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MRPL39 encodes one of 52 proteins comprising the large subunit of the mitochondrial ribosome (mitoribosome). In conjunction with 30 proteins in the small subunit, the mitoribosome synthesizes the 13 subunits of the mitochondrial oxidative phosphorylation (OXPHOS) system encoded by mitochondrial Deoxyribonucleic acid (DNA). We used multi-omics and gene matching to identify three unrelated individuals with biallelic variants in MRPL39 presenting with multisystem diseases with severity ranging from lethal, infantile-onset (Leigh syndrome spectrum) to milder with survival into adulthood. Clinical exome sequencing of known disease genes failed to diagnose these patients; however quantitative proteomics identified a specific decrease in the abundance of large but not small mitoribosomal subunits in fibroblasts from the two patients with severe phenotype. Re-analysis of exome sequencing led to the identification of candidate single heterozygous variants in mitoribosomal genes MRPL39 (both patients) and MRPL15. Genome sequencing identified a shared deep intronic MRPL39 variant predicted to generate a cryptic exon, with transcriptomics and targeted studies providing further functional evidence for causation. The patient with the milder disease was homozygous for a missense variant identified through trio exome sequencing. Our study highlights the utility of quantitative proteomics in detecting protein signatures and in characterizing gene-disease associations in exome-unsolved patients. We describe Relative Complex Abundance analysis of proteomics data, a sensitive method that can identify defects in OXPHOS disorders to a similar or greater sensitivity to the traditional enzymology. Relative Complex Abundance has potential utility for functional validation or prioritization in many hundreds of inherited rare diseases where protein complex assembly is disrupted.

De novo variants in QRICH1 (Glutamine-rich protein 1) has recently been reported in 11 individuals with intellectual disability (ID). The function of QRICH1 is largely unknown but it is likely to play a key role in the unfolded response of endoplasmic reticulum stress through transcriptional control of proteostasis. In this study, we present 27 additional individuals and delineate the clinical and molecular spectrum of the individuals (n = 38) with QRICH1 variants. The main clinical features were mild to moderate developmental delay/ID (71%), nonspecific facial dysmorphism (92%) and hypotonia (39%). Additional findings included poor weight gain (29%), short stature (29%), autism spectrum disorder (29%), seizures (24%) and scoliosis (18%). Minor structural brain abnormalities were reported in 52% of the individuals with brain imaging. Truncating or splice variants were found in 28 individuals and 10 had missense variants. Four variants were inherited from mildly affected parents. This study confirms that heterozygous QRICH1 variants cause a neurodevelopmental disorder including short stature and expands the phenotypic spectrum to include poor weight gain, scoliosis, hypotonia, minor structural brain anomalies, and seizures. Inherited variants from mildly affected parents are reported for the first time, suggesting variable expressivity.

Overlapping clinical phenotypes and an expanding breadth and complexity of genomic associations are a growing challenge in the diagnosis and clinical management of Mendelian disorders. The functional consequences and clinical impacts of genomic variation may involve unique, disorder-specific, genomic DNA methylation episignatures. In this study, we describe 19 novel episignature disorders and compare the findings alongside 38 previously established episignatures for a total of 57 episignatures associated with 65 genetic syndromes. We demonstrate increasing resolution and specificity ranging from protein complex, gene, sub-gene, protein domain, and even single nucleotide-level Mendelian episignatures. We show the power of multiclass modeling to develop highly accurate and disease-specific diagnostic classifiers. This study significantly expands the number and spectrum of disorders with detectable DNA methylation episignatures, improves the clinical diagnostic capabilities through the resolution of unsolved cases and the reclassification of variants of unknown clinical significance, and provides further insight into the molecular etiology of Mendelian conditions.

