Molecular characterization of β-thalassemia mutations in Egypt

Summary The relative frequency of different -thalassemia mutations and their association with -globin haplotypes were studied in patients from the Nile delta region, Egypt, by means of the polymerase chain reaction, oligonucleotide hybridization and restriction analysis. We found that 8 mutations account for 77% of -thalassemia chromosomes in this population, the commonest being IVS-1 nt 110, IVS-1 nt 6 and IVS-1 nt 1. Each mutation was associated with a specific haplotype, with the exception of IVS-1 nt 110, found on 3 different chromosomal backgrounds. Our data show that testing for the 8 detectable mutations makes feasible prenatal diagnosis in 65% of at risk couples and exclusion testing in an additional 25% of cases.     

The spectrum of mutations in erythrokeratodermias – novel and de novo mutations in GJB3

Intercellular channels in skin are a complex and functionally diverse system formed by at least eight connexins (Cx). Our recent molecular studies implicating Cx defects in inherited skin disorders emphasize the critical role of this signaling pathway in epidermal differentiation. Erythrokeratodermia variabilis (EKV) is an autosomal dominant genodermatosis with a striking phenotype characterized by the independent occurrence of transient localized erythema and hyperkeratosis. The disease maps to 1p34-p35, and recently we identified the causative gene GJB3 encoding Cx31. We have now investigated GJB3 in two families and three sporadic cases with EKV, and report three new heterozygous mutations. In a sporadic case, we detected a mutation leading to substitution of a conserved phenylalanine (F137L) in the third transmembrane domain, which likely interferes with the proper assembly or gating properties of connexons. In another family, all three affected individuals carried two distinct mutations on the same GJB3 allele. However, only a de novo heterozygous missense mutation replacing arginine 42 with proline (R42P) co-segregated with the disease, while a 12 bp deletion predicted to eliminate four amino acid residues in the variable carboxy terminal domain of Cx31 was also found in clinically unaffected relatives but not in 90 unaffected controls. Including the previously published mutations, in toto, five different missense mutations have now been detected in 6 out of 17 families investigated by our laboratory, all of which presumably affect the cytoplasmic amino terminal and transmembrane domains of Cx31. In contrast, two mutations linked to progressive high-tone hearing impairment were located in the second extracellular domain, suggesting that the character and position of Cx mutations determine their phenotypic expression in different tissues. However, the phenotypic spectrum of GJB3 mutations seems not to include progressive symmetric erythrokeratodermia, another dominant genodermatosis with overlapping features, since no mutations were found in six unrelated families tested.     

Identification of 28 novel mutations in the Bardet–Biedl syndrome genes: the burden of private mutations in an extensively heterogeneous disease

Bardet–Biedl syndrome (BBS), an emblematic disease in the rapidly evolving field of ciliopathies, is characterized by pleiotropic clinical features and extensive genetic heterogeneity. To date, 14 BBS genes have been identified, 3 of which have been found mutated only in a single BBS family each (BBS11/TRIM32, BBS13/MKS1 and BBS14/MKS4/NPHP6). Previous reports of systematic mutation detection in large cohorts of BBS families (n > 90) have dealt only with a single gene, or at most small subsets of the known BBS genes. Here we report extensive analysis of a cohort of 174 BBS families for 12/14 genes, leading to the identification of 28 novel mutations. Two pathogenic mutations in a single gene have been found in 117 families, and a single heterozygous mutation in 17 families (of which 8 involve the BBS1 recurrent mutation, M390R). We confirm that BBS1 and BBS10 are the most frequently mutated genes, followed by BBS12. No mutations have been found in BBS11/TRIM32, the identification of which as a BBS gene only relies on a single missense mutation in a single consanguineous family. While a third variant allele has been observed in a few families, they are in most cases missenses of uncertain pathogenicity, contrasting with the type of mutations observed as two alleles in a single gene. We discuss the various strategies for diagnostic mutation detection, including homozygosity mapping and targeted arrays for the detection of previously reported mutations.     

