Mitochondrial cristae shape determines respiratory chain supercomplexes assembly and respiratory efficiency.

Respiratory chain complexes assemble into functional quaternary structures called supercomplexes (RCS) within the folds of the inner mitochondrial membrane, or cristae. Here, we investigate the relationship between respiratory function and mitochondrial ultrastructure and provide evidence that cristae shape determines the assembly and stability of RCS and hence mitochondrial respiratory efficiency. Genetic and apoptotic manipulations of cristae structure affect assembly and activity of RCS in vitro and in vivo, independently of changes to mitochondrial protein synthesis or apoptotic outer mitochondrial membrane permeabilization. We demonstrate that, accordingly, the efficiency of mitochondria-dependent cell growth depends on cristae shape. Thus, RCS assembly emerges as a link between membrane morphology and function.

Transcriptional programming of lipid and amino acid metabolism by the skeletal muscle circadian clock.

Circadian clocks are fundamental physiological regulators of energy homeostasis, but direct transcriptional targets of the muscle clock machinery are unknown. To understand how the muscle clock directs rhythmic metabolism, we determined genome-wide binding of the master clock regulators brain and muscle ARNT-like protein 1 (BMAL1) and REV-ERBα in murine muscles. Integrating occupancy with 24-hr gene expression and metabolomics after muscle-specific loss of BMAL1 and REV-ERBα, here we unravel novel molecular mechanisms connecting muscle clock function to daily cycles of lipid and protein metabolism. Validating BMAL1 and REV-ERBα targets using luciferase assays and in vivo rescue, we demonstrate how a major role of the muscle clock is to promote diurnal cycles of neutral lipid storage while coordinately inhibiting lipid and protein catabolism prior to awakening. This occurs by BMAL1-dependent activation of Dgat2 and REV-ERBα-dependent repression of major targets involved in lipid metabolism and protein turnover (MuRF-1, Atrogin-1). Accordingly, muscle-specific loss of BMAL1 is associated with metabolic inefficiency, impaired muscle triglyceride biosynthesis, and accumulation of bioactive lipids and amino acids. Taken together, our data provide a comprehensive overview of how genomic binding of BMAL1 and REV-ERBα is related to temporal changes in gene expression and metabolite fluctuations.

The FSHD2 gene SMCHD1 is a modifier of disease severity in families affected by FSHD1.

Facioscapulohumeral muscular dystrophy type 1 (FSHD1) is caused by contraction of the D4Z4 repeat array on chromosome 4 to a size of 1-10 units. The residual number of D4Z4 units inversely correlates with clinical severity, but significant clinical variability exists. Each unit contains a copy of the DUX4 retrogene. Repeat contractions are associated with changes in D4Z4 chromatin structure that increase the likelihood of DUX4 expression in skeletal muscle, but only when the repeat resides in a genetic background that contains a DUX4 polyadenylation signal. Mutations in the structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1) gene, encoding a chromatin modifier of D4Z4, also result in the increased likelihood of DUX4 expression in individuals with a rare form of FSHD (FSHD2). Because SMCHD1 directly binds to D4Z4 and suppresses somatic expression of DUX4, we hypothesized that SMCHD1 may act as a genetic modifier in FSHD1. We describe three unrelated individuals with FSHD1 presenting an unusual high clinical severity based on their upper-sized FSHD1 repeat array of nine units. Each of these individuals also carries a mutation in the SMCHD1 gene. Familial carriers of the FSHD1 allele without the SMCHD1 mutation were only mildly affected, suggesting a modifier effect of the SMCHD1 mutation. Knocking down SMCHD1 in FSHD1 myotubes increased DUX4 expression, lending molecular support to a modifier role for SMCHD1 in FSHD1. We conclude that FSHD1 and FSHD2 share a common pathophysiological pathway in which the FSHD2 gene can act as modifier for disease severity in families affected by FSHD1.

Molecular characterization of the human COQ5 C-methyltransferase in coenzyme Q10 biosynthesis.

