A genetic cluster of patients with variant xeroderma pigmentosum with two different founder mutations.

BACKGROUND: Xeroderma pigmentosum (XP) is a rare human syndrome associated with hypersensitivity to sunlight and a high frequency of skin tumours at an early age. We identified a community in the state of Goias (central Brazil), a sunny and tropical region, with a high incidence of XP (17 patients among approximately 1000 inhabitants). OBJECTIVES: To identify gene mutations in the affected community and map the distribution of the affected alleles, correlating the mutations with clinical phenotypes. METHODS: Functional analyses of DNA repair capacity and cell-cycle responses after ultraviolet exposure were investigated in cells from local patients with XP, allowing the identification of the mutated gene, which was then sequenced to locate the mutations. A specific assay was designed for mapping the distribution of these mutations in the community. RESULTS: Skin primary fibroblasts showed normal DNA damage removal but abnormal DNA synthesis after ultraviolet irradiation and deficient expression of the Polη protein, which is encoded by POLH. We detected two different POLH mutations: one at the splice donor site of intron 6 (c.764 +1 G>A), and the other in exon 8 (c.907 C>T, p.Arg303X). The mutation at intron 6 is novel, whereas the mutation at exon 8 has been previously described in Europe. Thus, these mutations were likely brought to the community long ago, suggesting two founder effects for this rare disease. CONCLUSIONS: This work describes a genetic cluster involving POLH, and, particularly unexpected, with two independent founder mutations, including one that likely originated in Europe.

Ataxia and myoclonic epilepsy due to a heterozygous new mutation in KCNA2: proposal for a new channelopathy.

We have recently performed exome analysis in a 7 year boy who presented in infancy with an encephalopathy characterized by ataxia and myoclonic epilepsy. Parents were not consanguineous and there was no family history of the disease. Exome analysis did not show any pathogenic variants in genes known to be associated with seizures and/or ataxia in children, including all known human channelopathies. However, we have identified a mutation in KCNA2 that we believe to be responsible for the disease in our patient. This gene, which encodes a member of the potassium channel, voltage-gated, shaker-related subfamily, has not been previously described as a cause of disease in humans, but mutations of the orthologous gene in mice (Kcna2) are known to cause both ataxia and convulsions. The mutation is c.890C>A, leading to the amino acid substitution p.Arg297Gln, which involves the second of the critical arginines in the S4 voltage sensor. This mutation is characterized as pathogenic by five different prediction programs. RFLP analysis and Sanger sequencing confirmed the presence of the mutation in the patient, but not in his parents, characterizing it as de novo. We believe that this discovery characterizes a new channelopathy.

A multiplex panel of short-amplicon insertion-deletion DNA polymorphisms for forensic analysis.

We have previously developed a panel of 40 insertion-deletion (INDEL) human DNA polymorphisms that was proven to ad-equately cover the span of global human genetic diversity. The panel was found to have very low matching probabilities with respect to both the global and Brazilian populations. To optimize the panel for application with degraded DNA samples, which are commonly encountered in fo-rensic analysis, we have significantly reduced the amplicon size of the INDELs and developed a new multiplex panel. The panel has an ampli-con size ranging from 50 to 153 base pairs, with a mean of 93 base pairs. It could be amplified by polymerase chain reaction in two multiplex re-actions, which were then combined for electrophoretic separation and identification of the individual products in the ABI3130 four-color DNA analyzer. The results of the new panel were fully validated.

Pharmacogenomics: accessing important alleles by imputation from commercial genome-wide SNP arrays.

Personalized medicine is becoming a medical reality, as important genotype-phenotype relationships are being unraveled. The availability of pharmacogenomic data is a key element of individualized care. In this study, we explored genotype imputation as a means to infer important pharmacogenomic alleles from a regular commercially available genome-wide SNP array. Using these arrays as a starting point can reduce testing costs, increasing access to these pharmacogenomic data and still retain a larger amount of genome-wide information. IMPUTE2 and MaCH-Admix were used to perform genotype imputation with a dense reference panel from 1000 Genomes data. We were able to correctly infer genotypes for the warfarin-related loci VKORC1 and CYP2C9 alleles 2, 3, 5, and 11 and also clopidogrel-related CYP2C19 alleles 2 and 17 for a small sample of Brazilian individuals, as well as for HapMap samples. The success of an imputation approach in admixed samples using publicly available reference panels can encourage further imputation initiatives in those populations.

