Linkage Disequilibrium for Two X-Linked Genes in Sardinia and Its Bearing on the Statistical Mapping of the Human X Chromosome

The distribution of four X-linked mutants (G6PD, Deutan, Protan and Xg) among lowland and once highly malarial populations of Sardinia discloses a clear-cut example of linkage disequiligrium between two of them (G6PD and Protan). In the same populations the distribution of G6PD-deficiency versus colorblindness of the Deutan type and the Xg blood-group is not significantly different from that expected at equilibrium. These data suggest indirectly that the loci for G6PD and Protan may be nearer to one another than those for G6PD and Deutan.     

Frequency of glucose-6-phosphate dehydrogenase phenotypes and deficiency in Al-Baha

This investigation was conducted on 847 males and females in Al-Baha, the mountainous western province of Saudi Arabia, to determine the prevalence of glucose-6-phosphate dehydrogenase (G6PD) phenotypes and G6PD deficiency. Among the G6PD phenotypes, G6PD B+, G6PD A+, G6PD A-, G6PD Mediterranean and G6PD Mediterranean-like were identified with a gene frequency in the male population of 0.7769, 0.0119, 0.0020, 0.1255 and 0.0817, respectively, and in the females with a frequency of 0.722, 0.003, 0.003, 0.1128 and 0.1311, respectively. Heterozygous females with the phenotypes of G6PD B+/A+ and B+/A- were identified with a frequency of 0.0183 and 0.0090, respectively. The frequency of severe G6PD deficiency in this population was 0.1275 and 0.1158 in males and females, respectively.     

G6PD deficient alleles and haplotype analysis of human G6PD locus in São Tomé e Príncipe (West Africa)

A population sample from S?o Tomé e Príncipe (West Africa) was screened for the G6PD-deficient variants A- (376G/202A), Betica (376G/968C), and Santa Maria (376G/542T). G6PD locus haplotype diversity was also investigated using six intragenic RFLPs (FokI, PvuII, BspHI, PstI, BclI, NlaIII) and a (CTT)n microsatellite 18.61 kb within the G6PD locus. The estimated frequencies of the G6PD*B normal allele, the G6PD*A variant (376G), and the G6PD*A- allele were 0.698, 0.194, and 0.108, respectively. G6PD variants Betica and Santa Maria were not found. Similar levels of microsatellite diversity were found on variants G6PD*B and G6PD*A (H = 0.61 and 0.68, respectively), indicating a similar age for both alleles. All G6PD*A- alleles share the RFLP-microsatellite haplotype ++(-)+(-)+/195, the same haplotype described in nearly all the *A-alleles from sub-Saharan, Mexican Mestizo, and Portuguese populations, consistent with a single and recent origin of the G202A mutation on this *A haplotype.     

The extent of linkage disequilibrium caused by selection on G6PD in humans

The gene coding for glucose-6-phosphate dehydrogenase (G6PD) is subject to positive selection by malaria in some human populations. The G6PD A- allele, which is common in sub-Saharan Africa, is associated with deficient enzyme activity and protection from severe malaria. To delimit the impact of selection on patterns of linkage disequilibrium (LD) and nucleotide diversity, we resequenced 5.1 kb at G6PD and approximately 2-3 kb at each of eight loci in a 2.5-Mb region roughly centered on G6PD in a diverse sub-Saharan African panel of 51 unrelated men (including 20 G6PD A-, 11 G6PD A+, and 20 G6PD B chromosomes). The signature of selection is evident in the absence of genetic variation at G6PD and at three neighboring loci within 0.9 Mb from G6PD among all individuals bearing G6PD A- alleles. A genomic region of approximately 1.6 Mb around G6PD was characterized by long-range LD associated with the A- alleles. These patterns of nucleotide variability and LD suggest that G6PD A- is younger than previous age estimates and has increased in frequency in sub-Saharan Africa due to strong selection (0.1 < s < 0.2). These results also show that selection can lead to nonrandom associations among SNPs over great physical and genetic distances, even in African populations.     

A 1.6-Mb contig of yeast artificial chromosomes around the human factor VIII gene reveals three regions homologous to probes for the DXS115 locus and two for the DXYS64 locus.

