Mutations of human NARS2, encoding the mitochondrial asparaginyl-tRNA synthetase, cause nonsyndromic deafness and Leigh syndrome.

Here we demonstrate association of variants in the mitochondrial asparaginyl-tRNA synthetase NARS2 with human hearing loss and Leigh syndrome. A homozygous missense mutation ([c.637G>T; p.Val213Phe]) is the underlying cause of nonsyndromic hearing loss (DFNB94) and compound heterozygous mutations ([c.969T>A; p.Tyr323*] + [c.1142A>G; p.Asn381Ser]) result in mitochondrial respiratory chain deficiency and Leigh syndrome, which is a neurodegenerative disease characterized by symmetric, bilateral lesions in the basal ganglia, thalamus, and brain stem. The severity of the genetic lesions and their effects on NARS2 protein structure cosegregate with the phenotype. A hypothetical truncated NARS2 protein, secondary to the Leigh syndrome mutation p.Tyr323* is not detectable and p.Asn381Ser further decreases NARS2 protein levels in patient fibroblasts. p.Asn381Ser also disrupts dimerization of NARS2, while the hearing loss p.Val213Phe variant has no effect on NARS2 oligomerization. Additionally we demonstrate decreased steady-state levels of mt-tRNAAsn in fibroblasts from the Leigh syndrome patients. In these cells we show that a decrease in oxygen consumption rates (OCR) and electron transport chain (ETC) activity can be rescued by overexpression of wild type NARS2. However, overexpression of the hearing loss associated p.Val213Phe mutant protein in these fibroblasts cannot complement the OCR and ETC defects. Our findings establish lesions in NARS2 as a new cause for nonsyndromic hearing loss and Leigh syndrome.

Hypoxia-reoxygenation-induced endothelial barrier failure: role of RhoA, Rac1 and myosin light chain kinase.

Hypoxia-reoxygenation induces loss of endothelial barrier function and oedema formation, which presents a major impediment for recovery of the organ. The integrity of the endothelial barrier is highly dependent on its contractile machinery and actin dynamics, which are precisely regulated by Rho GTPases. Perturbed activities of these Rho-GTPases under hypoxia-reoxygenation lead to derangement of the actin cytoskeleton and therefore may affect the integrity of the endothelial barrier. The aim of the present study was to analyse the role of these GTPases in regulating endothelial barrier function during hypoxia-reoxygenation in cultured porcine aortic endothelial cells and isolated perfused rat hearts. Hypoxia-reoxygenation induced an increase in albumin permeability of endothelial monolayers accompanied by an activation of the endothelial contractile machinery, derangement of the actin cytoskeleton and loss of VE-cadherin from cellular junctions. Inhibition of contractile activation with ML-7 partially protected against hypoxia-reoxygenation-induced hyperpermeability. Likewise, reoxygenation caused an increase in RhoA and a reduction in Rac1 activity accompanied by enhanced stress fibre formation and loss of peripheral actin. Inhibition of RhoA/rho kinase (Rock) signalling with RhoA or Rock inhibitors led to a complete depolymerisation and derangement of the actin cytoskeleton and worsened hypoxia-reoxygenation-induced hyperpermeability. Activation of Rac1 using a cAMP analogue, 8-CPT-O-Me-cAMP, which specifically activates Epac/Rap1 signalling, restored peripheral localisation of actin and VE-cadherin at cellular junctions and abrogated reoxygenation-induced hyperpermeability. Similar results were reproduced in isolated saline-perfused rat hearts. These data show that activation of Rac1 but not the inhibition of RhoA preserves endothelial integrity against reoxygenation-induced loss of barrier function.

Mutation spectrum of MYO7A and evaluation of a novel nonsyndromic deafness DFNB2 allele with residual function.

Recessive mutations of MYO7A, encoding unconventional myosin VIIA, can cause either a deaf-blindness syndrome (type 1 Usher syndrome; USH1B) or nonsyndromic deafness (DFNB2). In our study, deafness segregating as a recessive trait in 24 consanguineous families showed linkage to markers for the DFNB2/USH1B locus on chromosome 11q13.5. A total of 23 of these families segregate USH1 due to 17 homozygous mutant MYO7A alleles, of which 14 are novel. One family segregated nonsyndromic hearing loss DFNB2 due to a novel three-nucleotide deletion in an exon of MYO7A (p.E1716del) encoding a region of the tail domain. We hypothesized that DFNB2 alleles of MYO7A have residual myosin VIIA. To address this question we investigated the effects of several mutant alleles by making green fluorescent protein (GFP) tagged cDNA expression constructs containing engineered mutations of mouse Myo7a at codons equivalent to pathogenic USH1B and DFNB2 alleles of human MYO7A. We show that in transfected mouse hair cells an USH1B mutant GFP-myosin VIIa does not localize properly to inner ear hair cell stereocilia. However, a GFP-myosin VIIa protein engineered to have an equivalent DFNB2 mutation to p.E1716del localizes correctly in transfected mouse hair cells. This finding is consistent with the hypothesis that p.E1716del causes a less severe phenotype (DFNB2) than the USH1B-associated alleles because the resulting protein retains some degree of normal function.

The autosomal recessive nonsyndromic deafness locus DFNB72 is located on chromosome 19p13.3.

We ascertained three consanguineous Pakistani families (PKDF291, PKDF335 and PKDF793) segregating nonsyndromic recessive hearing loss. The hearing loss segregating in PKDF335 and PKDF793 is moderate to severe, whereas it is profound in PKDF291. The maximum two-point LOD scores are 3.01 (D19S1034), 3.85 (D19S894) and 3.71 (D19S894) for PKDF291, PKDF335 and PKDF793, respectively. Haplotype analyses of the three families define a 1.16 Mb region of overlap of the homozygous linkage intervals bounded by markers D19S216 (20.01 cM) and D19S1034 (20.75 cM). These results define a novel locus, DFNB72, on chromosome 19p13.3. There are at least 22 genes in the 1.16 Mb interval, including PTPRS, ZNRF4 and CAPS. We identified no pathogenic variants in the exons and flanking intronic sequences of these three genes in affected members of the DFNB72 families. DFNB72 is telomeric to DFNB68, the only other known deafness locus with statistically significant support for linkage to chromosome 19p.

Mutations in TRIOBP, which encodes a putative cytoskeletal-organizing protein, are associated with nonsyndromic recessive deafness.

In seven families, six different mutant alleles of TRIOBP on chromosome 22q13 cosegregate with autosomal recessive nonsyndromic deafness. These alleles include four nonsense (Q297X, R788X, R1068X, and R1117X) and two frameshift (D1069fsX1082 and R1078fsX1083) mutations, all located in exon 6 of TRIOBP. There are several alternative splice isoforms of this gene, the longest of which, TRIOBP-6, comprises 23 exons. The linkage interval for the deafness segregating in these families includes DFNB28. Genetic heterogeneity at this locus is suggested by three additional families that show significant evidence of linkage of deafness to markers on chromosome 22q13 but that apparently have no mutations in the TRIOBP gene.