Phenotypic variability in two families with novel splice-site and frameshift NF2 mutations

Neurofibromatosis 2 (NF2) is a clinically variable autosomal dominant disorder, caused by mutations in the NF2 tumor suppressor gene on chromosome 22q12, that predisposes to nervous system tumors and ocular abnormalities. To assess intrafamilial phenotypic variability, we performed mutation analysis and clinical assessment on two multigeneration NF2 families with five patients and seven asymptomatic first-degree relatives of patients. One family had a point mutation of agCC→ggCC at position 1447–2 at the exon 13/14 boundary predicted to lead to an altered splice acceptor sequence and exon deletion. The other family had an insertion of 2 base pairs (TC) at position 761 in exon 8, leading to a frameshift. Both mild and severe phenotypes occurred in each family, indicating that phenotypic variability in NF2 can be caused by factors other than NF2 mutations. Genetic counseling of NF2 families should include the possibility that presymptomatic NF2 mutation carriers can develop a different phenotype than previously diagnosed patients. Received: 4 January 1996 / Revised: 26 March 1996     

Phenotypic variation associated with molecular alterations at a cluster of thymidine kinase genes.

Genetic variation was studied in several mouse L cell lines containing tandemly repeated herpes simplex virus thymidine kinase (TK) genes introduced by DNA-mediated gene transfer. Variants were obtained after alternate positive and negative selection for TK expression. Three classes of molecular alteration are described. One class consisted of a concerted wave of hypermethylation affecting many sites in all or nearly all of the TK genes. This resulted in genetically stable TK- variants. Of five TK+ transformants from independent transfer experiments, only one, named HM, showed this class of methylation. Hypermethylation was a reproducible phenomenon in HM, yielding TK- variants after selection with either bromodeoxyuridine or acycloguanosine [Acyclovir or 9-(2-hydroxyethy-oxymethyl)guanine]. A second class of alteration consisted of methylation affecting some, but not all, genes in the cluster. This happened in all TK+ (HAT [hypoxanthine-aminopterin-thymidine]-resistant) cell lines investigated, and this second class of methylation was incapable of generating TK- variants. Neither type of methylation was accompanied by genomic rearrangements. The third class of molecular alteration was found among TK+ (HAT-resistant) back revertants of hypermethylated HM TK- derivatives. It consisted of a 10-fold amplification of the hypermethylated TK genes. Demethylation of hypermethylated HM variants was not observed. Thus, hypermethylation in this system can be compensated for by amplification but cannot be reversed.     

Ligand-dependent differences in the internalization of endothelin A and endothelin B receptor heterodimers

Endothelin-1 (ET-1) is a potent vasoactive peptide that acts on endothelin A (ET(A)) and endothelin B (ET(B)) receptors. Although both receptor subtypes are co-expressed in numerous cells, little is known about their ability to form heterodimers. Here we show that both receptors were co-immunoprecipitated with an ET(B)-specific antibody using extracts from HEK293 cells stably co-expressing a fusion protein consisting of a myc-tagged ET(A) receptor and CFP (ET(A)myc.CFP) and a fusion protein consisting of an ET(B) receptor and YFP (ET(B).YFP). Co-immunoprecipitation was also observed with extracts from HEK293 cells transiently co-expressing FLAG-tagged ET(B) and myc-tagged ET(A) receptors, thereby excluding that heterodimerization is mediated by the CFP/YFP moieties. Heterodimerization was further confirmed in fluorescence resonance energy transfer (FRET) analysis of HEK293 cells transiently co-expressing ET(A)myc.CFP and ET(B).YFP receptors. FRET efficiencies were between 12 and 18% in untreated and antagonist- or ET-1-treated cells, indicating constitutive heterodimerization. Prolonged stimulation (30 min) with the ET(B) receptor-selective agonist BQ3020 decreased FRET efficiency by 50%. This decrease was not observed when internalization was inhibited by co-expression of dominant-negative K44A.dynamin I or incubation with 450 mm sucrose. Enzyme-linked immunosorbent assay and laser scanning microscopy of cell clones stably co-expressing ET(A)myc.CFP/ET(B)flag.YFP receptors revealed a slower sequestration of the ET(B)flag.YFP receptors upon stimulation with ET-1 than with BQ3020. No difference in ET-1 or BQ3020-mediated sequestration was observed with cell clones expressing ET(B)flag.YFP receptors alone. The data suggest that ET(A) and ET(B) receptors form constitutive heterodimers, which show a slower sequestration upon stimulation with ET-1 than with BQ3020. Heterodimer dissociation along the endocytic pathway only occurs upon ET(B)-selective stimulation.     

