RRM1 variants cause a mitochondrial DNA maintenance disorder via impaired de novo nucleotide synthesis

Genetic diseases; Mitochondria; Molecular pathologyEnfermedades genéticas; Mitocondrias; Patología molecularMalalties genètiques; Mitocondris; Patologia molecularMitochondrial DNA (mtDNA) depletion/deletions syndromes (MDDS) encompass a clinically and etiologically heterogenous group of mitochondrial disorders caused by impaired mtDNA maintenance. Among the most frequent causes of MDDS are defects in nucleoside/nucleotide metabolism, which is critical for synthesis and homeostasis of the deoxynucleoside triphosphate (dNTP) substrates of mtDNA replication. A central enzyme for generating dNTPs is ribonucleotide reductase, a critical mediator of de novo nucleotide synthesis composed of catalytic RRM1 subunits in complex with RRM2 or p53R2. Here, we report 5 probands from 4 families who presented with ptosis and ophthalmoplegia as well as other clinical manifestations and multiple mtDNA deletions in muscle. We identified 3 RRM1 loss-of-function variants, including a dominant catalytic site variant (NP_001024.1: p.N427K) and 2 homozygous recessive variants at p.R381, which has evolutionarily conserved interactions with the specificity site. Atomistic molecular dynamics simulations indicate mechanisms by which RRM1 variants affect protein structure. Cultured primary skin fibroblasts of probands manifested mtDNA depletion under cycling conditions, indicating impaired de novo nucleotide synthesis. Fibroblasts also exhibited aberrant nucleoside diphosphate and dNTP pools and mtDNA ribonucleotide incorporation. Our data reveal that primary RRM1 deficiency and, by extension, impaired de novo nucleotide synthesis are causes of MDDS.This work was supported by Department of Defense Focused Program Award W81XWH2010807 (to MH), NIH research grant P01 HD32062 (to MH), and NIH grant 35 GM139453 (to JF). MH is supported by the Arturo Estopinan TK2 Research Fund, Nicholas Nunno Foundation, JDM Fund for Mitochondrial Research, Shuman Mitochondrial Disease Fund, the Marriott Mitochondrial Disease Clinic Research Fund from the J. Willard and Alice S. Marriott Foundation, and NIH grant U54 NS078059. Work in Newcastle upon Tyne was supported by the Wellcome Centre for Mitochondrial Research (203105/Z/16/Z), Medical Research Council International Centre for Genomic Medicine in Neuromuscular Disease (MR/S005021/1), UK NIHR Biomedical Research Centre in Age and Age Related Diseases award to the Newcastle upon Tyne Hospitals NHS Foundation, the Lily Foundation, and the UK National Health Service Highly Specialised Service for Rare Mitochondrial Disorders. RWT receives financial support from the Pathological Society. EWS was funded by a Medical Research Council PhD studentship. This work used the Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by National Science Foundation grant ACI-1548562. JBGC is supported by grant BIO210070 from XSEDE. The authors thank the patients and their families for collaborating in this study and Saba Tadesse for technical support of mitochondrial respiratory chain enzyme activities. We also thank the Genome Technology Center at the Radboud University Medical Center and BGI Copenhagen for WES technical support

RRM1 variants cause a mitochondrial DNA maintenance disorder via impaired de novo nucleotide synthesis

Mitochondrial DNA (mtDNA) depletion/deletions syndromes (MDDS) encompass a clinically and etiologically heterogenous group of mitochondrial disorders caused by impaired mtDNA maintenance. Among the most frequent causes of MDDS are defects in nucleoside/nucleotide metabolism, which is critical for synthesis and homeostasis of the deoxynucleoside triphosphate (dNTP) substrates of mtDNA replication. A central enzyme for generating dNTPs is ribonucleotide reductase, a critical mediator of de novo nucleotide synthesis composed of catalytic RRM1 subunits in complex with RRM2 or p53R2. Here, we report 5 probands from 4 families who presented with ptosis and ophthalmoplegia as well as other clinical manifestations and multiple mtDNA deletions in muscle. We identified 3 RRM1 loss-of-function variants, including a dominant catalytic site variant (NP_001024.1: p.N427K) and 2 homozygous recessive variants at p.R381, which has evolutionarily conserved interactions with the specificity site. Atomistic molecular dynamics simulations indicate mechanisms by which RRM1 variants affect protein structure. Cultured primary skin fibroblasts of probands manifested mtDNA depletion under cycling conditions, indicating impaired de novo nucleotide synthesis. Fibroblasts also exhibited aberrant nucleoside diphosphate and dNTP pools and mtDNA ribonucleotide incorporation. Our data reveal that primary RRM1 deficiency and, by extension, impaired de novo nucleotide synthesis are causes of MDDS

Orthogonal projections and bootstrap resampling procedures in the study of infraspecific variation

The effect of an increase in quantitative continuous characters resulting from indeterminate growth upon the analysis of population differentiation was investigated using, as an example, a set of continuous characters measured as distance variables in 10 populations of a rodent species. The data before and after correction for allometric size effects using orthogonal projections were analyzed with a parametric bootstrap resampling procedure applied to canonical variate analysis. The variance component of the distance measures attributable to indeterminate growth within the populations was found to be substantial, although the ordination of the populations was not affected, as evidenced by the relative and absolute positions of the centroids. The covariance pattern of the distance variables used to infer the nature of the morphological differences was strongly influenced by indeterminate growth. The uncorrected data produced a misleading picture of morphological differentiation by indicating that groups of populations differed in size. However, the data corrected for allometric effects clearly demonstrated that populations differed morphologically both in size and shape. These results are discussed in terms of the analysis of morphological differentiation among populations and the definition of infraspecific geographic units.<br>A influência do aumento em caracteres quantitativos contínuos devido ao crescimento indeterminado sobre a análise de diferenciação entre populações foi investigado utilizando como exemplo um conjunto de dados de variáveis craniométricas em 10 populações de uma espécie de roedor. Dois conjuntos de dados, um não corrigido para o efeito alométrico do tamanho e um outro corrigido para o efeito alométrico do tamanho utilizando um método de projeção ortogonal, foram analisados por um procedimento "bootstrap" de reamostragem aplicado à análise de variáveis canônicas. O componente de variância devido ao crescimento indeterminado dentro das populações foi significativo para a maioria das medidas de distâncias, o que não influenciou a ordenação das populações, conforme evidenciado pela posição relativa dos centróides. O padrão de covariância entre as variáveis de distância que foi utilizado para inferir a natureza das diferenças morfológicas foi, no entanto, fortemente influenciado pela variação nas medidas de distâncias dentro das populações. O conjunto de dados não corrigido resultou em uma interpretação errônea sobre a natureza da diferenciação morfológica, sugerindo que as populações diferiram somente em tamanho. O conjunto de dados corrigido para o efeito alométrico, por sua vez, demonstrou claramente que as populações diferiram, não somente no tamanho, mas também na forma. Os resultados são discutidos em termos da diferenciação das populações em forma e tamanho no contexto da definição das unidades geográficas infraspecíficas