Mutations in NDUFB11, Encoding a Complex I Component of the Mitochondrial Respiratory Chain, Cause Microphthalmia with Linear Skin Defects Syndrome.

Microphthalmia with linear skin defects (MLS) syndrome is an X-linked male-lethal disorder also known as MIDAS (microphthalmia, dermal aplasia, and sclerocornea). Additional clinical features include neurological and cardiac abnormalities. MLS syndrome is genetically heterogeneous given that heterozygous mutations in HCCS or COX7B have been identified in MLS-affected females. Both genes encode proteins involved in the structure and function of complexes III and IV, which form the terminal segment of the mitochondrial respiratory chain (MRC). However, not all individuals with MLS syndrome carry a mutation in either HCCS or COX7B. The majority of MLS-affected females have severe skewing of X chromosome inactivation, suggesting that mutations in HCCS, COX7B, and other as-yet-unidentified X-linked gene(s) cause selective loss of cells in which the mutated X chromosome is active. By applying whole-exome sequencing and filtering for X-chromosomal variants, we identified a de novo nonsense mutation in NDUFB11 (Xp11.23) in one female individual and a heterozygous 1-bp deletion in a second individual, her asymptomatic mother, and an affected aborted fetus of the subject's mother. NDUFB11 encodes one of 30 poorly characterized supernumerary subunits of NADH:ubiquinone oxidoreductase, known as complex I (cI), the first and largest enzyme of the MRC. By shRNA-mediated NDUFB11 knockdown in HeLa cells, we demonstrate that NDUFB11 is essential for cI assembly and activity as well as cell growth and survival. These results demonstrate that X-linked genetic defects leading to the complete inactivation of complex I, III, or IV underlie MLS syndrome. Our data reveal an unexpected role of cI dysfunction in a developmental phenotype, further underscoring the existence of a group of mitochondrial diseases associated with neurocutaneous manifestations.

Mutations in LONP1, a mitochondrial matrix protease, cause CODAS syndrome.

Cerebral, ocular, dental, auricular, skeletal anomalies (CODAS) syndrome (MIM 600373) was first described and named by Shehib et al, in 1991 in a single patient. The anomalies referred to in the acronym are as follows: cerebral-developmental delay, ocular-cataracts, dental-aberrant cusp morphology and delayed eruption, auricular-malformations of the external ear, and skeletal-spondyloepiphyseal dysplasia. This distinctive constellation of anatomical findings should allow easy recognition but despite this only four apparently sporadic patients have been reported in the last 20 years indicating that the full phenotype is indeed very rare with perhaps milder or a typical presentations that are allelic but without sufficient phenotypic resemblance to permit clinical diagnosis. We performed exome sequencing in three patients (an isolated case and a brother and sister sib pair) with classical features of CODAS. Sanger sequencing was used to confirm results as well as for mutation discovery in a further four unrelated patients ascertained via their skeletal features. Compound heterozygous or homozygous mutations in LONP1 were found in all (8 separate mutations; 6 missense, 1 nonsense, 1 small in-frame deletion) thus establishing the genetic basis of CODAS and the pattern of inheritance (autosomal recessive). LONP1 encodes an enzyme of bacterial ancestry that participates in protein turnover within the mitochondrial matrix. The mutations cluster at the ATP-binding and proteolytic domains of the enzyme. Biallelic inheritance and clustering of mutations confirm dysfunction of LONP1 activity as the molecular basis of CODAS but the pathogenesis remains to be explored.

Clonal expansion of early to mid-life mitochondrial DNA point mutations drives mitochondrial dysfunction during human ageing