Coq5 catalyzes the only C-methylation involved in the biosynthesis of coenzyme Q (Q or ubiquinone) in humans and yeast Saccharomyces cerevisiae. As one of eleven polypeptides required for Q production in yeast, Coq5 has also been shown to assemble with the multi-subunit complex termed the CoQ-synthome. In humans, mutations in several COQ genes cause primary Q deficiency, and a decrease in Q biosynthesis is associated with mitochondrial, cardiovascular, kidney and neurodegenerative diseases. In this study, we characterize the human COQ5 polypeptide and examine its complementation of yeast coq5 point and null mutants. We show that human COQ5 RNA is expressed in all tissues and that the COQ5 polypeptide is associated with the mitochondrial inner membrane on the matrix side. Previous work in yeast has shown that point mutations within or adjacent to conserved COQ5 methyltransferase motifs result in a loss of Coq5 function but not Coq5 steady state levels. Here, we show that stabilization of the CoQ-synthome within coq5 point mutants or by over-expression of COQ8 in coq5 null mutants permits the human COQ5 homolog to partially restore coq5 mutant growth on respiratory media and Q6 content. Immunoblotting against the human COQ5 polypeptide in isolated yeast mitochondria shows that the human Coq5 polypeptide migrates in two-dimensional blue-native/SDS-PAGE at the same high molecular mass as other yeast Coq proteins. The results presented suggest that human and Escherichia coli Coq5 homologs expressed in yeast retain C-methyltransferase activity but are capable of rescuing the coq5 yeast mutants only when the CoQ-synthome is assembled.

Validation of CFTR intronic variants identified during cystic fibrosis population screening by a minigene splicing assay.

BACKGROUND: Cystic fibrosis, caused by mutations of the CFTR gene, is the most common autosomal recessive condition in the European population and there are specific screening programs aimed at investigating healthy carriers. They are usually articulated in two steps: initially individuals are screened with a panel of the 20-50 most common CFTR mutations; the second step is offered to partners of carriers who were found negative at the first test and consists in the analysis of the entire CFTR gene. This strategy provides high sensitivity, however, it often identifies novel variants (especially in introns) of unknown significance. Establishing the pathogenicity of these variants of the CFTR gene is not a simple task. METHODS: We have examined five CFTR intronic variants of unclear significance (c.274-6T>C, c.744-6T>G, c.1117-64G>A, c.2620-26A>G, and c.3468+51C>A) using a functional splicing assay based on hybrid minigenes. RESULTS: Four out of five variants (including c.2620-26A>G which was previously reported as a possible splice-site mutation) did not alter the correct splicing of the minigene and are likely to be neutral polymorphisms, whereas c.744-6T>G caused complete skipping of CFTR exon 7 and should be therefore regarded as a pathogenic CFTR mutation. CONCLUSIONS: Hybrid minigenes assay are a simple and rapid tool to evaluate the effects of intronic variants without the need of analyzing patient's mRNA, and are particularly suited to analyze variants identified during population screenings.

Haploinsufficiency of COQ4 causes coenzyme Q10 deficiency.

BACKGROUND: COQ4 encodes a protein that organises the multienzyme complex for the synthesis of coenzyme Q(10) (CoQ(10)). A 3.9 Mb deletion of chromosome 9q34.13 was identified in a 3-year-old boy with mental retardation, encephalomyopathy and dysmorphic features. Because the deletion encompassed COQ4, the patient was screened for CoQ(10) deficiency. METHODS: A complete molecular and biochemical characterisation of the patient's fibroblasts and of a yeast model were performed. RESULTS: The study found reduced COQ4 expression (48% of controls), CoQ(10) content and biosynthetic rate (44% and 43% of controls), and activities of respiratory chain complex II+III. Cells displayed a growth defect that was corrected by the addition of CoQ(10) to the culture medium. Knockdown of COQ4 in HeLa cells also resulted in a reduction of CoQ(10.) Diploid yeast haploinsufficient for COQ4 displayed similar CoQ deficiency. Haploinsufficency of other genes involved in CoQ(10) biosynthesis does not cause CoQ deficiency, underscoring the critical role of COQ4. Oral CoQ(10) supplementation resulted in a significant improvement of neuromuscular symptoms, which reappeared after supplementation was temporarily discontinued. CONCLUSION: Mutations of COQ4 should be searched for in patients with CoQ(10) deficiency and encephalomyopathy; patients with genomic rearrangements involving COQ4 should be screened for CoQ(10) deficiency, as they could benefit from supplementation.

