Highly preferential association of NonF508del CF mutations with the M470 allele.

BACKGROUND: On the basis of previous findings on random individuals, we hypothesized a preferential association of CF causing mutations with the M allele of the M470V polymorphic site of the CFTR gene. METHODS: We have determined the M/V-CF mutation haplotype in a series of 201 North East Italian and 73 Czech CF patients who were not F508del homozygotes, as F508del was already known to be fully associated with the M allele. RESULTS: Out of 358 not F508del CF genes, 84 carried the V allele and 274 the less common M allele. In the N-E Italian population, MM subjects have a risk of carrying a CF causing mutation 6.9x greater than VV subjects when F508del is excluded and 15.4x when F508del is included. In the Czech population a similar, although less pronounced, association is observed. CONCLUSIONS: Besides the possible biological significance of this association, the possibility of exploiting it for a pilot screening program has been explored in a local North East Italian population for which CF patients were characterized for their CF mutation. General M470V genotyping followed by common CF mutation screening limited to couples in which each partner carries at least one M allele would need testing only 39% of the couples, which contribute 89% of the total risk, with a cost benefit.

Haemoglobin C protects against clinical Plasmodium falciparum malaria.

Haemoglobin C (HbC; beta6Glu --> Lys) is common in malarious areas of West Africa, especially in Burkina Faso. Conclusive evidence exists on the protective role against severe malaria of haemoglobin S (HbS; beta6Glu --> Val) heterozygosity, whereas conflicting results for the HbC trait have been reported and no epidemiological data exist on the possible role of the HbCC genotype. In vitro studies suggested that HbCC erythrocytes fail to support the growth of P. falciparum but HbC homozygotes with high P. falciparum parasitaemias have been observed. Here we show, in a large case-control study performed in Burkina Faso on 4,348 Mossi subjects, that HbC is associated with a 29% reduction in risk of clinical malaria in HbAC heterozygotes (P = 0.0008) and of 93% in HbCC homozygotes (P = 0.0011). These findings, together with the limited pathology of HbAC and HbCC compared to the severely disadvantaged HbSS and HbSC genotypes and the low betaS gene frequency in the geographic epicentre of betaC, support the hypothesis that, in the long term and in the absence of malaria control, HbC would replace HbS in central West Africa.

The lower susceptibility to Plasmodium falciparum malaria of Fulani of Burkina Faso (west Africa) is associated with low frequencies of classic malaria-resistance genes.

The gene frequencies in 1993-94 for haemoglobin S, haemoglobin C, alpha-3.7 deletional thalassaemia, G6PDA-, HLAB*5301 were estimated in Fulani, Mossi and Rimaibé ethnic groups of Burkina Faso, West Africa. The aim of the study was to verify whether the previously reported Fulani lower susceptibility to Plasmodium falciparum malaria was associated with any of these malaria-resistance genes. Similar frequencies for haemoglobin S were recorded in the 3 ethnic groups (0.024 +/- 0.008, 0.030 +/- 0.011, 0.022 +/- 0.013; in Mossi, Rimaibé and Fulani, respectively). The Mossi and Rimaibé showed higher frequencies when compared to Fulani for haemoglobin C (0.117 +/- 0.018, 0.127 +/- 0.020, 0.059 +/- 0.020), alpha-3.7 deletional thalassaemia (0.227 +/- 0.040, 0.134 +/- 0.032, 0.103 +/- 0.028), G6PDA- (0.196 +/- 0.025, 0.187 +/- 0.044, 0.069 +/- 0.025) and HLA B*5301 (0.189 +/- 0.038, 0.202 +/- 0.041, 0.061 +/- 0.024). Among Fulani the proportion of individuals not having any of these protective alleles was more than 3-fold greater than in the Mossi-Rimaibé group (56.8% vs 16.7%; P < 0.001). These findings exclude the involvement of these genetic factors of resistance to P. falciparum in the lower susceptibility to malaria of Fulani. This evidence, in association with the previously reported higher immune reactivity to malaria of Fulani, further supports the existence in this ethnic group of unknown genetic factor(s) of resistance to malaria probably involved in the regulation of humoral immune responses.

HLA class I in three West African ethnic groups: genetic distances from sub-Saharan and Caucasoid populations.

