Population structure from NOS genes correlates with geographical differences in coronary incidence across Europe.

OBJECTIVES: The population analysis of cardiovascular risk and non-risk genetic variation can help to identify adaptive or random demographic processes that shaped coronary incidence variation across geography. MATERIAL AND METHODS: In this study, 114 single nucleotide polymorphisms and 17 tandem repeat polymorphisms from Nitric Oxide Synthases (NOS) regions were analyzed in 1686 individuals from 35 populations from Europe, North Africa, and the Middle East. NOS genes encode for key enzymes on nitric oxide availability, which is involved in several cardiovascular processes. These genetic variations were used to test for selection and to infer the population structure of NOS regions. Moreover, we tested whether the variation in the incidence of coronary events and in the levels of classical risk factors in 11 of these European populations could be explained by the population structure estimates. RESULTS: Our results supported, first, the absence of clear signs of selection for NOS genetic variants associated with cardiovascular diseases, and second, the presence of a continuous genetic pattern of variation across European and North African populations without a Mediterranean barrier for gene flow. Finally, population structure estimates from NOS regions are closely correlated with coronary event rates and classical risk parameters (explaining 39-98%) among European populations. CONCLUSION: Our results reinforce the hypothesis that genetic bases of cardiovascular diseases and associated complex phenotypes could be geographically shaped by random demographic processes.

Genetic drift of influenza A(H3N2) viruses during two consecutive seasons in 2011-2013 in Corsica, France.

The 2011-2012 and 2012-2013 post-pandemic influenza outbreaks were characterized by variability in the A(H3N2) influenza viruses, resulting in low to moderate vaccine effectiveness (VE). The aim of this study was to investigate the molecular evolution and vaccine strain match of the A(H3N2) influenza viruses, having been circulated throughout the population of the French Corsica Island in 2011-2012 and again in 2012-2013. Clinical samples from 31 patients with confirmed A(H3N2) influenza viruses were collected by general practitioners (GPs) over these two consecutive seasons. An analysis of genetic distance and antigenic drift was conducted. Based on a hemagglutinin (HA) aminoacid sequence analysis, the Corsican A(H3N2) viruses fell into the A/Victoria/208/2009 genetic clade, group 3. All influenza viruses were characterized by at least four fixed amino acid mutations which were: N145S (epitope A); Q156H and V186G (epitope B) Y219S (epitope D), with respect to the A/Perth/16/2009 (reference vaccine strain for the 2011-2012) and the A/Victoria/361/2011 (reference vaccine strain for the 2012-2013). Using the p(epitope) model, the percentages of the perfect match VE estimated against circulated strains declined within and between seasons, with estimations of <50%. Overall, these results seem to indicate an antigenic drift of the A(H3N2) influenza viruses which were circulated in Corsica. These findings highlight the importance of the continuous and careful surveillance of genetic changes in the HA domain during seasonal influenza epidemics, in order to provide information on newly emerging genetic variants.

Acute diarrhea in adults consulting a general practitioner in France during winter: incidence, clinical characteristics, management and risk factors.

BACKGROUND: Data describing the epidemiology and management of viral acute diarrhea (AD) in adults are scant. The objective of this study was to identify the incidence, clinical characteristics, management and risk factors of winter viral AD in adults. METHODS: The incidence of AD in adults during two consecutive winters (from December 2010 to April 2011 and from December 2011 to April 2012) was estimated from the French Sentinelles network. During these two winters, a subset of Sentinelles general practitioners (GPs) identified and included adult patients who presented with AD and who filled out a questionnaire and returned a stool specimen for virological examination. All stool specimens were tested for astrovirus, group A rotavirus, human enteric adenovirus, and norovirus of genogroup I and genogroup II. Age- and sex-matched controls were included to permit a case-control analysis with the aim of identifying risk factors for viral AD. RESULTS: During the studied winters, the average incidence of AD in adults was estimated to be 3,158 per 100,000 French adults (95% CI [2,321 - 3,997]). The most reported clinical signs were abdominal pain (91.1%), watery diarrhea (88.5%), and nausea (83.3%). GPs prescribed a treatment in 95% of the patients with AD, and 80% of the working patients with AD could not go to work. Stool examinations were positive for at least one enteric virus in 65% (95% CI [57 - 73]) of patients with AD with a predominance of noroviruses (49%). Having been in contact with a person who has suffered from AD in the last 7 days, whether within or outside the household, and having a job (or being a student) were risk factors significantly associated with acquiring viral AD. CONCLUSIONS: During the winter, AD of viral origin is a frequent disease in adults, and noroviruses are most often the cause. No preventable risk factor was identified other than contact with a person with AD. Thus, at the present time, reinforcement of education related to hand hygiene remains the only way to reduce the burden of disease.

