Mutant and misfolded human growth hormone is rapidly degraded through the proteasomal degradation pathway in a cellular model for isolated growth hormone deficiency type II.

Autosomal dominant isolated growth hormone deficiency type II (IGHD II) is mainly caused by splice site mutations of the GH-1 gene, leading to deletion of amino acids 32-71 of the human growth hormone (hGH). The severe hGH deficit in IGHD II suggests a dominant negative effect of the partially deleted del(32-71)-hGH on the production, storage or secretion of normal wild-type (wt)-hGH in somatotrophic cells of the pituitary. To shed more light on the cellular and molecular basis of IGHD II, we established and analysed diverse clones of the rat somatotrophic cell line GH(4)C(1) stably expressing either wt-hGH, del(32-71)-hGH, or both proteins concomitantly. The cellular morphology of all transfected GH(4)C(1) cell clones showed moderate differences to untransfected GH(4)C(1) cells. On the molecular level, both cDNA-constructs induced transcription but, under normal culture conditions, only wt-hGH protein was found to be synthesised and secreted in readily detectable amounts. By contrast, only after inhibition of proteasomes did high amounts of del(32-71)-hGH show up. The solubility of del(32-71)-hGH in nondenaturing buffer was poor compared to wt-hGH, hinting at molecular aggregation, and several epitopes recognised by monoclonal hGH antibodies were not present on del(32-71)-hGH, confirming the assumption that del(32-71)-hGH must be severely misfolded. Expression of both proteins in Escherichia coli mirrored the findings from the GH(4)C(1) cell clones in terms of solubility and immunological reactivity. The results of the present study indicate that, in IGHD II, somatotrophs continuously have to remove misfolded del(32-71)-hGH via the proteasomal degradation pathway, suggesting a mechanism that may result in chronic cellular stress.

Novel rhodopsin mutations and genotype-phenotype correlation in patients with autosomal dominant retinitis pigmentosa.

AIM: To identify novel or rare rhodopsin gene mutations in patients with autosomal dominant retinitis pigmentosa and description of their clinical phenotype. METHODS: The complete rhodopsin gene was screened for mutations by DNA sequencing in index patients. Mutation specific assays were used for segregation analysis and screening for controls. Eight patients from five families and their relatives were diagnosed with autosomal dominant retinitis pigmentosa (adRP) by means of clinical evaluation. RESULTS: Mutation screening identified five different rhodopsin mutations including three novel mutations: Ser176Phe, Arg314fs16, and Val20Gly and two missense mutations, Pro215Leu and Thr289Pro, that were only reported once in a mutation report. Electrophysiological and psychophysical testings provide evidence of an impaired rod system with additionally affected cone system in subjects from each genotype group. Visual function tended to be less affected in subjects with the Arg314fs16 and Val20Gly mutations than in the Ser176Phe phenotype. In contrast, Pro215Leu and Thr289Pro mutations caused a remarkably severe phenotype. CONCLUSION: The ophthalmic findings support a correlation between disease expression and structural alteration: (1) extracellular/intradiscal Val20Gly and cytoplasmic Arg314fs16 mutation-mild adRP phenotype; (2) Ser176Phe mutation-"mostly type 1" disease; (3) predicted alteration of transmembrane domains TM V and TM VII induced by Pro215Leu and Thr289Pro-severe phenotype. However, variation of phenotype expression in identical genotypes may still be a typical feature of RHO mutations.

A novel mutation of the RP1 gene (Lys778ter) associated with autosomal dominant retinitis pigmentosa.

BACKGROUND: Besides the three known genes (RHO, RDS/Peripherin, NRL) involved in autosomal dominant retinitis pigmentosa (adRP), a fourth gene, RP1, has been recently identified. Initial reports suggest that mutations in the RP1 gene are the second most frequent cause of adRP. The clinical findings were described in a family with adRP and a novel mutation in the RP1 gene. METHOD: Index patients from 15 independent families with adRP in which RHO mutations had been excluded in previous examinations were screened for mutations in the RP1 gene by means of direct DNA sequencing. Evaluation of the RP1 phenotype in patients included funduscopy, kinetic perimetry, dark adapted final threshold test, standard electroretinography and, in one case, multifocal electroretinography. RESULTS: One novel nonsense mutation (Lys778ter) in one of these 15 patients was detected. Cosegregation of the mutation with the disease phenotype could be established in the index patient's family. The phenotype comprises variable expression of clinical disease probably including one case of incomplete penetrance, a onset of symptoms beginning in adulthood, and evidence of regionally varying retinal function loss. CONCLUSION: The Lys778ter mutation localises inside the critical region harbouring all mutations described so far. The ophthalmic findings support previous observations that variation of disease expression appears as a typical feature of the RP1 phenotype.

CNGA3 mutations in hereditary cone photoreceptor disorders.

We recently showed that mutations in the CNGA3 gene encoding the alpha-subunit of the cone photoreceptor cGMP-gated channel cause autosomal recessive complete achromatopsia linked to chromosome 2q11. We now report the results of a first comprehensive screening for CNGA3 mutations in a cohort of 258 additional independent families with hereditary cone photoreceptor disorders. CNGA3 mutations were detected not only in patients with the complete form of achromatopsia but also in incomplete achromats with residual cone photoreceptor function and (rarely) in patients with evidence for severe progressive cone dystrophy. In total, mutations were identified in 53 independent families comprising 38 new CNGA3 mutations, in addition to the 8 mutations reported elsewhere. Apparently, both mutant alleles were identified in 47 families, including 16 families with presumed homozygous mutations and 31 families with two heterozygous mutations. Single heterozygous mutations were identified in six additional families. The majority of all known CNGA3 mutations (39/46) are amino acid substitutions compared with only four stop-codon mutations, two 1-bp insertions and one 3-bp in-frame deletion. The missense mutations mostly affect amino acids conserved among the members of the cyclic nucleotide gated (CNG) channel family and cluster at the cytoplasmic face of transmembrane domains (TM) S1 and S2, in TM S4, and in the cGMP-binding domain. Several mutations were identified recurrently (e.g., R277C, R283W, R436W, and F547L). These four mutations account for 41.8% of all detected mutant CNGA3 alleles. Haplotype analysis suggests that the R436W and F547L mutant alleles have multiple origins, whereas we found evidence that the R283W alleles, which are particularly frequent among patients from Scandinavia and northern Italy, have a common origin.