Modulation of expressivity in PDGFRB-related infantile myofibromatosis: a role for PTPRG?

Infantile myofibromatosis is a rare genetic disorder characterized by the development of benign tumors in the skin, muscle, bone, and viscera. The molecular pathogenesis is still incompletely known. An autosomal dominant form had been reported as causally related with mutations in the gene for platelet-derived growth factor receptor beta (PDGFRB). We report here two siblings with infantile myofibromatosis and with a PDGFRB mutation identified by exome sequence analysis. However, the unaffected mother also had the same PDGFRB mutation. We showed that both children had also inherited from their healthy father a heterozygous mutation in the gene for receptor protein tyrosine phosphatase gamma (PTPRG), an enzyme known to dephosphorylate PDGFRB. We suggest that in this family, the additional mutation in PTPRG may explain the full phenotypic penetrance in the siblings affected, in comparison with the unaffected mother.

MULTINDELS-BOV: Zebu traceback method based on DNA insertion-deletion polymorphisms.

Brazil is a major producer and exporter of beef, with a herd of approximately 210 million animals. For the meat industry, a reliable animal traceback from its origin to the consumer market is paramount. Of all available identification systems, DNA is the only one that survives the slaughterhouse and reaches the dish of the consumer. DNA polymorphisms are already used for cattle traceback, but primarily for the subspecies Bos taurus taurus. However, in Brazil, another subspecies, B. taurus indicus predominates. We describe here the development of a DNA traceback method designed primarily for B. taurus indicus (Zebu), without leaving B. taurus taurus aside. We used insertion/deletion (indel) polymorphisms, which have the advantage of being simple and easily automatable, since in most cases, the variable loci are biallelic. We studied 94 indels, with a difference of two or more base pairs, in DNA pools of 60 Zebu and 60 taurine animals. A set of 22 indels with heterozygosity greater than 0.3 were selected and used to construct two multiplex PCRs. On the basis of the allelic frequency of these indels, the probability of random match was calculated to be 1.12 x 10(-8) for B. taurus indicus and 1.60 x 10(-6) for B. taurus taurus. Moreover, we estimated that an analysis would cost less than US$15.00 per animal. Thus, this system (MULTINDELS-BOV) is perfectly suited for building large genetic databases and offering viable prospects of a national system for cattle traceback DNA in Brazil.

Global pharmacogenomics: Impact of population diversity on the distribution of polymorphisms in the CYP2C cluster among Brazilians.

The impact of biogeographical ancestry, self-reported 'race/color' and geographical origin on the frequency distribution of 10 CYP2C functional polymorphisms (CYP2C8*2, *3, *4, CYP2C9*2, *3, *5, *11, CYP2C19*2, *3 and *17) and their haplotypes was assessed in a representative cohort of the Brazilian population (n=1034). TaqMan assays were used for allele discrimination at each CYP2C locus investigated. Individual proportions of European, African and Amerindian biogeographical ancestry were estimated using a panel of insertion-deletion polymorphisms. Multinomial log-linear models were applied to infer the statistical association between the CYP2C alleles and haplotypes (response variables), and biogeographical ancestry, self-reported Color and geographical origin (explanatory variables). The results showed that CYP2C19*3, CYP2C9*5 and CYP2C9*11 were rare alleles (<1%), the frequency of other variants ranged from 3.4% (CYP2C8*4) to 17.3% (CYP2C19*17). Two distinct haplotype blocks were identified: block 1 consists of three single nucleotide polymorphisms (SNPs) (CYP2C19*17, CYP2C19*2 and CYP2C9*2) and block 2 of six SNPs (CYP2C9*11, CYP2C9*3, CYP2C9*5, CYP2C8*2, CYP2C8*4 and CYP2C8*3). Diplotype analysis generated 41 haplotypes, of which eight had frequencies greater than 1% and together accounted for 96.4% of the overall genetic diversity. The distribution of CYP2C8 and CYP2C9 (but not CYP2C19) alleles, and of CYP2C haplotypes was significantly associated with self-reported Color and with the individual proportions of European and African genetic ancestry, irrespective of Color self-identification. The individual odds of having alleles CYP2C8*2, CYP2C8*3, CYP2C9*2 and CYP2C9*3, and haplotypes including these alleles, varied continuously as the proportion of European ancestry increased. Collectively, these data strongly suggest that the intrinsic heterogeneity of the Brazilian population must be acknowledged in the design and interpretation of pharmacogenomic studies of the CYP2C cluster in order to avoid spurious conclusions based on improper matching of study cohorts. This conclusion extends to other polymorphic pharmacogenes among Brazilians, and most likely to other admixed populations of the Americas.