Age-related decline in the integrity of mitochondria is an important contributor to the human ageing process. In a number of ageing stem cell populations, this decline in mitochondrial function is due to clonal expansion of individual mitochondrial DNA (mtDNA) point mutations within single cells. However the dynamics of this process and when these mtDNA mutations occur initially are poorly understood. Using human colorectal epithelium as an exemplar tissue with a well-defined stem cell population, we analysed samples from 207 healthy participants aged 17-78 years using a combination of techniques (Random Mutation Capture, Next Generation Sequencing and mitochondrial enzyme histochemistry), and show that: 1) non-pathogenic mtDNA mutations are present from early embryogenesis or may be transmitted through the germline, whereas pathogenic mtDNA mutations are detected in the somatic cells, providing evidence for purifying selection in humans, 2) pathogenic mtDNA mutations are present from early adulthood (<20 years of age), at both low levels and as clonal expansions, 3) low level mtDNA mutation frequency does not change significantly with age, suggesting that mtDNA mutation rate does not increase significantly with age, and 4) clonally expanded mtDNA mutations increase dramatically with age. These data confirm that clonal expansion of mtDNA mutations, some of which are generated very early in life, is the major driving force behind the mitochondrial dysfunction associated with ageing of the human colorectal epithelium.

Mutations in the Mitochondrial Methionyl-tRNA Synthetase Cause a Neurodegenerative Phenotype in Flies and a Recessive Ataxia (ARSAL) in Humans

An increasing number of genes required for mitochondrial biogenesis, dynamics, or function have been found to be mutated in metabolic disorders and neurological diseases such as Leigh Syndrome. In a forward genetic screen to identify genes required for neuronal function and survival in Drosophila photoreceptor neurons, we have identified mutations in the mitochondrial methionyl-tRNA synthetase, Aats-met, the homologue of human MARS2. The fly mutants exhibit age-dependent degeneration of photoreceptors, shortened lifespan, and reduced cell proliferation in epithelial tissues. We further observed that these mutants display defects in oxidative phosphorylation, increased Reactive Oxygen Species (ROS), and an upregulated mitochondrial Unfolded Protein Response. With the aid of this knowledge, we identified MARS2 to be mutated in Autosomal Recessive Spastic Ataxia with Leukoencephalopathy (ARSAL) patients. We uncovered complex rearrangements in the MARS2 gene in all ARSAL patients. Analysis of patient cells revealed decreased levels of MARS2 protein and a reduced rate of mitochondrial protein synthesis. Patient cells also exhibited reduced Complex I activity, increased ROS, and a slower cell proliferation rate, similar to Drosophila Aats-met mutants.

Mutations in SDHD lead to autosomal recessive encephalomyopathy and isolated mitochondrial complex II deficiency

BACKGROUND Defects of the mitochondrial respiratory chain complex II (succinate dehydrogenase (SDH) complex) are extremely rare. Of the four nuclear encoded proteins composing complex II, only mutations in the 70 kDa flavoprotein (SDHA) and the recently identified complex II assembly factor (SDHAF1) have been found to be causative for mitochondrial respiratory chain diseases. Mutations in the other three subunits (SDHB, SDHC, SDHD) and the second assembly factor (SDHAF2) have so far only been associated with hereditary paragangliomas and phaeochromocytomas. Recessive germline mutations in SDHB have recently been associated with complex II deficiency and leukodystrophy in one patient. METHODS AND RESULTS We present the clinical and molecular investigations of the first patient with biochemical evidence of a severe isolated complex II deficiency due to compound heterozygous SDHD gene mutations. The patient presented with early progressive encephalomyopathy due to compound heterozygous p.E69 K and p.*164Lext*3 SDHD mutations. Native polyacrylamide gel electrophoresis and western blotting demonstrated an impaired complex II assembly. Complementation of a patient cell line additionally supported the pathogenicity of the novel identified mutations in SDHD. CONCLUSIONS This report describes the first case of isolated complex II deficiency due to recessive SDHD germline mutations. We therefore recommend screening for all SDH genes in isolated complex II deficiencies. It further emphasises the importance of appropriate genetic counselling to the family with regard to SDHD mutations and their role in tumorigenesis.

