Interaction between methionine synthase isoforms and MMACHC: characterization in cblG-variant, cblG and cblC inherited causes of megaloblastic anaemia

The cblG and cblC disorders of cobalamin (Cbl) metabolism are two inherited causes of megaloblastic anaemia. In cblG, mutations in methionine synthase (MTR) decrease conversion of hydroxocobalamin (HOCbl) to methylcobalamin, while in cblC, mutations in MMACHC disrupt formation of cob(II)alamin (detected as HOCbl). Cases with undetectable methionine synthase (MS) activity are extremely rare and classified as ‘cblG-variant'. In four ‘cblG-variant' cases, we observed a decreased conversion of cyanocobalamin to HOCbl that is also seen in cblC cases. To explore this observation, we studied the gene defects, splicing products and expression of MS, as well as MS/MMACHC protein interactions in cblG-variant, cblG, cblC and control fibroblasts. We observed a full-size MS encoded by MTR-001 and a 124 kDa truncated MS encoded by MTR-201 in cblG, cblC, control fibroblasts and HEK cells, but only the MTR-201 transcript and inactive truncated MS in cblG-variant cells. Co-immunoprecipitation and proximity ligation assay showed interaction between truncated MS and MMACHC in cblG-variant cells. This interaction decreased 2.2, 1.5 and 5.0-fold in the proximity ligation assay of cblC cells with p.R161Q and p.R206W mutations, and HEK cells with knock down expression of MS by siRNA, respectively, when compared with control cells. In 3D modelling and docking analysis, both truncated and full-size MS provide a loop anchored to MMACHC, which makes contacts with R-161 and R-206 residues. Our data suggest that the interaction of MS with MMACHC may play a role in the regulation of the cellular processing of Cbls that is required for Cbl cofactor synthesi

Publisher Correction: A PRDX1 mutant allele causes a MMACHC secondary epimutation in cblC patients

The original version of this Article contained an error in the title, which was incorrectly given as 'APRDX1 mutant allele causes a MMACHC secondary epimutation in cblC patients'. This has now been corrected in both the PDF and HTML versions of the Article to read 'A PRDX1 mutant allele causes a MMACHC secondary epimutation in cblC patients'

Next generation sequencing to discover genes for Mendelian disorders

Cobalamin is an essential vitamin in mammals. In cells, adenosylcobalamin and methylcobalamin act as cofactors to enzymes methylmalonyl CoA mutase and methionine synthase, respectively. Inborn errors of Cbl metabolism are rare Mendelian disorders associated with hematological and neurological manifestations, and elevations of methylmalonic acid and/or homocysteine in the blood and urine. These disorders have traditionally been exclusively diagnosed by somatic cell complementation analysis. 131 DNA samples from patients with elevated methylmalonic acid and no diagnosis following somatic cell complementation analysis were analyzed using a 24 gene extended cobalamin metabolism next generation sequencing based panel. This study identified two or more variants in a single gene in 16/131 patients, with eight patients having pathogenic findings, one a finding of uncertain significance, and seven benign findings. Of the patients with pathogenic findings, five had mutations in ACSF3, two in SUCLG1 and one in TCN2. It was suspected that the Cbl gene panel would not provide a diagnosis for one patient with elevated methylmalonic acid and homocysteine, who was suspected to have an inborn error of cobalamin metabolism. He had decreased incorporation of [14C] propionate and 5-methyltetrahydrofolate, but could not be diagnosed as any known complementation groups. Indeed, whole exome sequencing (WES) revealed that he did not have mutations in any of the genes known to be associated with Cbl metabolism. WES data identified c.851T>G (p.L284*) and c.1019C>T (p.T340I) in ZNF143 as causative for this patient's disease. ZNF143 is a transcription factor whose role in Cbl metabolism has not been previously studied. The patient had a unique cellular phenotype of accumulation of TC-Cbl in his fibroblasts. qRT-PCR analysis revealed low MMACHC expression levels both in patient fibroblasts, and in control fibroblasts incubated with ZNF143 siRNA. The work described in this thesis was performed as part of the Rare Disease Collaboration for Autosomal Loci (RaDiCAL), which is an initiative that aims to discover DNA variants causing Mendelian diseases. In addition to the identification of variants causing disease, this thesis describes RaDiCAL's informed consent and consideration for inclusion of the right not to know.Cobalamine est une vitamine essentielle chez les mammifères. Dans les cellules, l'adénosulcobalamine et la méthylcobalamine agissent comme cofacteurs pour les enzymes méthylmalonyl-CoA et méthionine synthase respectivement. Les erreurs innées du métabolisme de la Cobalamine sont de rares maladies héréditaire mendéliennes qui se manifestent par des complications hématologiques et neurologiques, ainsi que par une augmentation du taux d'acide méthylmalonique et/ou d'homocystéine dans le sang et l'urine. Traditionnellement, ces maladies génétiques sont exclusivement diagnostiquées par une analyse de complémentation des cellules somatiques. 131 échantillons d'ADN provenant de patients non-diagnostiqués après une analyse de complémentation des cellules somatiques et avec un taux élevé d'acide méthylmalonique ont été analysés utilisant une plateforme de séquençage nouvelle génération contenant les 24 gènes du métabolisme de la cobalamine. Cette analyse a identifié deux variantes ou plus dans un seul gène chez 16/131 patients. De ces 16 patients, huit avaient des résultats pathogéniques, un avait des résultats d'importance incertaine et sept démontraient des résultats bénins. Chez les patients avec des résultats pathogéniques, cinq patients possédaient une mutation dans le gène ACSF3, deux patients avaient une mutation dans le gène SUCLG1 et un patient avait une mutation dans le gène TCN2. Toutefois, la plateforme de gènes du métabolisme de cobalamine n'a pas fourni de diagnostic pour un patient ayant un taux élevé d'acide méthylmalonique et d'homocystéine et qui était soupçonné d'avoir une erreur de métabolisme de cobalamine. Ce patient avait une incorporation de [14C] propionate et 5-méthyltétrahydrofolate, mais ne pouvait pas être diagnostiqué selon les groupes de complémentation. Le séquençage de l'exome entier pour ce patient n'a révélé aucune mutation dans les gènes associés au métabolisme de cobalamine. Cependant, les données du séquençage de l'exome entier ont identifié deux mutations, c.851T>G (p.L284*) et c.1019C>T (p.T340I), dans le gène ZNF143 comme étant la cause de la maladie chez ce patient. ZNF143 est un facteur de transcription qui n'a jusqu'à présent pas été associé avec le métabolisme de cobalamine. Ce patient avait un phénotype cellulaire unique caractérisé par l'accumulation de TC-Cbl dans ses fibroblastes. Une analyse qRT-PCR a démontré une faible expression du gène MMACHC dans les fibroblastes de ce patient ainsi que dans des fibroblastes témoins incubés avec des ZNF143 pARNi. Le travail décrit dans cette thèse a été réalisé dans le cadre du projet collaboratif "Rare Disease Collaboration for Autosomal Loci (RaDiCAL)", qui vise a identifier des variantes d'ADN causant des maladies héréditaires mendéliennes. En plus d'identifier des variantes d'ADN causant des maladies, cette thèse décrit les formulaires de consentement développés pour le projet RaDiCAL incluant l'option de refuser une prise de connaissance