Phenotypic heterogeneity associated with identical mutations in residue 870 of the androgen receptor

BACKGROUND/AIMS: Mutations in the androgen receptor (AR) gene result in an X-linked recessive form of male pseudohermaphroditism known as the androgen-insensitivity syndrome (AIS). The alterations most frequently observed are missense or nonsense point mutations in exons 4-8 of the AR gene that affect the steroid-binding domain of the receptor in subjects with various degrees of androgen resistance. Despite the increasing number of AR mutations identified, a reliable genotype-phenotype correlation has not been established and individuals with the same molecular defect may exhibit different phenotypes. Here, we studied a patients with an AIS characterized by bilateral gynecomastia, normal male external genitalia, and normal sperm counts. METHODS: Exon-specific polymerase chain reaction, single-stranded conformational polymorphism, and sequencing analysis of the subject's AR gene were performed in addition to hormone-binding assays in skin fibroblasts from the patient. RESULTS: A point mutation at codon 870 of the AR, changing alanine to valine, was detected. CONCLUSION: As AR missense mutations changing alanine 870 to valine have been previously described in 3 unrelated patients showing severe AIS phenotypes, we conclude that phenotypic heterogeneity associated to identical mutations in the AR gene is probably due to individual functional differences in AR coregulator molecules.     

Regulated interaction of endothelin B receptor with caveolin-1.

The peptide hormone endothelin transmits various signals through G protein-coupled receptors, the endothelin type A (ETAR) and B (ETBR) receptors. Caveolae are specialized lipid rafts containing polymerized caveolins. We examined the interaction of ETBR with caveolin-1, expressed in Sf9, COS-1, and HEK293 cells, and its effects on the subcellular distribution and the signal transduction of ETBR. ETBR formed a complex with caveolin-1 in cells in which these two proteins were coexpressed and in the mixture after purification and reconstitution (as examined by immunoprecipitation) suggesting the direct binding of ETBR with caveolin-1. The complex formed efficiently only when the ETBR was ligand-free or bound to an antagonist, RES-701-1, whereas the addition of ET-1 or another antagonist, BQ788, dissociated the complex, suggesting the structural recognition of ETBR by caveolin-1. In contrast, the ETAR bound to caveolin-1 regardless of ligand binding. Caveolin-1 utilized its scaffolding domain (residues 82-101) and the C-terminal domain (residues 136-178) to bind to ETBR, as for other signalling molecules. Furthermore, the amount of ETBR localized in caveolae increased significantly with the expression of caveolin-1 and decreased with the addition of ET-1. The disruption of caveolae by filipin reduced the ET-1-derived phosphorylation of ERK1/2. These results suggest the possibility that the binding to caveolin-1 retains the ligand-free ETBR in caveolae and regulates the ET signal.     

Validation of endothelin B receptor antibodies reveals two distinct receptor-related bands on Western blot

Antibodies are important tools for the study of protein expression but are often used without full validation. In this study, we used Western blots to characterize antibodies targeted to the N or C terminal (NT or CT, respectively) and the second or third intracellular loop (IL2 or IL3, respectively) of the endothelin B receptor (ETB). The IL2-targeted antibody accurately detected endogenous ETB expression in rat brain and cultured rat astrocytes by labeling a 50-kDa band, the expected weight of full-length ETB. However, this antibody failed to detect transfected ETB in HEK293 cultures. In contrast, the NT-targeted antibody accurately detected endogenous ETB in rat astrocyte cultures and transfected ETB in HEK293 cultures by labeling a 37-kDa band but failed to detect endogenous ETB in rat brain. Bands detected by the CT- or IL3-targeted antibody were found to be unrelated to ETB. Our findings show that functional ETB can be detected at 50 or 37 kDa on Western blot, with drastic differences in antibody affinity for these bands. The 37-kDa band likely reflects ETB processing, which appears to be dependent on cell type and/or culture condition.     