Polymorphisms of the SCN1A gene in children and adolescents with primary headache and idiopathic or cryptogenic epilepsy: is there a linkage?

The purpose of this study was to evaluate the distribution of the polymorphisms of the SCN1A gene in a series of children and adolescents with primary headache and idiopathic or cryptogenic epilepsy compared to controls. Five non-synonymous exonic polymorphisms (1748A > T, 2656T > C, 3199A > G, 5771G > A, 5864T > C) of the SCN1A gene were selected and their genotyping was performed, by high resolution melting (HRM), in 49 cases and 100 controls. We found that among the five polymorphisms, only 3199A > G was a true polymorphism. We did not find a statistically significant difference between distribution of 3199A > G genotypes between cases and controls. We excluded the role of the SCN1A gene in the pathogenesis of comorbidity between headache (especially migraine) and epilepsy. The SCN1A gene is a major gene in different epilepsies and epilepsy syndromes; the HRM could be the new methodology, more rapid and efficacious, for molecular analysis of the SCN1A gene.

Functional complementation in yeast allows molecular characterization of missense argininosuccinate lyase mutations.

Deficiency of argininosuccinate lyase (ASL) causes argininosuccinic aciduria, an urea cycle defect that may present with a severe neonatal onset form or with a late onset phenotype. To date phenotype-genotype correlations are still not clear because biochemical assays of ASL activity correlate poorly with clinical severity in patients. We employed a yeast-based functional complementation assay to assess the pathogenicity of 12 missense ASL mutations, to establish genotype-phenotype correlations, and to screen for intragenic complementation. Rather than determining ASL enzyme activity directly, we have measured the growth rate in arginine-free medium of a yeast ASL(null) strain transformed with individual mutant ASL alleles. Individual haploid strains were also mated to obtain diploid, "compound heterozygous" yeast. We show that the late onset phenotypes arise in patients because they harbor individual alleles retaining high residual enzymatic activity or because of intragenic complementation among different mutated alleles. In these cases complementation occurs because in the hybrid tetrameric enzyme at least one active site without mutations can be formed or because the differently mutated alleles can stabilize each other, resulting in partial recovery of enzymatic activity. Functional complementation in yeast is simple and reproducible and allows the analysis of large numbers of mutant alleles. Moreover, it can be easily adapted for the analysis of mutations in other genes involved in urea cycle disorders.

LETM1, deleted in Wolf-Hirschhorn syndrome is required for normal mitochondrial morphology and cellular viability.

Wolf-Hirschhorn syndrome (WHS) is a complex congenital syndrome caused by a monoallelic deletion of the short arm of chromosome 4. Seizures in WHS have been associated with deletion of LETM1 gene. LETM1 encodes for the human homologue of yeast Mdm38p, a mitochondria-shaping protein of unclear function. Here we show that human LETM1 is located in the inner membrane, exposed to the matrix and oligomerized in higher molecular weight complexes of unknown composition. Down-regulation of LETM1 did not disrupt these complexes, but led to DRP1-independent fragmentation of the mitochondrial network. Fragmentation was not associated with changes in the levels of respiratory chain complexes, or with obvious or latent mitochondrial dysfunction, but was recovered by nigericin, which catalyzes the electroneutral exchange of K+ against H+. Down-regulation of LETM1 caused 'necrosis-like' death, without activation of caspases and not inhibited by overexpression of Bcl-2. Primary fibroblasts from a WHS patient displayed reduced LETM1 mRNA and protein, but mitochondrial morphology was surprisingly unaffected, raising the question of whether and how WHS patients counteract the consequences of monoallelic deletion of LETM1. LETM1 highlights the relationship between mitochondrial ion homeostasis, integrity of the mitochondrial network and cell viability.

A novel deletion in the GTPase domain of OPA1 causes defects in mitochondrial morphology and distribution, but not in function.