Analysis of 30 known cystic fibrosis mutations: 10 mutations account for 27% of non-ΔF508 chromosomes in Southern France

We have analyzed 131 unrelated families from Southern France for 29 known cystic fibrosis (CF) mutations identified in 8 exons of the cystic fibrosis transmembrane regulator gene. All these mutations were detected by amplification of DNA by the polymerase chain reaction (PCR) followed by restriction enzyme digestion or hybridization with allele specific oligoprobes. The most frequent mutations after the F508 deletion (frequency: 63%) were G542X (5.3%), AI507 (1.1%), and N1303K (0.76%). Seven other mutations (621 + 1G T, Y122X, R347P, R334W, S549N, G551D, R1162X) were each identified in only one CF chromosome. Apart from G542X, most of the other mutations identified in this study were found to be associated with 7-(GATT)-repeats allele of IVS6A. In Southern France, only 73% of CF chromosomes could be identified by the analysis of 30 mutations.     

Aromatic cluster mutations produce focal modulations of β-sheet structure

Site-directed mutagenesis is a powerful tool for altering the structure and function of proteins in a focused manner. Here, we examined how a model β-sheet protein could be tuned by mutation of numerous surface-exposed residues to aromatic amino acids. We designed these aromatic side chain “clusters” at highly solvent-exposed positions in the flat, single-layer β-sheet of Borrelia outer surface protein A (OspA). This unusual β-sheet scaffold allows us to interrogate the effects of these mutations in the context of well-defined structure but in the absence of the strong scaffolding effects of globular protein architecture. We anticipated that the introduction of a cluster of aromatic amino acid residues on the β-sheet surface would result in large conformational changes and/or stabilization and thereby provide new means of controlling the properties of β-sheets. Surprisingly, X-ray crystal structures revealed that the introduction of aromatic clusters produced only subtle conformational changes in the OspA β-sheet. Additionally, despite burying a large degree of hydrophobic surface area, the aromatic cluster mutants were slightly less stable than the wild-type scaffold. These results thereby demonstrate that the introduction of aromatic cluster mutations can serve as a means for subtly modulating β-sheet conformation in protein design.     

Slow accumulation of mutations in Xpc−/− mice upon induction of oxidative stress

XPC is one of the key DNA damage recognition proteins in the global genome repair route of the nucleotide excision repair (NER) pathway. Previously, we demonstrated that NER-deficient mouse models Xpa^?/? and Xpc^?/? exhibit a divergent spontaneous tumor spectrum and proposed that XPC might be functionally involved in the defense against oxidative DNA damage. Others have mechanistically dissected several functionalities of XPC to oxidative DNA damage sensitivity using in vitro studies. XPC has been linked to regulation of base excision repair (BER) activity, redox homeostasis and recruitment of ATM and ATR to damage sites, thereby possibly regulating cell cycle checkpoints and apoptosis. XPC has additionally been implicated in recognition of bulky (e.g. cyclopurines) and non-bulky DNA damage (8-oxodG). However, the ultimate contribution of the XPC functionality in vivo in the oxidative DNA damage response and subsequent mutagenesis process remains unclear. Our study indicates that Xpc^?/? mice, in contrary to Xpa^?/? and wild type mice, have an increased mutational load upon induction of oxidative stress and that mutations arise in a slowly accumulative fashion. The effect of non-functional XPC in vivo upon oxidative stress exposure appears to have implications in mutagenesis, which can contribute to the carcinogenesis process. The levels and rate of mutagenesis upon oxidative stress correlate with previous findings that lung tumors in Xpc^?/? mice overall arise late in the lifespan and that the incidence of internal tumors in XP-C patients is relatively low in comparison to skin cancer incidence.     

