Abstract Background Variants of the leucine-rich repeat kinase 2 (LRRK2) are associated with an increased susceptibility to Parkinson disease but also Crohn’s disease (CD). Aims The present research is designed to develop a comprehensive understanding of the role of LRRK2 in immune system modulation, and how dysfunction of this pathway may lead to the development of CD. Methods WT and LRRK2-deficient neutrophil were infected with Gram-positive Bacteria (Listeria monocytogenes-LM) in a gentamicin protection assays and colony-forming unit assessment will determine the competence of LRRK2 deficient cells for bacterial phagocytosis as well as killing capacity). To examine how LRRK2 is involved in the generation of ROS during the respiratory burst, we will first examine if neutrophil from LRRK2-KO mice have altered ROS generation upon infection with LM and addition of PMA. We evaluate in vitro the ability of neutrophils from LRRK2-KO versus WT mice to transmigrate in vitro in a transwell assay using fMLP as a chemattractant. Also, we investigate the peritoneal cells (by FACS analysis) after injection of different microbial stimuli including FK105 (NOD1 ligand), MDP (NOD2 ligand) and LPS (TLR4 ligand) and anti-cd3 model of ielitis. Results We found that LRRK2 KO mice have a defect in migration of neutrophils to the peritoneal cavity after injection of different microbial stimuli including FK10565 (NOD1 ligand), MDP (NOD2 ligand) and LPS (TLR4 ligand). Neutrophils from LRRK2 mice were compromised in their ability to transmigrate in vitro in a transwell assay using fMLP as a chemoattractant. Chemotaxis was also compromised. In parallel, we designed experiments to examine reactive oxygen species (ROS) produced in response to infection of myeloid cells with bacteria. Neutrophils from LRRK2 KO mice infected with Listeria monocytogenes were less able to restrict bacteria growth compared to WT cells. Consistent with these findings, cells from LRRK2 KO mice produced lower levels of ROS following bacterial infection. In order to determine whether myeloid cell migration is compromised in vivo during inflammation, we performed experiments in WT and KO mice looking at different models of ileitis/colitis. Conclusions With this work we will further characterize the role of LRRK2 in intestinal homeostasis and mucosal barrier maintenance, including how its deficiency may predispose an individual to developing CD. Funding Agencies CAG, CIHR

Variants of the leucine-rich repeat kinase 2 (LRRK2) are associated with an increased susceptibility to Parkinson disease but also Crohn’s disease (CD). Studies using a murine colitis model have pointed to the involvement of LRRK2 in regulating an NFAT-dependent pathway that dampens the production of certain inflammatory cytokines. Also, recent work showed that LRRK2 interacts with NOD2 in Paneth cells in order to properly secrete antimicrobial peptides into the intestinal lumen and promote gut-microbiota homeostasis. The present research is designed to develop a comprehensive understanding of the role of LRRK2 in immune system modulation, and how dysfunction of this pathway may lead to the development of Crohn’s disease (CD). WT and LRRK2-deficient neutrophil and macrophages were infected with different Gram-negative and a Gram-positive Bacteria (S. typhimurium and Listeria monocytogenes) in a gentamicin protection assays and colony-forming unit assessment will determine the competence of LRRK2 deficient cells for bacterial phagocytosis (4h) and killing capacity (24h). We evaluate the ability of neutrophils from LRRK2-KO versus WT mice to transmigrate in vitro in a transwell assay using fMLP as a chemattractant. Also, we investigate the peritoneal cells (by FACS analysis) phenotype after injection of different microbial stimuli including FK105 (NOD1 ligand), MDP (NOD2 ligand) and LPS (TLR4 ligand) in WT mice compared to LRRK2-KO and G2019S-KI mice. We found that LRRK2 KO mice have a defect in migration of immune cells (neutrophil and monocytes) to the peritoneal cavity after injection of different microbial stimuli including FK105 (NOD1 ligand), MDP (NOD2 ligand) and LPS (TLR4 ligand). In contrast, the G2019S knock-in mice show a higher rate of migration of immune cells compared to cells from wild-type animals. Neutrophils from LRRK2 mice were compromised in their ability to transmigrate in vitro in a transwell assay using fMLP as a chemattractant. In parallel, we designed experiments to examine reactive oxygen species (ROS) produced in response to infection of myeloid cells with bacteria. Neutrophils and bone marrow-derived macrophages from LRRK2 KO mice infected with Listeria monocytogenes or Salmonella typhimurium were less able to restrict bacteria growth compared to WT cells. Consistent with these findings, cells from LRRK2 KO mice produced lower levels of ROS following bacterial infection. In order to determine whether myeloid cell migration is compromised in vivo during inflammation, we are beginning experiments in WT and KO mice looking at different models of ileitis/colitis. With this work we will further characterize the role of LRRK2 in intestinal homeostasis and the mucosal barrier maintenance, including how its deficiency may predispose an individual to developing CD. CAG, CIHR