Analysis of microsatellite mutations in the mitochondrial DNA of Saccharomyces cerevisiae

In the nuclear genome of Saccharomyces cerevisiae, simple, repetitive DNA sequences (microsatellites) mutate at rates much higher than nonrepetitive sequences. Most of these mutations are deletions or additions of repeat units. The yeast mitochondrial genome also contains many microsatellites. To examine the stability of these sequences, we constructed a reporter gene (arg8m) containing out-of-frame insertions of either poly(AT) or poly(GT) tracts within the coding sequence. Yeast strains with this reporter gene inserted within the mitochondrial genome were constructed. Using these strains, we showed that poly(GT) tracts were considerably less stable than poly(AT) tracts and that alterations usually involved deletions rather than additions of repeat units. In contrast, in the nuclear genome, poly(GT) and poly(AT) tracts had similar stabilities, and alterations usually involved additions rather than deletions. Poly(GT) tracts were more stable in the mitochondria of diploid cells than in haploids. In addition, an msh1 mutation destabilized poly(GT) tracts in the mitochondrial genome.

No relationship found between point heteroplasmy in mitochondrial DNA control region and age range, sex and haplogroup in human hairs

The analysis of heteroplasmy (presence of more than one type of mitochondrial DNA in an individual) is used as a tool in human identification studies, anthropology, and most currently in studies that relate heteroplasmy with longevity. The frequency of heteroplasmy and its correlation with age has been analyzed using different tissues such as blood, muscle, heart, bone and brain and in different regions of mitochondrial DNA, but this analysis had never been performed using hair samples. In this study, samples of hair were sequenced in order to ascertain whether the presence or not of heteroplasmy varied according to age, sex and origin of haplogroup individuals. The samples were grouped by age (3 groups), gender (male and female) and haplogroup of origin (European, African and Native American), and analyzed using the chi-square statistical test (chi(2)). Based in statistical results obtained, we conclude that there is no relationship between heteroplasmy and sex, age and haplogroup origin using hair samples.

The spectrum of autoantibodies in IPEX syndrome is broad and includes anti-mitochondrial autoantibodies

IPEX syndrome is a congenital disorder of immune regulation caused by mutations in the FOXP3 gene, which is required for the suppressive function of naturally arising CD4 + CD25 + regulatory T cells. In this case series we evaluated serum samples from 12 patients with IPEX syndrome for the presence of common autoantibodies associated with a broad range of autoimmune disorders. We note that 75% of patients (9/12) had 1 or more autoantibodies, an incidence far above the cumulative rate observed in the general population. The range of autoantibodies differed between patients and there was no predominant autoantibody or pattern of autoantibodies present in this cohort. Surprisingly, one patient had high-titer anti-mitochondrial antibodies (AMA) typically associated with primary biliary cirrhosis (PBC) although the patient had no signs of cholestasis. PBC is a well-characterized autoimmune disease that occurs primarily in women and includes the serological hallmarks of serum AMA and elevated IgM which were both present in this patient. PBC is virtually absent in children with the exception of one reported child with interleukin 2 receptor a (CD25) deficiency which is associated with an IPEX-like regulatory T cell dysfunction. Based on the present data and the available literature we suggest a direct role for CD4 + CD25 + regulatory T cells in restraining B cell autoantibody production and that defects in regulatory T cells may be crucial to the development of PBC. (C) 2010 Elsevier Ltd. All rights reserved.

Multi-system neurological disease is common in patients with OPA1 mutations

Additional neurological features have recently been described in seven families transmitting pathogenic mutations in OPA1, the most common cause of autosomal dominant optic atrophy. However, the frequency of these syndromal `dominant optic atrophy plus` variants and the extent of neurological involvement have not been established. In this large multi-centre study of 104 patients from 45 independent families, including 60 new cases, we show that extra-ocular neurological complications are common in OPA1 disease, and affect up to 20% of all mutational carriers. Bilateral sensorineural deafness beginning in late childhood and early adulthood was a prominent manifestation, followed by a combination of ataxia, myopathy, peripheral neuropathy and progressive external ophthalmoplegia from the third decade of life onwards. We also identified novel clinical presentations with spastic paraparesis mimicking hereditary spastic paraplegia, and a multiple sclerosis-like illness. In contrast to initial reports, multi-system neurological disease was associated with all mutational subtypes, although there was an increased risk with missense mutations [odds ratio = 3.06, 95% confidence interval = 1.44-6.49; P = 0.0027], and mutations located within the guanosine triphosphate-ase region (odds ratio = 2.29, 95% confidence interval = 1.08-4.82; P = 0.0271). Histochemical and molecular characterization of skeletal muscle biopsies revealed the presence of cytochrome c oxidase-deficient fibres and multiple mitochondrial DNA deletions in the majority of patients harbouring OPA1 mutations, even in those with isolated optic nerve involvement. However, the cytochrome c oxidase-deficient load was over four times higher in the dominant optic atrophy + group compared to the pure optic neuropathy group, implicating a causal role for these secondary mitochondrial DNA defects in disease pathophysiology. Individuals with dominant optic atrophy plus phenotypes also had significantly worse visual outcomes, and careful surveillance is therefore mandatory to optimize the detection and management of neurological disability in a group of patients who already have significant visual impairment.