Simultaneous investigation of influenza and enteric viruses in the stools of adult patients consulting in general practice for acute diarrhea.

BACKGROUND: Gastrointestinal symptoms are not an uncommon manifestation of an influenza virus infection. In the present study, we aimed to investigate the presence of influenza viruses in the stools of adult patients consulting their general practitioner for uncomplicated acute diarrhea (AD) and the proportion of concurrent infections by enteric and influenza viruses. METHOD: A case-control study was conducted from December 2010 to April 2011. Stool specimens were collected and tested for influenza viruses A (seasonal A/H3N2 and pandemic A/H1N1) and B, and for four enteric viruses (astrovirus, group A rotavirus, human enteric adenovirus, norovirus of genogroups I - NoVGI - and genogroup II - NoVGII). RESULTS: General practitioners enrolled 138 cases and 93 controls. Of the 138 stool specimens collected, 92 (66.7%) were positive for at least one of the four enteric viruses analysed and 10 (7.2%) tested positive for one influenza virus. None of these 10 influenza positive patients reported respiratory symptoms. In five influenza-positive patients (3.6%), we also detected one enteric virus, with 4 of them being positive for influenza B (2 had co-detection with NoVGI, 1 with NoVGII, and 1 with astrovirus). None of the 93 controls tested positive for one of the enteric and/or other influenza viruses we investigated. CONCLUSIONS: In this study we showed that the simultaneous detection of influenza and enteric viruses is not a rare event. We have also reported, for the first time in general practice, the presence of seasonal and pandemic influenza viruses in the stools of adult patients consulting for uncomplicated AD. A simultaneous investigation of enteric and influenza viruses in patients complaining of gastrointestinal symptoms could be useful for future studies to better identify the agents responsible for AD.

Selective neutrality analysis of 17 STRs in Mediterranean populations.

Detection of genes that have been targeted by natural selection is a powerful tool for predicting regions of the genome potentially linked with diseases and of interest in the field of genetic epidemiology. In recent years, several methods to detect patterns of natural selection have been developed. In general, these tests are based on different assumptions and parameters; hence, the detection of outlier loci with more than one statistical approach simultaneously will support the candidate status of a particular locus. In this study, we evaluated the presence of patterns of positive selection in 17 short tandem repeat loci genotyped in six different human populations from the Mediterranean area, for a total of 429 individuals. To identify patterns of selective pressure, we applied three different neutrality tests on the basis of different models, performing pairwise comparisons between populations. Results show the presence of one marker, a (CA)n repeat located in exon 29 of the NOS1 gene, which seems significant in the three different tests in two pairwise comparisons: Sicily vs Morocco and Balearic Islands vs Morocco. This suggests that this locus and its genome localization are candidates for further studies to investigate selective pressure, as well as for association studies.

The value of some Corsican sub-populations for genetic association studies.

BACKGROUND: Genetic isolates with a history of a small founder population, long-lasting isolation and population bottlenecks represent exceptional resources in the identification of disease genes. In these populations the disease allele reveals Linkage Disequilibrium (LD) with markers over significant genetic intervals, therefore facilitating disease locus identification. In a previous study we examined the LD extension on the Xq13 region in three Corsican sub-populations from the inner mountainous region of the island. On the basis of those previous results we have proposed a multistep procedure to carry out studies aimed at the identification of genes involved in complex diseases in Corsica. A prerequisite to carry out the proposed multi-step procedure was the presence of different degrees of LD on the island and a common genetic derivation of the different Corsican sub-populations. In order to evaluate the existence of these conditions in the present paper we extended the analysis to the Corsican coastal populations. METHODS: Samples were analyzed using seven dinucleotide microsatellite markers on chromosome Xq13-21: DXS983, DXS986, DXS8092, DXS8082, DXS1225, DXS8037 and DXS995 spanning approximately 4.0 cM (13.3 Mb). We have also investigated the distribution of the DXS1225-DXS8082 haplotype which has been recently proposed as a good marker of population genetic history due to its low recombination rate. RESULTS: the results obtained indicate a decrease of LD on the island from the central mountainous toward the coastal sub-populations. In addition the analysis of the DXS1225-DXS8082 haplotype revealed: 1) the presence of a particular haplotype with high frequency; 2) the derivation from a common genetic pool of the sub-populations examined in the present study. CONCLUSION: These results indicate the Corsican sub-populations useful for the fine mapping of genes contributing to complex diseases.

