Destabilization of transthyretin by pathogenic mutations in the DE loop

Transthyretin single-amino-acid variants are responsible for familial amyloidotic polyneuropathy, in which transthyretin variants accumulate extracellularly in the form of fibrillar aggregates. We studied the structural stabilities of four transthyretin variants (L58H, L58R, T59K, and E61K), in which a positively charged amino acid is introduced in a loop region between the D- and E-strands. In addition to being located in the DE-loop, L58 and T59 are involved in the core of the transthyretin monomer. The L58H, L58R, and T59K substitutions destabilized transthyretin more than the E61K mutation did, indicating that transthyretin is substantially destabilized by the substitution of residues located in both the DE-loop and the monomer core. By utilizing hydrogen-deuterium exchange and nuclear magnetic resonance, we demonstrated that residues in the G-strand and the loop between the A- and B-strands were destabilized by these pathogenic mutations in the DE loop. At the quaternary structural level, the DE-loop mutations destabilized the dimer-dimer contact area, which may lead to transient dissociation into a dimer. Our results suggest that the destabilization of the dimer-dimer interface and the monomer core is important for the amyloidogenesis of transthyretin.     

Haplotype distribution and mutations at the PAH locus in Croatia

Summary Restriction fragment length polymorphism (RFLP) haplotypes and mutations at the phenylalanine hydroxylase (PAH) locus have been studied in 25 unrelated families from Croatia. The results of RFLP analysis demonstrated that 80% of the mutant alleles were associated with three haplotypes (1, 2 and 4). Eight mutations were detected on the background of six mutant haplotypes, comprising 68% of phenylketonuria (PKU) alleles in Croatia. The mutation in codon 408 was most frequent, as was the haplotype 2 allele with which it was associated. These data are in accordance with formerly published population genetic analyses at the PAH locus, and with studies revealing the molecular basis of the phenotypic heterogeneity of PKU. The codon 281 mutation was more frequent in Croatia than previously observed in other populations.     

Haplotype analysis of familial amyloidotic polyneuropathy

Summary Familial amyloidotic polyneuropathy (FAP) is an autosomal dominant genetic disease characterized by systemic accumulation of amyloid fibrils. A major component of FAP anyloid has been identified as variant transthyretin (TTR, also called prealbumin). In particular, a variant with the substitution ³⁰ValMet has been commonly found in FAP of various ethnic groups. To understand the origin and spread of the ValMet mutation, we analyzed DNA polymorphisms associated with the TTR gene in six Japanese FAP families and several Portuguese FAP patients. Three distinct haplotypes associated with the ValMet mutation were identified in Japanese FAP families, one of which was also found in Portuguese patients. On the other hand, it was found that the ValMet mutation can be explained by a C-T transition at the C_pG dinucleotide sequence of a mutation hot spot. Thus, our findings indicate that the ValMet mutation has probably recurred in the human population, to generate FAP families of independent origin.     

Haplotype analysis of DFNB8/10 locus reveals contribution of TMPRSS3 mutations in Pakistani deaf population

TMPRSS3 mutations are associated with non-syndromic recessive deafness (DFNB8/10). To evaluate the frequency of TMPRSS3 mutation in Pakistani population, highly consanguineous families were enrolled. A group of five consanguineous families without any history of associated environmental causes were found to be linked to DFNB8/10 locus. To correlate haplotypes and to evaluate founder affect 17 other families linked to DFNB8/10 were provided by NCEMB DNA bank. Haploytpe analysis revealed that out of 22 families, haplotypes of 8 families (42 %) were found similar to PKDF003 and PKDF311 having 207delC mutation, 5 (26 %) families had haplotypes similar to PKDF040 and 4 families (15.7 %) shared halpotypes similar to PKDF064, having C407R (1219T>C) and C194F (518G>T) mutations, respectively. Interestingly, PKDF321 and PKDF337 (10.5 %) showed different haplotypes and might harbor novel mutation. Taken together, these data imply that Punjab region is more affected by TMPRSS3 mutation, and the founder-effect mutation might be traced back to Punjab region.     

