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Mutation analysis in the FKRP gene provides an explanation for a rare cause of intrafamilial clinical variability in LGMD2I.

We report a limb-girdle muscular dystrophy 2I family with three affected sisters and a highly variable clinical course. FKRP gene sequencing showed that all three sisters carried a nonsense paternal mutation (W225X). The two oldest sisters with a severe phenotype carried two maternal mutations V79M and P89A. However, the youngest sister with a milder course carried the paternal and only the V79M maternal mutation, due to an intragenic recombination.

Adolescent↗

A matched set of cat vectors for rapid mutational analysis of eukaryotic promoters and enhancers.

The eukaryotic cat expression vectors, pBRAMScat1 and pBRAMScat2, were constructed to simplify the analysis of genomic fragments containing putative transcriptional regulatory elements. These vectors contain the f1 filamentous phage origin of replication for single-stranded DNA rescue, and permit site-directed mutagenesis, and dideoxy sequencing of nested deletion mutants using commercial T3, T7 and M13 universal forward/reverse primers. The above features eliminate the need to shuttle back and forth between a conventional cloning vector and the cat expression vector during the analysis of putative eukaryotic gene regulatory elements. Plasmid pBRAMScat1 contains the bacterial chloramphenicol acetyltransferase-encoding gene (cat) and no eukaryotic promoter and was designed for the analysis of eukaryotic promoters. Plasmid pBRAMScat2 contains the cat gene under the control of the Herpes simplex virus thymidine kinase promoter and was designed for the analysis of eukaryotic enhancers.

Animals↗

Biochemical and mutational analysis of the polyomavirus core promoter: involvement of nuclear factor-1 in early promoter function.

The polyomavirus enhancer is separated from the early RNA initiation sites by a 120 bp promoter region. To identify the core promoter elements, we introduced base-substitution mutations within the potential elements in the vicinity of the RNA initiation site. Three of these mutants, two with mutations within a putative nuclear factor-1 (NF-1) binding site and the other within the TATA box, exhibited reduced promoter activity by about threefold in the mouse NIH 3T3 cell line. The activity of the other three mutants was either little affected or remained unchanged. Mobility shift assays using specific competitors and antibodies against NF-1 demonstrated the binding of a protein of the NF-1 family at a site adjacent to the TATA box, suggesting a role for NF-1 binding in early promoter function. The effect of these mutations was also evaluated in undifferentiated mouse embryonal carcinoma (F9) cells in the presence of an additional mutation (F441) at nucleotide position 5233. This additional mutation creates a strong binding site for a transcription factor, TEF-1, and helps the virus to grow in this cell line. While the TATA box and the GC box mutants behaved qualitatively in a similar fashion, the NF-1 motif now played a minor role in F9 cells. Western blot experiments demonstrated low levels of NF-1 protein in this cell line. The NF-1 motif partially overlaps a T-antigen binding motif and this motif is not involved in T-antigen-mediated regulation of the early promoter. Our results suggest that a protein of the NF-1 family binds to the core promoter and is important for early transcription in vivo. We further demonstrate that undifferentiated F9 cells contain a very low level of NF-1 and the F441 mutant possibly follows a different mechanism for promoter function in these cells.

3T3 Cells↗

Mutational analysis of Vpr-induced G2 arrest, nuclear localization, and cell death in fission yeast.

Cell cycle G2 arrest, nuclear localization, and cell death induced by human immunodeficiency virus type 1 Vpr were examined in fission yeast by using a panel of Vpr mutations that have been studied previously in human cells. The effects of the mutations on Vpr functions were highly similar between fission yeast and human cells. Consistent with mammalian cell studies, induction of cell cycle G2 arrest by Vpr was found to be independent of nuclear localization. In addition, G2 arrest was also shown to be independent of cell killing, which only occurred when the mutant Vpr localized to the nucleus. The C-terminal end of Vpr is crucial for G2 arrest, the N-terminal alpha-helix is important for nuclear localization, and a large part of the Vpr protein is responsible for cell killing. It is evident that the overall structure of Vpr is essential for these cellular effects, as N- and C-terminal deletions affected all three cellular functions. Furthermore, two single point mutations (H33R and H71R), both of which reside at the end of each alpha-helix, disrupted all three Vpr functions, indicating that these two mutations may have strong effects on the overall Vpr structure. The similarity of the mutant effects on Vpr function in fission yeast and human cells suggests that fission yeast can be used as a model system to evaluate these Vpr functions in naturally occurring viral isolates.

Cell Death↗

Lattice corneal dystrophy type I without typical lattice lines: role of mutational analysis.