Functional characterization of mutations in inherited human cPLA₂ deficiency

Group IVA cytosolic phospholipase A(2) (cPLA(2)α) catalyzes the first step in the arachidonic acid cascade leading to the synthesis of important lipid mediators, the prostaglandins and leukotrienes. We previously described a patient deficient in cPLA(2)α activity, which was associated with mutations in both alleles encoding the enzyme. In this paper, we describe the biochemical characterization of each of these mutations. Using saturating concentrations of calcium, we showed that the R485H mutant was nearly devoid of any catalytic activity, that the S111P mutation did not affect the enzyme activity, and that the known K651R polymorphism was associated with activity slightly higher than that of the wild type. Using MDCK cells, we showed that translocation to the Golgi in response to serum activation was impaired for the S111P mutant but not for the other mutants. Using immortalized mouse lung fibroblasts lacking endogenous cPLA(2)α activity, we showed that both mutations S111P and R485H/K651R caused a profound defect in the enzyme catalytic activity in response to cell stimulation with serum. Taken together, our results show that the S111P mutation hampers calcium binding and membrane translocation without affecting the catalytic activity, and that the mutation R485H does not affect membrane translocation but blocks catalytic activity that leads to inactivation of the enzyme. Interestingly, our results show that the common K651R polymorphism confers slightly higher activity to the enzyme, suggesting a role of this residue in favoring a catalytically active conformation of cPLA(2)α. Our results define how the mutations negatively influence cPLA(2)α function and explain the inability of the proband to release arachidonic acid for eicosanoid production.     

Effects of point mutations in pVHL on the binding of HIF-1α

The von Hippel-Lindau tumor suppressor protein (pVHL) has an essential role in the regulation of the hypoxia response pathway in animal cells. Under normoxic conditions, the hypoxia-inducible factor (HIF) undergoes trans-4-prolyl hydroxylation and is subsequently recognised by the β-domain of pVHL, leading to the ubiquitination and degradation of HIF. Mutations of pVHL alter the binding of HIF. A subset of relevant clinically observed mutations to pVHL are thought to cause weaker binding of HIF-1α and are associated with cancer and cardiovascular diseases. Here, we present computational studies analyzing the interaction of HIF with mutant forms of pVHL, describing at atomic detail the local structural reorganization caused by substitution of certain residues of pVHL. The results reveal that the canonical configuration in the wild-type system is vital for the efficient functioning of the complex and that mutation of any of the residues implicated in the h-bond network in the binding site disrupts HIF binding. Although the experimentally observed ordering of binding energies for mutants of Tyr98 is reproduced, our examination of a broader range of mutations does not support the hypothesis of a correlation between the degree of disruption of the pVHL/HIF-1α interaction caused by a mutation and the phenotype with which the mutation is associated. We suggest that disruption of the binding interaction is one of many factors behind the manifestation of VHL disease.     

Suppressors of thermosensitive mutations in the DNA polymerase δ gene of Saccharomyces cerevisiae

DNA polymerases (Pol) α, δ and ε are necessary for replication of nuclear DNA. Po1δ interacts permanently or transiently with numerous accessory proteins whose identification may shed light on the function(s) of Po18. In vitro mutagenesis was used to induce thermosensitive (ts) mutations in the DNA polymerase δ gene (POL3). We have attempted to clone two recessive extragenic suppressors of such is mutants (sdp1 for mutation pol3-14 and sdp5-1 for mutation pol3-11) by transforming thermoresistant haploid strains pol3-14 sdpl and pol3-11 sdp5-1 with wild-type genomic libraries in singlecopy or multicopy vectors. None of the thermosensitive transformants so obtained was identified as being sdp1 or sdp5-1. Instead, three genes were cloned whose products interfere with the activity of suppressors. One of them is the type 1 protein phosphatase gene, D1S2. Another is a novel gene, ASM4, whose gene product is rich in asparagine and glutamine residues.     