Two yeast artificial chromosome (YAC) libraries were screened for probes in Xq28, around the gene for coagulation factor VIII (F8). A set of 30 YACs were recovered and assembled into a contig spanning at least 1.6 Mb from the DXYS64 locus to the glucose 6-phosphate dehydrogenase gene (G6PD). Overlaps among the YACs were determined by several fingerprinting techniques and by additional probes generated from YAC inserts by using Alu-vector or ligation-mediated PCR. Analysis of more than 30 probes and sequence-tagged sites (STSs) made from the region revealed the presence of several homologous genomic segments. For example, a probe for the DXYS64 locus, which maps less than 500 kb 5' of F8, detects a similar but not identical locus between F8 and G6PD. Also, a probe for the DXS115 locus detects at least three identical copies in this region, one in intron 22 of F8 and at least two more, which are upstream of the 5' end of the gene. Comparisons of genomic and YAC DNA suggest that the multiple loci are not created artifactually during cloning but reflect the structure of uncloned human DNA. On the basis of these data, the most likely order for the loci analyzed is tel-DXYS61-DXYS64-(DXS115-3-DXS115-2)-5'F8-(D XS115-1)-3'F8-G6PD.     

Localization of G6PD and HPRT to different arms of the X chromosome of the North American marsupial (Didelphis virginiana) by in situ hybridization and deletion mapping: evolutionary significance

We have mapped HPRT and G6PD loci on the X chromosome in the American opossum, Didelphis virginiana, by in situ hybridization to cells derived from two females by using genomic opossum DNA as probes. The localizations (G6PD to Xp13 and HPRT to Xq21), indicating that the two genes are separated by the centromere, were confirmed by results of hybridization to X chromosomes with deletions that include the HPRT locus and opossum-mouse cell hybrids containing the relevant fragment of the opossum X chromosome.     

Localization of G6PD and HPRT to different arms of the X chromosome of the North American marsupial (Didelphis virginiana) by in situ hybridization and delection mapping: Evolutionary significance

We have mapped HPRT and G6PD loci on the X chromosome in the American opossum, Didelphis virginiana, by in situ hybridization to cells derived from two females by using genomic opossum DNA as probes. The localizations (G6PD to Xp13 and HPRT to Xq21), indicating that the two genes are separated by the centromere, were confirmed by results of hybridization to X chromosomes with deletions that include the HPRT locus and opossum-mouse cell hybrids containing the relevant fragment of the opossum X chromosome.     

The origin of glucose-6-phosphate-dehydrogenase (G6PD) polymorphisms in African-Americans.

DNA samples from 54 male Afro-Americans were examined for glucose-6-phosphate dehydrogenase (G6PD) genotypes G6PD A(+)376G, G6PD A(-)202A/376G, and G6PD B and for polymorphisms in intron 5 (PvuII), at nucleotide 1311, and at nucleotide 1116 (PstI). In the G6PD B subjects, the nucleotide 1311 mutation and the PstI site appeared to be in linkage equilibrium. No PvuII+ G6PD men were encountered. The G6PD A(+) mutation was in disequilibrium with respect to both the nucleotide 1311 mutation and the PstI site. The G6PD A- nucleotide 202 mutation was in disequilibrium with all three polymorphic sites. No conclusion could be drawn with respect to the PvuII site, except that it preceded the nucleotide 202 (A-) mutation. We conclude from these and our previous studies that G6PD B is the most ancient genotype. The nucleotide 1311 mutation, with its worldwide distribution, probably occurred next. The PstI mutation, limited to Africans, probably arose next and is more ancient than the A(+) mutation, which occurred in a gene without either the PstI or the 1311 mutation. G6PD A-202A/376G is the most recent of these mutations and is still in linkage disequilibrium with all of the sites. Presumably it occurred in an individual with both the A(+) and PvuII mutations.     

Nucleotide variability at G6pd and the signature of malarial selection in humans

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy in humans. Deficiency alleles for this X-linked disorder are geographically correlated with historical patterns of malaria, and the most common deficiency allele in Africa (G6PD A-) has been shown to confer some resistance to malaria in both hemizygous males and heterozygous females. We studied DNA sequence variation in 5.1 kb of G6pd from 47 individuals representing a worldwide sample to examine the impact of selection on patterns of human nucleotide diversity and to infer the evolutionary history of the G6PD A- allele. We also sequenced 3.7 kb of a neighboring locus, L1cam, from the same set of individuals to study the effect of selection on patterns of linkage disequilibrium. Despite strong clinical evidence for malarial selection maintaining G6PD deficiency alleles in human populations, the overall level of nucleotide heterozygosity at G6pd is typical of other genes on the X chromosome. However, the signature of selection is evident in the absence of genetic variation among A- alleles from different parts of Africa and in the unusually high levels of linkage disequilibrium over a considerable distance of the X chromosome. In spite of a long-term association between Plasmodium falciparum and the ancestors of modern humans, patterns of nucleotide variability and linkage disequilibrium suggest that the A- allele arose in Africa only within the last 10,000 years and spread due to selection.     