Haplotype sharing analysis using mantel statistics

OBJECTIVE: The potential value of haplotypes has attracted widespread interest in the mapping of complex traits. Haplotype sharing methods take the linkage disequilibrium information between multiple markers into account, and may have good power to detect predisposing genes. We present a new approach based on Mantel statistics for spacetime clustering, which is developed in order to improve the power of haplotype sharing analysis for gene mapping in complex disease. METHODS: The new statistic correlates genetic similarity and phenotypic similarity across pairs of haplotypes for case-only and case-control studies. The genetic similarity is measured as the shared length between haplotypes around a putative disease locus. The phenotypic similarity is measured as the mean-corrected cross-product based on the respective phenotypes. We analyzed two tests for statistical significance with respect to type I error: (1) assuming asymptotic normality, and (2) using a Monte Carlo permutation procedure. The results were compared to the chi(2) test for association based on 3-marker haplotypes. RESULTS: The results of the type I error rates for the Mantel statistics using the permutational procedure yielded pointwise valid tests. The approach based on the assumption of asymptotic normality was seriously liberal. CONCLUSION: Power comparisons showed that the Mantel statistics were better than or equal to the chi(2) test for all simulated disease models.     

Haplotype and phenotype analysis of six recurrent BRCA1 mutations in 61 families: results of an international study.

Several BRCA1 mutations have now been found to occur in geographically diverse breast and ovarian cancer families. To investigate mutation origin and mutation-specific phenotypes due to BRCA1, we constructed a haplotype of nine polymorphic markers within or immediately flanking the BRCA1 locus in a set of 61 breast/ovarian cancer families selected for having one of six recurrent BRCA1 mutations. Tests of both mutations and family-specific differences in age at diagnosis were not significant. A comparison of the six mutations in the relative proportions of cases of breast and ovarian cancer was suggestive of an effect (P = .069), with 57% of women presumed affected because of the 1294 del 40 BRCA1 mutation having ovarian cancer, compared with 14% of affected women with the splice-site mutation in intron 5 of BRCA1. For the BRCA1 mutations studied here, the individual mutations are estimated to have arisen 9-170 generations ago. In general, a high degree of haplotype conservation across the region was observed, with haplotype differences most often due to mutations in the short-tandem-repeat markers, although some likely instances of recombination also were observed. For several of the instances, there was evidence for multiple, independent, BRCA1 mutational events.     

Haplotype and phenotype analysis of nine recurrent BRCA2 mutations in 111 families: results of an international study.

Several BRCA2 mutations are found to occur in geographically diverse breast and ovarian cancer families. To investigate both mutation origin and mutation-specific phenotypes due to BRCA2, we constructed a haplotype of 10 polymorphic short tandem-repeat (STR) markers flanking the BRCA2 locus, in a set of 111 breast or breast/ovarian cancer families selected for having one of nine recurrent BRCA2 mutations. Six of the individual mutations are estimated to have arisen 400-2,000 years ago. In particular, the 6174delT mutation, found in approximately 1% of individuals of Ashkenazi Jewish ancestry, was estimated to have arisen 29 generations ago (1-LOD support interval 22-38). This is substantially more recent than the estimated age of the BRCA1 185delAG mutation (46 generations), derived from our analogous study of BRCA1 mutations. In general, there was no evidence of multiple origins of identical BRCA2 mutations. Our study data were consistent with the previous report of a higher incidence of ovarian cancer in families with mutations in a 3.3-kb region of exon 11 (the ovarian cancer cluster region [OCCR]) (P=.10); but that higher incidence was not statistically significant. There was significant evidence that age at diagnosis of breast cancer varied by mutation (P<.001), although only 8% of the variance in age at diagnosis could be explained by the specific mutation, and there was no evidence of family-specific effects. When the age at diagnosis of the breast cancer cases was examined by OCCR, cases associated with mutations in the OCCR had a significantly older mean age at diagnosis than was seen in those outside this region (48 years vs. 42 years; P=.0005).     

