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A non-negligible proportion of human pathogenic variants are known to be present as wild type in at least some non-human mammalian species. The standard explanation for this finding is that molecular mechanisms of compensatory epistasis can alleviate the mutations' otherwise pathogenic effects. Examples of compensated variants have been described in the literature but the interacting residue(s) postulated to play a compensatory role have rarely been ascertained. In this study, the examination of five human X-chromosomally encoded proteins (FIX, GLA, HPRT1, NDP and OTC) allowed us to identify several candidate compensated variants. Strong evidence for a compensated/compensatory pair of amino acids in the coagulation FIXa protein (involving residues 270 and 271) was found in a variety of mammalian species. Both amino acid residues are located within the 60-loop, spatially close to the 39-loop that performs a key role in coagulation serine proteases. To understand the nature of the underlying interactions, molecular dynamics simulations were performed. The predicted conformational change in the 39-loop consequent to the Glu270Lys substitution (associated with hemophilia B) appears to impair the protein's interaction with its substrate but, importantly, such steric hindrance is largely mitigated in those proteins that carry the compensatory residue (Pro271) at the neighboring amino acid position.

Understanding the role of common polymorphisms in modulating the clinical phenotype when they co‐occur with a disease‐causing lesion is of critical importance in medical genetics. We explored the impact of apparently neutral common polymorphisms, using the gene encoding the urea cycle enzyme, ornithine transcarbamylase (OTC), as a model system. Distinct combinations of genetic backgrounds embracing two missense polymorphisms were created in cis with the pathogenic p.Arg40His replacement. In vitro enzymatic assays revealed that the polymorphic variants were able to modulate OTC activity both in the presence or absence of the pathogenic lesion. First, we found that the combination of the minor alleles of polymorphisms p.Lys46Arg and p.Gln270Arg significantly enhanced enzymatic activity in the wild‐type protein. Second, enzymatic assays revealed that the minor allele of the p.Gln270Arg polymorphism was capable of ameliorating OTC activity when combined in cis with the pathogenic p.Arg40His replacement. Structural analysis predicted that the minor allele of the p.Gln270Arg polymorphism would serve to stabilize the OTC wild‐type protein, thereby corroborating the results of the experimental assays. Our findings demonstrate the potential importance of cis‐interactions between common polymorphic variants and pathogenic missense mutations and illustrate how standing genetic variation can modulate protein function.

Gene duplication plays a significant role in evolution. Paralogous gene copies may be lost due to the successive accumulation of deleterious mutations or remain active in the genome. In this work, a partial duplication of an X-linked region in the Macaca genus is identified and explored. Genomic comparisons reveal that the duplication encompasses the genes encoding ornithine transcarbamylase (OTC) and retinitis pigmentosa GTPase regulator (RPGR), spanning over 0.1 Mb on the chromosome 9 of Macaca. According to our analyses, the duplicated region of chromosome 9 involves partial coding sequences of both OTC and RPGR genes. Analyses of the selective pressures did not reveal significant differences in the ratio between nonsynonymous and synonymous mutations (w1), suggesting that no selective pressures were acting in the evolutionary process. Reports for a biological role regarding some partial duplications exist in the literature, therefore, although being rare events, partial duplications of functionally important genes are worthy of study so that their impact can be explored.

Lysosomal Storage Disorders (LSDs) are a group of rare inherited metabolic diseases caused by the malfunction of the lysosomal system, which results in the accumulation of undegraded substrates inside the lysosomes and leads to severe and progressive pathology. Despite there is currently a broad understanding of the molecular defects behind LSDs, curative therapies have been approved for only few of these diseases whereas existing treatments are still mostly symptomatic with several limitations. Mucolipidosis type II alpha/beta (ML II) is one of most severe LSDs, which is caused by the total deficiency of the GlcNAc-1-phosphotransferase, a key enzyme for the formation of specific targeting signals on lysosomal hydrolases to lysosomes. GlcNAc-1-phosphotransferase is a multimeric enzyme complex encoded by two genes: GNPTAB and GNPTG. One of the most frequent ML II causal mutation is a dinucleotide deletion on exon 19 of GNPTAB (c.3503_3504del) that leads to the generation of a truncated protein, loss of GlcNAc-1-phosphotransferase activity, and missorting of multiple lysosomal enzymes. Presently, there is no therapy available for ML II. In this study we explored the possibility of an innovative therapeutic strategy for ML II based on the use of antisense oligonucleotides (AOs) capable to induce the skipping of GNPTAB exon 19 harboring the most common disease-causing mutation, c.3503_3504del. The approach confirmed the ability of specific AOs for RNA splicing modulation, thus paving the way for future studies on the therapeutic potential of this strategy. This work was financially supported by Fundação para a Ciência e Tecnologia (FCT) IP (project: PTDC /BBBBMD/6301/2014); and Asociación Nour de Mucolipidosis (project: Skip 19). info:eu-repo/semantics/publishedVersion

In the past few years, there has been an increasing neuroscientific interest in understanding the function of mammalian chromodomains helicase DNA-binding (CHD) proteins due to their association with severe developmental syndromes. Mammalian CHDs include nine members (CHD1 to CHD9), grouped into subfamilies according to the presence of specific functional domains, generally highly conserved in evolutionary terms. Mutations affecting these domains hold great potential to disrupt protein function, leading to meaningful pathogenic scenarios, such as embryonic defects incompatible with life. Here, we analysed the evolution of CHD proteins by performing a comparative study of the functional domains of CHD proteins between orthologous and paralogous protein sequences. Our findings show that the highest degree of inter-species conservation was observed at Group II (CHD3, CHD4, and CHD5) and that most of the pathological variations documented in humans involve amino acid residues that are conserved not only between species but also between paralogs. The parallel analysis of both orthologous and paralogous proteins, in cases where gene duplications have occurred, provided extra information showing patterns of flexibility as well as interchangeability between amino acid positions. This added complexity needs to be considered when the impact of novel mutations is assessed in terms of evolutionary conservation.

COMPETE 2020. POCI-01-0145-FEDER-007274 to i3S. Submitted by Maria da Luz Antunes (mluz.antunes@estesl.ipl.pt) on 2019-08-27T10:17:35Z No. of bitstreams: 1 Unusual β-globin haplotype distribution in newborns from Bengo, Angola.pdf: 1300567 bytes, checksum: 707e68d119bc13ff44b0bde94b8ffabc (MD5) Made available in DSpace on 2019-08-27T10:17:35Z (GMT). No. of bitstreams: 1 Unusual β-globin haplotype distribution in newborns from Bengo, Angola.pdf: 1300567 bytes, checksum: 707e68d119bc13ff44b0bde94b8ffabc (MD5) Previous issue date: 2019-08 info:eu-repo/semantics/publishedVersion

To obtain refreshed insights into the paternal lineages of Tunisian populations, Y-chromosome diversity was assessed in two populations belonging to an Arab genealogical lineage, Kairouan and Wesletia, as well as in four Tunisian Andalusian populations, Testour, Slouguia, Qalaat-El-Andalous and El Alia. The Arabs from Kairouan revealed 73.47% of E-M81 and close affinities with Berber groups, indicating they are likely arabized Berbers, clearly differentiated from the Arabs from Wesletia, who harbored the highest frequency (71.8%) of the Middle Eastern component ever observed in North Africa. In the Tunisian Andalusians, the North African component largely prevailed, followed by the Middle Eastern contribution. Global comparative analysis highlighted the heterogeneity of Tunisian populations, among which, as a whole, dominated a set of lineages ascribed to be of autochthonous Berber origin (71.67%), beside a component of essentially Middle Eastern extraction (18.35%), and signatures of Sub-Saharan (5.2%), European (3.45%) and Asiatic (1.33%) contributions. The remarkable frequency of T-M70 in Wesletia (17.4%) prompted to refine its phylogeographic analysis, allowing to confirm its Middle Eastern origin, though signs of local evolution in Northern Africa were also detected. Evidence was clear on the ancient introduction of T lineages into the region, probably since Neolithic times associated to spread of agriculture.