DNA polymerase kappa from Trypanosoma cruzi localizes to the mitochondria, bypasses 8-oxoguanine lesions and performs DNA synthesis in a recombination intermediate.

DNA polymerase kappa (Pol kappa) is a low-fidelity polymerase that has the ability to bypass several types of lesions. The biological role of this enzyme, a member of the DinB subfamily of Y-family DNA polymerases, has remained elusive. In this report, we studied one of the two copies of Pol kappa from the protozoan Trypanosoma cruzi (TcPol kappa). The role of this TcPol kappa copy was investigated by analysing its subcellular localization, its activities in vitro, and performing experiments with parasites that overexpress this polymerase. The TcPOLK sequence has the N-terminal extension which is present only in eukaryotic DinB members, but its C-terminal region is more similar to prokaryotic and archaeal counterparts since it lacks C(2)HC motifs and PCNA interaction domain. Our results indicate that in contrast to its previously described orthologues, this polymerase is localized to mitochondria. The overexpression of TcPOLK increases T. cruzi resistance to hydrogen peroxide, and in vitro polymerization assays revealed that TcPol kappa efficiently bypasses 8-oxoguanine lesions. Remarkably, our results also demonstrate that the DinB subfamily of polymerases can participate in homologous recombination, based on our findings that TcPol kappa increases T. cruzi resistance to high doses of gamma irradiation and zeocin and can catalyse DNA synthesis within recombination intermediates.

Worldwide diversity of the Y-chromosome tetra-local microsatellite DYS464.

Of all DNA markers on the human Y-chromosome, the tetra-local Y-linked microsatellite DYS464 is the most polymorphic. We genotyped DYS464 in 677 male samples collected worldwide, maintained in the HGDP-CEPH Human Genome Diversity Cell Line Panel. Fourteen different alleles were found, with allele lengths varying from 9 to 23 repeats. One hundred and seventy-five different genotypes were detected, of which 90 appeared to be continent-specific. The region with the highest percentage of unique genotypes was Africa. Genotype diversity was 0.98 for Europe, 0.97 for Central and East Asia, 0.95 for Africa, 0.94 for Oceania, 0.92 for the Middle East, and 0.90 for the Americas. A hierarchical analysis of molecular variance showed low levels of worldwide genetic structure; 88.42% of the genetic variance was found within populations, 9.62% between populations within regions and 1.96% between regions. Since the four DYS464 repeats are identical, one cannot assign each peak in the electropherogram to a specific locus. Thus, the same genotype may correspond to several haplotypes, with different permutations of alleles. Consequently, genotypes are degenerate, which limits phylogeographical analyses. Yet, because of its high variability, DYS464 still constitutes an informative tool for population and evolutionary studies.

Efficient human paternity testing with a panel of 40 short insertion-deletion polymorphisms.

We developed a panel of 40 multiplexed short insertion-deletion (indel) polymorphic loci with widespread chromosomal locations and allele frequencies close to 0.50 in the European population. We genotyped these markers in 360 unrelated self-classified White Brazilians and 50 mother-child-probable father trios with proven paternity. The average heterozygosity (gene diversity) per locus was 0.48, and the combined probability of identity (matching probability) for the 40-locus set was 3.48 x 10(-17). The combined power of exclusion of the indel panel was 0.9997. The efficiency of the 40 indel set in the exclusion of falsely accused individuals in paternity casework was equivalent to the CODIS set of 13 microsatellites. The geometric mean of the paternity indices of the 50 mother-child-probable father trios was 17,607. This panel of 40 short indels was found to have excellent performance. Thus, especially because of its simplicity and low cost, and the fact that it is composed of genomic markers that have very low mutation rates, it represents a useful new tool for human paternity testing.

