Bi-allelic variants in COL3A1 encoding the ligand to GPR56 are associated with cobblestone-like cortical malformation, white matter changes and cerebellar cysts.

BACKGROUND: Collagens are one of the major constituents of the pial membrane, which plays a crucial role in neuronal migration and cortical lamination during brain development. Type III procollagen, the chains of which are encoded by COL3A1, is the ligand of the G protein-coupled receptor 56 (GPR56), also known as adhesion G protein-coupled receptor G1. Bi-allelic mutations in GPR56 give rise to cobblestone-like malformation, white matter changes and cerebellar dysplasia. This report shows that bi-allelic mutations in COL3A1 are associated with a similar phenotype. METHODS: Exome analysis was performed in a family consisting of two affected and two non-affected siblings. Brain imaging studies of this family and of two previously reported individuals with bi-allelic mutations in COL3A1 were reviewed. Functional assays were performed on dermal fibroblasts. RESULTS: Exome analysis revealed a novel homozygous variant c.145C>G (p.Pro49Ala) in exon 2 of COL3A1. Brain MRI in the affected siblings as well as in the two previously reported individuals with bi-allelic COL3A1 mutations showed a brain phenotype similar to that associated with mutations in GPR56. CONCLUSION: Homozygous or compound heterozygous mutations in COL3A1 are associated with cobblestone-like malformation in all three families reported to date. The variability of the phenotype across patients suggests that genetic alterations in distinct domains of type III procollagen can lead to different outcomes. The presence of cobblestone-like malformation in patients with bi-allelic COL3A1 mutations emphasises the critical role of the type III collagen-GPR56 axis and the pial membrane in the regulation of brain development and cortical lamination.

I-PV: a CIRCOS module for interactive protein sequence visualization.

SUMMARY: Today's genome browsers and protein databanks supply vast amounts of information about proteins. The challenge is to concisely bring together this information in an interactive and easy to generate format. AVAILABILITY AND IMPLEMENTATION: We have developed an interactive CIRCOS module called i-PV to visualize user supplied protein sequence, conservation and SNV data in a live presentable format. I-PV can be downloaded from http://www.i-pv.org. CONTACT: ibrahim.tanyalcin@i-pv.org, itanyalc@vub.ac.be or support@i-pv.org SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Convert your favorite protein modeling program into a mutation predictor: "MODICT".

BACKGROUND: Predict whether a mutation is deleterious based on the custom 3D model of a protein. RESULTS: We have developed MODICT, a mutation prediction tool which is based on per residue RMSD (root mean square deviation) values of superimposed 3D protein models. Our mathematical algorithm was tested for 42 described mutations in multiple genes including renin (REN), beta-tubulin (TUBB2B), biotinidase (BTD), sphingomyelin phosphodiesterase-1 (SMPD1), phenylalanine hydroxylase (PAH) and medium chain Acyl-Coa dehydrogenase (ACADM). Moreover, MODICT scores corresponded to experimentally verified residual enzyme activities in mutated biotinidase, phenylalanine hydroxylase and medium chain Acyl-CoA dehydrogenase. Several commercially available prediction algorithms were tested and results were compared. The MODICT PERL package and the manual can be downloaded from https://github.com/IbrahimTanyalcin/MODICT . CONCLUSIONS: We show here that MODICT is capable tool for mutation effect prediction at the protein level, using superimposed 3D protein models instead of sequence based algorithms used by POLYPHEN and SIFT.

Characterization of CoQ10 biosynthesis in fibroblasts of patients with primary and secondary CoQ10 deficiency.