Autosomal dominant optic atrophy (ADOA), the commonest cause of inherited optic atrophy, is caused by mutations in the ubiquitously expressed gene optic atrophy 1 (OPA1), involved in fusion and biogenesis of the inner membrane of mitochondria. Bioenergetic failure, mitochondrial network abnormalities and increased apoptosis have all been proposed as possible causal factors. However, their relative contribution to pathogenesis as well as the prominent susceptibility of the retinal ganglion cell (RGC) in this disease remains uncertain. Here we identify a novel deletion of OPA1 gene in the GTPase domain in three patients affected by ADOA. Muscle biopsy of the patients showed neurogenic atrophy and abnormal morphology and distribution of mitochondria. Confocal microscopy revealed increased mitochondrial fragmentation in fibroblasts as well as in myotubes, where mitochondria were also unevenly distributed, with clustered organelles alternating with areas where mitochondria were sparse. These abnormalities were not associated with altered bioenergetics or increased susceptibility to pro-apoptotic stimuli. Therefore, changes in mitochondrial shape and distribution can be independent of other reported effects of OPA1 mutations, and therefore may be the primary cause of the disease. The arrangement of mitochondria in RGCs, which degenerate in ADOA, may be exquisitely sensitive to disturbance, and this may lead to bioenergetic crisis and/or induction of apoptosis. Our results highlight the importance of mitochondrial dynamics in the disease per se, and point to the loss of the fine positioning of mitochondria in the axons of RGCs as a possible explanation for their predominant degeneration in ADOA.

Is CFTR 621+3 A>G a cystic fibrosis causing mutation?

The 621+3 A>G variant of the CFTR gene was initially detected in four Greek patients with a severe form of cystic fibrosis, and it is reported to impair CFTR mRNA splicing. We present three lines of evidence that argue against the pathogenicity of this variant. First, its allelic frequency in the Italian population was 0.4%. Even considering the lowest value in the confidence interval we would expect 10% of Italian CF patients to be heterozygotes for this variant, whereas it has been reported only in one patient (0.04% of Italian CF patients). Second, expression of the 621+3 A>G variant in HeLa cells using a hybrid minigene showed that 39.5+/-1.1% of transcripts were correctly spliced, indicating that its effects on mRNA splicing are similar to those of the CFTR intron 8 5T variant, associated with congenital bilateral absence of vas deferens (CBAVD), but not with CF. Third, we have identified an asymptomatic individual who harbored the 621+3 A>G variant in trans with the Q552X mutation. Because 621+3 A>G is often included in population-screening programs, this information is critical to provide adequate counseling to patients. Further work should be aimed at investigating whether this variant may have a role in CBAVD or atypical CF.

X-linked brachytelephalangic chondrodysplasia punctata: a simple trait that is not so simple.

Brachytelephalangic chondrodysplasia punctata (CDPX1) is an X-linked recessive disorder caused by mutations in the arylsulfatase E (ARSE) gene, characterized by the presence of stippled epiphyses on radiograms in infancy and early childhood. Other features include hypoplasia of the midface and of the nasal bone, short stature, brachytelephalangy, and ectopic calcifications. Patients display marked clinical variability and there is no clear genotype-phenotype correlation. We report on a 14-month-old boy who presented with respiratory stridor due to tracheal calcifications. He had mild midface hypoplasia and brachytelephalangy, but lacked other features of CDPX1, such as short stature and epiphyseal stippling. Analysis of ARSE detected a deletion involving exons 7-10. His maternal grandfather harbored the same defect but lacked any clinical manifestation. These findings underscore two important points. First, the absence of stippled epiphyses on radiograms should not be considered an exclusion criteria for ARSE mutation screening in patients with other features of the disease, especially after the neonatal period. Second, counseling to parents of affected children should be cautious because although the theoretical risk of inheriting the ARSE mutation is 50% for every male child of a carrier mother, it is not possible to determine whether he will develop features of CDPX1 and the eventual severity of symptoms. The actual risk of developing the disease is probably lower than 50%. Conversely, normal prenatal sonography does not rule out potentially severe complications such as tracheal stenosis.