Vaz Rodrigues L, Costa F, Marques P, Mendonça C, Rocha J, Seixas S. Severe α‐1 antitrypsin deficiency caused by Q0Ourém allele: clinical features, haplotype characterization and history.α‐1 Antitrypsin deficiency (AATD) caused by null alleles is associated with the total lack of protein and generally it translates into more severe clinical features of pulmonary disease. This is the case of Q0Ourém, a rare variant found in several families of Central Portugal caused by the L353fsX376 mutation. A total of 41 patients carrying at least one copy of Q0Ourém were evaluated for SERPINA1 levels, respiratory function values and lung parenchyma status (chest X‐ray and computerized tomography scan). Q0Ourém haplotype background was characterized using seven microsatellites flanking SERPINA1 and Q0Ourém age was estimated by a statistical method relying on the decay of haplotype sharing at linked markers (DHSMAP).Homozygous patients showed a compromised lung function and extensive emphysema. SQ0Ourém, although having serum levels below the 11 µM threshold, did not necessarily result in signs of disease. MQ0Ourém were found to be a heterogeneous group, mainly composed of normal individuals. Eight Q0Ourém haplotypes were identified and the allele was estimated to have arisen 650 years ago. Q0Ourém was associated with mild to severe AATD and has a single origin, probably linked to the major Ourém settlements where the occurrence of severe AATD may not be explained by recent consanguinity.

A multiplex STR study using the Powerplex 16 System (Promega) in 503 unrelated individuals from the island of São Tomé (Gulf of Guinea, West Africa) revealed 10 male individuals presenting only the Y homologue of the Amelogenin locus (∼2%). These individuals were further typed with other commercial kits which also amplify the Amelogenin locus, namely the AmpFLSTR Identifiler (Applied Biosystems) and Y-Plex 12 (Reliagene) kits, and an X/Y genotype was only obtained with the primers used in Y-Plex 12. Although this X Amelogenin drop-out was only detected in males, this does not rule out the fact that females may also carry it. Sequencing of the X Amelogenin allele responsible for the amplification failure in these male individuals revealed a C to T substitution at position 294 of GeneBank sequence M55418, corresponding to the Powerplex 16 forward primer binding site.

The genetic variation at a compound nonrecombining haplotype system, consisting of the previously reported SB19.3 Alu insertion polymorphism and a newly identified adjacent short tandem repeat (STR), was studied in population samples from Portugal and São Tomé (Gulf of Guinea, West Africa). Age estimates based on the linked microsatellite variation suggest that the Alu insertion occurred about 190,000 years ago. In accordance with the global patterns of distribution of human genetic variation, the highest haplotype diversity was found in the African sample. This excess in African diversity was due to both a substantial reduction in heterozygosity at the Alu polymorphism and a lower STR variability associated with the predominant Alu insertion allele in the Portuguese sample. The high level of interpopulation differentiation observed at the Alu locus (F(ST) = 0.43) was interpreted under alternative selective and demographic scenarios. The need for compatibility between patterns of variation at the STR and Alu loci could be used to restrict the range of selection coefficients in selection-driven genetic hitchhiking frameworks and to favor demographic scenarios dominated by larger pre-expansion African population sizes. Taken together, the data show that the SB19.3 Alu-STR system is an informative marker that can be included in more extended batteries of compound haplotypes used in human evolutionary studies.

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.