Primary coenzyme Q10 (CoQ10) deficiencies are associated with mutations in genes encoding enzymes important for its biosynthesis and patients are responsive to CoQ10 supplementation. Early treatment allows better prognosis of the disease and therefore, early diagnosis is desirable. The complex phenotype and genotype and the frequent secondary CoQ10 deficiencies make it difficult to achieve a definitive diagnosis by direct quantification of CoQ10. We developed a non-radioactive methodology for the quantification of CoQ10 biosynthesis in fibroblasts that allows the identification of primary deficiencies. Fibroblasts were incubated 72 h with 28 µmol/L (2)H3-mevalonate or 1.65 mmol/L (13)C6-p-hydroxybenzoate. The newly synthesized (2)H3- and (13)C6- labelled CoQ10 were analysed by high performance liquid chromatography-tandem mass spectrometry. The mean and the reference range for (13)C6-CoQ10 and (2)H3-CoQ10 biosynthesis were 0.97 (0.83-1.1) and 0.13 (0.09-0.17) nmol/Unit of citrate synthase, respectively. We validated the methodology through the study of one patient with COQ2 mutations and six patients with CoQ10 deficiency secondary to other inborn errors of metabolism. Afterwards we investigated 16 patients' fibroblasts and nine showed decreased CoQ10 biosynthesis. Therefore, the next step is to study the COQ genes in order to reach a definitive diagnosis in these nine patients. In the patients with normal rates the deficiency is probably secondary. In conclusion, we have developed a non-invasive non-radioactive method suitable for the detection of defects in CoQ10 biosynthesis, which offers a good tool for the stratification of patients with these treatable mitochondrial diseases.

Effect of resveratrol on cultured skin fibroblasts from patients with oxidative phosphorylation defects.

Few therapeutic options are available to patients with oxidative phosphorylation disorders. Administering pharmacological agents that are able to stimulate mitochondrial biogenesis have been put forward as a possible treatment, yet the approach remains in need of thorough testing. We investigated the effect of resveratrol in an in vitro setting. Mitochondrial enzymatic activities were tested in cultured skin fibroblasts from patients harboring a nuclear defect in either complex II or complex IV (n = 11), and in fibroblasts from healthy controls (n = 11). In the latter, preincubation with resveratrol resulted in a significant increase of citrate synthase, complex II and complex IV enzyme activity. In patients with complex II or complex IV deficiency, however, activity of the deficient complex could not be substantially augmented, and response was dependent upon the residual activity. We conclude that resveratrol is not capable of normalizing oxidative phosphorylation activities in deficient cell lines.

Systematic detection of pathogenic alu element insertions in NGS-based diagnostic screens: the BRCA1/BRCA2 example.

Pathogenic Alu element insertions are rarely reported, whereas their occurrence is expected to be much higher. Alu containing alleles are usually out-competed during the PCR process and consequently undetectable with the classical screening methods. However, with the introduction of the next generation sequencing (NGS) technology in the diagnostic field, new opportunities are emerging. NGS data for a particular genomic region can be seen as the summation of all the individual sequences (reads) obtained for that region and no longer as the mean of this sum as it is the case for traditional Sanger sequencing. Because each single read covering that region is expected to be generated from a different template molecule, the presence of one single mutant read must theoretically be sufficient to identify the mutation. However, generation and identification of mutant reads bearing Alu insertions remains challenging and several wet/dry bench parameters need to be optimized. Hereby we present the proof of principle of a NGS-based mutation screening procedure allowing the detection of inherited Alu insertions within any predefined sequence by investigating 2 cases: c.1739_1740insAlu in BRCA1 and c.156_157insAlu in BRCA2.

X chromosomal mutations and spermatogenic failure.

The X and Y chromosomes, the sex chromosomes, are important key players in germ cell development. Both chromosomes contain genes that are uniquely expressed in male spermatogenesis. Furthermore, these chromosomes are special because men only have a single copy of them. These features make the sex chromosomes interesting for studying in view of spermatogenesis defects. The role of the Y chromosome, together with the presence of Yq microdeletions, in male infertility is well established. Less well-understood are the X-linked genes, their expression patterns and potential impact on male infertility. This review provides an overview of the current knowledge on potential spermatogenesis genes that are located on the mouse and human X chromosomes. A summary is given on knock-out mice models in which X-linked genes have been shown to alter spermatogenesis, and on genes that have been studied in humans. Finally, new research areas like miRNA analysis, Genome Wide Association Studies (GWAS) and array comparative genomic hybridisation (CGH) studies are discussed. This article is part of a Special Issue entitled: Molecular Genetics of Human Reproductive Failure.