Frequencies of promoter pentanucleotide (TTTTA)n of CYP11A gene in European and North African populations.

The present work attempts to determine the distribution of CYP11A (TTTTA)n genotype and allele frequencies in 10 European and North African populations. This polymorphism has been associated with hyperandrogenism by several association studies. To our knowledge, this is the first study investigating the ethnic variation of this polymorphism. DNA was extracted from 868 whole-blood samples with the standard phenol-chloroform technique, and PCR reactions were carried out using fluorescent primers as described previously. PCR products were analyzed by an ABI 3,730 DNA Analyzer. A total of six alleles were identified, ranging from 220 bp (4 repeats [4R]) to 250 bp (10R). The most frequent allelic fragment size in all populations was 4R, with frequencies ranging from 47.9% (Sicily) to 62.8% (Tuscany and Germany). Allelic frequencies showed high heterogeneity between analyzed populations. We detected a significant gradient for alleles 4R and 8R. In this study, we report the allele frequency distribution of CYP11A (TTTTA)n showing a north-south geographic gradient. This result could be useful for epidemiological studies about hyperandrogenism.

Population genetic data on four STR loci, PAI (CA)n, GpIIIa (CT)n, PLAT (TG)14 (CA)12, and NOS2A (CCTTT)n, in Mediterranean populations.

In the present study, we have analyzed four highly polymorphic regions (STRs) chosen from four candidate genes involved in: (1) Platelet aggregation: alpha subunit of the platelet GpIIb/GpIIIa integrin complex (GpIIIa (CT)n; 17q21.31), (2) Coagulation fibrinolysis: Plasminogen Activator Tissue (PLAT5 (TG)14(TA)12; 8p12-q11.2) and Plasminogen Activator Inhibitor-1 (PAI-1 (CA)n; 7q21.3-q22), (3) Oxidative metabolism: the inducible nitric oxide (NO) synthase (iNOS) gene (NOS2A (CCTTT)n; 17cen-q11.2). Allele frequencies for these four STR loci were investigated in several Mediterranean populations. The population data deviate from the Hardy-Weinberg equilibrium in all populations for GpIIIa (CT)n polymorphism.

Genetic isolates in Corsica (France): linkage disequilibrium extension analysis on the Xq13 region.

Genetic isolates with a history of a small founder population, long-lasting isolation and population bottlenecks represent exceptional resources in the identification of genes involved in the pathogenesis of multifactorial diseases. In these populations, the disease allele reveals linkage disequilibrium (LD) with markers over significant genetic intervals, therefore facilitating disease locus identification. This study has been designed to examine the background LD extension in some subpopulations of Corsica. Our interest in the island of Corsica is due to its geographical and genetic proximity to the other Mediterranean island of Sardinia. Sardinian isolates in which the extension of the background LD is particularly high have been recently identified and are now the object of studies aimed at the mapping of genes involved in complex diseases. Recent evidence has highlighted that the genetic proximity between the populations of Corsica and Sardinia is particularly true for the internal conservative populations. Given these considerations, Sardinia and Corsica may represent a unique system to carry out parallel association studies whose results could be validated by comparison. In the present study, we have analyzed the LD extension on the Xq13 genomic region in three subpopulations of Corsica: Corte, Niolo and Bozio, all located in the mountainous north-center of the island. Our results show a strong degree of LD over long distance for the population of Bozio and to a less extent for the population of Niolo. Their LD extent is comparable to or higher than that reported for other isolates.

A pentanucleotide repeat polymorphism (TTTTA) in the apolipoprotein (a) gene--its distribution and its association with the risk of cardiovascular disease.