Aims: This study aimed to investigate associations between renal and extrarenal manifestations of mitochondrial diseases and their natural history as well as predictors of renal disease severity and overall disease outcome. The secondary aim was to generate a protocol of presymptomatic assessment and monitoring of renal function in patients with a defined mitochondrial disease. Methods: A multicenter, retrospective cohort study was performed by the Mitochondrial Clinical and Research Network (MCRN). Patients of any age with renal manifestations associated with a genetically verified mitochondrial disease were included from 8 expert European centers specializing in mitochondrial diseases: Gothenburg, Oulu, Copenhagen, Bergen, Helsinki, Stockholm, Rotterdam, and Barcelona. Results: Of the 36 patients included, two-thirds had mitochondrial DNA-associated disease. Renal manifestations were the first sign of mitochondrial disease in 19%, and renal involvement was first identified by laboratory tests in 57% of patients. Acute kidney injury occurred in 19% of patients and was the first sign of renal disease in the majority of these. The most common renal manifestation was chronic kidney disease (75% with stage 2 or greater), followed by tubulopathy (44.4%), the latter seen mostly among patients with single large-scale mitochondrial DNA deletions. Acute kidney injury and tubulopathy correlated with worse survival outcome. The most common findings on renal imaging were increased echogenicity and renal dysplasia/hypoplasia. Renal histology revealed focal segmental glomerulosclerosis, nephrocalcinosis, and nephronophthisis. Conclusion: Acute kidney injury is a distinct renal phenotype in patients with mitochondrial disease. Our results highlight the importance to recognize renal disease as a sign of an underlying mitochondrial disease. Acute kidney injury and tubulopathy are 2 distinct indicators of poor survival in patients with mitochondrial diseases.

Objective: To study the impact of gender, puberty, and pregnancy on the expression of POLG disease, one of the most common mitochondrial diseases known. Methods: Clinical, laboratory, and genetic data were collected retrospectively from 155 patients with genetically confirmed POLG disease recruited from seven European countries. We used the available data to study the impact of gender, puberty, and pregnancy on disease onset and deterioration. Results: We found that disease onset early in life was common in both sexes but there was also a second peak in females around the time of puberty. Further, pregnancy had a negative impact with 10 of 14 women (71%) experiencing disease onset or deterioration during pregnancy. Interpretation: Gender clearly influences the expression of POLG disease. While onset very early in life was common in both males and females, puberty in females appeared associated both with disease onset and increased disease activity. Further, both disease onset and deterioration, including seizure aggravation and status epilepticus, appeared to be associated with pregnancy. Thus, whereas disease activity appears maximal early in life with no subsequent peaks in males, both menarche and pregnancy appear associated with disease onset or worsening in females. This suggests that hormonal changes may be a modulating factor.

Summary Background: Variants in POLG are one of the most common causes of inherited mitochondrial disease. Phenotypic classification of POLG disease has evolved haphazardly making it complicated and difficult to implement in everyday clinical practise. The aim of our study was to simplify the classification and facilitate better clinical recognition. Methods: A multinational, retrospective study using data from 155 patients with POLG variants recruited from seven European countries. Results: We describe the spectrum of clinical features associated with POLG variants in the largest known cohort of patients. While clinical features clearly form a continuum, stratifying patients simply according to age of onset—onset prior to age 12 years; onset between 12 and 40 years and onset after the age of 40 years, permitted us to identify clear phenotypic and prognostic differences. Prior to 12 years of age, liver involvement (87%), seizures (84%), and feeding difficulties (84%) were the major features. For those with onset between 12 and 40 years, ataxia (90%), peripheral neuropathy (84%), and seizures (71%) predominated, while for those with onset over 40 years, ptosis (95%), progressive external ophthalmoplegia (89%), and ataxia (58%) were the major clinical features. The earlier the onset the worse the prognosis. Patients with epilepsy and those with compound heterozygous variants carried significantly worse prognosis. Conclusions: Based on our data, we propose a simplified POLG disease classification, which can be used to guide diagnostic investigations and predict disease course.

Variants in PTCH2 have been described to be associated with Nevoid Basal Cell Carcinoma Syndrome (NBCCS). We report a family with a healthy female who is homozygous for a frameshift variant, c.269delG, p.(Gly90Alafs*4), in PTCH2 and her heterozygous daughter. The variant predicts a frameshift and a premature stop codon. A summary of reported heterozygous individuals with germline PTCH2 variants along with the existence of a healthy homozygous individual question whether variants in PTCH2 are associated with NBCCS.

Background: Mitochondrial acyl-CoA dehydrogenase family member 9 (ACAD9) is essential for the assembly of mitochondrial respiratory chain complex I. Disease causing biallelic variants in ACAD9 have been reported in individuals presenting with lactic acidosis and cardiomyopathy. Results: We describe the genetic, clinical and biochemical findings in a cohort of 70 patients, of whom 29 previously unpublished. We found 34 known and 18 previously unreported variants in ACAD9. No patients harbored biallelic loss of function mutations, indicating that this combination is unlikely to be compatible with life. Causal pathogenic variants were distributed throughout the entire gene, and there was no obvious genotype-phenotype correlation. Most of the patients presented in the first year of life. For this subgroup the survival was poor (50% not surviving the first 2 years) comparing to patients with a later presentation (more than 90% surviving 10 years). The most common clinical findings were cardiomyopathy (85%), muscular weakness (75%) and exercise intolerance (72%). Interestingly, severe intellectual deficits were only reported in one patient and severe developmental delays in four patients. More than 70% of the patients were able to perform the same activities of daily living when compared to peers. Conclusions: Our data show that riboflavin treatment improves complex I activity in the majority of patient-derived fibroblasts tested. This effect was also reported for most of the treated patients and is mirrored in the survival data. In the patient group with disease-onset below 1 year of age, we observed a statistically-significant better survival for patients treated with riboflavin.

