Mutations in NDUFB11, encoding a complex I component of the mitochondrial respiratory chain, cause microphthalmia with linear skin defects syndrome.

Microphthalmia with linear skin defects (MLS) syndrome is an X-linked male-lethal disorder also known as MIDAS (microphthalmia, dermal aplasia, and sclerocornea). Additional clinical features include neurological and cardiac abnormalities. MLS syndrome is genetically heterogeneous given that heterozygous mutations in HCCS or COX7B have been identified in MLS-affected females. Both genes encode proteins involved in the structure and function of complexes III and IV, which form the terminal segment of the mitochondrial respiratory chain (MRC). However, not all individuals with MLS syndrome carry a mutation in either HCCS or COX7B. The majority of MLS-affected females have severe skewing of X chromosome inactivation, suggesting that mutations in HCCS, COX7B, and other as-yet-unidentified X-linked gene(s) cause selective loss of cells in which the mutated X chromosome is active. By applying whole-exome sequencing and filtering for X-chromosomal variants, we identified a de novo nonsense mutation in NDUFB11 (Xp11.23) in one female individual and a heterozygous 1-bp deletion in a second individual, her asymptomatic mother, and an affected aborted fetus of the subject's mother. NDUFB11 encodes one of 30 poorly characterized supernumerary subunits of NADH:ubiquinone oxidoreductase, known as complex I (cI), the first and largest enzyme of the MRC. By shRNA-mediated NDUFB11 knockdown in HeLa cells, we demonstrate that NDUFB11 is essential for cI assembly and activity as well as cell growth and survival. These results demonstrate that X-linked genetic defects leading to the complete inactivation of complex I, III, or IV underlie MLS syndrome. Our data reveal an unexpected role of cI dysfunction in a developmental phenotype, further underscoring the existence of a group of mitochondrial diseases associated with neurocutaneous manifestations.

RASopathy-associated CBL germline mutations cause aberrant ubiquitylation and trafficking of EGFR.

Noonan syndrome, a congenital disorder comprising a characteristic face, short stature, heart defects, learning difficulties, and a predisposition to malignancies, is caused by heterozygous germline mutations in genes encoding components of RAS-MAPK signaling pathways. Mutations in the CBL tumor suppressor gene have been reported in patients with a Noonan syndrome-like phenotype. CBL encodes a multivalent adaptor protein with ubiquitin ligase activity, which promotes ubiquitylation and vesicle-mediated internalization and degradation of the epidermal growth factor (EGF) receptor (EGFR). We investigated the functional consequences of disease-associated CBL amino acid changes p.K382E, p.D390Y, and p.R420Q on ligand-induced EGFR trafficking. Expression of CBL(K382E), CBL(D390Y), or CBL(R420Q) in COS-7 cells resulted in increased levels of surface EGFR and reduced amounts of intracellular EGFR; both consequences indicate ineffective EGFR internalization. Accordingly, receptor-mediated uptake of EGF was decreased. Furthermore, the p.K382E, p.D390Y, and p.R420Q lesions impaired CBL-mediated EGFR ubiquitylation and degradation. Together, these data indicate that pathogenic CBL mutations severely affect vesicle-based EGFR trafficking. Since we detected enhanced ERK phosphorylation in cells expressing mutant CBL, we conclude that aberrant EGFR trafficking contributes to augmented RAS-MAPK signaling, the common trait of Noonan syndrome and related RASopathies. Thus, our data suggest that EGFR trafficking is a novel disease-relevant regulatory level in the RASopathy network.

The 5-phosphatase OCRL mediates retrograde transport of the mannose 6-phosphate receptor by regulating a Rac1-cofilin signalling module.

