Molecular Characterization of ß-Thalassemia Mutations in Central Vietnam.

The molecular basis of ß-thalassemia (ß-thal) mutations in North and in South Vietnam have been described during the past 15 years, whereas limited data were available concerning the central area of the country. In this study, we describe the molecular characterization and frequency of ß-globin gene mutations in the Thua Thien Hue Province of Central Vietnam as the result of a first survey conducted in 22 transfusion-dependent patients, and four unrelated heterozygotes. Nine different known mutations were identified (seven of the ß0 and two of the ß+ type) in a total of 48 chromosomes. The most common was codon 26 (G>A) or Hb E (HBB: c.79 G>A) accounting for 29.2% of the total studied chromosomes, followed by codon 17 (A>T) (HBB: c.52 A>T) (25.0%), and codons 41/42 (-TTCT) (HBB: c.126_129delCTTT) (18.8%). Other mutations with appreciable frequencies (6.3-8.3%) were IVS-I-1 (G>T) (HBB: c.92+1 G>T), codon 26 (G>T) (HBB: c.79 G>T) and codons 71/72 (+A) (HBB: c.216_217insA). Relatively rarer (2.0%) were the promoter -28 (A>G) (HBB: c.78 A>G) mutation, the codon 95 (+A) (HBB: c.287_288insA), which is reported only in the Vietnamese, and the codons 14/15 (+G) (HBB: c.45_46insG) mutation, thus far observed only in Thailand. Results are relevant for implementing appropriate measures for ß-thal prevention and control in the region as well as in the whole country.

Application of a new method for GWAS in a related case/control sample with known pedigree structure: identification of new loci for nephrolithiasis.

In contrast to large GWA studies based on thousands of individuals and large meta-analyses combining GWAS results, we analyzed a small case/control sample for uric acid nephrolithiasis. Our cohort of closely related individuals is derived from a small, genetically isolated village in Sardinia, with well-characterized genealogical data linking the extant population up to the 16(th) century. It is expected that the number of risk alleles involved in complex disorders is smaller in isolated founder populations than in more diverse populations, and the power to detect association with complex traits may be increased when related, homogeneous affected individuals are selected, as they are more likely to be enriched with and share specific risk variants than are unrelated, affected individuals from the general population. When related individuals are included in an association study, correlations among relatives must be accurately taken into account to ensure validity of the results. A recently proposed association method uses an empirical genotypic covariance matrix estimated from genome-screen data to allow for additional population structure and cryptic relatedness that may not be captured by the genealogical data. We apply the method to our data, and we also investigate the properties of the method, as well as other association methods, in our highly inbred population, as previous applications were to outbred samples. The more promising regions identified in our initial study in the genetic isolate were then further investigated in an independent sample collected from the Italian population. Among the loci that showed association in this study, we observed evidence of a possible involvement of the region encompassing the gene LRRC16A, already associated to serum uric acid levels in a large meta-analysis of 14 GWAS, suggesting that this locus might lead a pathway for uric acid metabolism that may be involved in gout as well as in nephrolithiasis.

Iodinated 4,4'-Bipyridines with Antiproliferative Activity Against Melanoma Cell Lines.

In the last decade, biological processes involving halogen bond (HaB) as a leading interaction attracted great interest. However, although bound iodine atoms are considered powerful HaB donors, few iodinated new drugs were reported so far. Recently, iodinated 4,4'-bipyridines showed interesting properties as HaB donors in solution and in the solid state. In this paper, a study on the inhibition activity of seven halogenated 4,4'-bipyridines against malignant melanoma (MM) cell proliferation is described. Explorative dose/response proliferation assays were first performed with three 4,4'-bipyridines by using four MM cell lines and the normal BJ fibroblast cell line as control. Among them, the A375 MM cell line was the most sensitive, as determined by MTT assays, which was selected to evaluate the antiproliferative activity of all 4,4'-bipyridines. Significantly, the presence of an electrophilic iodine impacted the biological activity of the corresponding compounds. The 3,3',5,5'-tetrachloro-2-iodo-4,4'-bipyridine showed significant antiproliferation activity against the A375 cell line, and lower toxicity on BJ fibroblasts. Through in silico studies, the stereoelectronic features of possible sites determining the bioactivity were explored. These results pave the way for the utilization of iodinated 4,4'-bipyridines as templates to design new promising HaB-enabled inhibitors of MM cell proliferation.

Emergence of Talanin protein associated with human uric acid nephrolithiasis in the Hominidae lineage.