Proteomic analysis in giant axonal neuropathy: new insights into disease mechanisms.

INTRODUCTION: Giant axonal neuropathy (GAN) is a progressive hereditary disease that affects the peripheral and central nervous systems. It is characterized morphologically by aggregates of intermediate filaments in different tissues. Mutations have been reported in the gene that codes for gigaxonin. Nevertheless, the underlying molecular mechanism remains obscure. METHODS: Cell lines from 4 GAN patients and 4 controls were analyzed by iTRAQ. RESULTS: Among the dysregulated proteins were ribosomal protein L29, ribosomal protein L37, galectin-1, glia-derived nexin, and aminopeptidase N. Also, nuclear proteins linked to formin-binding proteins were found to be dysregulated. Although the major role of gigaxonin is reported to be degradation of cytoskeleton-associated proteins, the amount of 76 structural cytoskeletal proteins was unaltered. CONCLUSIONS: Several of the dysregulated proteins play a role in cytoskeletal reorganization. Based on these findings, we speculate that disturbed cytoskeletal regulation is responsible for formation of aggregates of intermediate filaments.

Fluorescence imaging of mitochondria in cultured skin fibroblasts: a useful method for the detection of oxidative phosphorylation defects.

BACKGROUND: Protons are pumped from the mitochondrial matrix via oxidative phosphorylation (OXPHOS) into the intermembrane space, creating an electric membrane potential (ΔΨ) that is used for adenosine triphosphate (ATP) production. Defects in one or more of the OXPHOS complexes are associated with a variety of clinical symptoms, often making it difficult to pinpoint the causal mutation. METHODS: In this article, a microscopic method for the quantitative evaluation of ΔΨ in cultured skin fibroblasts is described. The method using 5,5',6,6'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) fluorescence staining was tested in a selection of OXPHOS-deficient cell lines. RESULTS: A significant reduction of ΔΨ was found in the cell lines of patients with either an isolated defect in complex I, II, or IV or a combined defect (complex I + complex IV). ΔΨ was not reduced in the fibroblasts of two patients with severe complex V deficiency. Addition of the complex I inhibitor rotenone induced a significant reduction of ΔΨ and perinuclear relocalization of the mitochondria. In cells with a heteroplasmic mitochondrial DNA (mtDNA) defect, a more heterogeneous reduction of ΔΨ was detected. CONCLUSION: Our data show that imaging of ΔΨ in cultured skin fibroblasts is a useful method for the evaluation of OXPHOS functioning in cultured cell lines.

Defining the pathogenesis of the human Atp12p W94R mutation using a Saccharomyces cerevisiae yeast model.

Studies in yeast have shown that a deficiency in Atp12p prevents assembly of the extrinsic domain (F(1)) of complex V and renders cells unable to make ATP through oxidative phosphorylation. De Meirleir et al. (De Meirleir, L., Seneca, S., Lissens, W., De Clercq, I., Eyskens, F., Gerlo, E., Smet, J., and Van Coster, R. (2004) J. Med. Genet. 41, 120-124) have reported that a homozygous missense mutation in the gene for human Atp12p (HuAtp12p), which replaces Trp-94 with Arg, was linked to the death of a 14-month-old patient. We have investigated the impact of the pathogenic W94R mutation on Atp12p structure/function. Plasmid-borne wild type human Atp12p rescues the respiratory defect of a yeast ATP12 deletion mutant (Deltaatp12). The W94R mutation alters the protein at the most highly conserved position in the Pfam sequence and renders HuAtp12p insoluble in the background of Deltaatp12. In contrast, the yeast protein harboring the corresponding mutation, ScAtp12p(W103R), is soluble in the background of Deltaatp12 but not in the background of Deltaatp12Deltafmc1, a strain that also lacks Fmc1p. Fmc1p is a yeast mitochondrial protein not found in higher eukaryotes. Tryptophan 94 (human) or 103 (yeast) is located in a positively charged region of Atp12p, and hence its mutation to arginine does not alter significantly the electrostatic properties of the protein. Instead, we provide evidence that the primary effect of the substitution is on the dynamic properties of Atp12p.