Mutations in the OCRL gene encoding the phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) 5-phosphatase OCRL cause Lowe syndrome (LS), which is characterized by intellectual disability, cataracts and selective proximal tubulopathy. OCRL localizes membrane-bound compartments and is implicated in intracellular transport. Comprehensive analysis of clathrin-mediated endocytosis in fibroblasts of patients with LS did not reveal any difference in trafficking of epidermal growth factor, low density lipoprotein or transferrin, compared with normal fibroblasts. However, LS fibroblasts displayed reduced mannose 6-phosphate receptor (MPR)-mediated re-uptake of the lysosomal enzyme arylsulfatase B. In addition, endosome-to-trans Golgi network (TGN) transport of MPRs was decreased significantly, leading to higher levels of cell surface MPRs and their enrichment in enlarged, retromer-positive endosomes in OCRL-depleted HeLa cells. In line with the higher steady-state concentration of MPRs in the endosomal compartment in equilibrium with the cell surface, anterograde transport of the lysosomal enzyme, cathepsin D was impaired. Wild-type OCRL counteracted accumulation of MPR in endosomes in an activity-dependent manner, suggesting that PI(4,5)P(2) modulates the activity state of proteins regulated by this phosphoinositide. Indeed, we detected an increased amount of the inactive, phosphorylated form of cofilin and lower levels of the active form of PAK3 upon OCRL depletion. Levels of active Rac1 and RhoA were reduced or enhanced, respectively. Overexpression of Rac1 rescued both enhanced levels of phosphorylated cofilin and MPR accumulation in enlarged endosomes. Our data suggest that PI(4,5)P(2) dephosphorylation through OCRL regulates a Rac1-cofilin signalling cascade implicated in MPR trafficking from endosomes to the TGN.

De novo desmin-mutation N116S is associated with arrhythmogenic right ventricular cardiomyopathy.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited heart muscle disease, frequently accompanied by sudden cardiac death and terminal heart failure. Genotyping of ARVC patients might be used for palliative treatment of the affected family. We genotyped a cohort of 22 ARVC patients referred to molecular genetic screening in our heart center for mutations in the desmosomal candidate genes JUP, DSG2, DSC2, DSP and PKP2 known to be associated with ARVC. In 43% of the cohort, we found disease-associated sequence variants. In addition, we screened for desmin mutations and found a novel desmin-mutation p.N116S in a patient with ARVC and terminal heart failure, which is located in segment 1A of the desmin rod domain. The mutation leads to the aggresome formation in cardiac and skeletal muscle without signs of an overt clinical myopathy. Cardiac aggresomes appear to be prominent, especially in the right ventricle of the heart. Viscosimetry and atomic force microscopy of the desmin wild-type and N116S mutant isolated from recombinant Escherichia coli revealed severe impairment of the filament formation, which was supported by transfections in SW13 cells. Thus, the gene coding for desmin appears to be a novel ARVC gene, which should be included in molecular genetic screening of ARVC patients.

Phenotypic and molecular insights into CASK-related disorders in males.

BACKGROUND: Heterozygous loss-of-function mutations in the X-linked CASK gene cause progressive microcephaly with pontine and cerebellar hypoplasia (MICPCH) and severe intellectual disability (ID) in females. Different CASK mutations have also been reported in males. The associated phenotypes range from nonsyndromic ID to Ohtahara syndrome with cerebellar hypoplasia. However, the phenotypic spectrum in males has not been systematically evaluated to date. METHODS: We identified a CASK alteration in 8 novel unrelated male patients by targeted Sanger sequencing, copy number analysis (MLPA and/or FISH) and array CGH. CASK transcripts were investigated by RT-PCR followed by sequencing. Immunoblotting was used to detect CASK protein in patient-derived cells. The clinical phenotype and natural history of the 8 patients and 28 CASK-mutation positive males reported previously were reviewed and correlated with available molecular data. RESULTS: CASK alterations include one nonsense mutation, one 5-bp deletion, one mutation of the start codon, and five partial gene deletions and duplications; seven were de novo, including three somatic mosaicisms, and one was familial. In three subjects, specific mRNA junction fragments indicated in tandem duplication of CASK exons disrupting the integrity of the gene. The 5-bp deletion resulted in multiple aberrant CASK mRNAs. In fibroblasts from patients with a CASK loss-of-function mutation, no CASK protein could be detected. Individuals who are mosaic for a severe CASK mutation or carry a hypomorphic mutation still showed detectable amount of protein. CONCLUSIONS: Based on eight novel patients and all CASK-mutation positive males reported previously three phenotypic groups can be distinguished that represent a clinical continuum: (i) MICPCH with severe epileptic encephalopathy caused by hemizygous loss-of-function mutations, (ii) MICPCH associated with inactivating alterations in the mosaic state or a partly penetrant mutation, and (iii) syndromic/nonsyndromic mild to severe ID with or without nystagmus caused by CASK missense and splice mutations that leave the CASK protein intact but likely alter its function or reduce the amount of normal protein. Our findings facilitate focused testing of the CASK gene and interpreting sequence variants identified by next-generation sequencing in cases with a phenotype resembling either of the three groups.