Review More than two thirds of Lysosomal Storage Diseases (LSDs) present central nervous system involvement. Nevertheless, only one of the currently approved therapies has an impact on neuropathology. Therefore, alternative approaches are under development, either addressing the underlying enzymatic defect or its downstream consequences. Also under study is the possibility to block substrate accumulation upstream, by promoting a decrease of its synthesis. This concept is known as substrate reduction therapy and may be triggered by several molecules, such as small interfering RNAs (siRNAs). siRNAs promote RNA interference, a naturally occurring sequence-specific post-transcriptional gene-silencing mechanism, and may target virtually any gene of interest, inhibiting its expression. Still, naked siRNAs have limited cellular uptake, low biological stability, and unfavorable pharmacokinetics. Thus, their translation into clinics requires proper delivery methods. One promising platform is a special class of liposomes called stable nucleic acid lipid particles (SNALPs), which are characterized by high cargo encapsulation efficiency and may be engineered to promote targeted delivery to specific receptors. Here, we review the concept of SNALPs, presenting a series of examples on their efficacy as siRNA nanodelivery systems. By doing so, we hope to unveil the therapeutic potential of these nanosystems for targeted brain delivery of siRNAs in LSDs. This work was supported by the Portuguese Society for Metabolic Disorders (Sociedade Portuguesa de Doenças Metabólicas, SPDM—Bolsa SPDM de apoio à investigação Dr. Aguinaldo Cabral 2018; 2019DGH1629/ SPDM2018I&D) and Sanfilippo Children’s Foundation (SCF Incubator Grant 2019; 2019DGH1656/SCF2019I&D). Additional support came from the European Regional Development Fund (ERDF), through the Centro 2020 Regional Operational Programme (project CENTRO-01-0145-FEDER-000008: BrainHealth 2020), and the COMPETE 2020—Operational Programme for Competitiveness and Internationalisation and Portuguese national funds via FCT—Fundação para a Ciência e a Tecnologia (projects POCI-01-0145-FEDER-016390: CANCEL STEM and POCI-01-0145-FEDER-007440; UIDB/04539/2020). L.S.M. is funded by FCT/MCTES National Funds under project CEECIND/04242/2017 info:eu-repo/semantics/publishedVersion

The gene encoding the cytosolic ß-glucosidase GBA3 shows pseudogenization due to a truncated allele (rs358231) that is polymorphic in humans. Since this enzyme is involved in the transformation of many plant ß-glycosides, this particular case of gene loss may have been influenced by dietary adaptations during evolution. In humans, apart from the inactivating allele, we found that GBA3 accumulated additional damaging mutations, implying an extensive GBA3 loss. The allelic distribution of loss-of-function alleles revealed significant differences between human populations which can be partially related with their staple diet. The analysis of mammalian orthologs disclosed that GBA3 underwent at least nine pseudogenization events. Most events of pseudogenization occurred in carnivorous lineages, suggesting a possible link to a ß-glycoside poor diet. However, GBA3 was also lost in omnivorous and herbivorous species, hinting that the physiological role of GBA3 is not fully understood and other unknown causes may underlie GBA3 pseudogenization. Such possibility relies upon a putative role in sialic acid biology, where GBA3 participates in a cellular network involving NEU2 and CMAH. Overall, our data shows that the recurrent loss of GBA3 in mammals is likely to represent an evolutionary endpoint of the relaxation of selective constraints triggered by diet-related factors. This work was supported by FEDER - Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 - Operacional Programme for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through FCT - Fundação para a Ciência e a Tecnologia, in the framework of the Project POCI-01–0145-FEDER-007274 to i3S and by FCT research Project POCI-01–0145-FEDER-29723. CS holds FCT PhD fellowship (SFRH/BD/137925/2018). ARC holds FCT PhD fellowship (SFRH/BD/141702/2018). Funders had no role in the design, collection, analysis, interpretation of the data, and writing of the manuscript. The authors would like to thank the reviewers for their comments and suggestions.

Taste perception is crucial in monitoring food intake and, hence, is thought to play a significant role in human evolution. To gain insights into possible adaptive signatures in genes encoding bitter, sweet, and umami taste receptors, we surveyed the available sequence variation data from the 1000 Genomes Project Phase 3 for TAS1R (TAS1R1-3) and TAS2R (TAS2R16 and TAS2R38) families. Our study demonstrated that genes from these two families have experienced contrasting evolutionary histories: While TAS1R1 and TAS1R3 showed worldwide evidence of positive selection, probably correlated with improved umami and sweet perception, the patterns of variation displayed by TAS2R16 and TAS2R38 were more consistent with scenarios of balancing selection that possibly conferred a heterozygous advantage associated with better capacity to perceive a wide range of bitter compounds. In TAS2R16, such adaptive events appear to have occurred restrictively in mainland Africa, whereas the strongest evidence in TAS2R38 was detected in Europe. Despite plausible associations between taste perception and the TAS1R and TAS2R selective signatures, we cannot discount other biological mechanisms as driving the evolutionary trajectories of those TAS1R and TAS2R members, especially given recent findings of taste receptors behaving as the products of pleiotropic genes involved in many functions outside the gustatory system. IPATIMUP is included in the i3S Consortium, which is partially supported by the Portuguese Foundation for Science and Technology (FCT). This work is also funded by FEDER funds through the Operational Programme Competitiveness Factors (COMPETE) and National Funds through the FCT (projects PEst-C/SAU/LA0003/2013 and POCI-01-0145-FEDER-007274, fellowships SFRH/BD/63343/2009 to C.V., SFRH/ BPD/65000/2009 to L.A., and SFRH/BPD/120777/2016 to P.I.M.), and by Programa Operacional Regional do Norte (Norte 2020), through FEDER funds under the Quadro de Referência Estratégico Nacional (QREN; NORTE-01-0145-FEDER-000029). We thank Jacquelyn Beals for the careful editing of the work. We also thank Sònia Casillas for the helpful instructions about the tracks from the PopHuman browser, and Miguel Arenas and Eduardo Conde Sousa for the aid in extracting data from those tracks.