Phenotypic variation and promiscuity in a wild population of pure dingoes (Canis dingo)

Phenotypic diversity occurs in natural populations as a result of the interaction between an individual's genotype and the environment. Nevertheless, individual variation in phenotypic traits such as coat colour and body size is routinely used to differentiate between “pure” dingoes Canis dingo and dingo‐dog hybrids. Extensive anthropogenic impacts and widespread hybridization with domestic dogs has hindered our ability to study intact dingo populations and, therefore, most of our basic understanding of dingo biology (e.g., phenotypic variation, mating systems, genetic diversity) stems from observational studies on perturbed populations. We sampled a relatively undisturbed population of dingoes, from arid Australia, to determine their purity and genetic diversity. We explored their mating strategy using a pedigree built from genetic data and examined how phenotypic variation was influenced by age, sex, heterozygosity, and relatedness. Coat colour was our measure of phenotype and our population displayed four types (sandy, black & tan, white, and sable). All dingoes (n = 83) possessed a high level of dingo ancestry (mean purity > 90%) and were closely related to each other; with all but one individual related as full‐sibling or parent–offspring. Our pedigree shows both monogamous and promiscuous mating strategies exist within an undisturbed population. Variation in coat colour or body size cannot be used to infer a dingo's level of purity because the phenotype of pure dingoes is intrinsically variable. The breeding system of dingoes was long thought to be monogamous, but we provide genetic evidence for numerous mating strategies including both long‐term monogamy and extreme promiscuity.     

Benzofuro[3,2-b]pyridines as mixed ET(A)/ET(B) and selective ET(B) endothelin receptor antagonists

The discovery, synthesis and structure-activity relationships of a series of novel benzofuro[3,2-b]pyridines as non-selective endothelin ET(A)/ET(B) as well as selective ET(B) receptor antagonists are described. The most potent non-selective inhibitor 7s displayed an IC50 of 21 nM and 41 nM for ET(A) and ET(B) receptors, respectively, whereas 7ee merely showed affinity for the ET(B) receptor (IC50 = 3.6 nM).     

Localization of Bacillus subtilis sacU(Hy) mutations to two linked genes with similarities to the conserved procaryotic family of two-component signalling systems.

Mutations in the sacU region have a pleiotropic phenotype. Certain mutations designated sacU(Hy), for example, express degradative enzymes at high levels, are able to sporulate in the presence of glucose, have severely reduced transformation efficiencies, and are nonmotile. We isolated and sequenced the sacU gene region of Bacillus subtilis. Two open reading frames were found in the sacU region, and sacU(Hy) mutations were localized to both of these open reading frames. The two open reading frames have similarities to two widespread families of proteins that mediate responses to environmental stimuli.     

Renal aplasia in humans is associated with RET mutations

In animal models, kidney formation is known to be controlled by the proteins RET, GDNF, and GFRA1; however, no human studies to date have shown an association between abnormal kidney development and mutation of these genes. We hypothesized that stillborn fetuses with congenital renal agenesis or severe dysplasia would possess mutations in RET, GDNF, or GFRA1. We assayed for mutations in these genes in 33 stillborn fetuses that had bilateral or unilateral renal agenesis (29 subjects) or severe congenital renal dysplasia (4 subjects). Mutations in RET were found in 7 of 19 fetuses with bilateral renal agenesis (37%) and 2 of 10 fetuses (20%) with unilateral agenesis. In two fetuses, there were two different RET mutations found, and a total of ten different sequence variations were identified. We also investigated whether these mutations affected RET activation; in each case, RET phosphorylation was either absent or constitutively activated. A GNDF mutation was identified in only one fetus with unilateral agenesis; this subject also had two RET mutations. No GFRA1 mutations were seen in any fetuses. These data suggest that in humans, mutations in RET and GDNF may contribute significantly to abnormal kidney development.     

Hearing impairment caused by mutations in two different genes responsible for nonsyndromic and syndromic hearing loss within a single family

Usher syndrome is rare genetic disorder impairing two human senses, hearing and vision, with the characteristic late onset of vision loss. This syndrome is divided into three types. In all cases, the vision loss is postlingual, while loss of hearing is usually prelingual. The vestibular functions may also be disturbed in Usher type 1 and sometimes in type 3. Vestibular areflexia is helpful in making a proper diagnosis of the syndrome, but, often, the syndrome is misdiagnosed as a nonsyndromic hearing loss. Here, we present a Polish family with hearing loss, which was clinically classified as nonsyndromic. After excluding mutations in the DFNB1 locus, we implemented the next-generation sequencing method and revealed that hearing loss was syndromic and mutations in the USH2A gene indicate Usher syndrome. This research highlights the importance of molecular analysis in establishing a clinical diagnosis of congenital hearing loss.     