A RaDiCAL gene hunt

في السنوات العديدة الماضية، شرعت الاتحادات للأمراض النادرة في استهداف اكتشاف الجينات المسببة للأمراض في الأمراض المندلية، باستخدام أساليب تسلسل الجيل القادم. وعلى الرغم من نجاح هذه المبادرات الواسعة التطبيق، لم يتعرف الباحثون على المسببات الجينية للكثير من الأمراض. يدرس “التعاون للأمراض النادرة لمواضع الصبغيات الجسدية” (راديكال) أندر الأمراض، التي قد لا يتوفر بها سوى مستلفت واحد، في سبيل التعرف على الجينات المفترَضة المسببة للأمراض. تستعرض هذه المقالة الكيفية التي تعامل بها التعاون للأمراض النادرة لمواضع الصبغيات الجسدية مع بعض التحديات لاستحداث وثائق الموافقة المسبقة المطلوبة للمشاركين الدوليين. كما أنها تأخذ في الاعتبار، الموضوع الناشئ “حق ألا يعلم” في تصميم الدراسة

Gain-of-function mutation in TRPV4 identified in patients with osteonecrosis of the femoral head

BACKGROUND: Osteonecrosis of the femoral head is a debilitating disease that involves impaired blood supply to the femoral head and leads to femoral head collapse. METHODS: We use whole-exome sequencing and Sanger sequencing to analyse a family with inherited osteonecrosis of the femoral head and fluorescent Ca(2+) imaging to functionally characterise the variant protein. RESULTS: We report a family with four siblings affected with inherited osteonecrosis of the femoral head and the identification of a c.2480_2483delCCCG frameshift deletion followed by a c.2486T>A substitution in one allele of the transient receptor potential vanilloid 4 (TRPV4) gene. TRPV4 encodes a Ca(2+)-permeable cation channel known to play a role in vasoregulation and osteoclast differentiation. While pathogenic TRPV4 mutations affect the skeletal or nervous systems, association with osteonecrosis of the femoral head is novel. Functional measurements of Ca(2+) influx through mutant TRPV4 channels in HEK293 cells and patient-derived dermal fibroblasts identified a TRPV4 gain of function. Analysis of channel open times, determined indirectly from measurement of TRPV4 activity within a cluster of TRPV4 channels, revealed that the TRPV4 gain of function was caused by longer channel openings. CONCLUSIONS: These findings identify a novel TRPV4 mutation implicating TRPV4 and altered calcium homeostasis in the pathogenesis of osteonecrosis while reinforcing the importance of TRPV4 in bone diseases and vascular endothelium

