Locus Heterogeneity for Waardenburg Syndrome is Predictive of Clinical Subtypes

Waardenburg syndrome (WS) is a dominantly inherited and clinically variable syndrome of deafness, pigmentary changes, and distinctive facial features. Clinically, WS type I (WS1) is differentiated from WS type II (WS2) by the high frequency of dystopia canthorum in the family. In some families, WS is caused by mutations in the PAX3 gene on chromosome 2q. We have typed microsatellite markers within and flanking PAX3 in 41 WS1 kindreds and 26 WS2 kindreds in order to estimate the proportion of families with probable mutations in PAX3 and to study the relationship between phenotypic and genotypic heterogeneity. Evaluation of heterogeneity in location scores obtained by multilocus analysis indicated that WS is linked to PAX3 in 60% of all WS families and in 100% of WS1 families. None of the WS2 families were linked. In those families in which equivocal lod scores (between −2 and +1) were found, PAX3 mutations have been identified in 5 of the 15 WS1 families but in none of the 4 WS2 families. Although preliminary studies do not suggest any association between the phenotype and the molecular pathology in 20 families with known PAX3 mutations and in four patients with chromosomal abnormalities in the vicinity of PAX3, the presence of dystopia in multiple family members is a reliable indicator for identifying families likely to have a defect in PAX3.     

MYH9-siRNA and MYH9 mutant alleles: expression in cultured cell lines and their effects upon cell structure and function

MYH9 encodes a class II nonmuscle myosin heavy chain-A (NMHC-IIA), a widely expressed 1960 amino acid polypeptide, with translated molecular weight of 220 kDa. From studies of type II myosin in invertebrates and analogy with the skeletal and smooth muscle myosin II, NMHC-IIA is considered to be involved in diverse cellular functions, including cell shape, motility and division. The current study assessed the consequences of two separate, naturally occurring MYH9 dominant mutant alleles, MYH9(R702C) and MYH9(R705H) linked to syndromic and nonsyndromic hearing loss, respectively, upon diverse NMHC-IIA related functions in two separate cultured cell lines. MYH9-siRNA-induced inhibition of NMHC-IIA in HeLa cells or HEK293 cells resulted in alterations in their shape, actin cytoskeleton and adhesion properties. However, HeLa or HEK293 cells transfected with naturally occurring MYH9 mutant alleles, MYH9(R702C) or MYH9(R705H), as well as in vitro generated deletion derivatives, MYH9(DeltaN592) or MYH9(DeltaC570), were unaffected. The effects of MYH9-siRNA-induced suppression underline the critical role of NMHC-IIA in maintenance of cell shape and adhesion. However, the results also indicate that the NMHC-IIA mutants, R702C and R705H do not inactivate or suppress the endogenous wild type NMHC-IIA within the HeLa or HEK293 cell assay system.     

LncRNA VEAL2 regulates PRKCB2 to modulate endothelial permeability in diabetic retinopathy

Long non‐coding RNAs (lncRNAs) are emerging as key regulators of endothelial cell function. Here, we investigated the role of a novel vascular endothelial‐associated lncRNA (VEAL2) in regulating endothelial permeability. Precise editing of veal2 loci in zebrafish (veal2 ^gib005Δ8/+) induced cranial hemorrhage. In vitro and in vivo studies revealed that veal2 competes with diacylglycerol for interaction with protein kinase C beta‐b (Prkcbb) and regulates its kinase activity. Using PRKCB2 as bait, we identified functional ortholog of veal2 in humans from HUVECs and named it as VEAL2. Overexpression and knockdown of VEAL2 affected tubulogenesis and permeability in HUVECs. VEAL2 was differentially expressed in choroid tissue in eye and blood from patients with diabetic retinopathy, a disease where PRKCB2 is known to be hyperactivated. Further, VEAL2 could rescue the effects of PRKCB2‐mediated turnover of endothelial junctional proteins thus reducing hyperpermeability in hyperglycemic HUVEC model of diabetic retinopathy. Based on evidence from zebrafish and hyperglycemic HUVEC models and diabetic retinopathy patients, we report a hitherto unknown VEAL2 lncRNA‐mediated regulation of PRKCB2, for modulating junctional dynamics and maintenance of endothelial permeability.     

Correlation between Waardenburg syndrome phenotype and genotype in a population of individuals with identified PAX3 mutations