Apolipoprotein (a) is a component of lipoprotein (a). Several studies have shown the association between risk of coronary heart diseases and the size of apo(a) isoforms, although this issue is still controversial. Recent researches focused the attention on the pentanucleotide (TTTTA), highlighting a statistical correlation between low Lp(a) levels and high repeat numbers. In the present paper we studied the distribution of the apo(a) pentanucleotide polymorphism among populations from Corsica, and we then compared it with other populations from Europe, Africa and Asia. The results stressed out the usefulness of these markers in population genetics analysis. We later investigated the possible association of the apo(a) pentanucleotide polymorphism with serum lipid levels in two samples from Corsica (France): one comprises patients or individuals with high risk of future coronary heart disease and the other is a control sample. No significant differences between the two groups have been found, but the analysis of variance showed a significant association between different genotypes and cholesterol and LDL serum levels.

Influenza vaccine effectiveness: best practice and current limitations of the screening method and their implications for the clinic.

Is there a role for the screening method in estimating influenza vaccine effectiveness (VE)? The answer is yes, but the simplicity of the method used has raised concerns about its validity, and several cautions should be noted. The screening method provides an approximation of influenza VE by comparing the proportion of cases vaccinated (PCV) with the proportion of persons vaccinated (PPV) in the general population. This method has an important disadvantage: VE estimation could be inaccurate if the values for PCV and PPV are drawn from different populations, but it has an important strength, compared with other observational studies, in providing an early indication of VE in the field. Thus, when an infrastructure, such as routine surveillance, is in place to collect robust PCV values, and PPV can be obtained from routine vaccine uptake monitoring systems, the screening method can provide early estimates of influenza VE in target groups.

Epidemiology and viral etiology of the influenza-like illness in corsica during the 2012-2013 Winter: an analysis of several sentinel surveillance systems.

Influenza-like illness (ILI) surveillance is important to identify circulating and emerging/reemerging strains and unusual epidemiological trends. The present study aimed to give an accurate picture of the 2012-2013 ILI outbreak in Corsica by combining data from several surveillance systems: general practice, emergency general practice, hospital emergency units, intensive care units, and nursing homes. Twenty-eight respiratory viruses were retrospectively investigated from patients in general practice with ILI. Sequence analysis of the genetic changes in the hemagglutinin gene of influenza viruses (A(H1N1)pdm2009, A(H3N2) and B) was performed. The trends in ILI/influenza consultation rates and the relative illness ratios (RIRs) of having an ILI consultation were estimated by age group for the different surveillance systems analyzed. Of the 182 ILI patients enrolled by general practitioners, 57.7% tested positive for influenza viruses. Phylogenetic analyses suggested a genetic drift for influenza B and A(H3N2) viruses. The ILI/influenza surveillance systems showed similar trends and were well correlated. In accordance with virological data, the RIRs of having an ILI consultation were highest among the young (<15 years old) and decreased with age. No clusters of acute respiratory illness were declared by the sentinel nursing homes. This study is noteworthy in that it is the first extensive description of the 2012-2013 ILI outbreak in Corsica as monitored through several surveillance systems. To improve ILI surveillance in Corsica, a consortium that links together the complementary regional surveillance ILI systems described here is being implemented.

Field seasonal influenza vaccine effectiveness: evaluation of the screening method using different sources of data during the 2010/2011 French influenza season.

Thanks to the screening method, we estimated among target groups the 2010/2011 field vaccine effectiveness (FVE) against laboratory confirmed influenza cases seen in general practice. We also compared the values of FVE estimations obtained by using three sources of the population vaccination coverage (VC) based on three different methodologies: (1) administrative data from the main social security scheme (Caisse Nationale d'Assurance Maladie des Travailleurs Salariés--CNAMTS) covering about 85% of the French population, (2) a cross-sectional national telephone survey in the general population, and (3) a declarative survey in the population seen in a one-day general practitioner (GP) consultations. The FVE estimates among target groups were stratified by age (< 65 y old with reported chronic illness; ≥65 y old and overall). Using the VC of the CNAMTS, the FVE of the 2010/2011 seasonal trivalent vaccine against laboratory confirmed infection with any influenza virus was 59% (95% Confidence Interval, 17 to 81). It was 85% (17 to 99) and 50% (-16 to 80) for A(H1N1)pdm09 and B influenza infections, respectively. The values of FVE using the influenza VC obtained in a sample of the general population and of the population of GPs' patients were 73% (45 to 87) and 82% (63 to 92), respectively. We estimated a moderate influenza FVE in preventing confirmed influenza viruses in target groups by using the VC of the CNAMTS. We also observed that the screening method generates FVE values dependent on the choice of the source of VC and thus should be used cautiously.

Chemical and genetic differentiation of two Mediterranean subspecies of Teucrium scorodonia L.