Disease-associated mutations impacting BC-loop flexibility trigger long-range transthyretin tetramer destabilization and aggregation

Hereditary transthyretin amyloidosis (ATTR) is an autosomal dominant disease characterized by the extracellular deposition of the transport protein transthyretin (TTR) as amyloid fibrils. Despite the progress achieved in recent years, understanding why different TTR residue substitutions lead to different clinical manifestations remains elusive. Here, we studied the molecular basis of disease-causing missense mutations affecting residues R34 and K35. R34G and K35T variants cause vitreous amyloidosis, whereas R34T and K35N mutations result in amyloid polyneuropathy and restrictive cardiomyopathy. All variants are more sensitive to pH-induced dissociation and amyloid formation than the wild-type (WT)-TTR counterpart, specifically in the variants deposited in the eyes amyloid formation occurs close to physiological pHs. Chemical denaturation experiments indicate that all the mutants are less stable than WT-TTR, with the vitreous amyloidosis variants, R34G and K35T, being highly destabilized. Sequence-induced stabilization of the dimer–dimer interface with T119M rendered tetramers containing R34G or K35T mutations resistant to pH-induced aggregation. Because R34 and K35 are among the residues more distant to the TTR interface, their impact in this region is therefore theorized to occur at long range. The crystal structures of double mutants, R34G/T119M and K35T/T119M, together with molecular dynamics simulations indicate that their strong destabilizing effect is initiated locally at the BC loop, increasing its flexibility in a mutation-dependent manner. Overall, the present findings help us to understand the sequence-dynamic-structural mechanistic details of TTR amyloid aggregation triggered by R34 and K35 variants and to link the degree of mutation-induced conformational flexibility to protein aggregation propensity.     

Rapid detection of common southeast Asian beta-thalassemia mutations by nonisotopic multiplex PCR-SSCP analysis

This report describes the detection of seven beta-thalassemia mutations common in Southeast Asia by amplifying three short PCR fragments in two separate tubes, followed by single-strand conformation polymorphism (SSCP) analysis in single lanes. These mutations are -28 A --> G, codon 17 A --> T, IVS1 + 5 G --> C, codon 41/42 -CTTT, codon 43 G --> T, codon 71/72 + A, and IVS2 + 654 C --> T, and account for 70% to over 95% of the cases in this region. This rapid nonisotopic method was also found capable of detecting other mutations within the amplified fragments. It is simple, rapid, and cheap, and thus suitable for carrier screening and prenatal diagnosis in Southeast Asia.     

Fine mapping of the human biotinidase gene and haplotype analysis of five common mutations

Biotinidase deficiency is an autosomal recessive defect in the recycling of biotin that can lead to a variety of neurologic and cutaneous symptoms. The disease can be prevented or effectively treated with exogenous biotin. The biotinidase locus (BTD) has been maped to 3p25 by in situ hybridization. The gene has been cloned, the coding region sequenced, the genomic organization determined, and a spectrum of mutations has been characterized in more than 90 individuals with profound or partial biotinidase deficiency. We have conducted haplotype analysis of 10 consanguineous and 39 nonconsanguineous probands from the United States and 8 consanguineous probands from Turkey to localize BTD with respect to polymorphic markers on 3p and to investigate the origins of five common mutations. The inbred probands were homozygous for overlapping regions of 3p ranging in size from 1.1 to 80 cM which were flanked most narrowly by D3S1259 and D3S1293. Radiation hybrids and haplotype analysis of markers within this region suggest that BTD is located within a 0.1-cM region flanked by D3S3510 and D3S1286. The radiation hybrid data suggest that the BTD gene is oriented 5' to 3' between the centromere and the 3p telomere. Association studies indicate that the gene is closer to a third locus D3S3613 than D3S3510, two markers which cannot be resolved by existing linkage data. The BTD locus and D3S3613 must therefore lie between D3S3510 and D3S1286. Comparison of haplotypes reveals evidence for possible founder effects for four of the five common mutations.     

mtDNA mutations and common neurodegenerative disorders

The incidence and prevalence of Alzheimer's disease (AD) and Parkinson's disease (PD) are increasing as the population ages. Both disorders have been associated with oxidative stress and mitochondrial dysfunction, and it has been proposed that mutations in the mitochondrial genome have a key role in neurodegeneration in AD and PD patients. Two recent publications propose that heteroplasmic mtDNA mutations are involved in AD and PD. However, when these new studies are considered in relation to the sum of previous evidence, the role of mtDNA mutations in the development of either AD or PD still remains to be established.     