Vitamin K2 cannot substitute Coenzyme Q10 as electron carrier in the mitochondrial respiratory chain of mammalian cells.

Coenzyme Q10 (CoQ10) deficiencies are a group of heterogeneous conditions that respond to ubiquinone administration if treated soon after the onset of symptoms. However, this treatment is only partially effective due to its poor bioavailability. We tested whether vitamin K2, which was reported to act as a mitochondrial electron carrier in D. melanogaster, could mimic ubiquinone function in human CoQ10 deficient cell lines, and in yeast carrying mutations in genes required for coenzyme Q6 (CoQ6) biosynthesis. We found that vitamin K2, despite entering into mitochondria, restored neither electron flow in the respiratory chain, nor ATP synthesis. Conversely, coenzyme Q4 (CoQ4), an analog of CoQ10 with a shorter isoprenoid side chain, could efficiently substitute its function. Given its better solubility, CoQ4 could represent an alternative to CoQ10 in patients with both primary and secondary CoQ10 deficiencies.

Functional characterization of human COQ4, a gene required for Coenzyme Q10 biosynthesis.

Defects in genes involved in coenzyme Q (CoQ) biosynthesis cause primary CoQ deficiency, a severe multisystem disorders presenting as progressive encephalomyopathy and nephropathy. The COQ4 gene encodes an essential factor for biosynthesis in Saccharomyces cerevisiae. We have identified and cloned its human ortholog, COQ4, which is located on chromosome 9q34.13, and is transcribed into a 795 base-pair open reading frame, encoding a 265 amino acid (aa) protein (Isoform 1) with a predicted N-terminal mitochondrial targeting sequence. It shares 39% identity and 55% similarity with the yeast protein. Coq4 protein has no known enzymatic function, but may be a core component of multisubunit complex required for CoQ biosynthesis. The human transcript is detected in Northern blots as a approximately 1.4 kb single band and is expressed ubiquitously, but at high levels in liver, lung, and pancreas. Transcription initiates at multiple sites, located 333-23 nucleotides upstream of the ATG. A second group of transcripts originating inside intron 1 of the gene encodes a 241 aa protein, which lacks the mitochondrial targeting sequence (isoform 2). Expression of GFP-fusion proteins in HeLa cells confirmed that only isoform 1 is targeted to mitochondria. The functional significance of the second isoform is unknown. Human COQ4 isoform 1, expressed from a multicopy plasmid, efficiently restores both growth in glycerol, and CoQ content in COQ4(null) yeast strains. Human COQ4 is an interesting candidate gene for patients with isolated CoQ(10) deficiency.

Argininosuccinate lyase deficiency: mutational spectrum in Italian patients and identification of a novel ASL pseudogene.

Argininosuccinic aciduria (ASAuria) is an inborn error of metabolism caused by mutations in the argininosuccinate lyase (ASL) gene, which leads to the accumulation of argininosuccinic acid (ASA) in body fluids and severe hyperammonemia. A severe neonatal form and a milder late-onset variant are described. We report a novel ASL pseudogene located in the centromeric region of chromosome 7, 14 novel mutations in the ASL gene, and a novel intronic polymorphism found in a cohort of Italian patients. Our approach relied exclusively on genomic DNA analysis. We found seven missense mutations, two nonsense, three small insertions/deletions, and two splicing mutations. Only two patients harbored previously described mutations, and among the novel variants only two were present in more than one kindred. The pathogenicity of the splicing mutations was demonstrated by a functional splicing assay that employed a hybrid minigene. We also performed molecular modeling using the reported three-dimensional structure of ASL to predict the functional consequences of the missense mutations. There was no genotype-phenotype correlation. Patients with neonatal onset display developmental delay and seizures despite adequate metabolic control. Moreover, hepatomegaly, fibrosis, and abnormal liver function tests are common complications in these patients, but not in patients with the late infancy form. We stress the importance of mutation analysis in patients with ASAuria, to confirm the clinical diagnosis, and to perform DNA-based prenatal diagnosis in future pregnancies of these families.

