adPEO, and aging. Hum Mutat 10:381–386, 1997. © 1997 Wiley-Liss, Inc.     

Stimulated muscle force assessment of the sternocleidomastoid muscle in humans

The aim of this study was to configure a force assessment device and determine potential testing protocols for quantitative evaluation of human neck muscles. The study design consisted of non-randomized control trials, with repeated measures; data from 12 normal subjects were obtained. Several apparatuses were designed, constructed and tested, i.e. single or short trains of supramaximal stimuli were used to activate sternocleidomastoid muscles in a seated position with strain gauges (6.2% variability with double-pulse stimulations) or in supine positions with load cells (5.2% variability with similar activation). Using a final configuration, maximum elicited peak forces were 1742 +/- 323 g for single-pulse and 3976 +/- 484 g for double-pulse stimulations (n = 12). There were no significant differences in maximum recorded peak torques between sessions per individual. Yet, detectable muscle activities were simultaneously recorded in the contralateral sternocleidomastoid muscles. This non-invasive, quantitative assessment approach has novel value for determining treatment efficacy, disease progression, and/or approach has novel value for determining determining treatment efficacy, disease progression, and/or relative distribution of muscle strength in patients with abnormal neck muscle function.     

High proportion of large genomic STK11 deletions in Peutz-Jeghers syndrome

Germline mutations in the STK11 gene have been identified in 10-70% of patients with Peutz-Jeghers syndrome (PJS), an autosomal-dominant hamartomatous polyposis syndrome. A second locus was assumed in a large proportion of PJS patients. To date, STK11 alterations comprise mainly point mutations; only a small number of large deletions have been reported. We performed a mutation analysis for the STK11 gene in 71 patients. Of these, 56 met the clinical criteria for PJS and 12 were presumed to have PJS because of mucocutaneous pigmentation only or bowel problems due to isolated PJS polyps. No clinical information was available for the remaining three patients. By direct sequencing of the coding region of the STK11 gene, we identified point mutations in 37 of 71 patients (52%). We examined the remaining 34 patients by means of the multiplex ligation-dependent probe amplification (MLPA) method, and detected deletions in 17 patients. In four patients the deletion extended over all 10 exons, and in eight patients only the promoter region and exon 1 were deleted. The remaining deletions encompassed exons 2-10 (in two patients), exons 2-3, exons 4-5, or exon 8. When only patients who met the clinical criteria for PJS are considered, the overall mutation detection rate increases to 94% (64% point mutations and 30% large deletions). No mutation was identified in any of the 12 presumed cases. In conclusion, we found that approximately one-third of the patients who met the clinical PJS criteria exhibited large genomic deletions that were readily detectable by MLPA. Screening for point mutations and large deletions by direct sequencing or MLPA, respectively, increased the mutation detection rate in the STK11 gene up to 94%. There may be still other mutations in the STK11 gene that are not detectable by the methods applied here. Therefore, it is questionable whether a second PJS locus exists at all.