Fulani of Burkina Faso (West Africa) are a particularly interesting ethnic group because of their lower susceptibility to Plasmodium falciparum malaria as compared to sympatric populations, Mossi and Rimaibé. Moreover, the occurrence of a Caucasoid component in their genetic make-up has been suggested on the basis of their physical traits and cultural traditions even though this view was not supported by genetic studies. A total of 149 unrelated subjects (53 Mossi, 47 Rimaibé and 49 Fulani) have been typed for 97 HLA class I alleles with the amplification refractory mutation system/polymerase chain reaction (ARMS/PCR) technique. Mossi and Rimaibé data were pooled since none of the 42 statistically testable alleles exhibited a significant heterogeneity. These pooled gene frequencies were found to be very different from those of Fulani: a certain (P<0.001) or a likely (0.001

Latitude-correlated genetic polymorphisms: selection or gene flow?

Latitude-correlated polymorphisms can be due to either selection-driven evolution or gene flow. To discriminate between them, we propose an approach that studies subpopulations springing from a single population that have lived for generations at different latitudes and have had a low genetic admixture. These requirements are fulfilled to a large extent by Ashkenazi and Sephardi Jews. The original population lived at a latitude of 35 degrees N, where the Sephardis still live. The Ashkenazis, however, moved to a latitude of 50 degrees N, starting about 10 centuries ago. The present study examines 3 latitude-correlated polymorphisms: PGP, PGM1, and AHSG. We found that PGP*2 and AHSG*2 alleles most likely underwent selection-driven evolution, but that PGM1*ts allele was not similarly affected. Since temperature might have been considered a reasonable selective factor, we also studied a population living at >800 m above sea level from Aosta Valley (Italy).

Serum levels of erythropoietin and soluble transferrin receptor in the course of pregnancy in non beta thalassemic and beta thalassemic women.

BACKGROUND AND OBJECTIVES: In non-thalassaemic women serum erythropoietin (Epo) level increases during pregnancy, whereas that of soluble transferrin receptor (STFR) drops slightly in the first two trimesters to attain the original values in the third trimester. In this study the time-course of these two parameters was explored in b-thalassemic and non-b-thalassemic women, both pregnant and not. DESIGN AND METHODS: Two hundred and fifty-seven women were studied: 64 non-b-thalassemic, non-pregnant women made up the reference group, 89 were non-b-thalassemic pregnant women, and 104 were b-thalassemic pregnant or non-pregnant women. The full blood count, hemoglobin levels and iron status (serum iron and serum ferritin levels) were explored by traditional methods. Serum Epo and STFR levels were measured with standard commercial kits. RESULTS: In non-b-thalassemic women the mean non-pregnant Epo level (10.95 +/-4. 7 mU/mL) increased in the first trimester (17.12 +/-5.18 mU/mL), was stationary in the second, and increased again in the third (31. 43+/-14.13 mU/mL). STFR mean value dropped in early pregnancy from 2. 40+/-0.72 mg/L to 1.78 +/- 0.64 mg/L, and then returned to the original value (2.38 +/- 0.94 mg/L). In b-thalassemic women the mean non-pregnant Epo level (15.00 +/-6.56 mU/mL) was higher than in non-thal non-pregnant women. During pregnancy it progressively increased to 35.60+/-25.46 mU/mL. STFR (non-pregnant level 3.37+/-1. 07 mg/L) gradually increased throughout the whole gestation period and by the third trimester its level was markedly higher than that of non-b-thal women at the corresponding stage of gestation (9. 41+/-5.39 mg/L vs 2.40+/-0.72 mg/L). INTERPRETATION AND CONCLUSIONS: The STFR level changed to different extents in non-b-thal and b-thal women during their pregnancies. In the former STFR markedly decreased in early pregnancy; in the latter it showed no decrease in the first trimester, increased in the second and reached very high values in the third. This time course is likely to be the consequence of erythroid bone marrow hyperplasia and hyperactivity, which are usually present in all b-thalassemic patients and in heterozygous carriers as well.

A new approach for identifying non-pathogenic mutations. An analysis of the cystic fibrosis transmembrane regulator gene in normal individuals.

Given q as the global frequency of the alleles causing a disease, any allele with a frequency higher than q minus the cumulative frequency of the previously known disease-causing mutations (threshold) cannot be the cause of that disease. This principle was applied to the analysis of cystic fibrosis transmembrane conductance regulator (CFTR) mutations in order to decide whether they are the cause of cystic fibrosis. A total of 191 DNA samples from random individuals from Italy, France, and Spain were investigated by DGGE (denaturing gradient gel electrophoresis) analysis of all the coding and proximal non-coding regions of the gene. The mutations detected by DGGE were identified by sequencing. The sample size was sufficient to select essentially all mutations with a frequency of at least 0.01. A total of 46 mutations was detected, 20 of which were missense mutations. Four new mutations were identified: 1341+28 C/T, 2082 C/T, L1096R, and I11131V. Thirteen mutations (125 G/C, 875+40 A/G, TTGAn, IVS8-6 5T, IVS8-6 9T, 1525-61 A/G, M470V, 2694 T/G, 3061-65 C/A, 4002 A/G, 4521 G/A, IVS8 TG10, IVS8 TG12) were classified as non-CF-causing alleles on the basis of their frequency. The remaining mutations have a cumulative frequency far exceeding q; therefore, most of them cannot be CF-causing mutations. This is the first random survey capable of detecting all the polymorphisms of the coding sequence of a gene.