The Y chromosome carries several genes involved in spermatogenesis, which are distributed in three regions in the euchromatic part of the long arm, called AZFa (azoospermia factor a), AZFb, and AZFc. Microdeletions in these regions have been seen in 10-15% of sterile males with azoospermia or severe oligozoospermia. The relatively high de novo occurrence of these microdeletion events might be due to particular chromosome arrangements associated with certain Y chromosome haplogroups. To test whether there is any association between Y chromosome types and male infertility, we studied a sample of 84 Japanese oligozoospermic or azoospermic males. The patients were analyzed for the presence of Yq microdeletions and also typed with a battery of unique event polymorphisms (UEPs) to define their Y haplogroups. Six of the infertile patients presented likely pathological microdeletions detectable with the sequence tagged sites (STS) markers used. There was no significant association between Y chromosome haplogroups and the microdeletions. We also compared the Y haplogroup frequencies in our subset sample of 51 idiopathic azoospermia patients with 57 fertile control Japanese males, and did not observe any significant differences. Contrary to previous reports, our data suggest that Y microdeletions and other molecular events causally associated with male infertility in Japan occur independently of the Y chromosome background.

Aim: The main objective of the study was to determine whether risk factors associated with obesity are influenced by genetic variation of apolipoprotein E (ApoE). Methods: 81 obese children (mean age 9.4 ± 2.8 years) and an age-matched control group were included. Body composition, lipid profile, and glucose and insulin levels were evaluated according to international recommendations, and the blood pressure was measured by an oscillometric method. Results: The calculated frequencies of the ApoE alleles *2, *3, and *4 (0.04, 0.88 and 0.08) in obese children were similar to those of eutrophic age-matched controls (0.07, 0.82, and 0.11) and fitted the range of variation generally observed in southern European populations. Age, anthropometric parameters, body fat mass, and blood pressure were similar in E2/3, E3/3 and E4/3 genotypes. Total/high-density lipoprotein cholesterol and low-density lipoprotein/high-density lipoprotein cholesterol ratios were higher in the E4/3 group as compared with E3/3 (p < 0.01) and E2/3 (p < 0.05) groups. No differences concerning clusters of risk factors were observed among the three genotypes. No associations were found between ApoE polymorphism and glucose levels (fasting and at 2 h) and between fasting insulin levels and HOMAIR results. Higher levels of fasting and 2-hour insulin and higher HOMAIR values were significantly associated with a higher fat mass. Conclusions: ApoE polymorphism seems to influence some lipid profile abnormalities associated with obesity in childhood. However, clustering of risk factors and insulin resistance seem not to be dependent on ApoE polymorphism.

human Duffy blood group (FY) antigens are transmembrane glycoproteins that function as receptors for chemokines and for the malarial parasite Plasmodium vivax Most of the FY antigenic variation is determined by three common alleles (FY*A, FY*B, and FY*O) in a gene on chromosome 1q21-q22. DNA sequence char-acterization of these alleles and interspecies compari-sons with the orthologous genes from nonhuman primates FY*A and FY*O are derived variants, each resulting from a single mutation in an ancestral FY*B background the FY*A gene prod-uct is a functional protein with a Gly44Asp substitution, FY*O has a T-46C promoter disrupts a binding site for the GATA1 erythroid transcription factor to a tissue-specific loss of expression of FY antigens in red blood cells human autosomal polymorphisms common alleles to be shared by different, geographically distant populations, the distribution of FY alleles is FY*O has near fixation

The geographic origins of African slave settlers and the Portuguese genetic contribution to the population of São Tomé (Gulf of Guinea) were assessed through the analysis of b-globin haplotypes in 44 chromosomes bearing the bS allele and through the study of the genetic variation in eight autosomal markers (APOA1, AT3, FY, LPL, OCA2, RB1, Sb19.3, and GC) informative for admixture in a sample of 224 individuals. The observed bS haplotype distribution (36.4% Bantu, 52.3% Benin, 4.5% Cameroon, 4.5% Senegal, and 2.3% atypical) is in accordance with the historical information on the major geographic sources of slave settlers of São Tomé, although it captures a more important contribution of Central-West Africa regions than previously anticipated. European admixture, estimated to be 10.7 ± 0.9%, has created a considerable level of genetic structure, as indicated by the finding of significant linkage disequilibrium between 33% of unlinked marker loci pairs. Recent admixture was found to have an important contribution to these values, since removal of individuals with Portuguese or Cape Verdian parents or grandparents from the sample dropped the miscegenation level to 6.5 ± 0.8% and reduced significant linkage disequilibrium to 11% of unlinked marker pairs. Taken together, these results indicate that the peopling of São Tomé might have provided one of the first examples of the combination of diverse African contributions and European admixture that emerged from the overseas population relocations promoted by the Atlantic slave trade.