Recently, we identified a susceptibility locus for human uric acid nephrolithiasis (UAN) on 10q21-q22 and demonstrated that a novel gene (ZNF365) included in this region produces through alternative splicing several transcripts coding for four protein isoforms. Mutation analysis showed that one of them (Talanin) is associated with UAN. We examined the evolutionary conservation of ZNF365 gene through a comparative genomic approach. Searching for mouse homologs of ZNF365 transcripts, we identified a highly conserved mouse ortholog of ZNF365A transcript, expressed specifically in brain. We did not found a mouse homolog for ZNF365D transcript encoding the Talanin protein, even if we were able to identify the corresponding genomic region in mouse and rat not yet organized in canonical gene structure suggesting that ZNF365D was originated after the branching of hominoid from rodent lineage. In mouse and in most mammals, a functional uricase degrades the uric acid to allantoin, but uricase activity was lost during the Miocene epoch in hominoids. Searching for the presence of Talanin in Primates, we found a canonical intron-exon structure with several stop codons preventing protein production in Old World and New World monkeys. In humans, we observe expression and we have evidence that ZNF365D transcript produces a functional protein. It seems therefore that ZNF365D transcript emerged during primate evolution from a noncoding genomic sequence that evolved in a standard gene structure and assumed its role in parallel with the disappearance of uricase, probably against a disadvantageous excessive hyperuricemia.

Phylogeny and patterns of diversity of goat mtDNA haplogroup A revealed by resequencing complete mitogenomes.

We sequenced to near completion the entire mtDNA of 28 Sardinian goats, selected to represent the widest possible diversity of the most widespread mitochondrial evolutionary lineage, haplogroup (Hg) A. These specimens were reporters of the diversity in the island but also elsewhere, as inferred from their affiliation to each of 11 clades defined by D-loop variation. Two reference sequences completed the dataset. Overall, 206 variations were found in the full set of 30 sequences, of which 23 were protein-coding non-synonymous single nucleotide substitutions. Many polymorphic sites within Hg A were informative for the reconstruction of its internal phylogeny. Bayesian and network clustering revealed a general similarity over the entire molecule of sequences previously assigned to the same D-loop clade, indicating evolutionarily meaningful lineages. Two major sister groupings emerged within Hg A, which parallel distinct geographical distributions of D-loop clades in extant stocks. The pattern of variation in protein-coding genes revealed an overwhelming role of purifying selection, with the quota of surviving variants approaching neutrality. However, a simple model of relaxation of selection for the bulk of variants here reported should be rejected. Non-synonymous diversity of Hg's A, B and C denoted that a proportion of variants not greater than that allowed in the wild was given the opportunity to spread into domesticated stocks. Our results also confirmed that a remarkable proportion of pre-existing Hg A diversity became incorporated into domestic stocks. Our results confirm clade A11 as a well differentiated and ancient lineage peculiar of Sardinia.

Haplotype affinities resolve a major component of goat (Capra hircus) MtDNA D-loop diversity and reveal specific features of the Sardinian stock.

Goat mtDNA haplogroup A is a poorly resolved lineage absorbing most of the overall diversity and is found in locations as distant as Eastern Asia and Southern Africa. Its phylogenetic dissection would cast light on an important portion of the spread of goat breeding. The aims of this work were 1) to provide an operational definition of meaningful mtDNA units within haplogroup A, 2) to investigate the mechanisms underlying the maintenance of diversity by considering the modes of selection operated by breeders and 3) to identify the peculiarities of Sardinian mtDNA types. We sequenced the mtDNA D-loop in a large sample of animals (1,591) which represents a non-trivial quota of the entire goat population of Sardinia. We found that Sardinia mirrors a large quota of mtDNA diversity of Western Eurasia in the number of variable sites, their mutational pattern and allele frequency. By using bayesian analysis, a distance-based tree and a network analysis, we recognized demographically coherent groups of sequences identified by particular subsets of the variable positions. The results showed that this assignment system could be reproduced in other studies, capturing the greatest part of haplotype diversity.We identified haplotype groups overrepresented in Sardinian goats as a result of founder effects. We found that breeders maintain diversity of matrilines most likely through equalization of the reproductive potential. Moreover, the relevant amount of inter-farm mtDNA diversity found does not increase proportionally with distance. Our results illustrate the effects of breeding practices on the composition of maternal gene pool and identify mtDNA types that may be considered in projects aimed at retrieving the maternal component of the oldest breeds of Sardinia.

A new essential hypertension susceptibility locus on chromosome 2p24-p25, detected by genomewide search.