PURPOSE: To describe a Japanese patient with lattice corneal dystrophy type I (LCD I) who lacked the typical lattice lines. DESIGN: Interventional case report. METHODS: A complete ophthalmologic examination was performed on a 54-year-old woman, and the TGFBI gene was analyzed by direct genomic sequencing. RESULTS: The patient had diffuse opacification of the central corneal stroma but without lattice lines and corneal epithelial erosions bilaterally. Molecular genetic analysis identified a lattice corneal dystrophy I-associated heterozygous missense alteration (C417T) that changed arginine in codon 124 to cysteine (R124C) in the TGFBI gene. CONCLUSIONS: The cornea of the patient appeared to represent late-stage lattice corneal dystrophy I, which suggests the existence of interactions of modifier genes, environmental factors during corneal aging, or both. The molecular genetic analysis of TGFBI can offer rapid, accurate diagnosis of patients with atypical corneal appearance.

Corneal Dystrophies, Hereditary↗

Mutational analysis of the OA1 gene in ocular albinism.

Ocular albinism type 1 (OA1) is an X-linked disorder, mainly characterized by a severe reduction in visual acuity, foveal hypoplasia, nystagmus, hypopigmentation of the retina, the presence of macromelanosomes in the skin and eyes, and the misrouting of optic pathways, resulting in the loss of stereoscopic vision. We screened the OA1 gene for mutations in three unrelated Canadian and French families and in two isolated patients with OA1. We found three different missense mutations and two different nonsense mutations, three of which were novel. To date, 41 mutations (including missense mutations, insertions, and deletions) have been reported in the OA1 gene. Mutation and polymorphism data for this gene are available from the international albinism center albinism database website: http://www.cbc.umn.edu/tad/oa1map.htm.

Albinism, Ocular↗

Mutation analysis of 3 genes in patients with Leber congenital amaurosis.

OBJECTIVE: To assess the frequency of mutations in the CRX, GUCY2D, and RPE65 genes in patients with Leber congenital amaurosis (LCA). PATIENTS: One hundred seventy-six probands with a clinical diagnosis of LCA were from 9 countries, with the largest subgroup being 39 probands from India. METHODS: Samples were screened with single-strand conformation polymorphism analysis followed by DNA sequencing of 3 genes (CRX, GUCY2D, and RPE65) known to be associated with LCA. RESULTS: Of the 176 probands, 28 (15.9%) harbored possible disease-causing mutations. The relative contribution of each gene to the total number of mutations was as follows: CRX, 2.8%; GUCY2D, 6.3%; and RPE65, 6.8%. No patients who harbored mutations in these genes had associated systemic abnormalities. Molecular diagnosis allowed definitive genetic counseling in a family affected with Best disease and LCA. CONCLUSIONS: Molecular diagnosis may be of benefit to patients affected with LCA. The relative paucity of mutations found in this study suggests that more LCA-associated genes remain to be discovered. CLINICAL RELEVANCE: Molecular diagnosis can confirm and clarify the diagnosis of LCA. As genotype data accumulate, clinical phenotypes associated with specific mutations will be established. This will facilitate the counseling of patients on their visual prognosis and the likelihood of associated systemic anomalies.

Adolescent↗

Mutational analysis of hepatitis C virus NS3-associated helicase.

Nonstructural protein 3 (NS3) of hepatitis C virus contains a bipartite structure consisting of an N-terminal serine protease and a C-terminal DEXH box helicase. To investigate the roles of individual amino acid residues in the overall mechanism of unwinding, a mutational-functional analysis was performed based on a molecular model of the NS3 helicase domain bound to ssDNA, which has largely been confirmed by a recently published crystal structure of the NS3 helicase-ssDNA complex. Three full-length mutated NS3 proteins containing Tyr(392)Ala, Val(432)Gly and Trp(501)Ala single substitutions, respectively, together with a Tyr(392)Ala/Trp(501)Ala double-substituted protein were expressed in Escherichia coli and purified to homogeneity. All individually mutated forms showed a reduction in duplex unwinding activity, single-stranded polynucleotide binding capacity and polynucleotide-stimulated ATPase activity compared to wild-type, though to different extents. Simultaneous replacement of both Tyr(392) and Trp(501) with Ala completely abolished all these enzymatic functions. On the other hand, the introduced amino acid substitutions had no influence on NS3 intrinsic ATPase activity and proteolytic efficiency. The results obtained with Trp(501)Ala and Val(432)Gly single-substituted enzymes are in agreement with a recently proposed model for NS3 unwinding activity. The mutant phenotype of the Tyr(392)Ala and Tyr(392)Ala/Trp(501)Ala enzymes, however, represents a completely novel finding.