PedExpert: a computer program for the application of Bayesian networks to human paternity testing.

PedExpert is a Windows-based Bayesian network software, especially constructed to solve problems in parentage testing that are complex because of missing genetic information on the alleged father and/or because they involve genetic mutations. PedExpert automates the creation and manipulation of Bayesian networks, implementing algorithms that convert pedigrees and sets of indispensable information (genotypes, allele frequencies, mutation rates) into Bayesian networks. This program has a novel feature that can incorporate information about gene mutations into tables of conditional probabilities of transmission of alleles from the alleged father to the child, without adding new nodes to the network. This permits using the same Bayesian network in different modes, for analysis of cases that include mutations or not. PedExpert is user-friendly and greatly reduces the time of analysis for complex cases of paternity testing, eliminating most sources of logical and operational error.

Isolation and characterization of HC1: a novel human DNA repair gene.

Nucleotide excision repair (NER) acts on a broad spectrum of large lesions, while base excision repair removes individual modified bases. Although both processes have been well studied in human cells, novel genes involved in these DNA repair pathways have been described. Using a heterologous complementation approach, we identified a fetal human cDNA that complemented two Escherichia coli mutants that are defective in 3-methyl adenine glycosylase and in three endonucleases, all of which are enzymes with important roles in base excision repair. The central cDNA open reading frame complemented NER mutant strains and promoted an increase in survival rate of bacteria exposed to UV light. The corresponding protein was able to restore nucleotide-excision-repair activity when added to a cell extract from Chinese hamster ovary cells deficient in the ERCC1 protein, an enzyme known to promote incision at the 5' end of the lesion during NER. In contrast, that protein was not able to complement XPG Chinese hamster ovary cells deficient in the 3' incision step of NER. These data indicate a new human repair gene, which we named HC1; it is involved in the recognition of two kinds of DNA lesions and it contributes to the 5' DNA incision step in NER.

Cell culture and animal infection with distinct Trypanosoma cruzi strains expressing red and green fluorescent proteins.

Different strains of Trypanosoma cruzi were transfected with an expression vector that allows the integration of green fluorescent protein (GFP) and red fluorescent protein (RFP) genes into the beta-tubulin locus by homologous recombination. The sites of integration of the GFP and RFP markers were determined by pulse-field gel electrophoresis and Southern blot analyses. Cloned cell lines selected from transfected epimastigote populations maintained high levels of fluorescent protein expression even after 6 months of in vitro culture of epimastigotes in the absence of drug selection. Fluorescent trypomastigotes and amastigotes were observed within Vero cells in culture as well as in hearts and diaphragms of infected mice. The infectivity of the GFP- and RFP-expressing parasites in tissue culture cells was comparable to wild type populations. Furthermore, GFP- and RFP-expressing parasites were able to produce similar levels of parasitemia in mice compared with wild type parasites. Cell cultures infected simultaneously with two cloned cell lines from the same parasite strain, each one expressing a distinct fluorescent marker, showed that at least two different parasites are able to infect the same cell. Double-infected cells were also detected when GFP- and RFP-expressing parasites were derived from strains belonging to two distinct T. cruzi lineages. These results show the usefulness of parasites expressing GFP and RFP for the study of various aspects of T. cruzi infection including the mechanisms of cell invasion, genetic exchange among parasites and the differential tissue distribution in animal models of Chagas disease.

Huntington disease-like 2: the first patient with apparent European ancestry.

Huntington disease-like 2 (HDL2) is a rare autosomal dominant disorder of the nervous system, apparently indistinguishable from Huntington disease (HD). HDL2 is caused by the expansion above 40 CTG/CAG repeats, in a variably spliced exon of the junctophilin-3 gene, on chromosome 16q24.3. All patients described so far have been of African ancestry. A clinical evaluation, including the Unified Huntington's Disease Rating Scale, and brain Magnetic resonance imaging were achieved in a 48-year-old Brazilian man of apparent European extraction, and presenting a picture very suggestive of HD. Gene mutation analysis (HD, HDL1, HDL2, dentatorubralpallidoluysian atrophy and spinocerebellar ataxia 17) was performed. After exclusion of the HD mutation and other HDL disorders, we identified an expansion of 47 CTG/CAG at the HDL2 locus. To clarify the origin of the mutation and estimate the patient's ancestry, we performed haplotype studies and used the insertion/deletion polymorphisms method. Despite the fact that this patient had an estimated likelihood of 97.4% of being of European ancestry, the haplotype containing the expanded allele has been found only in Africans. Thus, this is the first HDL2 case reported in a patient with an apparent European ancestry, although bearing an African HDL2 haplotype. This work stresses the importance of performing the diagnosis of HDL2 in HD-like patients of various ethnicities, and particularly in highly mixed populations.