PROTEIN FOLDING AND DISEASE THE MITOCHONDRIAL PROTEIN FRATAXIN

This dissertation focuses on the study of frataxin, a small mitochondrial protein whose deficiency is associated with the neurodegenerative disease Friedreich's ataxia (FRDA). Aiming at a better understanding of frataxin conformational and functional properties, two lines of research were followed: first, the effect of FRDA-related mutations in human frataxin (FXN) were studied and the role of oxidative stress related modification addressed; second, yeast frataxin (Yfh1) orthologue was used to explore the conformational and functional properties of the protein.(...)

Proteomic and Metabolomic Analyses of Mitochondrial Complex I-deficient Mouse Model Generated by Spontaneous B2 Short Interspersed Nuclear Element (SINE) Insertion into NADH Dehydrogenase (Ubiquinone) Fe-S Protein 4 (Ndufs4) Gene.

Eukaryotic cells generate energy in the form of ATP, through a network of mitochondrial complexes and electron carriers known as the oxidative phosphorylation system. In mammals, mitochondrial complex I (CI) is the largest component of this system, comprising 45 different subunits encoded by mitochondrial and nuclear DNA. Humans diagnosed with mutations in the gene NDUFS4, encoding a nuclear DNA-encoded subunit of CI (NADH dehydrogenase ubiquinone Fe-S protein 4), typically suffer from Leigh syndrome, a neurodegenerative disease with onset in infancy or early childhood. Mitochondria from NDUFS4 patients usually lack detectable NDUFS4 protein and show a CI stability/assembly defect. Here, we describe a recessive mouse phenotype caused by the insertion of a transposable element into Ndufs4, identified by a novel combined linkage and expression analysis. Designated Ndufs4(fky), the mutation leads to aberrant transcript splicing and absence of NDUFS4 protein in all tissues tested of homozygous mice. Physical and behavioral symptoms displayed by Ndufs4(fky/fky) mice include temporary fur loss, growth retardation, unsteady gait, and abnormal body posture when suspended by the tail. Analysis of CI in Ndufs4(fky/fky) mice using blue native PAGE revealed the presence of a faster migrating crippled complex. This crippled CI was shown to lack subunits of the "N assembly module", which contains the NADH binding site, but contained two assembly factors not present in intact CI. Metabolomic analysis of the blood by tandem mass spectrometry showed increased hydroxyacylcarnitine species, implying that the CI defect leads to an imbalanced NADH/NAD(+) ratio that inhibits mitochondrial fatty acid β-oxidation.

Methylation profile of TP53 regulatory pathway and mtDNA alterations in breast cancer patients lacking TP53 mutations.