The Roma population is the largest transnational ethnic minority in Europe, characterized by a linguistic, cultural and historical heterogeneity. Comparative linguistics and genetic studies have placed the origin of European Roma in the Northwest of India. After their migration across Persia, they entered into the Balkan Peninsula, from where they spread into Europe, arriving in the Iberian Peninsula in the 15th century. Their particular demographic history has genetic implications linked to rare and common diseases. However, the South Asian source of the proto-Roma remains still untargeted and the West Eurasian Roma component has not been yet deeply characterized. Here, in order to describe both the South Asian and West Eurasian ancestries, we analyze previously published genome-wide data of 152 European Roma and 34 new Iberian Roma samples at a fine-scale and haplotype-based level, with special focus on the Iberian Roma genetic substructure. Our results suggest that the putative origin of the proto-Roma involves a Punjabi group with low levels of West Eurasian ancestry. In addition, we have identified a complex West Eurasian component (around 65%) in the Roma, as a result of the admixture events occurred with non-proto-Roma populations between 1270–1580. Particularly, we have detected the Balkan genetic footprint in all European Roma, and the Baltic and Iberian components in the Northern and Western Roma groups, respectively. Finally, our results show genetic substructure within the Iberian Roma, with different levels of West Eurasian admixture, as a result of the complex historical events occurred in the Peninsula., Author summary Human demographic processes and admixture events leave traceable footprints in the genomes of the populations and they can modulate the genetic architecture of complex diseases. Here, we aim to study the Roma people, an admixed population with a particular demographic history recognized as the largest ethnic minority in Europe. Previous studies suggest that they originated in South Asia 1,500 years ago and followed a diaspora towards Europe with extensive admixture with non-Roma West Eurasian groups. However, the genetic components of the Roma have not been deeply characterized. Our study reveals a common South Asian origin of all European Roma, closely related to a Punjabi group from Northwestern India. Through fine-scale haplotype-based methods, we describe a complex West Eurasian genetic component in the Roma groups, identifying a common Balkan ancestry and country-specific admixture footprints consistent with the dispersion through Europe. Our findings provide new insights into the demographic history and recent admixture events that have shaped the genetic composition of European Roma groups and could enable a better genetic characterization of complex disease in this population.

Neurodevelopmental disorders (NDDs) represent a growing medical challenge in modern societies. Ever-increasing sophisticated diagnostic tools have been continuously revealing a remarkably complex architecture that embraces genetic mutations of distinct types (chromosomal rearrangements, copy number variants, small indels, and nucleotide substitutions) with distinct frequencies in the population (common, rare, de novo). Such a network of interacting players creates difficulties in establishing rigorous genotype-phenotype correlations. Furthermore, individual lifestyles may also contribute to the severity of the symptoms fueling a large spectrum of gene-environment interactions that have a key role on the relationships between genotypes and phenotypes.Herein, a review of the genetic discoveries related to NDDs is presented with the aim to provide useful general information for the medical community. This work was financed by FEDER - Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 - Operacional Programme for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through FCT - Fundação para a Ciência e a Tecnologia, in the framework of the project POCI-01-0145-FEDER-007274 to i3S and UID/ BIM/04501/2013 and UID/BIM/04501/2019 to iBiMED, as well as by national funds (OE), through FCT, in the scope of the framework contract foreseen in the numbers 4, 5, and 6 of the article 23, of the Decree-Law 57/2016, of August 29, changed by Law 57/2017, of July 19 to RMS, and by FCT research project POCI-01-0145-FEDER-29723. ARC and CS hold FCT PhD fellowships (SFRH/BD/141702/2018-ARC and SFRH/BD/137925/2018-CS). Funders had no role in the design, collection, analysis, interpretation of the data, and writing of the manuscript.

Recent genomic analyses suggest that the current North African gene pool was mainly influenced by population flow coming from the East that altered the genetic structure of autochthonous Berber populations. Such genetic flow has not been extensively addressed yet using North African populations of Middle-eastern origin as reference.To discern the Middle-eastern component in the genetic background of Tunisian Arabs and evaluate the extent of gene flow from the Middle East into North African autochthonous Berber populations.This study has examined 113 Tunisians of well-known Arabian origin from Kairouan region, using 15 autosomal Short Tandem Repeats (STRs) loci.No deviations from Hardy-Weinberg equilibrium were observed and all loci presented high levels of heterozygosity. Principal coordinate and STRUCTURE analyses were consistent in clustering together North African and Middle Eastern populations, likely reflecting the recent gene flow from the East dating back to the Arab conquest period. This demographic migration and the Arabisation process that submerged the original Berber language and customs seems to have be accompanied by substantial gene flow and genetic admixture.This study represents an additional step to obtain a comprehensive understanding of the complex demographic history of North African populations.

Objectives The purpose of this study was to genetically characterize the male lineages of people who speak Mirandese, an interesting case of a linguistic relict that can still be found in the municipality of Miranda do Douro, NE Portugal. This region lies within the area of the Leonese dialects, which are remnants of the Romance dialects spoken in the Kingdom of Leon currently grouped in the Astur–Leonese linguistic continuum. We intended to disclose affinities with surrounding populations, namely from Spain where the Astur–Leonese is also spoken. Methods Eighty-eight unrelated males (58 from Miranda and 30 from Braganca, the broad Portuguese region where Miranda is located) were genotyped with the combined use of 17 Y chromosome short tandem repeats (Y-STRs) and a high resolution Y chromosome single nucleotide polymorphism (Y-SNPs) strategy. Moreover, 236 males from Miranda and neighboring regions, previously classified as R-M269, were also genotyped. Results R-P312 was the most frequent haplogroup in the Mirandese, followed by J-12f2.1 and T-M70. The male lineages J-12f2.1 and T-M70 were also well represented, and both were shared with descendants of Sephardic Jews. No signs of diversity reduction were detected. Conclusions Mirandese speakers display a Y chromosome gene pool that shows a subtle differentiation from neighboring populations, mainly attributable to the assimilation of lineages ascribed to be of Jewish ancestry. Although not revealing signs of geographic/linguistic isolation, no clear affinities with other Astur–Leonese populations were detected. The results suggest that in Miranda language sharing is not accompanied by significant gene flow between populations from both sides of the political border. Am. J. Hum. Biol. 28:671–680, 2016. © 2016 Wiley Periodicals, Inc.

The newly-synthesized lysosomal enzymes travel to the trans-Golgi network (TGN) and are then driven to the acidic organelle. While the best-known pathway for TGN-to-endosome transport is the delivery of soluble hydrolases by the M6P receptors (MPRs), additional pathways do exist, as showed by the identification of two alternative receptors: LIMP-2, implicated in the delivery of β-glucocerebrosidase; and sortilin, involved in the transport of the sphingolipid activator proteins prosaposin and GM2AP, acid sphingomyelinase and cathepsins D and H. Disruption of the intracellular transport and delivery pathways to the lysosomes may result in lysosomal dysfunction, predictably leading to a range of clinical manifestations of lysosomal storage diseases. However, for a great percentage of patients presenting such manifestations, no condition is successfully diagnosed. To analyse if, in this group, phenotypes could be determined by impairments in the known M6P-independent receptors, we screened the genes that encode for LIMP-2 and sortilin. No pathogenic mutations were identified. Other approaches will be needed to clarify whether sortilin dysfunction may cause disease.

