Mitochondrial genome variant m.3250T>C as a possible risk factor for mitochondrial cardiomyopathy.

The MT-TL1 gene codes for the mitochondrial leucine transfer RNA (tRNALeu(UUR) ) necessary for mitochondrial translation. Pathogenic variants in the MT-TL1 gene result in mitochondriopathy in humans. The m.3250T>C variant in the MT-TL1 gene has been previously associated with exercise intolerance and mitochondrial myopathy, yet disease classification for this variant has not been consistently reported. Molecular studies suggest the m.3250T>C variant does not alter tRNALeu(UUR) structure but may have a modest impact on aminoacylation capacity. However, functional studies are limited. Our study aimed to further define the clinical presentation, inheritance pattern, and molecular pathology of the m.3250T>C variant. Families with the m.3250T>C variant were recruited from the Mitochondrial Disease Clinic at Cincinnati Children's Hospital Medical Center and GeneDx laboratory database. Affected individuals most frequently presented with cardiac findings, exercise intolerance, and muscle weakness. Hypertrophic cardiomyopathy was the most frequent cardiac finding. Many asymptomatic individuals had homoplasmic or near homoplasmic levels of the m.3250T>C variant, suggesting the penetrance is incomplete. Patient-derived fibroblasts demonstrated lowered ATP production and increased levels of reactive oxygen species. Our results demonstrate that the m.3250T>C variant exhibits incomplete penetrance and may be a possible cause of cardiomyopathy by impacting cellular respiration in mitochondria.

A dominant autoinflammatory disease caused by non-cleavable variants of RIPK1.

Activation of RIPK1 controls TNF-mediated apoptosis, necroptosis and inflammatory pathways1. Cleavage of human and mouse RIPK1 after residues D324 and D325, respectively, by caspase-8 separates the RIPK1 kinase domain from the intermediate and death domains. The D325A mutation in mouse RIPK1 leads to embryonic lethality during mouse development2,3. However, the functional importance of blocking caspase-8-mediated cleavage of RIPK1 on RIPK1 activation in humans is unknown. Here we identify two families with variants in RIPK1 (D324V and D324H) that lead to distinct symptoms of recurrent fevers and lymphadenopathy in an autosomal-dominant manner. Impaired cleavage of RIPK1 D324 variants by caspase-8 sensitized patients' peripheral blood mononuclear cells to RIPK1 activation, apoptosis and necroptosis induced by TNF. The patients showed strong RIPK1-dependent activation of inflammatory signalling pathways and overproduction of inflammatory cytokines and chemokines compared with unaffected controls. Furthermore, we show that expression of the RIPK1 mutants D325V or D325H in mouse embryonic fibroblasts confers not only increased sensitivity to RIPK1 activation-mediated apoptosis and necroptosis, but also induction of pro-inflammatory cytokines such as IL-6 and TNF. By contrast, patient-derived fibroblasts showed reduced expression of RIPK1 and downregulated production of reactive oxygen species, resulting in resistance to necroptosis and ferroptosis. Together, these data suggest that human non-cleavable RIPK1 variants promote activation of RIPK1, and lead to an autoinflammatory disease characterized by hypersensitivity to apoptosis and necroptosis and increased inflammatory response in peripheral blood mononuclear cells, as well as a compensatory mechanism to protect against several pro-death stimuli in fibroblasts.

Novel pathogenic COX20 variants causing dysarthria, ataxia, and sensory neuropathy.

COX20/FAM36A encodes a mitochondrial complex IV assembly factor important for COX2 activation. Only one homozygous COX20 missense mutation has been previously described in two separate consanguineous families. We report four subjects with features that include childhood hypotonia, areflexia, ataxia, dysarthria, dystonia, and sensory neuropathy. Exome sequencing in all four subjects identified the same novel COX20 variants. One variant affected the splice donor site of intron-one (c.41A>G), while the other variant (c.157+3G>C) affected the splice donor site of intron-two. cDNA and protein analysis indicated that no full-length cDNA or protein was generated. These subjects expand the phenotype associated with COX20 deficiency.

Aberrant Drp1-mediated mitochondrial division presents in humans with variable outcomes.

Mitochondrial dynamics, including mitochondrial division, fusion and transport, are integral parts of mitochondrial and cellular function. DNM1L encodes dynamin-related protein 1 (Drp1), a member of the dynamin-related protein family that is required for mitochondrial division. Several de novo mutations in DNM1L are associated with a range of disease states. Here we report the identification of five patients with pathogenic or likely pathogenic variants of DNM1L, including two novel variants. Interestingly, all of the positions identified in these Drp1 variants are fully conserved among all members of the dynamin-related protein family that are involved in membrane division and organelle division events. This work builds upon and expands the clinical spectrum associated with Drp1 variants in patients and their impact on mitochondrial division in model cells.

Biallelic Mutations in MRPS34 Lead to Instability of the Small Mitoribosomal Subunit and Leigh Syndrome.