Essential hypertension (EH) is a complex disorder that results from the interaction of a number of susceptibility genes and environmental factors. We studied an isolated Sardinian village (Talana) in which the prevalence of hypertension is comparable to that in most Western populations. Talana exhibits features, such as slow demographic growth, high inbreeding, a low number of founders, stable lifestyle and culture, and accurate genealogical records, that make it suitable for the study of complex disorders. Clinical assessment of the entire adult population (N= approximately 1,000) identified approximately 100 hypertensive subjects. For our study, we selected the individuals with the most-severe EH (i.e., diastolic blood pressure >100 mm Hg), belonging to a single deep-rooted pedigree (12 generations), whose common ancestors lived in the 17th century. We performed a three-stage genomewide search using 36 affected individuals, by means of parametric linkage and allele-sharing approaches. LOD scores >1 were observed on chromosomes 1, 2, 13, 15, 17, and 19 (stage I). The most striking result was found in a 7.57-cM region on chromosome 2p24-p25. All five nonparametric linkage statistics estimated by the SimWalk2 program lie above the significance threshold of P<.008 for the whole region. Similar significance was obtained for 2p24-25 when parametric linkage (LOD score 1.99) and linkage disequilibrium mapping (P=.00006) were used, suggesting that a hypertension-susceptibility locus is located between D2S2278 and D2S168. This finding is strengthened by a recent report of linkage with marker D2S168 in a hypertensive sib-pair sample from China.

Not all isolates are equal: linkage disequilibrium analysis on Xq13.3 reveals different patterns in Sardinian sub-populations.

Recent studies indicate that, whereas the Sardinian population as a whole is comparable to outbred populations for linkage disequilibrium (LD) mapping of common variants, LD in Sardinian sub-isolates is more extended, making these populations particularly suitable for this approach. To evaluate the extent of LD between microsatellite markers, we compared different sub-populations within Sardinia selected on the basis of their geographical position and isolation: two small isolated villages (Talana, Urzulei), two larger but remote areas (Ogliastra, Nuoro province) and a cohort of samples representing the wider Sardinian population. LD analysis was carried out by using six microsatellite markers that are located on Xq13.3 and that have been extensively studied in different populations. We found different extents and patterns of LD in the sub-population samples depending on their degree of isolation and demographic history. All LD measurements and haplotype analyses indicate that there is a decreasing trend from Talana (the most inbred population, LD up to 9.5-11.5 Mb) to the more outbred Sardinian population (LD only for intervals <2 Mb). In one village (Talana), five haplotype classes accounting for 80% of the entire sample perfectly matched five Ogliastra clusters, supporting the origin of the village from the Ogliastra genetic pool. In contrast, the other village (Urzulei) showed a different pattern of haplotypes with a closer relationship to the Nuoro region sub-population. LD analyses therefore show that even neighbouring isolate villages may differ in their genetic background. Here, we highlight the importance of selecting appropriate populations and/or sub-populations for the analysis of complex traits. Isolated sub-populations showing different extents of LD can provide a powerful method for mapping complex traits by LD scanning at relatively low marker density.

Identification of a novel gene and a common variant associated with uric acid nephrolithiasis in a Sardinian genetic isolate.

Uric acid nephrolithiasis (UAN) is a common disease with an established genetic component that presents a complex mode of inheritance. While studying an ancient founder population in Talana, a village in Sardinia, we recently identified a susceptibility locus of approximately 2.5 cM for UAN on 10q21-q22 in a relatively small sample that was carefully selected through genealogical information. To refine the critical region and to identify the susceptibility gene, we extended our analysis to severely affected subjects from the same village. We confirm the involvement of this region in UAN through identical-by-descent sharing and autozygosity mapping, and we refine the critical region to an interval of approximately 67 kb associated with UAN by linkage-disequilibrium mapping. After inspecting the genomic sequences available in public databases, we determined that a novel gene overlaps this interval. This gene is divided into 15 exons, spanning a region of approximately 300 kb and generating at least four different proteins (407, 333, 462, and 216 amino acids). Interestingly, the last isoform was completely included in the 67-kb associated interval. Computer-assisted analysis of this isoform revealed at least one membrane-spanning domain and several N- and O-glycosylation consensus sites at N-termini, suggesting that it could be an integral membrane protein. Mutational analysis shows that a coding nucleotide variant (Ala62Thr), causing a missense in exon 12, is in strong association with UAN (P=.0051). Moreover, Ala62Thr modifies predicted protein secondary structure, suggesting that it may have a role in UAN etiology. The present study underscores the value of our small, genealogically well-characterized, isolated population as a model for the identification of susceptibility genes underlying complex diseases. Indeed, using a relatively small sample of affected and unaffected subjects, we identified a candidate gene for multifactorial UAN.