Construction and behavior of strains with mutations in two chemotaxis genes.

Double mutants have been constructed by transducing each of the six che genes from the main che gene cluster into the cheC mutant with reversed behavior. The behavioral properties of these double-mutant strains were examined. The results are interpreted in terms of a model based on the cheC gene product being the component of the flagellar basal body that generates tumbling or smooth swimming in response to changes in the level of the response regulator. The properties of the double mutants can then be explained in ways which provide further understanding of the bacterial sensing system.     

Phenotypic expression of familial hypobetalipoproteinemia in three kindreds with mutations of apolipoprotein B gene

We report the clinical phenotype in three kindreds with familial heterozygous hypobetalipoproteinemia (FHBL) carrying novel truncated apolipoprotein Bs (apoBs) of different sizes (apoB-8.15, apoB-33.4 and apoB-75.7). In D.A. kindred, we found three carriers of a C-deletion in exon 10 leading to the synthesis of apoB-8.15 not detectable in plasma. They showed steatorrhea and fatty liver. In N.L. kindred, the proband is heterozygous for a nonsense mutation in exon 26, leading to the formation of apoB-33.4. He had premature cerebrovascular disease and fatty liver; two apoB-33.4 carriers in this kindred showed only fatty liver. In B.E. kindred, the proband is heterozygous for a T-deletion in exon 26, which converts tyrosine at codon 3435 into a stop codon, resulting in apoB-75.7. The proband, a heavy alcohol drinker, had steatohepatitis, whereas his teetotaller daughter, an apoB-75.7 carrier, had no detectable fatty liver. This study suggests that: i) fatty liver invariably develops in FHBL carriers of short and medium-size truncated apoBs (< apoB-48), but its occurrence needs additional environmental factors in carriers of longer apoB forms; ii) intestinal lipid malabsorption develops only in carriers of short truncated apoBs, which are not secreted into the plasma; and iii) cerebrovascular disease due to premature atherosclerosis may occur even in FHBL subjects.     

The extracellular N terminus of the endothelin B (ETB) receptor is cleaved by a metalloprotease in an agonist-dependent process

The extracellular N terminus of the endothelin B (ET(B)) receptor is susceptible to limited proteolysis (cleavage at R64 downward arrow S65), but the regulation and the functional consequences of the proteolysis remain elusive. We analyzed the ET(B) receptor or an ET(B)-GFP fusion protein stably or transiently expressed in HEK293 cells. After incubation of cells at 4 degrees C, only the full-length ET(B) receptor was detected at the cell surface. However, when cells were incubated at 37 degrees C, N-terminal cleavage was observed, provided endothelin 1 was present during the incubation. Cleavage was not inhibited by internalization inhibitors (sucrose, phenylarsine oxide). However, in cells incubated with both internalization inhibitors and metalloprotease inhibitors (batimastat, inhibitor of TNFalpha-convertase) or metal chelators (EDTA, phenanthroline), the cleavage was blocked, indicating that metalloproteases cleave the agonist-occupied ET(B) receptor at the cell surface. Functional analysis of a mutant ET(B) receptor lacking the first 64 amino acids ([Delta2-64]ET(B) receptor) revealed normal functional properties, but a 15-fold reduced cell surface expression. The results suggest a role of the N-terminal proteolysis in the regulation of cell surface expression of the ET(B) receptor. This is the first example of a multispanning membrane protein, which is cleaved by a metalloprotease, but retains its functional activity and overall structure.     

Expression and purification of an engineered human endothelin receptor B in a monomeric form

In humans, two endothelin receptors, ETa and ETb, are activated by three endogenous 21-mer cyclic peptides, ET-1, ET-2, and ET-3, which control various physiological processes, including vasoconstriction, vasodilation, and stimulation of cell proliferation. The first stage of this study it to produce a stable solubilized and purified receptor in a monodisperse state. This article is focused on the engineering, expression, purification, and characterization of the endothelin receptor B for subsequent structural and functional studies.