Chemical and genetic diversity of Teucrium scorodonia L. subsp. scorodonia from Corsica and T. scorodonia L. subsp. baeticum from western Algeria were investigated. Diversity within and among the two populations of subspecies was assessed according to the chemical composition of their essential oils, and genetic diversity was evaluated using three polymorphic genetic markers. Chemical analysis was performed using a combination of capillary GC-RI and GC/MS after fractionation using column chromatography. Genetic structures were mapped using two chloroplast markers (RPL32-TRNL and TRNL-F) and ribosomal nuclear markers (ITS region). The statistical analysis showed that the two subspecies were clearly distinguished by these chemical and genetic markers. The chemical composition of oil differed qualitatively and quantitatively between the subspecies. Corsican oil samples contained germacrene B (4.2-8.8%) and γ-elemene (2.6-5.7%), which were not detected in Algerian oil samples. The oils of the scorodonia and baeticum subspecies were dominated by sesquiterpene hydrocarbon compounds (75.6-82.9% and 69.6-79.4%, respectively), but they differed in oxygenated sesquiterpene content (3.1-8.9% and 8.4-20.3%, respectively). Neighbor-joining trees constructed from chloroplast DNA and ITS region sequences showed the existence of two groups associated with taxonomic and chemical characteristics. One group consisted of T. scorodonia subsp. scorodonia and the other of T. scorodonia subsp. baeticum, indicating that variation in the essential oil composition of T. scorodonia subspecies depends more on genetic background than environmental characteristics.

Chemical and genetic diversity of two Mediterranean subspecies of Teucrium polium L.

Chemical and genetic diversity of Teucrium polium L. subsp. polium from western Algeria and T. polium L. subsp. capitatum from Corsica were investigated. Diversity within and among the two populations of subspecies was assessed according to the chemical composition of their essential oils and the genetic diversity. Chemical analysis was performed using a combination of capillary GC-RI and GC/MS after fractionation using column chromatography. Genetic structures were mapped using three polymorphic genetic markers: two chloroplast markers (RPL32-TRNL and TRNL-F) and ribosomal nuclear markers (ITS region). The statistical analysis showed that both subspecies were clearly distinguished by these chemical and genetic markers. The oil chemical compositions differed qualitatively and quantitatively between the subspecies. Both collective oils were dominated by hydrocarbon compounds however the Algerian sample oils exhibited higher amounts of hydrocarbon sesquiterpenes than those of Corsica (31.2 g/100 g vs. 4.4 g/100 g) while the latter displayed higher amounts of hydrocarbon monoterpenes than the first (59.3 g/100 g vs. 34.3 g/100 g). Neighbor-joining, Maximum likelihood and Bayesian trees constructed from chloroplast markers and nuclear ITS region sequences showed the existence of two groups associated with taxonomic and chemical characteristics. The study indicated that variation in the essential oil composition within subspecies depends on genetic background. The samples of subsp. capitatum from Corsica are a homogeneous group, in contrast to samples of subsp. polium from Algeria which were clustered in two groups. Chemical and genetic diversity of Algerian populations could be explained by geographical isolation of the populations. In addition, the morphological polymorphism observed throughout the colour of flowers could be explained by environmental parameters as well as the soil pH.

Chemical and genetic differentiation of Corsican subspecies of Teucrium flavum L.

Corsica Island exhibited the particularity to display Teucrium flavum subsp. glaucum and subsp. flavum on the same territory with the same bioclimatic conditions. For the first time, volatile components extracted from aerial parts and genetic diversity of both Corsican T. flavum L. subspecies have been investigated through (i) the characterization of the chemical composition of essential oils and (ii) the study of three polymorphic genetic markers. Chemical analysis were performed using combination of capillary GC/RI, GC-MS after fractionation on column chromatography and the definition of the genetic structure were carried out using two chlororoplast markers (RPL32-TRNL and TRNL-F) and ribosomal nuclear markers (ITS region). According to statistical analysis, both subspecies were clearly distinguished by the chemical and genetic studies. Chemical compositions of oils from both subspecies were qualitatively similar but they differed by the normalized% abundances of their major components; oils from subsp. flavum were dominated by large amounts of hydrocarbon monoterpenes while oils obtained from subsp. glaucum were characterized by higher amounts of oxygenated compounds. The genetic analysis divided T. flavum L. populations in two groups, the first displayed subsp. glaucum populations and the latter group exhibited subsp. flavum populations. The presence of two groups is weakly consistent with chemical differentiation. These data suggest that the differences in the volatile composition of the two T. flavum subspecies depends more on the genetic background and less on environmental factors.