Detecting Local Haplotype Sharing and Haplotype Association

A novel haplotype association method is presented, and its power is demonstrated. Relying on a statistical model for linkage disequilibrium (LD), the method first infers ancestral haplotypes and their loadings at each marker for each individual. The loadings are then used to quantify local haplotype sharing between individuals at each marker. A statistical model was developed to link the local haplotype sharing and phenotypes to test for association. We devised a novel method to fit the LD model, reducing the complexity from putatively quadratic to linear (in the number of ancestral haplotypes). Therefore, the LD model can be fitted to all study samples simultaneously, and, consequently, our method is applicable to big data sets. Compared to existing haplotype association methods, our method integrated out phase uncertainty, avoided arbitrariness in specifying haplotypes, and had the same number of tests as the single-SNP analysis. We applied our method to data from the Wellcome Trust Case Control Consortium and discovered eight novel associations between seven gene regions and five disease phenotypes. Among these, GRIK4, which encodes a protein that belongs to the glutamate-gated ionic channel family, is strongly associated with both coronary artery disease and rheumatoid arthritis. A software package implementing methods described in this article is freely available at http://www.haplotype.org.     

Mechanism of rescue of common p53 cancer mutations by second-site suppressor mutations

The core domain of p53 is extremely susceptible to mutations that lead to loss of function. We analysed the stability and DNA-binding activity of such mutants to understand the mechanism of second-site suppressor mutations. Double-mutant cycles show that N239Y and N268D act as 'global stability' suppressors by increasing the stability of the cancer mutants G245S and V143A-the free energy changes are additive. Conversely, the suppressor H168R is specific for the R249S mutation: despite destabilizing wild type, H168R has virtually no effect on the stability of R249S, but restores its binding affinity for the gadd45 promoter. NMR structural comparisons of R249S/H168R and R249S/T123A/H168R with wild type and R249S show that H168R reverts some of the structural changes induced by R249S. These results have implications for possible drug therapy to restore the function of tumorigenic mutants of p53: the function of mutants such as V143A and G245S is theoretically possible to restore by small molecules that simply bind to and hence stabilize the native structure, whereas R249S requires alteration of its mutant native structure.     

Haplotype and multipoint linkage analysis in Finnish choroideremia families

Summary Multipoint linkage analysis of choroideremia (TCD) and seven X chromosomal restriction fragment length polymorphisms (RFLPs) was carried out in 18 Finnish TCD families. The data place TCD distal to PGK and DXS72, very close to DXYS1 and DXYS5 (Zmax = 24 at = 0) and proximal to DXYS4 and DXYS12. This agrees with the data obtained from other linkage studies and from physical mapping. All the TCD males and carrier females studied have the same DXYS1 allele in coupling with TCD. In Northeastern Finland, 66/69 chromosomes carrying TCD had the same haplotype at loci DXS72, DXYS1, DXYS4, and DXYS12. The same haplotype is seen in only 15/99 chromosomes not carrying TCD. Moreover, in 71/104 non-TCD chromosomes, the haplotype at six marker loci is different from those seen in any of the 76 TCD chromosomes. This supports the previously described hypothesis that the large Northern Finnish choroideremia pedigrees, comprising a total of over 80 living patients representing more than a fifth of all TCD patients described worldwide, carry the same mutation. These linkage and haplotype data provide improved opportunities for prenatal diagnosis based on RFLP studies.     

Haplotype Parsing

While the shared consensus genetic sequence of our species contains a great deal of information about our common biology, there is also much to be learned from the subtle genetic variations across our species. These variations are believed to be generally of little or no direct functional significance and predominantly reflect the chance accumulation of small genetic changes since our emergence as a species. Therefore, they carry little useful information when observed in a single individual. When tallied across a whole population though, these chance mutations can teach us a great deal about our evolutionary history and the patterns of inheritance in particular individuals. In particular, frequently observed patterns of single nucleotide polymorphisms (SNPs) in a population can identify segments of chromosome that have been passed down largely intact through long stretches of our evolution. Finding these frequently conserved chromosomal segments, or haplotypes, and developing methods to identify haplotype patterns in particular individuals, will in turn help us to identify those particular segments that carry genetic factors influencing risk for many common human diseases. To make the best use of this data, we will need to develop new models for the encoding of information in genome variations--the "language of genetic variation"--and new algorithms for fitting datasets to those models. This article surveys past work by the author and colleagues on this problem, utilising computational methods for locating frequent patterns in haploid sequence data, and "parsing" sequences so as to optimally explain them given the knowledge of the general population structure. The author's recent work in this area has been compiled into a set of computational tools available at http://www-2.cs.cmu.edu/~russells/software/hapmotif.html.     