This work was undertaken to ascertain to what degree the physical appearance of a Brazilian individual was predictive of genomic African ancestry. Using a panel of 10 population-specific alleles, we assigned to each person an African ancestry index (AAI). The procedure was able to tell apart, with no overlaps, 20 males from northern Portugal from 20 males from São Tomé Island on the west coast of Africa. We also tested 10 Brazilian Amerindians and observed that their AAI values fell in the same range as the Europeans. Finally, we studied two different Brazilian population samples. The first consisted of 173 individuals from a rural Southeastern community, clinically classified according to their Color (white, black, or intermediate) with a multivariate evaluation based on skin pigmentation in the medial part of the arm, hair color and texture, and the shape of the nose and lips. In contrast to the clear-cut results with the African and European samples, our results showed large variances and extensive overlaps among the three Color categories. We next embarked on a study of 200 unrelated Brazilian white males who originated from cosmopolitan centers of the four major geographic regions of the country. The results showed AAI values intermediate between Europeans and Africans, even in southern Brazil, a region predominantly peopled by European immigrants. Our data suggest that in Brazil, at an individual level, color, as determined by physical evaluation, is a poor predictor of genomic African ancestry, estimated by molecular markers.

α1‐Antitrypsin (PI) deficiency is a common autosomal recessive disorder associated with emphysema and liver disease, which may result from a wide spectrum of mutations causing a reduction of serum levels (deficient alleles) or a total lack of circulating protein (null alleles). We report two different alleles associated with the absence of isoelectric focusing banding patterns in Portuguese patients with emphysema. The first allele, Q0ourém, results from the recurrence of the defining mutation of the Q0mattawa variant (L353fsX376) on a M3 normal background. The second allele, Q0porto, has a novel G→A mutation at position +1 of the intron IC (IVS1C+1G→A), which restricts mononuclear phagocyte transcripts to mRNA species resulting from the direct splice of exon IA to exon II. The absence of this normal splice alternative in the liver, where PI is primarily synthesized, provides a basis for the pathogenic effects of this mutation.

We examined DNA polymorphisms in the nonrecombining portion of the Y-chromosome to investigate the contribution of distinct patrilineages to the present-day white Brazilian population. Twelve unique-event polymorphisms were typed in 200 unrelated males from four geographical regions of Brazil and in 93 Portuguese males. In our Brazilian sample, the vast majority of Y-chromosomes proved to be of European origin. Indeed, there were no significant differences when the haplogroup frequencies in Brazil and Portugal were compared by means of an exact test of population differentiation. Y-chromosome typing was quite sensitive in the detection of regional immigration events. Distinct footprints of Italian immigration to southern Brazil, migration of Moroccan Jews to the Amazon region, and possible relies of the 17th-century Dutch invasion of northeast Brazil could be seen in the data. In sharp contrast with our mtDNA data in white Brazilians, which showed that greater than or equal to 60% of the matrilineages were Amerindian or African, only 2.5% of the Y-chromosome lineages were from sub-Saharan Africa, and none were Amerindian. Together, these results configure a picture of strong directional mating between European males and Amerindian and African females, which agrees with the known history of the peopling of Brazil since 1500.

Editor—α1-antitrypsin (PI), the major inhibitor of neutrophil elastase in the lower respiratory tract, is a highly polymorphic glycoprotein synthesised in the liver that has several rare gene products in which serum protein levels are reduced or even undetectable.1 Early onset pulmonary emphysema, resulting from unopposed elastase activity, and neonatal cholestasis probably resulting from the retention of the defective protein in the liver,2 are the two most common clinical manifestations of PI deficiency and are mainly associated with PI*Z, the most common deficient allele. In addition, other rare alleles occasionally associated with liver injury have been shown to share with PI*Z an increased tendency for intracellular accumulation. Recently, a complete intracellular transport block has been reported for a newly identified3 4 defective Pro369Ser allele (Mwurzburg) by in vitro expression studies in human cell cultures. Adenovirus mediated transfer of the mutant gene into the mouse reproduced the consequences of this block and no traceable amounts of the variant protein could be detected in the plasma after in vivo recombinant expression.3 However, no detectable intrahepatocytic PI inclusions were found in the mice expressing the Mwurzburg mutant3 and no liver biopsy material has yet been presented from patients with this defective allele. We report a carrier of the Mwurzburg allele with evidence for in vivo intrahepatic accumulation of …