Two ethnic-specific polymorphisms in the human beta pseudogene of hemoglobin.

Two polymorphic sites, -107 C-->T and -100 G-->C with respect to the cap site of the human beta pseudogene of the hemoglobin gene, are described. They have been studied in five European, one Indian, two Asian, and two sub-Saharan African populations. The -107 C-->T site turned out to be polymorphic in all five European populations and the Indian population (pooled q = 0.142 +/- 0.018) and in the two Asian populations (pooled q = 0.073 +/- 0.025), but it was monomorphic in the two sub-Saharan populations. On the contrary, the -100 G-->C site was polymorphic in the two sub-Saharan samples (q = 0.093 +/- 0.024), but the variant allele was not found in any of the European, Indian, or Asian samples. Thus this only 8-bp-long stretch of DNA is informative for estimating the extent of genetic admixture in sub-Saharan Africans.

Detection of alpha-globin gene disorders by a simple PCR methodology.

BACKGROUND: alpha thalassemias are very common in all thalassemic areas; however, complete knowledge of the phenotypic, genotypic and epidemiological features of these thalassemias has not yet been achieved for a number of reasons: the frequent absence of a thalassemic hematologic picture, the lack of a specific characteristic comparable to the Hb A2 increase for beta thalassemias, and the almost complete homology between the two alpha genes. METHODS AND RESULTS: A new set of PCR techniques, each based on primer(s) specific for a particular type of alpha globin gene disorder, has been devised in our laboratory. The procedures are simple, and non-radioactive. They lead to the identification of all alpha globin disorders common in the Mediterranean area [-alpha 3.7, -alpha 4.2, alpha Hphl, alpha Ncol, --MED, -(alpha)20.5, alpha alpha alpha anti3.7]. The electrophoretic patterns specific for the main alpha globin alterations as observed with this set of techniques, are presented. CONCLUSIONS: Owing to their advantageous properties, these techniques are suitable for precise molecular characterization of the numerous subjects selected through mass population screenings.

mtDNA provides the first known marker distinguishing proto-Indians from the other Caucasoids; it probably predates the diversification between Indians and Orientals.

The concomitant presence of the two sites Ddel at 10,394 and Alul at 10,397 has been considered an East-Asian marker of ancient origin (it was also observed in Australians, Melanesians and Native Americans). Unexpectedly, it was found in more than 50% of Indians (133 Hindus and 30 Tribals) who had shown Caucasoid characteristics not only at nuclear DNA but also at mtDNA level. It can therefore no longer be considered an exclusively East-Asian mtDNA feature. The analysis of more than 200 Caucasoids, mainly from the Mediterranean basin, showed that it is only sporadically present in these people. Thus it represents the first known marker which distinguishes Indians from the other Caucasoids. The lack of this marker in Indian mtDNA molecules carrying Caucasoid characteristics suggests that it predates the invasion of India by speakers of an Indo-European language and, if it is valid to extrapolate from Near Eastern data, the arrival in India of the farmers who spread the Dravidian language. If this polymorphism had a common origin in both Orientals and Indians, it should also predate the diversification between ancient Indians and Mongoloids.

Allele and haplotype frequency distribution of the EcoRI, RsaI, and MspI COL1A2 RFLPs among various human populations.

The EcoRI, RsaI, and MspI RFLPs (restriction fragment length polymorphisms) of the COL1A2 gene, one of the two genes that encode for the polypeptides of type I collagen, have been studied in four West African and two Asian populations to evaluate their potential effectiveness as anthropological markers. All three RFLPs were in Hardy-Weinberg equilibrium. The comparisons between present data on two of the major human groups and those on Europeans and Amerindians show a considerable heterogeneity for each of the three RFLPs under study. EcoRI, in particular, appears to be highly effective in distinguishing Africans, Europeans, and Asians from each other. As expected, the analysis at the haplotype level considerably improves the discriminating efficiency of these three markers by creating a clear-cut distinction between Tharus and Indonesians, the two Asian populations of the present survey. In fact, even though these two populations exhibit the same frequencies for the RsaI and MspI alleles, the frequency of the MspI(-) allele among the RsaI(-) chromosomes is 0.5 +/- 0.14 in the Indonesian sample and 0 + 0.04 in the Tharu sample.