The present study investigated promoter hypermethylation of TP53 regulatory pathways providing a potential link between epigenetic changes and mitochondrial DNA (mtDNA) alterations in breast cancer patients lacking a TP53 mutation. The possibility of using the cancer-specific alterations in serum samples as a blood-based test was also explored. Triple-matched samples (cancerous tissues, matched adjacent normal tissues and serum samples) from breast cancer patients were screened for TP53 mutations, and the promoter methylation profile of P14(ARF), MDM2, TP53 and PTEN genes was analyzed as well as mtDNA alterations, including D-loop mutations and mtDNA content. In the studied cohort, no mutation was found in TP53 (DNA-binding domain). Comparison of P14(ARF) and PTEN methylation patterns showed significant hypermethylation levels in tumor tissues (P < 0.05 and <0.01, respectively) whereas the TP53 tumor suppressor gene was not hypermethylated (P < 0.511). The proportion of PTEN methylation was significantly higher in serum than in the normal tissues and it has a significant correlation to tumor tissues (P < 0.05). mtDNA analysis revealed 36.36% somatic and 90.91% germline mutations in the D-loop region and also significant mtDNA depletion in tumor tissues (P < 0.01). In addition, the mtDNA content in matched serum was significantly lower than in the normal tissues (P < 0.05). These data can provide an insight into the management of a therapeutic approach based on the reversal of epigenetic silencing of the crucial genes involved in regulatory pathways of the tumor suppressor TP53. Additionally, release of significant aberrant methylated PTEN in matched serum samples might represent a promising biomarker for breast cancer.

A NOVEL MUTATION IN BCS1L IN A PATIENT WITH AN ISOLATED MITOCHONDRIAL COMPLEX III DEFICIENCY

Background: Isolated complex III deficiencies are caused by mutations in the mitochondrial CytB gene, in the BCS1L gene coding for a CIII assembly factor and in the UQCRQ gene that codes for the ubiquinone binding protein of complex III. Objective: Description of clinical features, mitochondrial function and molecular genetic analysis in a patient with an isolated complex III deficiency. Patient: A 17 year old boy, born to consanguineous parents who presented with hypoglycemia, glycosuria, deafness, growth retardation, Fanconi Syndrome and severe lactic acidosis in the neonatal period. Methods: Activities and assembly of OXPHOS complexes were investigated spectrophotometrically and by BN-PAGE. mt-DNAwas screened for deletions. Cytochrome b (CytB) and the BCS1L gene were sequenced. Results: Isolated complex III deficiency was detected in the patient's skeletal muscle. Using BN-PAGE blotting a complex III of lower molecular weight was detected. Staining the 2D reveals a missing subunit. No mutation was detected in the mitochondrial CytB gene. Sequence analysis of BCS1L revealed a novel homozygous point mutation p.M48V. Conclusion: The patients decreased complex III activity is most likely caused by incomplete assembly of complex III due to the homozygous p. M48V mutation in the BCS1L gene.

Repercussion of a deficiency in mitochondrial ß-oxidation on the carbon flux of short-chain fatty acids to the peroxisomal ß-oxidation cycle in Aspergillus nidulans.

The fungus Aspergillus nidulans contains both a mitochondrial and peroxisomal ß-oxidation pathway. This work was aimed at studying the influence of mutations in the foxA gene, encoding a peroxisomal multifunctional protein, or in the scdA/echA genes, encoding a mitochondrial short-chain dehydrogenase and an enoyl-CoA hydratase, respectively, on the carbon flux to the peroxisomal ß-oxidation pathway. A. nidulans transformed with a peroxisomal polyhydroxyalkanoate (PHA) synthase produced PHA from the polymerization of 3-hydroxyacyl-CoA intermediates derived from the peroxisomal ß-oxidation of external fatty acids. PHA produced from erucic acid or heptadecanoic acid contained a broad spectrum of monomers, ranging from 5 to 14 carbons, revealing that the peroxisomal ß-oxidation cycle can handle both long and short-chain intermediates. While the ∆foxA mutant grown on erucic acid or oleic acid synthesized 10-fold less PHA compared to wild type, the same mutant grown on octanoic acid or heptanoic acid produced 3- to 6-fold more PHA. Thus, while FoxA has an important contribution to the degradation of long-chain fatty acids, the flux of short-chain fatty acids to peroxisomal ß-oxidation is actually enhanced in its absence. While no change in PHA was observed in the ∆scdA∆echA mutant grown on erucic acid or oleic acid compared to wild type, there was a 2- to 4-fold increased synthesis of PHA in ∆scdA∆echA cells grown in octanoic acid or heptanoic acid. These results reveal that a compensatory mechanism exists in A. nidulans that increases the flux of short-chain fatty acids towards the peroxisomal ß-oxidation cycle when the mitochondrial ß-oxidation pathway is defective.