Waardenburg syndrome (WS) type 1 is an autosomal dominant disorder characterized by sensorineural hearing loss, pigmentary abnormalities of the eye, hair, and skin, and dystopia canthorum. The phenotype is variable and affected individuals may exhibit only one or a combination of several of the associated features. To assess the relationship between phenotype and gene defect, clinical and genotype data on 48 families (271 WS individuals) collected by members of the Waardenburg Consortium were pooled. Forty-two unique mutations in the PAX3 gene, previously identified in these families, were grouped in five mutation categories: amino acid (AA) substitution in the paired domain, AA substitution in the homeodomain, deletion of the Ser-Thr-Pro-rich region, deletion of the homeodomain and the Ser-Thr-Pro-rich region, and deletion of the entire gene. These mutation classes are based on the structure of the PAX3 gene and were chosen to group mutations predicted to have similar defects in the gene product. Association between mutation class and the presence of hearing loss, eye pigment abnormality, skin hypopigmentation, or white forelock was evaluated using generalized estimating equations, which allowed for incorporation of a correlation structure that accounts for potential similarity among members of the same family. Odds for the presence of eye pigment abnormality, white forelock, and skin hypopigmentation were 2, 8, and 5 times greater, respectively, for individuals with deletions of the homeodomain and the Pro-Ser-Thr-rich region compared to individuals with an AA substitution in the homeodomain. Odds ratios that differ significantly from 1.0 for these traits may indicate that the gene products resulting from different classes of mutations act differently in the expression of WS. Although a suggestive association was detected for hearing loss with an odds ratio of 2.6 for AA substitution in the paired domain compared with AA substitution in the homeodomain, this odds ratio did not differ significantly from 1.0. Received: 27 July 1997 / Accepted: 9 December 1997     

Genetic mapping refines DFNB3 to 17p11.2, suggests multiple alleles of DFNB3, and supports homology to the mouse model shaker-2.

The nonsyndromic congenital recessive deafness gene, DFNB3, first identified in Bengkala, Bali, was mapped to a approximately 12-cM interval on chromosome 17. New short tandem repeats (STRs) and additional DNA samples were used to identify recombinants that constrain the DFNB3 interval to less, similar6 cM on 17p11.2. Affected individuals from Bengkala and affected members of a family with hereditary deafness who were from Bila, a village neighboring Bengkala, were homozygous for the same alleles for six adjacent STRs in the DFNB3 region and were heterozygous for other distal markers, thus limiting DFNB3 to an approximately 3-cM interval. Nonsyndromic deafness segregating in two unrelated consanguineous Indian families, M21 and I-1924, were also linked to the DFNB3 region. Haplotype analysis indicates that the DFNB3 mutations in the three pedigrees most likely arose independently and suggests that DFNB3 makes a significant contribution to hereditary deafness worldwide. On the basis of conserved synteny, mouse deafness mutations shaker-2 (sh2) and sh2J are proposed as models of DFNB3. Genetic mapping has refined sh2 to a 0.6-cM interval of chromosome 11. Three homologous genes map within the sh2 and DFNB3 intervals, suggesting that sh2 is the homologue of DFNB3.     

HDL and CER-001 Inverse-Dose Dependent Inhibition of Atherosclerotic Plaque Formation in apoE-/- Mice: Evidence of ABCA1 Down-Regulation

Objective

CER-001 is a novel engineered HDL-mimetic comprised of recombinant human apoA-I and charged phospholipids that was designed to mimic the beneficial properties of nascent pre-ß HDL. In this study, we have evaluated the dose-dependent regulation of ABCA1 expression in vitro and in vivo in the presence of CER-001 and native HDL (HDL3).

Methods and Results

CER-001 induced cholesterol efflux from J774 macrophages in a dose-dependent manner similar to natural HDL. A strong down-regulation of the ATP-binding cassette A1 (ABCA1) transporter mRNA (- 50%) as well as the ABCA1 membrane protein expression (- 50%) was observed at higher doses of CER-001 and HDL₃ compared to non-lipidated apoA-I. In vivo, in an apoE^-/- mouse “flow cessation model,” in which the left carotid artery was ligatured to induce local inflammation, the inhibition of atherosclerotic plaque burden progression in response to a dose-range of every-other-day CER-001 or HDL in the presence of a high-fat diet for two weeks was assessed. We observed a U-shaped dose-response curve: inhibition of the plaque total cholesterol content increased with increasing doses of CER-001 or HDL3 up to a maximum inhibition (- 51%) at 5 mg/kg; however, as the dose was increased above this threshold, a progressively less pronounced inhibition of progression was observed, reaching a complete absence of inhibition of progression at doses of 20 mg/kg and over. ABCA1 protein expression in the same atherosclerotic plaque was decreased by-45% and-68% at 50 mg/kg for CER-001 and HDL respectively. Conversely, a-12% and 0% decrease in ABCA1 protein expression was observed at the 5 mg/kg dose for CER-001 and HDL respectively.

Conclusions

These data demonstrate that high doses of HDL and CER-001 are less effective at slowing progression of atherosclerotic plaque in apoE^-/- mice compared to lower doses, following a U-shaped dose-response curve. A potential mechanism for this phenomenon is supported by the observation that high doses of HDL and CER-001 induce a rapid and strong down-regulation of ABCA1 both in vitro and in vivo. In conclusion, maximally efficient HDL- or CER-001-mediated cholesterol removal from atherosclerotic plaque is achieved by maximizing macrophage-mediated efflux from the plaque while minimizing dose-dependent down-regulation of ABCA1 expression. These observations may help define the optimal dose of HDL mimetics for testing in clinical trials of atherosclerotic burden regression.