Is there a biological cost of protein disorder? Analysis of cancer-associated mutations

As many diseases can be traced back to altered protein function, studying the effect of genetic variations at the level of proteins can provide a clue to understand how changes at the DNA level lead to various diseases. Cellular processes rely not only on proteins with well-defined structure but can also involve intrinsically disordered proteins (IDPs) that exist as highly flexible ensembles of conformations. Disordered proteins are mostly involved in signaling and regulatory processes, and their functional repertoire largely complements that of globular proteins. However, it was also suggested that protein disorder entails an increased biological cost. This notion was supported by a set of individual IDPs involved in various diseases, especially in cancer, and the increased amount of disorder observed among disease-associated proteins. In this work, we tested if there is any biological risk associated with protein disorder at the level of single nucleotide mutations. Specifically, we analyzed the distribution of mutations within ordered and disordered segments. Our results demonstrated that while neutral polymorphisms were more likely to occur within disordered segments, cancer-associated mutations had a preference for ordered regions. Additionally, we proposed an alternative explanation for the association of protein disorder and the involvement in cancer with the consideration of functional annotations. Individual examples also suggested that although disordered segments are fundamental functional elements, their presence is not necessarily accompanied with an increased mutation rate in cancer. The presented study can help to understand how the different structural properties of proteins influence the consequences of genetic mutations.     

The spectrum of β-thalassemia mutations in northern and northeastern Thailand

Summary A total of 123 -thalassemia genes from northern (n = 113) and northeastern (n = 10) Thailand were examined. Using five oligonucleotide probes, the mutation in 108 genes (88%) was identified: 50 nonsense 17, 49 frameshift 41-42, 4-28(AG), 2IVS1 nt5(GC), 2IVS2 nt654, and 1 deletion removing the entire -globin gene. The nonsense 17 mutation (n = 39) was linked to a single haplotype, whereas the frameshift 41-42 mutation occurred with several haplotypes. The results of the present study indicate that prenatal diagnosis of clinically important -thalassemia syndromes using a limited set of oligonucleotides is feasible in approximately 80% of affected families in northern Thailand and most of the families with -thalassemia-Hb E disease in northeastern Thailand.     

Impact of β-galactosidase mutations on the expression of the canine lysosomal multienzyme complex

β-galactosidase (GLB1) forms a functional lysosomal multienzyme complex with lysosomal protective protein (PPCA) and neuraminidase 1 (NEU1) which is important for its intracellular processing and activity. Mutations in the β-galactosidase gene cause the lysosomal storage disease G_M1-gangliosidosis. In order to identify additional molecular changes associated with the presence of β-galactosidase mutations, the expression of canine lysosomal multienzyme complex components in GLB1^+/+, GLB1^+/? and GLB1^?/? fibroblasts was investigated by quantitative RT-PCR, Western blot and enzymatic assays. Quantitative RT-PCR revealed differential regulation of total β-galactosidase, β-galactosidase variants and protective protein for β-galactosidase gene (PPGB) in GLB1^+/? and GLB1^?/? compared to GLB1^+/+ fibroblasts. Furthermore, it was shown that PPGB levels gradually increased with the number of mutant β-galactosidase alleles while no change in the NEU1 expression was observed. This is the first study that simultaneously examine the effect of GLB1^+/+, GLB1^+/? and GLB1^?/? genotypes on the expression of lysosomal multienzyme complex components. The findings reveal a possible adaptive process in GLB1 homozygous mutant and heterozygous individuals that could facilitate the design of efficient therapeutic strategies.     

Frequency of mutations in “thrombophilic state” genes in Uzbekistan

The frequency of mutations in a number of genetic markers, specifically factor V gene (G1691A), blood coagulation factor II gene (G20210A), and the methylenetetrahydrofolate reductase (MTHFR) gene (C677T), is studied in ethnic Uzbek patients with deep vein thrombosis of the lower extremities and in healthy donors. It is established that the incidence of mutant alleles among patients in Uzbekistan for FV Leiden is 12.9%; for prothrombin, 4%; and for MTHFR, 47.8%. The mutant allele C677T of the MTHFR gene has the highest expressivity in the appearance of MTHFR (47.8%). It is noted that this mutation in the MTHFR gene is encountered significantly more frequently in females with deep vein thrombosis than in males with deep vein thrombosis. The G20210A mutation in the prothrombin gene is encountered more rarely in the Uzbek population. The penetrance is studied and the role of these mutations in the appearance of deep vein thrombosis is estimated.     