Contains fulltext : 153932.pdf (publisher's version ) (Closed access), Protein synthesis in mitochondria is initiated by formylmethionyl-tRNA(Met) (fMet-tRNA(Met)), which requires the activity of the enzyme MTFMT to formylate the methionyl group. We investigated the molecular consequences of mutations in MTFMT in patients with Leigh syndrome or cardiomyopathy. All patients studied were compound heterozygotes. Levels of MTFMT in patient fibroblasts were almost undetectable by immunoblot analysis, and BN-PAGE analysis showed a combined oxidative phosphorylation (OXPHOS) assembly defect involving complexes I, IV and V. The synthesis of only a subset of mitochondrial polypeptides (ND5, ND4, ND1, COXII) was decreased, whereas all others were translated at normal or even increased rates. Expression of the wild-type cDNA rescued the biochemical phenotype when MTFMT was expressed near control levels, but overexpression produced a dominant-negative phenotype, completely abrogating assembly of the OXPHOS complexes, suggesting that MTFMT activity must be tightly regulated. fMet-tRNA(Met) was almost undetectable in control cells and absent in patient cells by high-resolution northern blot analysis, but accumulated in cells overexpressing MTFMT. Newly synthesized COXI was under-represented in complex IV immunoprecipitates from patient fibroblasts, and two-dimensional BN-PAGE analysis of newly synthesized mitochondrial translation products showed an accumulation of free COXI. Quantitative mass spectrophotometry of an N-terminal COXI peptide showed that the ratio of formylated to unmodified N-termini in the assembled complex IV was approximately 350:1 in controls and 4:1 in patient cells. These results show that mitochondrial protein synthesis can occur with inefficient formylation of methionyl-tRNA(Met), but that assembly of complex IV is impaired if the COXI N-terminus is not formylated.

Background: Leigh syndrome is a progressive neurodegenerative disorder, associated with primary or secondary dysfunction of the mitochondrial oxidative phosphorylation. Despite the fact that Leigh syndrome is the most common phenotype of mitochondrial disorders in children, longitudinal natural history data is missing. This study was undertaken to assess the phenotypic and genotypic spectrum of patients with Leigh syndrome, characterise the clinical course and identify predictors of survival in a large cohort of patients. Methods: This is a retrospective study of patients with Leigh syndrome that have been followed at eight centers specialising in mitochondrial diseases in Europe; Gothenburg, Rotterdam, Helsinki, Copenhagen, Stockholm, Brussels, Bergen and Oulu. Results: A total of 130 patients were included (78 males; 52 females), of whom 77 patients had identified pathogenic mutations. The median age of disease onset was 7 months, with 80.8% of patients presenting by the age of 2 years. The most common clinical features were abnormal motor findings, followed by abnormal ocular findings. Epileptic seizures were reported in 40% of patients. Approximately 44% of patients experienced acute exacerbations requiring hospitalisation during the previous year, mainly due to infections. The presence of pathological signs at birth and a history of epileptic seizures were associated with higher occurrence of acute exacerbations and/or relapses. Increased lactate in the cerebrospinal fluid was significantly correlated to a more severe disease course, characterised by early onset before 6 months of age, acute exacerbations and/or relapses, as well as brainstem involvement. 39% of patients had died by the age of 21 years, at a median age of 2.4 years. Disease onset before 6 months of age, failure to thrive, brainstem lesions on neuroimaging and intensive care treatment were significantly associated with poorer survival. Conclusions: This is a multicenter study performed in a larg

Osteogenesis imperfecta is a genetic condition characterized by bone fragility and recurrent fractures, which in the large majority of patients are caused by defects in the production of type I collagen. Mutations in the gene encoding bone morphogenetic protein 1 (BMP1, also known as procollagen C-endopeptidase) have been associated with osteogenesis imperfecta in two sib pairs. In this report, we describe an additional patient with osteogenesis imperfecta with normal bone density and a recurrent, homozygous c.34G > C mutation in BMP1. Western blot analysis of dermal fibroblasts from this patient showed decreased protein levels of the two alternatively spliced products of BMP1 and abnormal cleavage of the C-terminal propeptide of type I procollagen. In addition, fluorescence and electron microscopy showed impaired assembly of type I collagen fibrils in the extracellular matrix of cultured fibroblasts derived from two patients: the patient described here and a previously reported patient with a homozygous BMP1 c.747C > G mutation. We conclude that BMP1 is essential for human type I collagen fibrilogenesis. © 2014 Wiley Periodicals, Inc.