Low frequency of VHL gene mutations in young individuals with polycythemia and high serum erythropoietin.

In one out of six young individuals with polycythemia and high erythropoietin levels we found a heterozygous VHL gene mutation (430G->A;Gly144Arg). The man's unaffected mother and sister carry the same mutation. No other VHL genomic or expression alterations were found. In one other patient different genetic conditions were found.

Primary coenzyme Q10 deficiency presenting as fatal neonatal multiorgan failure.

Coenzyme Q10 deficiency is a clinically and genetically heterogeneous disorder, with manifestations that may range from fatal neonatal multisystem failure, to adult-onset encephalopathy. We report a patient who presented at birth with severe lactic acidosis, proteinuria, dicarboxylic aciduria, and hepatic insufficiency. She also had dilation of left ventricle on echocardiography. Her neurological condition rapidly worsened and despite aggressive care she died at 23 h of life. Muscle histology displayed lipid accumulation. Electron microscopy showed markedly swollen mitochondria with fragmented cristae. Respiratory-chain enzymatic assays showed a reduction of combined activities of complex I+III and II+III with normal activities of isolated complexes. The defect was confirmed in fibroblasts, where it could be rescued by supplementing the culture medium with 10 µM coenzyme Q10. Coenzyme Q10 levels were reduced (28% of controls) in these cells. We performed exome sequencing and focused the analysis on genes involved in coenzyme Q10 biosynthesis. The patient harbored a homozygous c.545T>G, p.(Met182Arg) alteration in COQ2, which was validated by functional complementation in yeast. In this case the biochemical and morphological features were essential to direct the genetic diagnosis. The parents had another pregnancy after the biochemical diagnosis was established, but before the identification of the genetic defect. Because of the potentially high recurrence risk, and given the importance of early CoQ10 supplementation, we decided to treat with CoQ10 the newborn child pending the results of the biochemical assays. Clinicians should consider a similar management in siblings of patients with CoQ10 deficiency without a genetic diagnosis.

Complexity of the 5'UTR region of the CLCN5 gene: eleven 5'UTR ends are differentially expressed in the human kidney.

BACKGROUND: Dent disease 1 represents a hereditary disorder of renal tubular epithelial function associated with mutations in the CLCN5 gene that encoded the ClC-5 Cl-/H+ antiporter. All of the reported disease-causing mutations are localized in the coding region except for one recently identified in the 5'UTR region of a single patient. This finding highlighted the possible role for genetic variability in this region in the pathogenesis of Dent disease 1.The structural complexity of the CLCN5 5'UTR region has not yet been fully characterized. To date 6 different 5' alternatively used exons--1a, 1b, 1b1 and I-IV with an alternatively spliced exon II (IIa, IIb)--have been described, but their significance and differential expression in the human kidney have not been investigated. Therefore our aim was to better characterize the CLCN5 5'UTR region in the human kidney and other tissues. METHODS: To clone more of the 5' end portion of the human CLCN5 cDNA, total human kidney RNA was utilized as template and RNA ligase-mediated rapid amplification of cDNA 5' ends was applied.The expression of the different CLCN5 isoforms was studied in the kidney, leucocytes and in different tissues by quantitative comparative RT/PCR and Real--Time RT/PCR. RESULTS: Eleven transcripts initiating at 3 different nucleotide positions having 3 distinct promoters of varying strength were identified. Previously identified 5'UTR isoforms were confirmed, but their ends were extended. Six additional 5'UTR ends characterized by the presence of new untranslated exons (c, V and VI) were also identified. Exon c originates exon c.1 by alternative splicing. The kidney uniquely expresses all isoforms, and the isoform containing exon c appears kidney specific. The most abundant isoforms contain exon 1a, exon IIa and exons 1b1 and c. ORF analysis predicts that all isoforms except 3 encode for the canonical 746 amino acid ClC-5 protein. CONCLUSIONS: Our results confirm the structural complexity of the CLCN5 5'UTR region. Characterization of this crucial region could allow a clear genetic classification of a greater number of Dent disease patients, but also provide the basis for highlighting some as yet unexplored functions of the ClC-5 proton exchanger.