Complex III staining in blue native polyacrylamide gels.

For more than a decade now blue native polyacrylamide gel electrophoresis (BN-PAGE) has been used for the study of the oxidative phosphorylation (OXPHOS) complexes. Catalytic activities of complexes I, II, IV and V can be assessed, after separation by gel electrophoresis, by incubation of the BN-PAGE gel in specific staining solutions. However, until now, a reliable staining method for testing ubiquinol cytochrome c oxidoreductase (complex III) activity by BN-PAGE gel techniques was not available. In addition, spectrophotometric methods currently in use for detection of complex III deficiency in patients are not very sensitive. Here, we describe a newly developed diagnostic method for visualization of complex III activity by direct in-gel evaluation of ubiquinol cytochrome oxidoreductase activity. We validated the method by reporting the results in six patients with previously characterised complex III defects.

Discordance for retinitis pigmentosa in two monozygotic twin pairs.

BACKGROUND: Retinitis pigmentosa (RP) is a group of genetically heterogeneous diseases with progressive degeneration of the retina. The condition can be inherited as an autosomal dominant, autosomal recessive, and X-linked trait. METHODS: We report on two female twin pairs. One twin of each pair is affected with RP, the other twin is unaffected, both clinically and functionally.Molecular analysis in both twins included zygosity determination, arrayed primer extension chip analysis for autosomal recessive and dominant RP, sequencing of the entire RPGR gene, and analysis of X-chromosome inactivation status. RESULTS: Both unrelated twin pairs were genetically identical. Of the potential pathogenetic mechanisms, skewed X-inactivation was excluded on leukocytes. Autosomal recessive RP and autosomal dominant RP arrayed primer extension chip analysis result was completely normal, excluding known mutations in known genes as the cause of disease in the affected twins. Sequencing excluded mutations in RPGR. A postzygotic recessive or dominant genetic mutation of an RP gene is not impossible. A postfertilization error as a potential cause of uniparental isodisomy is unlikely albeit not entirely impossible. CONCLUSION: The authors report on the second and third unrelated identical twin pair discordant for RP. The exact cause of the condition and the explanation of the clinical discordance remain elusive.

Cancer predisposing missense and protein truncating BARD1 mutations in non-BRCA1 or BRCA2 breast cancer families.

Fifteen years ago BRCA1 and BRCA2 were reported as high penetrant breast cancer predisposing genes. However, mutations in these genes are found in only a fraction of high risk families. BARD1 is a candidate breast cancer gene, but only a limited number of missense mutations with rather unclear pathogenic consequences have been reported.We screened 196 high risk breast cancer families for the occurrence of BARD1 variants. All genetic variants were analyzed using clinical information as well as IN SILICO predictive tools, including protein modeling. We found three candidate pathogenic mutations in seven families including a first case of a protein truncating mutation (p.Glu652fs) removing the entire second BRCT domain of BARD1. In conclusion, we provide evidence for an increased breast cancer risk associated to specific BARD1 germline mutations. However, these BARD1 mutations occur in a minority of hereditary breast cancer families.

A homozygous inactivating calcium-sensing receptor mutation, Pro339Thr, is associated with isolated primary hyperparathyroidism: correlation between location of mutations and severity of hypercalcaemia.