Interaction between methionine synthase isoforms and MMACHC: characterization in cblG-variant, cblG and cblC inherited causes of megaloblastic anaemia

The cblG and cblC disorders of cobalamin (Cbl) metabolism are two inherited causes of megaloblastic anaemia. In cblG, mutations in methionine synthase (MTR) decrease conversion of hydroxocobalamin  (HOCbl) to methylcobalamin, while in cblC, mutations in MMACHC disrupt formation of cob(II)alamin (detected as HOCbl). Cases with undetectable methionine synthase (MS) activity are extremely rare and classified as 'cblG-variant'. In four 'cblG-variant' cases, we observed a decreased conversion of cyanocobalamin to HOCbl that is also seen in cblC cases. To explore this observation, we studied the gene defects, splicing products and expression of MS, as well as MS/MMACHC protein interactions in cblG-variant, cblG, cblC and control fibroblasts. We observed a full-size MS encoded by MTR-001 and a 124 kDa truncated MS encoded by MTR-201 in cblG, cblC, control fibroblasts and HEK cells, but only the MTR-201 transcript and inactive truncated MS in cblG-variant cells. Co-immunoprecipitation and proximity ligation assay showed interaction between truncated MS and MMACHC in cblG-variant cells. This interaction decreased 2.2, 1.5 and 5.0-fold in the proximity ligation assay of cblC cells with p.R161Q and p.R206W mutations, and HEK cells with knock down expression of MS by siRNA, respectively, when compared with control cells. In 3D modelling and docking analysis, both truncated and full-size MS provide a loop anchored to MMACHC, which makes contacts with R-161 and R-206 residues. Our data suggest that the interaction of MS with MMACHC may play a role in the regulation of the cellular processing of Cbls that is required for Cbl cofactor synthesis

Epimutations in both the TESK2 and MMACHC promoters in the Epi-cblC inherited disorder of intracellular metabolism of vitamin B12

BACKGROUND: epi-cblC is a recently discovered inherited disorder of intracellular vitamin B12 metabolism associating hematological, neurological, and cardiometabolic outcomes. It is produced by an epimutation at the promoter common to CCDC163P and MMACHC, which results from an aberrant antisense transcription due to splicing mutations in the antisense PRDX1 gene neighboring MMACHC. We studied whether the aberrant transcription produced a second epimutation by encompassing the CpG island of the TESK2 gene neighboring CCDC163P. METHODS: We unraveled the methylome architecture of the CCDC163P-MMACHC CpG island (CpG:33) and the TESK2 CpG island (CpG:51) of 17 epi-cblC cases. We performed an integrative analysis of the DNA methylome profiling, transcriptome reconstruction of RNA-sequencing (RNA-seq), chromatin immunoprecipitation sequencing (ChIP-Seq) of histone H3, and transcription expression of MMACHC and TESK2. RESULTS: The PRDX1 splice mutations and activation of numerous cryptic splice sites produced antisense readthrough transcripts encompassing the bidirectional MMACHC/CCDC163P promoter and the TESK2 promoter, resulting in the silencing of both the MMACHC and TESK2 genes through the deposition of SETD2-dependent H3K36me3 marks and the generation of epimutations in the CpG islands of the two promoters. CONCLUSIONS: The antisense readthrough transcription of the mutated PRDX1 produces an epigenetic silencing of MMACHC and TESK2. We propose using the term 'epi-digenism' to define this epigenetic disorder that affects two genes. Epi-cblC is an entity that differs from cblC. Indeed, the PRDX1 and TESK2 altered expressions are observed in epi-cblC but not in cblC, suggesting further evaluating the potential consequences on cancer risk and spermatogenesis.Isite LU

A PRDX1 mutant allele causes a MMACHC secondary epimutation in cblC patients

To date, epimutations reported in man have been somatic and erased in germlines. Here, we identify a cause of the autosomal recessive cblC class of inborn errors of vitamin B12 metabolism that we name “epi-cblC”. The subjects are compound heterozygotes for a genetic mutation and for a promoter epimutation, detected in blood, fibroblasts, and sperm, at the MMACHC locus; 5-azacytidine restores the expression of MMACHC in fibroblasts. MMACHC is flanked by CCDC163P and PRDX1, which are in the opposite orientation. The epimutation is present in three generations and results from PRDX1 mutations that force antisense transcription of MMACHC thereby possibly generating a H3K36me3 mark. The silencing of PRDX1 transcription leads to partial hypomethylation of the epiallele and restores the expression of MMACHC. This example of epi-cblC demonstrates the need to search for compound epigenetic-genetic heterozygosity in patients with typical disease manifestation and genetic heterozygosity in disease-causing genes located in other gene trios