The synthesis of all 13 mitochondrial DNA (mtDNA)-encoded protein subunits of the human oxidative phosphorylation (OXPHOS) system is carried out by mitochondrial ribosomes (mitoribosomes). Defects in the stability of mitoribosomal proteins or mitoribosome assembly impair mitochondrial protein translation, causing combined OXPHOS enzyme deficiency and clinical disease. Here we report four autosomal-recessive pathogenic mutations in the gene encoding the small mitoribosomal subunit protein, MRPS34, in six subjects from four unrelated families with Leigh syndrome and combined OXPHOS defects. Whole-exome sequencing was used to independently identify all variants. Two splice-site mutations were identified, including homozygous c.321+1G>T in a subject of Italian ancestry and homozygous c.322-10G>A in affected sibling pairs from two unrelated families of Puerto Rican descent. In addition, compound heterozygous MRPS34 mutations were identified in a proband of French ancestry; a missense (c.37G>A [p.Glu13Lys]) and a nonsense (c.94C>T [p.Gln32∗]) variant. We demonstrated that these mutations reduce MRPS34 protein levels and the synthesis of OXPHOS subunits encoded by mtDNA. Examination of the mitoribosome profile and quantitative proteomics showed that the mitochondrial translation defect was caused by destabilization of the small mitoribosomal subunit and impaired monosome assembly. Lentiviral-mediated expression of wild-type MRPS34 rescued the defect in mitochondrial translation observed in skin fibroblasts from affected subjects, confirming the pathogenicity of MRPS34 mutations. Our data establish that MRPS34 is required for normal function of the mitoribosome in humans and furthermore demonstrate the power of quantitative proteomic analysis to identify signatures of defects in specific cellular pathways in fibroblasts from subjects with inherited disease.

De Novo Mutations in CHD4, an ATP-Dependent Chromatin Remodeler Gene, Cause an Intellectual Disability Syndrome with Distinctive Dysmorphisms.

Chromodomain helicase DNA-binding protein 4 (CHD4) is an ATP-dependent chromatin remodeler involved in epigenetic regulation of gene transcription, DNA repair, and cell cycle progression. Also known as Mi2ß, CHD4 is an integral subunit of a well-characterized histone deacetylase complex. Here we report five individuals with de novo missense substitutions in CHD4 identified through whole-exome sequencing and web-based gene matching. These individuals have overlapping phenotypes including developmental delay, intellectual disability, hearing loss, macrocephaly, distinct facial dysmorphisms, palatal abnormalities, ventriculomegaly, and hypogonadism as well as additional findings such as bone fusions. The variants, c.3380G>A (p.Arg1127Gln), c.3443G>T (p.Trp1148Leu), c.3518G>T (p.Arg1173Leu), and c.3008G>A, (p.Gly1003Asp) (GenBank: NM_001273.3), affect evolutionarily highly conserved residues and are predicted to be deleterious. Previous studies in yeast showed the equivalent Arg1127 and Trp1148 residues to be crucial for SNF2 function. Furthermore, mutations in the same positions were reported in malignant tumors, and a de novo missense substitution in an equivalent arginine residue in the C-terminal helicase domain of SMARCA4 is associated with Coffin Siris syndrome. Cell-based studies of the p.Arg1127Gln and p.Arg1173Leu mutants demonstrate normal localization to the nucleus and HDAC1 interaction. Based on these findings, the mutations potentially alter the complex activity but not its formation. This report provides evidence for the role of CHD4 in human development and expands an increasingly recognized group of Mendelian disorders involving chromatin remodeling and modification.

De novo missense variants in PPP2R5D are associated with intellectual disability, macrocephaly, hypotonia, and autism.

Protein phosphatase 2A (PP2A) is a heterotrimeric protein serine/threonine phosphatase and is involved in a broad range of cellular processes. PPP2R5D is a regulatory B subunit of PP2A and plays an important role in regulating key neuronal and developmental regulation processes such as PI3K/AKT and glycogen synthase kinase 3 beta (GSK3ß)-mediated cell growth, chromatin remodeling, and gene transcriptional regulation. Using whole-exome sequencing (WES), we identified four de novo variants in PPP2R5D in a total of seven unrelated individuals with intellectual disability (ID) and other shared clinical characteristics, including autism spectrum disorder, macrocephaly, hypotonia, seizures, and dysmorphic features. Among the four variants, two have been previously reported and two are novel. All four amino acids are highly conserved among the PP2A subunit family, and all change a negatively charged acidic glutamic acid (E) to a positively charged basic lysine (K) and are predicted to disrupt the PP2A subunit binding and impair the dephosphorylation capacity. Our data provides further support for PPP2R5D as a genetic cause of ID.

Analyzing the role of receptor internalization in the regulation of melanin-concentrating hormone signaling.

The regulation of appetite is complex, though our understanding of the process is improving. The potential role for the melanin-concentrating hormone (MCH) signaling pathway in the treatment of obesity is being explored by many. It was hypothesized that internalization of MCH receptors would act to potently desensitize cells to MCH. Despite potent desensitization of ERK signaling by MCH in BHK-570 cells, we were unable to observe MCH-mediated internalization of MCH receptor 1 (MCHR1) by fluorescence microscopy. A more quantitative approach using a cell-based ELISA indicated only 15% of receptors internalized, which is much lower than that reported in the literature. When ß-arrestins were overexpressed in our system, removal of receptors from the cell surface was facilitated and signaling to a leptin promoter was diminished, suggesting that internalization of MCHR1 is sensitive to cellular ß-arrestin levels. A dominant-negative GRK construct completely inhibited loss of receptors from the cell surface in response to MCH, suggesting that the internalization observed is phosphorylation-dependent. Since desensitization of MCH-mediated ERK signaling did not correlate with significant loss of MCHR1 from the cell surface, we hypothesize that in this model system regulation of MCH signaling may be the result of segregation of receptors from signaling components at the plasma membrane.