Sex-biased gene flow in African Americans but not in American Caucasians.

We have previously shown evidence of strong sex-biased genetic blending in the founding and ongoing history of the Brazilian population, with the African and Amerindian contribution being highest from maternal lineages (as measured by mitochondrial DNA) and the European contribution foremost from paternal lineages (estimated from Y-chromosome haplogroups). The same phenomenon has been observed in several other Latin American countries, suggesting that it might constitute a universal characteristic of the Iberian colonization of the Americas. However, it has also recently been detected in the Black population of the United States. We thus wondered if the same could be observed in American Caucasians. To answer that question, we retrieved 1387 hypervariable I Caucasian mitochondrial DNA sequences from the FBI population database and established their haplogroups and continental geographical sources. In sharp contrast with the situation of the Caucasian population of Latin American countries, only 3.1% of the American Caucasian sequences had African and/or Amerindian origin. To explain this discrepancy we propose that the finding of elevated genomic contributions from European males and Amerindian or African females depends not only on the occurrence of directional mating, but also on the "racial" categorization of the children born from these relations. In this respect, social practices in Latin America and in the United States diverge considerably; in the former socially significant "races" are normally designated according to physical appearance, while in the latter descent appears to be the most important factor.

Analysis of DNA polymerase activity in vitro using non-radioactive primer extension assay in an automated DNA sequencer.

Although different DNA polymerases have distinct functions and substrate affinities, their general mechanism of action is similar. Thus, they can all be studied using the same technical principle, the primer extension assay employing radioactive tags. Even though fluorescence has been used routinely for many years for DNA sequencing, it has not been used in the in vitro primer extension assay. The use of fluorescence labels has obvious advantages over radioactivity, including safety, speed and ease of manipulation. In the present study, we demonstrated the potential of non-radioactive in vitro primer extension for DNA polymerase studies. By using an M13 tag in the substrate, we can use the same fluorescent M13 primer to study different substrate sequences. This technique allows quantification of the DNA polymerase activity of the Klenow fragment using different templates and under different conditions with similar sensitivity to the radioactive assay.

Determination of ancestral proportions in synthetic bovine breeds using commonly employed microsatellite markers.

The International Society of Animal Genetics (ISAG) has chosen nine microsatellites (international marker set) as a standard that should be included in all cattle parentage studies. They are BM1824, BM2113, INRA023, SPS115, TGLA122, TGLA126, TGLA227, ETH10, and ETH225. We decided to ascertain whether this microsatellite set could be used to determine ancestral proportions in individual animals of synthetic breeds produced by crossing zebu and taurine cattle. Since the genotypes of these markers are routinely available, this would constitute a practical and cost-free method to estimate the ancestry of synthetic breed animals. Genotypes of 100 Gir and 100 Holstein animals were examined for this ISAG marker set. As expected, there were very significant allele frequency differences between the two breeds at most loci. We also typed 20 Girolando animals for which there was complete genealogical information. "Structure" software easily distinguished Holstein and Gir animals based on their microsatellite genotypes; it also attributed the genomic proportion of zebu and taurine of each of the 20 Girolando animals. The proportion of Holstein ancestry was then regressed on the genealogical data; there was a highly significant correlation (r = 0.84, P < 0.0001). The nine microsatellites that compose the ISAG international marker set were capable of estimating the ancestral Gir and Holstein genomic proportions in individual Girolando animals within narrow confidence limits. This microsatellite set might also be useful for estimating the proportions of taurine and zebu origins in commercial meat products.

Real time PCR strategy for the identification of major lineages of Trypanosoma cruzi directly in chronically infected human tissues.