Adenosine Triphosphatases↗

A mutational analysis of dishevelled in Drosophila defines novel domains in the dishevelled protein as well as novel suppressing alleles of axin.

Drosophila dishevelled (dsh) functions in two pathways: it is necessary to transduce Wingless (Wg) signaling and it is required in planar cell polarity. To learn more about how Dsh can discriminate between these functions, we performed genetic screens to isolate additional dsh alleles and we examined the potential role of protein phosphorylation by site-directed mutagenesis. We identified two alleles with point mutations in the Dsh DEP domain that specifically disrupt planar polarity signaling. When positioned in the structure of the DEP domain, these mutations are located close to each other and to a previously identified planar polarity mutation. In addition to the requirement for the DEP domain, we found that a cluster of potential phosphorylation sites in a binding domain for the protein kinase PAR-1 is also essential for planar polarity signaling. To identify regions of dsh that are necessary for Wg signaling, we screened for mutations that modified a GMR-GAL4;UAS-dsh overexpression phenotype in the eye. We recovered many alleles of the transgene containing missense mutations, including mutations in the DIX domain and in the DEP domain, the latter group mapping separately from the planar polarity mutations. In addition, several transgenes had mutations within a domain containing a consensus sequence for an SH3-binding protein. We also recovered second-site-suppressing mutations in axin, mapping at a region that may specifically interact with overexpressed Dsh.

Adaptor Proteins, Signal Transducing↗

Mutation analysis of hereditary multiple exostoses in the Chinese.

Hereditary multiple exostoses (EXT; MIM 133700) is an autosomal dominant bone disorder. It is genetically heterogeneous with at least three chromosomal loci: EXT1 on 8q24.1, EXT2 on 11p11, and EXT3 on 19p. EXT1 and EXT2, the two genes responsible for EXT1 and EXT2, respectively, have been cloned. Recently, three other members of the EXT gene family, named the EXT-like genes (EXTL: EXTL1, EXTL2, and EXTL3), have been isolated. EXT1, EXT2, and the three EXTLs are homologous with one another. We have identified the intron-exon boundaries of EXTL1 and EXTL3 and analyzed EXT1, EXT2, EXTL1, and EXTL3, in 36 Chinese families with EXT, to identify underlying disease-related mutations in the Chinese population. Of the 36 families, five and 12 family groups have mutations in EXT1 and EXT2, respectively. No disease-related mutation has been found in either EXTL1 or EXTL2, although one polymorphism has been detected in EXTL1. Of the 15 different mutations (three families share a common mutation in EXT2), 12 are novel. Most of the mutations are either frameshift or nonsense mutations (12/15). These mutations lead directly or indirectly to premature stop codons, and the mutations generate truncated proteins. This finding is consistent with the hypothesis that the development of EXT is mainly attributable to loss of gene function. Missense mutations are rare in our families, but these mutations may reflect some functionally crucial regions of these proteins. EXT1 is the most frequent single cause of EXT in the Caucasian population in Europe and North America. It accounts for about 40% of cases of EXT. Our study of 36 EXT Chinese families has found that EXT1 seems much less common in the Chinese population, although the frequency of the EXT2 mutation is similar in the Caucasian and Chinese populations. Our findings suggest a possibly different genetic spectrum of this disease in different populations.

China↗

Mutational analysis of the disulfide catalysts DsbA and DsbB.

In prokaryotes, disulfides are generated by the DsbA-DsbB system. DsbB functions to generate disulfides by quinone reduction. These disulfides are passed to the DsbA protein and then to folding proteins. To investigate the DsbA-DsbB catalytic system, we performed an in vivo selection for chromosomal dsbA and dsbB mutants. We rediscovered many residues previously shown to be important for the activity of these proteins. In addition, we obtained one novel DsbA mutant (M153R) and four novel DsbB mutants (L43P, H91Y, R133C, and L146R). We also mutated residues that are highly conserved within the DsbB family in an effort to identify residues important for DsbB function. We found classes of mutants that specifically affect the apparent K(m) of DsbB for either DsbA or quinones, suggesting that quinone and DsbA may interact with different regions of the DsbB protein. Our results are consistent with the interpretation that the residues Q33 and Y46 of DsbB interact with DsbA, Q95 and R48 interact with quinones, and that residue M153 of DsbA interacts with DsbB. All of these interactions could be due to direct amino acid interactions or could be indirect through, for instance, their effect on protein structure. In addition, we find that the DsbB H91Y mutant severely affects the k(cat) of the reaction between DsbA and DsbB and that the DsbB L43P mutant is inactive, suggesting that both L43 and H91 are important for the activity of DsbB. These experiments help to better define the residues important for the function of these two protein-folding catalysts.