BACKGROUND: Inactivating mutations of the calcium-sensing receptor (CaSR), a G-protein-coupled receptor with extracellular (ECD), transmembrane (TMD) and intracellular (ICD) domains, cause familial hypocalciuric hypercalcaemia, neonatal severe primary hyperparathyroidism and occasionally primary hyperparathyroidism in adults. OBJECTIVE: To investigate a patient with typical symptomatic primary hyperparathyroidism for CaSR abnormalities. PATIENT AND DESIGN: A 51-year-old woman with primary hyperparathyroidism was investigated for CaSR abnormalities as her severe hypercalcaemia (3·75 mm) persisted after the removal of two large parathyroid adenomas and she was the daughter of normocalcaemic consanguineous parents. Following informed consent, CASR mutational analysis was undertaken using leucocyte DNA. Wild-type and mutant CaSR constructs were expressed in human embryonic kidney (HEK) 293 cells and assessed by measuring their intracellular calcium responses to changes in extracellular calcium. Clinical data were pooled with previous studies to search for genotype-phenotype correlations. RESULTS: The proband was homozygous for a Pro339Thr CaSR missense mutation, located in the ECD, and her normocalcaemic relatives were heterozygous. The mutant Thr339 CaSR had a rightward shift in its dose-response curve with a significantly higher EC(50) = 3·18 mm ± 0·19 compared to the wild-type EC(50) = 2·16 mm ± 0·1 (P < 0·01), consistent with a loss-of-function mutation. An analysis of CaSR mutations in patients with primary hyperparathyroidism revealed that those of the ECD were associated with a significantly greater hypercalcaemia that was less likely to be corrected after removal of the parathyroid tumours. CONCLUSIONS: A CaSR missense mutation causing a loss-of-receptor-function can cause symptomatic primary hyperparathyroidism in adulthood.

Giant axonal neuropathy caused by compound heterozygosity for a maternally inherited microdeletion and a paternal mutation within the GAN gene.

Different missense, nonsense and frameshift mutations in the GAN gene encoding gigaxonin have been described to cause giant axonal neuropathy, a severe early-onset progressive neurological disease with autosomal recessive inheritance. By oligonucleotide array CGH analysis, we identified a 57-131 kb microdeletion affecting this gene in a patient with developmental delay, ataxia, areflexia, macrocephaly, and strikingly frizzy hair. The microdeletion was inherited from the mother and mutation analysis revealed a paternally inherited missense mutation c.1456G>A in exon 9 on the other allele. Our findings illustrate the power of higher resolution array CGH studies and highlight the importance of considering copy number variations in autosomal recessive diseases.

Subcomplexes of mitochondrial complex V reveal mutations in mitochondrial DNA.

Complex V, site of the final step in oxidative phosphorylation, uses the proton gradient across the inner mitochondrial membrane for the production of ATP. It is a multi-subunit complex composed of a catalytic domain (F(1)) and a membrane domain (F(0)) linked by two stalks. Subcomplexes of complex V containing the F(1) domain have previously been reported in small series of patients. We report the results in tissue samples and/or cultured skin fibroblasts studied by blue native PAGE followed by activity staining in the gel. Catalytically active subcomplexes of complex V were detected in 66 tissues originating from 53 patients. In 29 of the latter (55%), a mitochondrial DNA (mtDNA) defect was identified. Twelve patients had a pathogenic point mutation in a mitochondrial tRNA, one a large mtDNA deletion, 12 showed mtDNA depletion and four had a mutation in the MT-ATP6 gene. We conclude that the presence of subcomplexes of complex V is a valuable indicator in the detection of mtDNA defects.

Lactic acidosis in a newborn with adrenal calcifications.

A patient is reported who presented in the newborn period with an unusual combination of congenital lactic acidosis and bilateral calcifications in the adrenal medulla, visible on standard abdominal x-ray and ultrasound examination. At birth, the proband was hypotonic and dystrophic. She developed respiratory insufficiency, cardiomegaly, and hepatomegaly and died at the age of 38 d. Examination of postmortem heart muscle revealed multiple areas of myocardial infarction with dystrophic calcifications. In the medulla of the adrenal glands, foci of necrosis and calcifications, and in the liver, multiple zones of necrosis and iron deposition were detected. Biochemical analysis in heart muscle revealed a decreased activity of complex IV of the oxidative phosphorylation (OXPHOS) and in liver a combined deficiency involving the complexes I, III, IV, and V. The findings were suggestive of a defect in biosynthesis of the mitochondrially encoded subunits of the OXPHOS complexes. Extensive analysis of the proband's mitochondrial DNA revealed neither pathogenic deletions and point mutations nor copy number alterations. Relative amounts of mitochondrial transcripts for the ribosomal mitochondrial 12S rRNA (12S) and mitochondrial 16S rRNA (16S) were significantly increased suggesting a compensatory mechanism involving the transcription machinery to low levels of translation. The underlying molecular defect has not been identified yet.