There is still much uncertainty about the demographic history of East Asia human populations, especially from China, where the Han is by far the main ethnic group. We have performed the genetic characterization of 130 Y-chromosomes from two Han Chinese populations (Macau and Shanghai). 47 Y-SNPs and 16 Y-STR markers were genotyped. As typically found in East Asia populations, in the two studied samples the large majority of the Y-chromosomes fell into haplogroup O-M175: 89.5% in Macau and 81.5% in Shanghai. High haplogroup and haplotype diversities were detected in both populations, reaching values commonly found in other Han Chinese populations. Comparative analyses also revealed a remarkable degree of population substructure inside China.

Miranda do Douro, located in the northeastern region of Portugal, has notable characteristics not only from a geographic or naturalistic point of view, but also from a cultural perspective. A remarkable one is the coexistence of two different languages: Portuguese and Mirandese, the second being an Astur-Leonese dialect. The current persistence of the Astur-Leonese dialect in this population falls on the singularity of the region: relative isolation, implying difficulties to communicate with other Portuguese regions, while the same location facilitated the establishment of social and commercial relationships with adjacent Spanish territories, origin of the Astur-Leonese language. The objective of this study was to characterize the population from Miranda through the analysis of maternal lineages in order to evaluate whether its mitochondrial DNA diversity fitted the patterns previously reported for other populations from the Iberian Peninsula. Viewing that, the entire control region of mitochondrial DNA from 121 individuals was examined. Miranda showed a haplogroup composition usual for a Western European population, in the sense that as high as 63.6% of sequences belonged to macro-haplogroup R0. Lineages ascribed to have an African (L2a and L1b) origin, were detected, but reaching an amount commonly found in Portugal. Miranda also presented a few haplogroups typically found in Jewish populations, while rarely observed in other Iberian populations. The finding can be explained by gene flow with crypto-Jew communities that since long are known to be established in the region where Miranda is located. In Miranda, both genetic and nucleotide diversities presented low values (0.9292 ± 0.0180 and 0.01101 ± 0.00614 respectively) when compared to populations from its micro-geographical framework, which constitute a sign of population isolation that certainly provided conditions for the survival of the Astur-Leonese dialect in the region.

Plasma low-density lipoprotein cholesterol (LDL-C) levels are a key determinant of the risk of cardiovascular disease, which is why many studies have attempted to elucidate the pathways that regulate its metabolism. Novel latest-generation sequencing techniques have identified a strong association between the 1p13 locus and the risk of cardiovascular disease caused by changes in plasma LDL-C levels. As expected for a complex phenotype, the effects of variation in this locus are only moderate. Even so, knowledge of the association is of major importance, since it has unveiled a new metabolic pathway regulating plasma cholesterol levels. Crucial to this discovery was the work of three independent teams seeking to clarify the biological basis of this association, who succeeded in proving that SORT1, encoding sortilin, was the gene in the 1p13 locus involved in LDL metabolism. SORT1 was the first gene identified as determining plasma LDL levels to be mechanistically evaluated and, although the three teams used different, though appropriate, experimental methods, their results were in some ways contradictory. Here we review all the experiments that led to the identification of the new pathway connecting sortilin with plasma LDL levels and risk of myocardial infarction. The regulatory mechanism underlying this association remains unclear, but its discovery has paved the way for considering previously unsuspected therapeutic targets and approaches. Resumo: O nível plasmático de c-LDL constitui um determinante chave para o risco de doença cardiovascular, razão pela qual muitos estudos têm procurado elucidar as vias que regulam o seu metabolismo. As novas técnicas de sequenciação de última geração permitiram identificar um forte sinal de associação entre o locus 1p13 e o risco de doença cardiovascular causada por alteração dos níveis de LDL no plasma. Como seria de esperar para um fenótipo complexo, os efeitos da variação nesse locus são apenas moderados, ainda assim, o conhecimento da associação foi de grande importância uma vez que conduziu à descoberta de uma nova via metabólica reguladora dos níveis de colesterol no plasma. Para tal, foram fundamentais os trabalhos efetuados por três equipas independentes, que ao procurarem esclarecer as bases biológicas da associação em causa conseguiram provar que o gene SORT1, codificador da sortilina, era o gene do locus 1p13 implicado no metabolismo das LDL. SORT1 foi o primeiro dos genes identificados como determinantes dos níveis plasmáticos de LDL a ser alvo de avaliação mecanística e embora cada uma das equipas recorresse a metodologias experimentais diferentes, mas igualmente apropriadas face à questão em investigação, os resultados que obtiveram foram contraditórios em alguns aspetos. Neste trabalho, revemos o caminho percorrido até à descoberta da nova via que relaciona a sortilina com os níveis plasmáticos de LDL e com o risco de enfarte do miocárdio. Ainda por esclarecer permanece o mecanismo regulador dessa ligação, mas a sua descoberta sugere novos alvos terapêuticos até há bem pouco tempo desconhecidos. Keywords: Genome-wide association studies, Coronary artery disease, Low-density lipoprotein cholesterol, Sortilin, Functional genetics, Lipoprotein metabolism, Palavras-chave: Genome wide association studies, Doença das artérias coronárias, Colesterol lipoproteína de baixa densidade, Sortilina, Genómica funcional, Metabolismo das lipoproteínas

While being well known that the diagnosis of many genetic disorders relies on a combination of clinical suspicion and confirmatory genetic testing, not rarely, however, genetic testing needs much perseverance and cunning strategies to identify the causative mutation(s). Here we present a case of a thorny molecular diagnosis of mucolipidosis type III alpha/beta, which is an autosomal recessive lysosomal storage disorder, caused by a defect in the GNPTAB gene that codes for the α/β-subunits of the GlcNAc-1-phosphotransferase. We used both cDNA and gDNA analyses to characterize a mucolipidosis type III alpha/beta patient whose clinical diagnosis was already confirmed biochemically. In a first stage only one causal mutation was identified in heterozygosity, the already described missense mutation c.1196C>T(p.S399F), both at cDNA and gDNA levels. Only after conducting inhibition of nonsense-mediated mRNA decay (NMD) assays and after the utilization of another pair of primers the second mutation, the c.3503_3504delTC deletion, was identified. Our findings illustrate that allelic dropout due to the presence of polymorphisms and/or of mutations that trigger the NMD pathway can cause difficulties in current molecular diagnosis tests. M.F. Coutinho is grantee from the FCT (SFRH/BPD/101965/2014). info:eu-repo/semantics/publishedVersion

Due to differences in transmission between X-chromosomal and autosomal DNA, the comparison of data derived from both markers allows deeper insight into the forces that shape the patterns of genetic diversity in populations. In this study, we applied this comparative approach to a sample of Portuguese Roma (Gypsies) by analyzing 43 X-chromosomal markers and 53 autosomal markers. Portuguese individuals of non-Gypsy ancestry were also studied. Compared with the host population, reduced levels of diversity on the X chromosome and autosomes were detected in Gypsies; this result was in line with known patterns of genetic diversity typical of Roma groups. As a consequence of the complex demographic past of the Roma, during which admixture and genetic drift played major roles, the amount of linkage disequilibrium (LD) on the X chromosome in Gypsies was considerably higher than that observed in non-Gypsies. When the pattern of differentiation on the X chromosome was compared with that of autosomes, there was evidence for asymmetries in female and male effective population sizes during the admixture between Roma and non-Roma. This result supplements previous data provided by mtDNA and the Y chromosome, underlining the importance of using combined information from the X chromosome and autosomes to dissect patterns of genetic diversity. Following the out-of-India dispersion, the Roma acquired a complex genetic pattern that was influenced by drift and introgression with surrounding populations, with important contributions from both males and females. We provide evidence that a sex-biased admixture with Europeans is probably associated with the founding of the Portuguese Gypsies. Am J Phys Anthropol 148:389–394, 2012. © 2012 Wiley Periodicals, Inc.