Two evolutionary lineages, called Trypanosoma cruzi I and II, have been identified in T. cruzi, the etiologic agent of human Chagas disease. Here, we describe a molecular strategy for direct genetic typing of these major groups of T. cruzi directly in human tissues. The protocol is based on heminested PCR amplification of the D7 region of the 24Salpha ribosomal DNA (rDNA), followed by identification of the products using denaturation curves in real time PCR. The repetitive nature of the gene, and the heminested PCR format insured the high sensitivity necessary to detect the presence of the very scarce T. cruzi DNA present in the chronically infected human tissues. There is 80% DNA sequence homology between the two 24Salpha rDNA alleles that define the T. cruzi I and II groups, sufficient to produce different thermal denaturation curves with melting temperature (TM) values of 81.7+/-0.43 and 78.2+/-0.33 degrees C (mean+/-SEM). Using this technical approach, we analysed tissue samples (esophagi, hearts and colon) from 25 different patients with the gastrointestinal or cardiac forms of Chagas disease; in all of them we found only the presence of T cruzi II. Previous epidemiological and immunological findings had already led to the idea that chronic human infections occurring in Brazil and Argentina might be primarily due to T. cruzi II strains, but all the evidence available had been indirect. Our findings provide definitive proof of this hypothesis and will also allow the establishment of which group of T. cruzi is responsible for Chagas disease in other countries.

Y-box binding protein from Schistosoma mansoni: interaction with DNA and RNA.

A Schistosoma mansoni homologue of the human Y-box binding protein (SMYB1), as well as truncated proteins containing its N-terminal Cold Shock Domain (CSD) or its C-terminal domain (TAIL) were cloned into the p-MAL-c2 expression vector and produced in Escherichia coli. In order to characterize the interactions of these proteins to an inverted CCAAT motif present in a number of gene promoters, their binding to DNA was measured by Electrophoretic Mobility Shift Assays. SMYB1 bound to single- and double-stranded DNA containing the CCAAT motif and could bind also to RNA. The truncated CSD and TAIL domain proteins bound to dsDNA and RNA, but exhibited distinct binding patterns. Protein-DNA interaction was also investigated in vivo, using the Yeast One-Hybrid System. The plasmid constructs were GSTTRI, a DNA fragment composed of three copies of the CCAAT motif of the S. mansoni glutathione S-transferase gene promoter and four oligonucleotides spanning different regions of the S. mansoni p14 gene promoter. None of the yeast clones transformed with the above plasmids was able to grow in selective medium or to activate the transcription of the HIS3 reporter gene, suggesting that SMYB1 could not interact with these promoters in vivo.

Nucleic acid binding properties of SmZF1, a zinc finger protein of Schistosoma mansoni.

During its life cycle, the flat worm Schistosoma mansoni is exposed to diverse environmental conditions and changes its morphological form. Each change calls for distinct patterns of gene expression. In order to understand the regulation of gene expression, it is necessary to identify regulatory elements in the promoter region of genes, and DNA transacting factors that control transcription. Zinc finger protein domains are responsible for transcription regulation of diverse genes in a wide range of organisms and are also involved in the promotion of protein-protein interactions. A transcript homologous to zinc finger gene sequences was isolated from a S. mansoni adult worm cDNA library and named SmZF1. It codes for a protein of 164 amino acids presenting three C(2)H(2) type zinc finger motifs. The recombinant SmZF1 protein was expressed and used on electrophoretic mobility shift assays to investigate the binding specificity of SmZF1 for DNA and RNA oligonucleotides. Our results demonstrated that SmZF1 binds both ds and ss DNA oligonucleotides, with an apparent preference for the specific D1-3DNA oligonucleotide, and also binds RNA oligonucleotides with lower affinity. Although we found that SmZF1 recognises DNA and RNA oligonucleotides not containing putative target sites, SmZF1 binds preferentially to sequence specific sites. Furthermore, unrelated oligonucleotides are not able to abolish this interaction. In silico studies identified putative SmZF1 binding sites in the complete genome of three model organisms and in partial genome sequences of S. mansoni. Six Drosophila genes presented these binding sites in their promoter region, indicating that they might be controlled by transcription factors containing zinc fingers motifs. Taken together, these results suggest that SmZF1 acts as a putative transcription factor of S. mansoni.