Contains fulltext : 96286.pdf (publisher's version ) (Closed access), BACKGROUND: This study investigated a girl with Leigh syndrome born to first-cousin parents of Pakistani descent with an isolated respiratory chain complex I deficiency in muscle and fibroblasts. Her early development was delayed, and from age 2 years she started losing motor abilities. Cerebral MRI showed basal ganglia lesions typical of Leigh syndrome. METHODS AND RESULTS: A genome-wide search for homozygosity was performed with the Affymetrix GeneChip 50K Xba array. The analysis revealed several homozygous regions. Three candidate genes were identified, and in one of the genes, NDUFA12, a homozygous c.178C-->T mutation leading to a premature stop codon (p.Arg60X) was found. Western blot analysis showed absence of NDUFA12 protein in patient fibroblasts and functional complementation by a baculovirus system showed restoration of complex I activity. CONCLUSION: NDUFA12 mutations are apparently not a frequent cause of complex I deficiency, since mutations were not found by screening altogether 122 complex I deficient patients in two different studies. NDUFA12 encodes an accessory subunit of complex I and is a paralogue of NDUFAF2. Despite the complete absence of NDUFA12 protein, a fully assembled and enzymatically active complex I could be found, albeit in reduced amounts. This suggests that NDUFA12 is required either at a late step in the assembly of complex I, or in the stability of complex I.

Contains fulltext : 89368.pdf (publisher's version ) (Closed access), We investigated the genetic basis for a global and uniform decrease in mitochondrial translation in fibroblasts from patients in two unrelated pedigrees who developed Leigh syndrome, optic atrophy, and ophthalmoplegia. Analysis of the assembly of the oxidative phosphorylation complexes showed severe decreases of complexes I, IV, and V and a smaller decrease in complex III. The steady-state levels of mitochondrial mRNAs, tRNAs, and rRNAs were not reduced, nor were those of the mitochondrial translation elongation factors or the protein components of the mitochondrial ribosome. Using homozygosity mapping, we identified a 1 bp deletion in C12orf65 in one patient, and DNA sequence analysis showed a different 1 bp deletion in the second patient. Both mutations predict the same premature stop codon. C12orf65 belongs to a family of four mitochondrial class I peptide release factors, which also includes mtRF1a, mtRF1, and Ict1, all characterized by the presence of a GGQ motif at the active site. However, C12orf65 does not exhibit peptidyl-tRNA hydrolase activity in an in vitro assay with bacterial ribosomes. We suggest that it might play a role in recycling abortive peptidyl-tRNA species, released from the ribosome during the elongation phase of translation.

Contains fulltext : 81004.pdf (publisher's version ) (Closed access), Contiguous gene syndromes affecting the mitochondrial oxidative phosphorylation system have been rarely reported. Here, we describe a patient with apparent mitochondrial encephalomyopathy accompanied by several unusual features, including dysmorphism and hepatopathy, caused by a homozygous triple gene deletion on chromosome 5. The deletion encompassed the NDUFAF2, ERCC8 and ELOVL7 genes, encoding complex I assembly factor 2 (also known as human B17.2L), a protein of the transcription-coupled nucleotide excision repair (TC-NER) machinery, and a putative elongase of very long-chain fatty acid synthesis, respectively. Detailed evaluation of cultured skin fibroblasts revealed disturbed complex I assembly, depolarization of the mitochondrial membrane, elevated cellular NAD(P)H level, increased superoxide production and defective TC-NER. ELOVL7 mRNA was not detectable in these cells and no alterations in fatty acid synthesis were found. By means of baculoviral complementation we were able to restore the aberrations, thereby establishing causative links between genotype and cell-physiological phenotype. This first chromosomal microdeletion illustrates that beside primary defects in mitochondrial genes also additional genes possibly contribute to the disease phenotype, providing an additional explanation for the broad clinical symptoms associated with these disorders.

Professor Gösta Samuelson samlade trycksaker; 64