A 3-bp insertion/deletion polymorphism in intron 6 of GSTM3 (rs1799735, GSTM3*A/*B) affects the activity of the phase 2 xenobiotic metabolizing enzyme GSTM3 and has been associated with increased cancer risk. The GSTM3*B allele is rare or absent in Southeast Asians, occurs in 5-20% of Europeans but was detected in 80% of Bantu from South Africa. The wide genetic diversity among Africans led us to investigate whether the high frequency of GSTM3*B prevailed in other sub-Saharan African populations. In 168 healthy individuals from Angola, Mozambique and the São Tomé e Príncipe islands, the GSTM3*B allele was three times more frequent (0.74-0.78) than the GSTM3*A allele (0.22-0.26), with no significant differences in allele frequency across the three groups. We combined these data with previously published results to carry out a multidimensional scaling analysis, which provided a visualization of the worldwide population affinities based on the GSTM3 *A/*B polymorphism.

In this study, 123 unrelated Portuguese Gypsies were analyzed for 15 highly polymorphic autosomal short tandem repeats (STRs). Average gene diversity across the 15 markers was 76.7%, which is lower than that observed in the non-Gypsy Portuguese population. Subsets of STRs were used to perform comparisons with other Gypsy and corresponding host populations. Interestingly, diversity reduction in Gypsy groups compared to their non-Gypsy surrounding populations apparently varied according to an East-West gradient, which parallels their dispersion in Europe as well as a decrease in complexity of their internal structure. Analysis of genetic distances revealed that the average level of genetic differentiation between Gypsy groups was much larger than that observed between the corresponding non-Gypsy populations. The high rate of heterogeneity among Gypsies can be explained by strong genetic drift and limited intergroup gene flow. However, when genetic relationships were addressed through principal component analysis, all Gypsy populations clustered together and was clearly distinguished from other populations, a pattern that suggests their common origin. Concerning the putative ancestral genetic component, admixture analysis did not reveal strong Indian ancestry in the current Gypsy gene pools, in contrast to the high admixture estimates for either Europeans or Western Asians.

Cigarette smoking is the most important risk factor for bladder cancer. Moreover, epidemiologic studies have implicated several genetic variations interfering with methyl group metabolisms in susceptibility for a variety of cancers. Examples of these variations can be found in genes of the folate metabolic pathway, which is crucial in the provision of methyl groups for DNA, RNA, and protein methylation, as well as in purine and pyrimidine synthesis. We conducted a case–control study to examine the relationship between the methylenetetrahydrofolate reductase ( MTHFR C677 T and MTHFR A1298C), methionine synthase (5-methyltetrahydrofolate-homocysteine methyltransferase, MTR A2756 G), methionine synthase reductase (5-methyltetrahydrofolate-homocysteine methyltransferase reductase, MTRR A66 G and MTRR C524 T), and thymidylate synthase ( TYMS 2R→3R and G/C) genotypes and the risk for bladder cancer in a Tunisian population. The isolated MTHFR 677∗T, MTRR 66∗G and MTRR 524∗T variants did not appear to influence bladder cancer susceptibility. The 3R∗C/3R∗C genotype for the TYMS gene appears to be a protective factor against bladder cancer development ( P =0.0001; OR=0.12; 95% CI=0.03–0.40). However, patients heterozygous for MTHFR A1298C or MTR A2756 G genotypes have 1.62- and 2.13-fold higher risk, respectively, of developing bladder cancer. Moreover, the combined study of MTHFR 1298∗C and MTR 2756∗G variants with either or both MTRR 66GG and TYMS 3R∗G/3R∗G genotypes suggests a cumulative effect. Finally, this study evidenced that interaction between gene variations involved in folate metabolism and risk of bladder cancer increased dramatically among smokers.

Maple syrup urine disease (MSUD) is a rare autosomal recessive disorder of branched-chain amino acid metabolism. In the context of the wide mutational spectrum known for this disease, a few common mutations have been described in populations where founder effects played a major role in modeling diversities. In Portugal, for instance, a high proportion of patients are of Gypsy origin and all share the same mutation (c.117delC-alpha; p.R40GfsX23), causing the neonatal severe form of MSUD. In this study, we used four microsatellite markers closely flanking the BCKDHA gene (E1alpha protein) to demonstrate that c.117delC-alpha is a founder mutation responsible for the high incidence of the disorder among Portuguese Gypsies. These results are of medical relevance since carrier tests and prenatal diagnosis can be offered to families at risk, particularly because the carrier frequency of c.117delC-alpha was estimated at 1.4% among the healthy Portuguese Gypsies from the South of the country. Finally we present evidence that the genomic region of the BCKDHA gene where c.117delC-alpha is located is likely a mutational hotspot, since recurrence of c.117delC-alpha was observed in two distinct population groups.

Maple syrup urine disease (MSUD) is an autosomal recessive disorder, caused by the defective function of the branched-chain alpha-ketoacid dehydrogenase complex (BCKD). BCKD is a mitochondrial complex, encoded by four nuclear genes (BCKDHA, BCKDHB, DBT and DLD), involved in the metabolism of branched-chain amino acids (BCAAs). Since the MSUD mutational spectrum has not been previously assessed in Portugal, in this study we present the molecular characterization of 30 MSUD Portuguese patients. Seventeen putative mutations have been identified (six in BCKDHA, five in BCKDHB and six in DBT); seven of them are here described for the first time. The most common mutation identified was a C deletion in BCKDHA gene (c.117delC; p.R40GfsX23), already reported in the Spanish population. Interestingly, it was found in all patients of a Gypsy community from South of the country, so a founder effect is probably responsible for the high incidence of the disease in this community. Structural models of MSUD missense mutations have been performed to understand their pathogenic effect, in order to elucidate and often to predict the severity of a mutation clinical consequence.

Among the settlers that, from 1432 onwards, arrived to the Azores Islands were individuals of North and sub-Saharan African origin. A previous study of markers of the Y chromosome revealed that haplogroup E is the second more frequent in the Azores (13%). Since this haplogroup is heterogeneous and may contain subtypes of African or non-African origin, we analyzed an extended sample of 319 Azoreans, originating from the three groups of islands (Eastern, Central, and Western), to evaluate the African contribution to the present-day population of the Azores. Samples belonging to the E clade were distributed into six haplogroups, from which the most frequent was E3b1a, representing 47.2% of the E chromosomes (6.3% of the total sample). The sub-Saharan haplogroup E3a was found in 7.1% of E chromosomes (0.9% of the total), corresponding to the highest frequency reported so far in a Portuguese population. No significant differences were detected in the haplogroup distribution among groups of islands, as well as between Azores and most of other European populations compared. The present-day representation of sub-Saharan lineages in Azores, although reduced, is higher than in other Portuguese populations, where the demographic representation of sub-Saharan slaves is reported as similar.