Amino Acid Substitution↗

Morphological-histochemical study of intestinal carcinoids and K-ras mutation analysis in appendiceal carcinoids.

Intestinal carcinoids are potentially malignant neoplasms. Their histogenesis and pathogenesis are currently uncertain. The morphological and histochemical characteristics of twenty intestinal carcinoids are studied. The primary sites of three mucin-producing tumors were examined by electron microscope. Furthermore 11 appendiceal carcinoids were analysed by the polymerase chain reaction (PCR) for the detection of ras and p53 point mutations. Microscopically all carcinoids were of mixed type. Focal mucin production was evident in three carcinoids that metastasised to regional lymph nodes. HID-Alcian blue staining proved that mucin in both primary and secondary foci did not belong to the sulphated group. The secretory granules and mucin droplets found in a single neoplastic cell suggest that carcinoids of the small intestine and some of the appendix arise from the endoderm. Neither ras nor p53 mutations were detected. It seems that ras oncogenes are probably not involved in the pathogenesis of appendiceal carcinoids.

Adolescent↗

Mutation analysis of the calpastatin gene (CAST) in patients with Alzheimer's disease.

The calpains, a family of calcium-dependent cysteine proteinases, and calpastatin, their endogenous inhibitor protein, are involved in the proteolysis of amyloid precursor protein, which is thought to be abnormal in patients with Alzheimer's disease (AD). Specific inhibitors of calpains attenuate amyloid beta peptide-induced neuronal death. We hypothesized that some AD patients have functionally deficient mutation(s) of the CAST gene encoding calpastatin, and we screened 40 Japanese patients with AD for mutations in the coding region of CAST. Nine polymorphisms, -82A/G, IVS7-96A/G, 669A/G, 1223C/G (Ser408Cys), IVS20-10C/T, IVS21-65G/A, IVS22+31T/C, IVS24+38Ins/DelA, and IVS25-32A/G, were identified. The 669A allele causes skipping of exon 11, leading to the loss of 13 residues. Comparisons between 101 patients and 90 controls revealed no significant association between CAST polymorphisms and risk for AD, indicating that genomic variations of CAST are not likely to be substantially involved in the etiology of AD.

Aged↗

Mutational analysis in Lebanese patients with congenital adrenal hyperplasia due to a deficit in 21-hydroxylase.

Molecular defects in the gene encoding steroid 21-hydroxylase (CYP21) result in impairment of adrenal steroid synthesis in patients affected with autosomal-recessive congenital adrenal hyperplasias (CAH). In this study, we report on the molecular screening of six point mutations, large deletions, gene conversion events and duplications in 25 unrelated Lebanese families affected by CAH due to steroid 21-hydroxylase. The methods used (PCR-digestion and southern blot) allowed the detection of 96% of the disease chromosomes. In classical forms, the most frequent mutation was the splice site mutation in intron 2 accounting for 39% of the disease alleles. Gene conversion events accounted for 14% of the alleles, but no large deletions were found. In nonclassical forms, the V281L mutation in exon 7 represent 86% of the tested alleles. Genotype-phenotype correlations were as expected: Delta 8nt, Q318X and gene conversion correspond to SW forms, whereas the intron 2 splice site mutation may give either SW or SV forms; the V281L mutation was responsible for nonclassical forms. The spectrum of mutations underlines the genetic diversity of the Lebanese population. No correlation could be drawn out between mutations and some specific religious communities, except for the Delta 8nt mutation, which is present only in the Christian Maronite group. Molecular study of the CYP21 gene might constitute a good support for clinicians, especially in consanguineous families, for whom we could provide genetic counselling.

Adolescent↗

Mutational analysis of nicotinic acetylcholine receptor beta2 subunit gene (CHRNB2) in a representative cohort of Italian probands affected by autosomal dominant nocturnal frontal lobe epilepsy.

Twenty-four autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) probands were analyzed for the presence of V287L and V287M mutations in the CHRNB2 gene, which have been recently associated with the disease. In all patients, the involvement of the two additional loci reported as being associated with ADNFLE (CHRNA4 gene and chromosome 15q24 region) had been previously excluded. Mutational screening was performed by sequencing a polymerase chain reaction-amplified CHRNB2 DNA fragment, spanning the whole exon 5, which contains the V287L and V287M mutations and codes for approximately 65% of the mature protein. In none of the patients were mutations in the analyzed region of CHRNB2 found. These data, obtained in the largest ADNFLE cohort so far analyzed, demonstrate the rarity of the identified CHRNB2 mutations in ADNFLE patients.

Base Sequence↗