Genetic structure of human A/H1N1 and A/H3N2 influenza virus on Corsica Island: phylogenetic analysis and vaccine strain match, 2006-2010.

BACKGROUND: The aim of this study was to analyse the genetic patterns of Hemagglutinin (HA) genes of influenza A strains circulating on Corsica Island during the 2006-2009 epidemic seasons and the 2009-2010 pandemic season. METHODS: Nasopharyngeal samples from 371 patients with influenza-like illness (ILI) were collected by General Practitioners (GPs) of the Sentinelles Network through a randomised selection routine. RESULTS: Phylogenetic analysis of HA revealed that A/H3N2 strains circulating on Corsica were closely related to the WHO recommended vaccine strains in each analyzed season (2006-2007 to 2008-2009). Seasonal Corsican influenza A/H1N1 isolated during the 2007-2008 season had drifted towards the A/Brisbane/59/2007 lineage, the A/H1N1 vaccine strain for the 2008-2009 season. The A/H1N1 2009 (A/H1N1pdm) strains isolated on Corsica Island were characterized by the S220T mutation specific to clade 7 isolates. It should be noted that Corsican isolates formed a separate sub-clade of clade 7 as a consequence of the presence of the fixed substitution D222E. The percentages of the perfect match vaccine efficacy, estimated by using the p(epitope) model, against influenza viruses circulating on Corsica Island varied substantially across the four seasons analyzed, and tend to be highest for A/H1N1 compared with A/H3N2 vaccines, suggesting that cross-immunity seems to be stronger for the H1 HA gene. CONCLUSION: The molecular analysis of the HA gene of influenza viruses that circulated on Corsica Island between 2006-2010 showed for each season the presence of a dominant lineage characterized by at least one fixed mutation. The A/H3N2 and A/H1N1pdm isolates were characterized by multiples fixation at antigenic sites. The fixation of specific mutations at each outbreak could be explained by the combination of a neutral phenomenon and a founder effect, favoring the presence of a dominant lineage in a closed environment such as Corsica Island.

Chemical composition of fatty acid and unsaponifiable fractions of leaves, stems and roots of Arbutus unedo and in vitro antimicrobial activity of unsaponifiable extracts.

The chemical composition of the fatty acid and unsaponifiable fractions of the leaves, stems and roots of Arbutus unedo L. were determined using gas chromatography and gas chromatography-mass spectrometry. The fatty acid fractions of the leaves, stems and roots contained 38.5%, 31.3% and 14.1% palmitic acid, respectively, along with other long-chain fatty acids (up to C22). The chemical composition of the unsaponifiable fractions differed: the leaf and stem fractions contained high levels of aliphatic (32.1% and 62.6%, respectively) and terpenic compounds (49.6% and 25.7%, respectively), and the root fraction mainly contained esters, of which the most abundant was benzyl cinnamate (36.6%). The antimicrobial activities of the unsaponifiable fractions against nine species of microorganisms were assessed. The unsaponifiable leaf and stem extracts inhibited the growth of Klebsiella pneumoniae, Enterococcus faecalis and Candida albicans.

A gradient of NOS1 overproduction alleles in European and Mediterranean populations.

AIM: A (CA)n repeat located in the 3' UTR region of exon 29 of the NOS1 gene (encoding for neuronal nitric oxide synthase) has been shown to affect the size of mRNA. NOS1 mRNA is highly diverse, contributing to changes in transcript generation, degradation, processing, or subcellular targeting. In the present work, we analyzed allele frequencies of this (CA)n repeat in nine populations of the Mediterranean area and Middle Europe. We aimed at testing the presence of a north-south positive gradient of frequencies of ≤17 allele repeats, compatible with the hypothesis of positive selection suggested in two of our previous works, related to the past prevalence of malaria infection in Europe. RESULTS: Results show significant negative correlations of latitude with frequencies of alleles S and genotypes S/S and S/L (p < 0.01). CONCLUSIONS: In conclusion, the north-south gradient of S alleles found in the present work would confirm our previous observation about the NOS1 gene, reinforcing the hypothesis of a selective action of malaria infection. This hypothesis is strengthened by the role of nitric oxide in the immunity system.