Objective: Most drugs are developed based on data from European-derived ‘reference’ populations; however, clinically relevant DNA polymorphisms often demonstrate population-specific patterns of allele frequencies. Given that the knowledge of the frequency distribution of functional polymorphisms in a population may guide national planning for selection of therapeutic options, in the present study we examined the allele frequencies of enzymes responsible for drug disposition in Portugal. Patients & methods: Using PCR- and Pyrosequencing™-based methods, the current study assessed the frequencies of 15 key polymorphisms from genes encoding enzymes involved in Phases I, II and III of drug metabolism, DNA repair and intracellular metabolism in 135 healthy individuals from Portugal. Results: Allele frequencies were derived for cytochrome P450 (CYP)2C9*2 (13.2%), CYP2C9*3 (8%), CYP2C19*2 (14%), CYP3A4*1B (7%), CYP3A5*3C (87.5%), glutathione S-transferase (GST)M1*0 (77.9%), GSTP1 313A>G (33%), inosine triphosphatase 94C>A (7%), UDP-glucuronosyltransferase (UGT)1A1*28 (28%), UGT1A1 -3156G>A (23%), ATP-binding cassette (ABC)B1 1236C>T (46%), ABCB1 2677G>A/T (2 and 42%), ABCG2 421C>A (8%), excision repair cross-complementing rodent repair deficiency 2 2251A>C (3%) and thymidylate synthetase 1494del (31%). Conclusion: Although, on the whole, the frequency distributions among the Portuguese fitted the patterns commonly found in other Europeans well, evidence for some degree of African influence was observed. This is the most comprehensive study on pharmacogenetically relevant variations in Portugal to date, and the baseline of pharmacogenetic data might be important for determining policy guidelines for cancer prevention and drug treatments in the Portuguese population.

Genetic data on the thiopurine S-methyltransferase (TPMT) polymorphism were obtained in population samples from Cabinda and Mozambique (located in the western and eastern coasts of sub-Saharan Africa, respectively). The overall frequency of TPMT-deficient alleles was 5.6% in Mozambique and 6.3% in Cabinda. Accordingly, one out of the 103 individuals from Cabinda tested had a genotype associated with TPMT deficiency, yielding a frequency that is threefold higher than heretofore reported in any population. In addition, in both Cabinda or Mozambique, TPMT*8 accounted for a significant proportion of non-functional alleles (nearly 40% in Cabinda). Since the substitution defining TPMT*8 seems to be highly specific of sub-Saharan Africa populations and given the fact it has not been integrated into the set of single nucleotide polymorphisms routinely tested for TPMT, a re-design of molecular screenings should be considered in the future in order to avoid serious underestimates of TPMT deficiency when the enzymatic profiles in populations are unknown.

The purpose of this study was to genetically characterize the male lineages of people who speak Mirandese, an interesting case of a linguistic relict that can still be found in the municipality of Miranda do Douro, NE Portugal. This region lies within the area of the Leonese dialects, which are remnants of the Romance dialects spoken in the Kingdom of Leon currently grouped in the Astur-Leonese linguistic continuum. We intended to disclose affinities with surrounding populations, namely from Spain where the Astur-Leonese is also spoken.Eighty-eight unrelated males (58 from Miranda and 30 from Bragança, the broad Portuguese region where Miranda is located) were genotyped with the combined use of 17 Y chromosome short tandem repeats (Y-STRs) and a high resolution Y chromosome single nucleotide polymorphism (Y-SNPs) strategy. Moreover, 236 males from Miranda and neighboring regions, previously classified as R-M269, were also genotyped.R-P312 was the most frequent haplogroup in the Mirandese, followed by J-12f2.1 and T-M70. The male lineages J-12f2.1 and T-M70 were also well represented, and both were shared with descendants of Sephardic Jews. No signs of diversity reduction were detected.Mirandese speakers display a Y chromosome gene pool that shows a subtle differentiation from neighboring populations, mainly attributable to the assimilation of lineages ascribed to be of Jewish ancestry. Although not revealing signs of geographic/linguistic isolation, no clear affinities with other Astur-Leonese populations were detected. The results suggest that in Miranda language sharing is not accompanied by significant gene flow between populations from both sides of the political border. Am. J. Hum. Biol. 28:671-680, 2016. © 2016 Wiley Periodicals, Inc.

© 2015. Mucopolysaccharidosis II is a lysosomal storage disorder caused by mutations in the IDS gene, including exonic alterations associated with aberrant splicing. In the present work, cell-based splicing assays were performed to study the effects of two splicing mutations in exon 3 of IDS, i.e., c.241C > T and c.257C > T, whose presence activates a cryptic splice site in exon 3 and one in exon 8, i.e., c.1122C > T that despite being a synonymous mutation is responsible for the creation of a new splice site in exon 8 leading to a transcript shorter than usual. Mutant minigene analysis and overexpression assays revealed that SRSF2 and hnRNP E1 might be involved in the use and repression of the constitutive 3' splice site of exon 3 respectively. For the c.1122C >. T the use of antisense therapy to correct the splicing defect was explored, but transfection of patient fibroblasts with antisense morpholino oligonucleotides (n = 3) and a locked nucleic acid failed to abolish the abnormal transcript; indeed, it resulted in the appearance of yet another aberrant splicing product. Interestingly, the oligonucleotides transfection in control fibroblasts led to the appearance of the aberrant transcript observed in patients' cells after treatment, which shows that the oligonucleotides are masking an important cis-acting element for 5' splice site regulation of exon 8. These results highlight the importance of functional studies for understanding the pathogenic consequences of mis-splicing and highlight the difficulty in developing antisense therapies involving gene regions under complex splicing regulation., Fundação para a Ciência e Tecnologia IP (FCT)/POPH/FSE, Portugal

In most vertebrates, hemoglobin (Hb) is a heterotetramer composed of two dissimilar globin chains, which change during development according to the patterns of expression of α- and β-globin family members. In placental mammals, the β-globin cluster includes three early-expressed genes, e(HBE)-γ(HBG)-ψβ(HBBP1), and the late expressed genes, δ (HBD) and β (HBB). While HBB encodes the major adult β-globin chain, HBD is weakly expressed or totally silent. Paradoxically, in human populations HBD shows high levels of conservation typical of genes under strong evolutionary constraints, possibly due to a regulatory role in the fetal-to-adult switch unique of Anthropoid primates. In this study, we have performed a comprehensive phylogenetic and comparative analysis of the two adult β-like globin genes in a set of diverse mammalian taxa, focusing on the evolution and functional divergence of HBD in primates. Our analysis revealed that anthropoids are an exception to a general pattern of concerted evolution in placental mammals, showing a high level of sequence conservation at HBD, less frequent and shorter gene conversion events. Moreover, this lineage is unique in the retention of a functional GATA-1 motif, known to be involved in the control of the developmental expression of the β-like globin genes. We further show that not only the mode but also the rate of evolution of the δ-globin gene in higher primates are strictly associated with the fetal/adult β-cluster developmental switch. To gain further insight into the possible functional constraints that have been shaping the evolutionary history of HBD in primates, we calculated dN/dS (ω) ratios under alternative models of gene evolution. Although our results indicate that HBD might have experienced different selective pressures throughout primate evolution, as shown by different ω values between apes and Old World Monkeys + New World Monkeys (0.06 versus 0.43, respectively), these estimates corroborated a constrained evolution for HBD in Anthropoid lineages, which is unlikely to be related to protein function. Collectively, these findings suggest that sequence change at the δ-globin gene has been under strong selective constraints over 65 Myr of primate evolution, likely due to a regulatory role in ontogenic switches of gene expression.