Clinical implementation of gene panel testing for lysosomal storage diseases.

BACKGROUND: The diagnostic workup in patients with a clinical suspicion of lysosomal storage diseases (LSD) is often difficult due to the variability in the clinical phenotype. The gold standard for diagnosis of LSDs consists of enzymatic testing. However, due to the sequential nature of this methodology and inconsistent genotype-phenotype correlations of certain LSDs, finding a diagnosis can be challenging. METHOD: We developed and clinically implemented a gene panel covering 50 genes known to cause LSDs when mutated. Over a period of 18 months, we analyzed 150 patients who were referred for LSD testing and compared these results with the data of patients who were previously enrolled in a scheme of classical biochemical testing. RESULTS: Our panel was able to determine the molecular cause of the disease in 22 cases (15%), representing an increase in diagnostic yield compared to biochemical tests developed for 21 LSDs (4.6%). We were furthermore able to redirect the diagnosis of a mucolipidosis patient who was initially suspected to be affected with galactosialidosis. Several patients were identified as being affected with neuronal ceroid lipofuscinosis, which cannot readily be detected by enzyme testing. Finally, several carriers of pathogenic mutations in LSD genes related to the disease phenotype were identified as well, thus potentially increasing the diagnostic yield of the panel as heterozygous deletions cannot be detected. CONCLUSION: We show that the implementation of a gene panel for LSD diagnostics results in an increased yield in comparison to classical biochemical testing. As the panel is able to cover a wider range of diseases, we propose to implement this methodology as a first-tier test in cases of an aspecific LSD presentation, while enzymatic testing remains the first choice in patients with a more distinctive clinical presentation. Positive panel results should however still be enzymatically confirmed whenever possible.

Do we need to search for gr/gr deletions in infertile men in a clinical setting?

BACKGROUND: Partial deletions of the AZFc region of the Y chromosome such as gr/gr deletions have been detected in infertile patients as well as in control groups. The impact of these gr/gr deletions on the etiology of male infertility remains unknown. In the present study, we investigated the presence of gr/gr deletions in Caucasian men. METHODS: gr/gr deletions were analyzed by using markers sY1291, sY1191 and sY1197 and by investigating the presence of single nucleotide variants (SNV) in DAZ and CDY1 genes in patients with azoospermia (n = 44), cryptozoospermia (n = 51) or severe oligozoospermia (n = 92). Control groups consisted of men with normal spermatogenesis on testicular biopsy (n = 33), normozoospermia (n = 278) or proven fertility (n = 83). RESULTS: We observed 20 gr/gr deletions, with eight in infertile patients (4.3%) and 12 in the control groups (3.0%), which was not significantly different. DAZ SNV analysis revealed eight different deletion patterns in patients and controls. CONCLUSIONS: In the present study, no significant differences in the frequency of gr/gr deletions between different patient and control groups were observed. We concluded that the relationship between gr/gr deletions and male infertility remains unclear and that it is too early to systematically test for gr/gr deletions for infertile couples seeking assisted reproduction treatment.

A single mutation in the GALC gene is responsible for the majority of late onset Krabbe disease patients in the Catania (Sicily, Italy) region.

A high proportion of patients with late onset forms of Krabbe disease is observed in a region north of Catania in Sicily. Molecular analysis in five families from this region shows that this condition is mainly due to a not previously described p.Gly41Ser substitution in the GALC gene that abolishes catalytic activity of the galactocerebrosidase enzyme, as shown by expression studies. Three patients were homozygous for this mutation, the other two were heterozygous, one with a frameshift mutation and one with a missense mutation on the second allele. Therefore, the mutation must be a mild one since it leads to late onset disease in all patients. In addition, it is on a unique haplotype indicating that it represents a founder mutation. This is also supported by the fact that the mutation was not found in three late onset patients from other regions in Sicily, in whom four novel mutations were identified.