Fluorescent multicolor multiplex homogeneous assay for the simultaneous analysis of the two most common hemochromatosis mutations

We report the development of a qualitative fluorescent multiplex homogeneous assay designed for the detection of the two most common hemochromatosis mutations using dual-labeled fluorescent probes. The assay is able to detect four allelic variants in a single closed tube using a single thermocycling protocol. The procedure combines the great sensitivity of the polymerase chain reaction, the specificity provided by allele-specific oligonucleotide hybridization using the 5(') nuclease assay format, and the higher throughput of a multicolor fluorescence detection procedure. Genomic DNA was prepared from whole blood specimens using standard procedures. Following DNA sample preparation, two regions of the hemochromatosis gene (HFE) including the H63D and C282Y mutations were coamplified and detected in real-time by four different fluorescently labeled allele-specific oligonucleotide probes. Assay specificity was demonstrated by a blind methods comparison study that included 37 DNA samples from individuals with a known HFE genotype. Results from the study showed that the multicolor multiplex HFE assay unambiguously classified all possible genotypes for the HFE gene C282Y and H63D mutations(1). This technique will be useful for research and molecular diagnostic laboratories and can be easily adapted for the detection of other single nucleotide polymorphisms.     

Clinical and molecular analysis of common MEFV gene mutations in familial Mediterranean fever in Sivas population

Familial Mediterranean fever (FMF) is the most frequent hereditary inflammatory disease characterized by self-limited recurrent attacks of fever and serositis. The aim of the current study is to determine the frequency of the mutations in 365 suspected FMF patients and to reveal whether there is a correlation between genotype and phenotype of these patients. All patients were clinically examined according to Tell-Hashomer FMF criteria and were screened genetically in terms of common 12 Mediterranean fever gene (MEFV) mutations. Various point mutations were detected in 270 (74%) patients. The most frequent mutation was M694V (26.85% of the alleles) and was followed by E148Q (15.55%), M680I (G/C) (9.62%) and V726A (7.96%). Patients who bear M694V homozygous mutation had most severe disease phenotype and high risk for amyloidosis (P = 0.04). Our results indicate that Sivas population has a wide range of heterozygous mutated carriers of MEFV gene and there is a high frequency of E148Q allele when compared to the other Mediterranean groups.     

Haplotype analyses, mechanism and evolution of common double mutants in the human LDL receptor gene

Familial hypercholesterolemia (FH), an autosomal dominant inherited disorder resulting in increased levels of circulating plasma low-density lipoprotein (LDL), tendon xanthomas and premature coronary artery disease (CAD), is caused by defects in the LDL receptor gene (LDLR). Three widespread LDLR alterations not causing FH (c.1061-8T>C, c.2177C>T and c.829G>A) and one mutation (c.12G>A) with narrow geographical distribution and thought to cause disease were investigated. In an attempt to improve knowledge on their origin, spread and possible selective effects, estimations of the ages of these variants (t generations) and haplotype analysis were performed by genotyping 86 healthy individuals and 98 FH patients in Spain for five LDLR SNPs: c.81T>C, c.1413G>A, c.1725C>T, c.1959T>C, and c.2232G>A; most patients carried two of these LDLR variants simultaneously. It was found that both the c.1061-8T>C (t = 54) and c.2177C>T alterations (t = 62) arose at about the same time (54 and 62 generations ago, respectively) in the CGCTG haplotype, while the c.12G>A mutation (t = 70) appeared in a CGCCG haplotype carrying an earlier c.829G>A alteration (t = 83). The estimated ages of selectively neutral alterations could explain their distribution by migrations. The origin of the c.12G>A mutation could be in the Iberian Peninsula; despite its estimated age, a low selective pressure could explain its conservation in Spain from where it could have spread to China and Mexico, since the sixteenth century through the Spanish/Portuguese colonial expeditions.