The Great Lakes lie within a region of East Africa with very high human genetic diversity, home of many ethno-linguistic groups usually assumed to be the product of a small number of major dispersals. However, our knowledge of these dispersals relies primarily on the inferences of historical, linguistics and oral traditions, with attempts to match up the archaeological evidence where possible. This is an obvious area to which archaeogenetics can contribute, yet Uganda, at the heart of these developments, has not been studied for mitochondrial DNA (mtDNA) variation. Here, we compare mtDNA lineages at this putative genetic crossroads across 409 representatives of the major language groups: Bantu speakers and Eastern and Western Nilotic speakers. We show that Uganda harbours one of the highest mtDNA diversities within and between linguistic groups, with the various groups significantly differentiated from each other. Despite an inferred linguistic origin in South Sudan, the data from the two Nilotic-speaking groups point to a much more complex history, involving not only possible dispersals from Sudan and the Horn but also large-scale assimilation of autochthonous lineages within East Africa and even Uganda itself. The Eastern Nilotic group also carries signals characteristic of West-Central Africa, primarily due to Bantu influence, whereas a much stronger signal in the Western Nilotic group suggests direct West-Central African ancestry. Bantu speakers share lineages with both Nilotic groups, and also harbour East African lineages not found in Western Nilotic speakers, likely due to assimilating indigenous populations since arriving in the region ~3000 years ago., This study was supported by Fundacao para a Ciencia e Tecnologia (FCT) and co-financed by the European Social Fund (Human Potential Thematic Operational Programme) through grants SFRH/BPD/76207/2011 (VG) and SFRH/BPD/81986/2011 (RP). PS is supported by FCT, European Social Fund, Programa Operacional Potencial Humano and the FCT Investigator Programme (IF/01641/2013) and acknowledges FCT/MEC for support to CBMA through Portuguese funds (PIDDAC)-PEst-OE/BIA/UI4050/2014. AS is supported by "Ministerio de Ciencia e Innovacion" (SAF2011-26983), the Plan Galego IDT (EM 2012/045) and the grant from the Sistema Universitario Gallego-Modalidad REDES (2012-PG226) from the Xunta de Galicia. IPATIMUP integrates the i3S Research Unit, which is partially supported by FCT, the Portuguese Foundation for Science and Technology. This work is funded by FEDER funds through the Operational Programme for Competitiveness Factors-COMPETE and National Funds through the FCT-Foundation for Science and Technology, under the project "PEst-C/SAU/LA0003/2013". NORTE-07-0162-FEDER-00018-Contributos para o reforco da capacidade do IPATIMUP enquanto actor do sistema regional de inovacao and NORTE-07-0162-FEDER-000067 Reforco e consolidacao da capacidade infraestrutural do IPATIMUP para o sistema regional de inovacao, both supported by Programa Operacional Regional do Norte (ON. 2-O Novo Norte), through FEDER funds under the Quadro de Referencia Estrategico Nacional (QREN). We would like to acknowledge the contribution of Magdalen Awor, Fr. Germano Serra and Iva Gomes for collecting the Karamoja samples. We thank Eric Bridgeland for collecting the Kabale samples, Dr Narcis Kabatereine (Vector Control Division, Uganda Ministry of Health) for the Piida samples, and Abigail Enaburekhan, Sadie Anderson- Mann, Rebecca Cole and Steven Groom for preliminary work on the Kabale and Piida sequences.

Mucopolysaccharidosis type II (MPS II) is an X-linked recessive lysosomal storage disease caused by a defect in the iduronate-2-sulfatase gene (IDS). Alternative splicing of the IDS gene can occur and the underlying regulatory mechanism may be rather complex. Nevertheless, little information is available on the role of variations at the IDS locus in the splicing process. Here we report that splice mutations at the IDS locus are an important source of MPS II pathogenicity, accounting for almost 56% of Portuguese cases. Among 16 unrelated Portuguese MPS II patients, 15 different mutations were identified: six intronic splice mutations (c.104−2AG, c.241−2A>G, c.241−1G>A, c.418+1G>A, c.880−8AG and c.1181−1G>C); two exonic splice mutations (c.1006G>lC and c.1122C>T); five missense mutations (D269V, D69V, D148N, R88C and P86L); one nonsense mutation (Q465Ter); one total IDS gene deletion; and one rearrangement involving a IDS gene inversion. Furthermore, nine of the 15 detected mutations affected the usual splicing pattern at the locus. Some of them are responsible for dramatic changes in the splicing mechanism. For example, the substitution mutation, c.418+1G>A, revealed the presence of an exonic sequence inside intron 3. Our study provides evidence that the IDS locus is prone to splicing mutations and that such susceptibility is particularly high in exon 3 and neighbouring regions. Consequently, mutation screening of the IDS gene cannot be restricted to gDNA examination. Unless cDNA analysis is also conducted, misclassifications as silent or missense mutations can be produced and even uncharacteristic splice-site mutations can be misinterpreted as classic splicing defects that may generate severe, unconventional splicing alterations.

Twenty biallelic Y chromosome markers were analyzed in Angolares, Forros and Tongas, three population groups from the African archipelago of Sao Tome e Principe. While most male lineages belonged to sub-Saharan haplogroups, the component of European origin added up 23.9% in the archipelago. This contrasts with the reported absence of European mtDNA lineages, and the combined findings testify to a strong sex-biased admixture process during the long-lasting colonial period in Sao Tome e Principe. Furthermore, the male mediated European component was clearly found to be out of proportion to the small demographic impact of the Portuguese on the islands, reflecting high variance in the reproductive success of the individuals that contributed to its peopling. The male portion of European ancestry was 33.3% in Forros, 27.3% in Tongas and approximately two-fold less, 14.5%, in Angolares. The Angolares also showed the lowest haplogroup diversity and the most reduced number of different haplogroups. The current results reinforce our previous evidence pointing to remarkable restrictions in gene flow between Angolares and other Sao Tomean inhabitants, in agreement with their considerable isolation and confinement to the south-eastern tip of Sao Tome until recently.

Thiopurine methyltransferase (TPMT) is an essential enzyme for normal metabolism of thiopurine drugs. In humans TPMT activity is largely dependent upon genetic variation at the TPMT locus, with TPMT*3A and TPMT*3C being the most frequent mutant alleles associated with reduced activity. TPMT*3C is a widespread allele reaching the highest frequencies in Africans, whereas TPMT*3A is virtually restricted to Caucasian descendent populations. To estimate the time of origin of these two alleles, we analyzed the levels of diversity at two CA repeats flanking the TPMT gene. In accordance to its pattern of geographical distribution, the study of the decay in linkage disequilibrium over time indicated that TPMT*3A was the younger allele. The estimated age was 5700 years, which coincides with the Neolithic, a period characterized by major population expansion that could have been responsible for the spread of TPMT*3A from its place of origin, maybe a western Eurasian population. TPMT*3C was found to have arisen earlier, roughly 14000 years ago, which could explain the worldwide dispersal of TPMT*3C.