Are splicing mutations the most frequent cause of hereditary disease?

Disease-causing point mutations are assumed to act predominantly through subsequent individual changes in the amino acid sequence that impair the normal function of proteins. However, point mutations can have a more dramatic effect by altering the splicing pattern of the gene. Here, we describe an approach to estimate the overall importance of splicing mutations. This approach takes into account the complete set of genes known to be involved in disease and suggest that, contrary to current assumptions, many mutations causing disease may actually be affecting the splicing pattern of the genes.     

Molecular basis of β-thalassemia in Turkey: detection of rare mutations by direct sequencing

Summary Using restriction endonuclease analysis, oligonucleotide hybridization, and direct sequencing of amplified genomic DNA, we characterized 11 different mutations in the DNA of 26 patients from Turkey homozygous for -thalassemia. We found that mutations IVS-1 nt110, IVS-1 nt6, and the frameshift at codon 8 were the most frequent. By direct sequencing we characterized two very rare mutations not previously reported in the Turkish population: a frameshift +1 at codons 9/10 and a nonsense mutation at codon 15.     

A high proportion of mutations in the BRCA1 gene in German breast/ovarian cancer families with clustering of mutations in the 3′ third of the gene

We have analyzed 61 German breast and breast/ovarian cancer families for BRCA1 mutations using single-strand conformation polymorphism analysis (SSCP) followed by sequencing. Forty-seven of the families had at least three cases (at least two under 60 years) and 14 families had only two cases of breast/ovarian cancer (at least one under 50 years). Twenty-eight families were breast/ovarian and 33 were breast cancer-only families. Eighteen mutations in BRCA1 were detected in 11/28 breast/ovarian cancer families and 7/33 breast cancer families and none in the families with only two cases. We identified 17 truncation mutations (8 frameshift, 7 nonsense and 2 splice variants) and one missense mutation. Seven of these are novel and two, the 5382insC and 5622C→T mutations, occurred in two apparently unrelated families. The genotype of the two families with the 5382insC mutation is compatible with the rare haplotype segregating with the 5382insC mutation in different populations, further supporting its European origin. One unclassified missense alteration, R841W, was found in one family but did not segregate with the disease, suggesting that it is more likely a polymorphism. We also report and discuss the sequence of several new unclassified single-nucleotide changes first identified by SSCP. Of the 18 mutations, 13 occurred in the 3′ third of the gene (end of exon 11–24) and ovarian cancers were found in eight of these families. Received: 5 February 1998 / Accepted: 7 April 1998     

Structural stability of human protein tyrosine phosphatase ρ catalytic domain: effect of point mutations

Protein tyrosine phosphatase ρ (PTPρ) belongs to the classical receptor type IIB family of protein tyrosine phosphatase, the most frequently mutated tyrosine phosphatase in human cancer. There are evidences to suggest that PTPρ may act as a tumor suppressor gene and dysregulation of Tyr phosphorylation can be observed in diverse diseases, such as diabetes, immune deficiencies and cancer. PTPρ variants in the catalytic domain have been identified in cancer tissues. These natural variants are nonsynonymous single nucleotide polymorphisms, variations of a single nucleotide occurring in the coding region and leading to amino acid substitutions. In this study we investigated the effect of amino acid substitution on the structural stability and on the activity of the membrane-proximal catalytic domain of PTPρ. We expressed and purified as soluble recombinant proteins some of the mutants of the membrane-proximal catalytic domain of PTPρ identified in colorectal cancer and in the single nucleotide polymorphisms database. The mutants show a decreased thermal and thermodynamic stability and decreased activation energy relative to phosphatase activity, when compared to wild- type. All the variants show three-state equilibrium unfolding transitions similar to that of the wild- type, with the accumulation of a folding intermediate populated at ~4.0 M urea.