Human MutationVolume 15, Issue 1 p. 121-122 Mutation and Polymorphism ReportFree Access A novel alpha-1-antitrypsin R281del variant found in a population sample from the Basque country † Susana Seixas, Susana Seixas Instituto de Patologia e Imunologia Molecular da Universidade do Porto (IPATIMUP), PortugalSearch for more papers by this authorOscar Garcia, Oscar Garcia Instituto de Patologia e Imunologia Molecular da Universidade do Porto (IPATIMUP), PortugalSearch for more papers by this authorAntónio Amorim, António Amorim Instituto de Patologia e Imunologia Molecular da Universidade do Porto (IPATIMUP), PortugalSearch for more papers by this authorJorge Rocha, Jorge Rocha Instituto de Patologia e Imunologia Molecular da Universidade do Porto (IPATIMUP), PortugalSearch for more papers by this author Susana Seixas, Susana Seixas Instituto de Patologia e Imunologia Molecular da Universidade do Porto (IPATIMUP), PortugalSearch for more papers by this authorOscar Garcia, Oscar Garcia Instituto de Patologia e Imunologia Molecular da Universidade do Porto (IPATIMUP), PortugalSearch for more papers by this authorAntónio Amorim, António Amorim Instituto de Patologia e Imunologia Molecular da Universidade do Porto (IPATIMUP), PortugalSearch for more papers by this authorJorge Rocha, Jorge Rocha Instituto de Patologia e Imunologia Molecular da Universidade do Porto (IPATIMUP), PortugalSearch for more papers by this author First published: 27 December 1999 https://doi.org/10.1002/(SICI)1098-1004(200001)15:1<121::AID-HUMU37>3.0.CO;2-UCitations: 3 † Communicated by: Mark H. Paalman ‡ Online Citation: Human Mutation, Mutation and Polymorphism Report #90 (1999) Online http://journals.wiley.com/1059-7794/pdf/mutation/mpr90.pdf § Acknowledgments:Susana Seixas is supported by grant BD/13885/97 from Praxis XXI. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article.Citing Literature Volume15, Issue1Special Issue: The HUGO Mutation Database Initiative: Issues, Databases, and Perspectives for the New MillenniumJanuary 2000Pages 121-122 RelatedInformation

Factor XIII a subunit (F13A) is the last enzyme in the blood coagulation cascade. It is characterized by extensive genetic polymorphism defined by 4 common alleles, F13A*1A, 1B, 2A and 2B and a few rare variants, some responsible for severe coagulation deficiencies. In order to infer the evolutionary affinities between the common F13A alleles we have applied PCR techniques to study, in a Northern Portuguese sample, a short tandem repeat polymorphism located within the 5′ untranslated region of the F13A gene. The analysis of the molecular heterogeneity within the F13A gene products revealed that the four biochemical variants shared very similar, truncated, distributions of STR alleles and showed no signs of predominant haplotypic associations. These findings seem to support both the inferences that intragenic recombination played an important role in the generation of molecular diversity within each of the four main F13A alleles and that all the four F13A alleles must be rather old. Molecular heterogeneity levels allowed the identification of 1B as the oldest F13A allelic state, and 2A as the most recently generated allele, but were not different enough to accurately track the divergence of alleles 1A and 2B. However, additional analysis of linkage disequilibrium patterns indicates that 1B→2B→1A→2A is the most likely evolutionary order of appearance of F13A main protein alleles, confirming and extending a previous hypothetical model inferred from their molecular features.

The joint distributions of phenotypes from the apolipoprotein E gene (APOE) and from a closely linked restriction site polymorphism at the apolipoprotein C1 locus (APOC1) were studied in population samples from Portugal and São Tomé e Príncipe (Gulf of Guinea), a former Portuguese colony that was originally populated by slaves imported from the African mainland. The frequencies of the APOE alleles (*2, *3, and *4) in Portugal and São Tomé fitted the ranges of variation generally observed in European and African populations, respectively. Haplotype analysis showed that in both populations the strength of linkage disequilibrium was highest for the APOE*2 allele and lowest for the APOE*4 allele, suggesting that the origin of the APOE alleles followed a 4-->3-->2 pathway and thus providing independent confirmation of the results from sequence homology studies with nonhuman primates. In accordance with global trends in the distribution of human genetic variation, the European sample from Portugal presented more intense linkage disequilibrium between APOE and APOC1 than the African sample from São Tomé where, despite the short 4-kb distance that separates the 2 loci, the level of association between the APOC1 alleles and APOE*4 was nonsignificant.