Genetic heterogeneity among the Hindus and their relationships with the other "Caucasoid" populations: new data on Punjab-Haryana and Rajasthan Indian states.

The genetic structure of Rajasthan Hindus and Punjab-Haryana Hindus and Sikhs has been studied for ABO, RH, APOC2, C6, C7, F13A, F13B, HP, ORM1, ACP1, ADA, AK1, ESD, GLO1, PGD, PGM1 subtyping, and PGP. This is the first genetic survey on Hindus of Rajasthan. Furthermore, many of these markers have never been studied on Hindus before (APOC2, C6, C7, F13A, F13B, ORM1, PGP). These data, together with those previously available for Hindus, have been utilized to analyze the within-Hindus genetic heterogeneity by RST statistic and correspondence analysis. The genetic relationships of Hindus to other Causcasoid populations were also investigated. In the first analysis, two eastern states (Orissa and Andhra Pradesh) were found to be quite separate from each other and clearly distinct from the northwestern and western states. Out of the markers which could not be utilized in this analysis, PGM1 subtyping turned out to discriminate between the Dravidian-speaking and the Indo-Aryan-speaking Hindus. The second analysis shows a clear-cut separation of Hindus from Europeans, with Near Eastern and Middle Eastern populations genetically in an intermediate position.

Are the SSTR alleles stable enough to be considered monophyletic and hence reliable anthropogenetic markers? Linkage disequilibrium study on the (ACT)n COL1A2 SSTR.

An extremely low production rate of a polymorphic allele (formally called the mutation rate)--a prerequisite for using the allele as a marker (particularly for anthropogenetic purposes where the alleles must be assumed to be monophyletic)--cannot be taken for granted for alleles of highly polymorphic VNTRs, but a low production rate can be used to identify alleles produced by a single nucleotide substitution. This property was indirectly tested for the (ACT)n COL1A2 (of type I collagen) microsatellite SSTR (degree of heterozygosity H = 0.72) by searching for linkage disequilibria between the SSTR's four common alleles (n = 6, 8, 9, or 10) and three RFLPs of the same gene. A strong linkage disequilibrium between at least three of the four SSTR alleles and two of the three closely linked RFLPs has been demonstrated in a Sardinian population (Italy), a finding that suggests a low production rate of these alleles. Thus it seems that this highly polymorphic system and, by a reasonable extrapolation, other VNTRs with a comparable degree of heterozygosity may be valuable anthropogenetic markers, at least in distinguishing subgroups of a major ethnic group.

COL1A2 gene (alpha 2 gene of type I collagen) at the haplotype level as a new valuable anthropogenetic marker: a study on Sardinians.

Sardinians, a population with many distinct anthropogenetic features, has been studied for the COLIA1 and COLIA2 genes at the DNA level for two purposes: to look for new RFLPs (restriction fragment length polymorphisms) and to study the distribution of three known COLIA2 RFLPs (EcoRI, RsaI, MspI) at both the allele and the haplotype levels. None of the eleven enzyme-probe systems examined led to the discovery of a new polymorphism. The following frequency q was found for the less common allele of the three RFLPs: EcoRI, q(+) = 0.178 +/- 0.031; RsaI, q(-) = 0.316 +/- 0.038; MspI, q(-) = 0.046 +/- 0.017. EcoRI turned out to be the most discriminant of the three polymorphisms because the frequency of the (+) allele in Sardinians was about half that estimated for a large homogeneous white sample (0.18 +/- 0.03 vs. 0.30 +/- 0.01). So far as the haplotype level is concerned, the sample is made up of triplets (parents and child). Therefore all the haplotype frequencies and delta values (degrees of disequilibrium, D) were obtained by direct counting of the unambiguously identified haplotypes rather than being based on their maximum-likelihood estimates. This together with their analytical and detailed presentation makes these data comparable with future findings, provided that the two data sets are presented in a comparable way. At this level the three RFLPs are efficient in distinguishing Sardinians from Calabrians (southern Italy) but not from the central Italian population. The present results, besides adding a further discriminative criterion between Sardinians and Italians (and whites on the whole), identify the complex COLIA2 locus as a valuable anthropogenetic marker.

COII/tRNA(Lys) intergenic 9-bp deletion and other mtDNA markers clearly reveal that the Tharus (southern Nepal) have Oriental affinities.