We present an approach to the evaluation of the probability of Y-STR haplotypes identity by state (IBS, in opposition to identity by descent, IBD) as an alternative to the empirical one for which it is necessary to have simultaneous information on STRs and SNPs, which is rare in the forensic field. It is based on the mismatch distribution analyses for the number of repeat unit differences between pairs of Y-STR haplotypes. The estimates of the IBS by both methods in a sample where STR and SNP information are available are compared and also the relevance of population structure in determining widely different proportions of IBS vs. IBD.

Within historical times, the forced displacement of Mozambicans as part of the slave trade, documented as being mainly to the Americas, generated a differential input of eastern African sequences into the mtDNA pools of the Americas and of Europe, as testified by the greater number of sequences matches between Mozambique and the Americas compared to those between Mozambique and Europe. D 2003 Elsevier Science B.V. All rights reserved.

Objectives We examined internal lineages and haplotype diversity in Portuguese samples belonging to J-M304 to improve the spatial and temporal understanding of the introduction of this haplogroup in Iberia, using the available knowledge about the phylogeography of its main branches, J1-M267 and J2-M172. Methods A total of 110 males of Portuguese descent were analyzed for 17 Y-chromosome bi-allelic markers and seven Y-chromosome short tandem repeats (Y-STR) loci. Results Among J1-M267 individuals (n = 36), five different sub-haplogroups were identified, with the most common being J1a2b2-L147.1 (∼72%), which encompassed the majority of representatives of the J1a2b-P58 subclade. One sample belonged to the rare J1a1-M365.1 lineage and presented a core Y-STR haplotype consistent with the Iberian settlement during the fifth century by the Alans, a people of Iranian heritage. The analysis of J2-M172 Portuguese males (n = 74) enabled the detection of the two main subclades at very dissimilar frequencies, J2a-M410 (∼80%) and J2b-M12 (∼20%), among which the most common branches were J2a1(xJ2a1b,h)-L26 (22.9%), J2a1b(xJ2a1b1)-M67 (20.3%), J2a1h-L24 (27%), and J2b2-M241 (20.3%). Conclusions While previous inferences based on modern haplogroup J Y-chromosomes implicated a main Neolithic dissemination, here we propose a later arrival of J lineages into Iberia using a combination of novel Portuguese Y-chromosomal data and recent evidence from ancient DNA. Our analysis suggests that a substantial tranche of J1-M267 lineages was likely carried into the Iberian Peninsula as a consequence of the trans-Mediterranean contacts during the first millennium BC, while most of the J2-M172 lineages may be associated with post-Neolithic population movements within Europe.

In the Iberian Peninsula, the Mirandese dialect, spoken in Miranda do Douro (Portugal) close to the north-eastern border with Spain, has attracted much attention. Aim, subjects and methods: This study focuses on providing further insight into the connections forged between Miranda do Douro and regions in the nearby Province of Zamora. This is in order to better assess the extent to which such relations could have been detained by the current patterns of genetic diversity of the populations, whilst contributing to refining the knowledge on patterns of micro-differentiation within the Peninsula. The genetic characterization of both populations was performed through the analysis of X-chromosomal markers: X-STRs and X-indels.The results showed that Miranda do Douro tended to present slightly lower levels of diversity in comparison to the other studied regions, which can be a discreet sign of isolation of that population over the years that might have led the way to the preservation of a language not spoken anywhere else in the country. The analysis of X-STRs particularly brought to light the presence of a subtle population sub-structure at the micro-geographical area encompassing the north-eastern border, which seems to portray the importance of the political border as a mechanism withholding gene flow between the two countries.

Background Mutations affecting RNA splicing represent more than 20% of the mutant alleles in Sanfilippo syndrome type C, a rare lysosomal storage disorder that causes severe neurodegeneration. Many of these mutations are localized in the conserved donor or acceptor splice sites, while few are found in the nearby nucleotides. Methods In this study we tested several therapeutic approaches specifically designed for different splicing mutations depending on how the mutations affect mRNA processing. For three mutations that affect the donor site (c.234 + 1G > A, c.633 + 1G > A and c.1542 + 4dupA), different modified U1 snRNAs recognizing the mutated donor sites, have been developed in an attempt to rescue the normal splicing process. For another mutation that affects an acceptor splice site (c.372-2A > G) and gives rise to a protein lacking four amino acids, a competitive inhibitor of the HGSNAT protein, glucosamine, was tested as a pharmacological chaperone to correct the aberrant folding and to restore the normal trafficking of the protein to the lysosome. Results Partial correction of c.234 + 1G > A mutation was achieved with a modified U1 snRNA that completely matches the splice donor site suggesting that these molecules may have a therapeutic potential for some splicing mutations. Furthermore, the importance of the splice site sequence context is highlighted as a key factor in the success of this type of therapy. Additionally, glucosamine treatment resulted in an increase in the enzymatic activity, indicating a partial recovery of the correct folding. Conclusions We have assayed two therapeutic strategies for different splicing mutations with promising results for the future applications. Electronic supplementary material The online version of this article (doi:10.1186/s13023-014-0180-y) contains supplementary material, which is available to authorized users.

In recent years a large amount of mitochondrial population data for forensic purposes has been produced. Current efforts are focused at increasing the number of studied populations while generating updated genetic information of forensic quality. However, complete mitochondrial control region sequences are still scarce for most populations and even more so for complete mitochondrial genomes. In the case of Portugal, previous population genetics studies have already revealed the general portrait of HVS-I and HVS-II mitochondrial diversity, becoming now important to update and expand the mitochondrial region analysed. Accordingly, a total of 292 complete control region sequences from continental Portugal were obtained, under a stringent experimental design to ensure the quality of data through double sequencing of each target region. Furthermore, H-specific coding region SNPs were examined to detail haplogroup classification and complete mitogenomes were obtained for all sequences belonging to haplogroups U4 and U5. In general, a typical Western European haplogroup composition was found in mainland Portugal, associated to high level of mitochondrial genetic diversity. Within the country, no signs of substructure were detected. The typing of extra coding region SNPs has provided the refinement or confirmation of the previous classification obtained with EMMA tool in 96% of the cases. Finally, it was also possible to enlarge haplogroup U phylogeny with 28 new U4 and U5 mitogenomes.

We report a neonate who was diagnosed as a case of skeletal dysplasia during pregnancy, and was subsequently diagnosed as a case of MLII alpha/beta on the basis of clinical and radiological findings and molecular testing of the parents. A novel GNPTAB mutation c.1701delC [p.F566LfsX5] was identified in the father. The case reiterates the severe prenatal phenotype of MLII alpha/beta which mimics skeletal dysplasia and illustrates the utility of molecular genetic analysis in confirmation of diagnosis and subsequent genetic counselling. This work was supported by FCT — project PIC/IC/83252/2007(http://alfa.fct.mctes.pt/). Coutinho MF is a grantee from the FCT (SFRH/BD/48103/2008).