We searched for the East Asian mtDNA 9-bp deletion in the intergenic COII/tRNA(Lys) region in a sample of 107 Tharus (50 from central Terai and 57 from eastern Terai), a population whose anthropological origin has yet to be completely clarified. The deletion, detected by electrophoresis of the PCR-amplified nt 7392-8628 mtDNA fragment after digestion with HaeIII, was found in about 8% of both Tharu groups but was found in none of the 76 Hindus who were examined as a non-Oriental neighboring control population. A complete triplication of the 9-bp unit, the second case so far reported, was also observed in one eastern Tharu. All the mtDNAs with the deletion, and that with the triplication, were further characterized (by PCR amplification of the relevant mtDNA fragments and their digestion with the appropriate enzymes) to locate them in the Ballinger et al. phylogeny of East Asian mtDNA haplotypes. The deletion was found to be associated with four different haplotypes, two of which are reported for the first time. One of the deletions and especially the triplication could be best explained by the assumption of novel length-change events. Ballinger's classification of East Asian mtDNA haplotypes is mainly based on the phenotypes for the DdeI site at nt 10394 and the AluI site at nt 10397. Analysis of the entire Tharu sample revealed that more than 70% of the Tharus have both sites, the association of which has been suggested as an ancient East Asian peculiarity. These results conclusively indicate that the Tharus have a predominantly maternal Oriental ancestry. Moreover, they show at least one and perhaps two further distinct length mutations, and this suggests that the examined region is a hot spot of rearrangements.

The common, Near-Eastern origin of Ashkenazi and Sephardi Jews supported by Y-chromosome similarity.

About 80 Sephardim, 80 Ashkenazim and 100 Czechoslovaks were examined for the Y-specific RFLPs revealed by the probes p12f2 and p49a,f on TaqI DNA digests. The aim of the study was to investigate the origin of the Ashkenazi gene pool through the analysis of markers which, having an exclusively holoandric transmission, are useful to estimate paternal gene flow. The comparison of the two groups of Jews with each other and with Czechoslovaks (which have been taken as a representative source of foreign Y-chromosomes for Ashkenazim) shows a great similarity between Sephardim and Ashkenazim who are very different from Czechoslovaks. On the other hand both groups of Jews appear to be closely related to Lebanese. A preliminary evaluation suggests that the contribution of foreign males to the Ashkenazi gene pool has been very low (1% or less per generation).

MtDNA polymorphisms among Tharus of eastern Terai (Nepal).

Tharus--a population of Terai (a region with a severe malarial morbidity in the past)--can be subdivided into three main groups: Western, Central and Southern Tharus. They have usually been considered a Mongoloid population and this has been further substantiated by mtDNA findings on Central Tharus. Studies on the distribution of malaria-related genes have shown an extremely high frequency (0.8) of the alpha-thal gene among Western and Central Tharus. This frequency, however, unexpectedly turned out to be only 0.04 in a sample of Eastern Tharus. This raised doubts on the common notion that Tharus are a single anthropological entity. In the present investigation mtDNA markers were studied in the same sample of Eastern Tharus previously examined for the alpha-thal gene. The findings were: 1. the same three features which confirmed the classification of Central Tharus as Mongoloids (i.e., the common occurrence of HpaI-1/HincII-1 and HaeII-5 morphs, and the lack of BamHI polymorphism) were also present in this sample. Since the only neighbouring population accessible to Tharus, until recently, has been Hindu (Caucasoids), this result strongly supports the notion that Tharus are indeed a single anthropological entity; 2. two statistically significant differences between Eastern and Central Tharus--namely, a much higher HaeII morph 5 frequency among Central Tharus, and the absence in the same group of the mutation at 15.487 bp (very common among Eastern Tharus)--together with the results on alpha-tal gene, suggested that Tharu subgroups underwent an effective reproductive isolation.

A survey of six genetic markers on the populations of Punjab and Rajasthan (India).

190 Punjabis (Hindus and Sikhs) of Chandigarh and 152 Hindus of Jodhpur (Rajasthan) were examined for six genetic markers, four of which (APO C-II, C6, C7 and FXIIIA) were not studied before in Asiatic Indians. For APO C-II and C7 only the common phenotype was found in a total of 229 and 99 subjects, respectively. For the remaining four markers the two samples were pooled since the gene frequency estimates were not significantly different: FXIIIA*2 = 0.205 +/- 0.016; C6*B = 0.366 +/- 0.037; PGM1*2 = 0.247 +/- 0.017; PGD*C = 0.041 +/- 0.008. These data may contribute to evaluate the extent of the Mongoloid genetic admixture into the Caucasoid gene pool of the Punjab and Rajasthan Hindu population.