High-resolution mapping of the X-linked hypohidrotic ectodermal dysplasia (EDA) locus

The X-linked hypohidrotic ectodermal dysplasia (EDA) locus has been previously localized to the subchromosomal region Xq11-q21.1. We have extended our previous linkage studies and analyzed linkage between the EDA locus and 10 marker loci, including five new loci, in 41 families. Four of the marker loci showed no recombination with the EDA locus, and six other loci were also linked to the EDA locus with recombination fractions of .009-.075. Multipoint analyses gave support to the placement of the PGK1P1 locus proximal to the EDA locus and the DXS453 and PGK1 loci distal to EDA. Further ordering of the loci could be inferred from a human/rodent somatic cell hybrid derived from an affected female with EDA and an X;9 translocation and from studies of an affected male with EDA and a submicroscopic deletion. Three of the proximal marker loci, which showed no recombination with the EDA locus, when used in combination, were informative in 92% of females. The closely linked flanking polymorphic loci DXS339 and DXS453 had heterozygosities of 72% and 76%, respectively, and when used jointly, they were doubly informative in 52% of females. The human DXS732 locus was defined by a conserved mouse probe pcos169E/4 (DXCrc169 locus) that cosegregates with the mouse tabby (Ta) locus, a potential homologue to the EDA locus. The absence of recombination between EDA and the DXS732 locus lends support to the hypothesis that the DXCrc169 locus in the mouse and the DXS732 locus in humans may contain candidate sequences for the Ta and EDA genes, respectively.     

Phenotypic variability in X-linked ocular albinism: relationship to linkage genotypes.

One hundred nineteen individuals from 11 families with X-linked ocular albinism (OA1) were studied with respect to both their clinical phenotypes and their linkage genotypes. In a four-generation Australian family, two affected males and an obligatory carrier lacked cutaneous melanin macroglobules (MMGs); ocular features were identical to those of Nettleship-Falls OA1. Four other families had more unusual phenotypic features in addition to OA1. All OA1 families were genotyped at DXS16, DXS85, DXS143, STS, and DXS452 and for a CA-repeat polymorphism at the Kallmann syndrome locus (KAL). Separate two-point linkage analyses were performed for the following: group A, six families with biopsy-proved MMGs in at least one affected male; group B, four families whose biopsy status was not known; and group C, OA-9 only (16 samples), the family without MMGs. At the set of loci closest to OA1, there is no clear evidence in our data set for locus heterogeneity between groups A and C or among the four other families with complex phenotypes. Combined multipoint analysis (LINKMAP) in the 11 families and analysis of individual recombination events confirms that the major locus for OA1 resides within the DXS85-DXS143 interval. We suggest that more detailed clinical evaluations of OA1 individuals and families should be performed for future correlation with specific mutations in candidate OA1 genes.     

Evidence for linkage between glucose 6-phosphate dehydrogenase and hypoxanthine-guanine-phosphoribosyl transferase loci in Chinese hamster cells

G6PD and 6PGD activities were determined in diploid, hyperdiploid, tetraploid, and hybrid cells all originating from the same Chinese hamster cell line (the DON line). A relationship between gene multiplicity and enzyme activity has been observed. The same enzymes were studied in hybrid cells cultivated in selective media. Selection was carried out against and for the HGPRT⁺ locus. The differences in G6PD and 6PGD activities between the cell lines found under these conditions indicate a positive linkage of the G6PD and HGPRT loci and negative linkage of the 6PGD and HGPRT loci in these Chinese hamster cells.     

Linkage between a PvuII restriction fragment length polymorphism and G6PD A-202A/376G: evidence for a single origin of the common G6PD A — mutation

Summary DNA samples from 29 males with the G6PD A-phenotype and 14 males with a G6PD B phenotype were studied for the presence of each of four polymorphic restriction sites in the glucose-6-phosphate dehydrogenase locus. All G6PD A-subjects with the G6PD A-^202A/376G genotype, regardless of population origin, shared identical haplotypes. In view of the fact that at least one of the restriction sites, the PvuII site in the intron between exon 5 and 6, has thus far been uncommon in the populations studied, it seems likely that the G6PD A-mutation at nucleotide 202 arose relatively recently and in a single individual.     

African Glucose-6-Phosphate Dehydrogenase Alleles Associated with Protection from Severe Malaria in Heterozygous Females in Tanzania

X-linked Glucose-6-phosphate dehydrogenase (G6PD) A- deficiency is prevalent in sub-Saharan Africa populations, and has been associated with protection from severe malaria. Whether females and/or males are protected by G6PD deficiency is uncertain, due in part to G6PD and malaria phenotypic complexity and misclassification. Almost all large association studies have genotyped a limited number of G6PD SNPs (e.g. G6PD202 / G6PD376), and this approach has been too blunt to capture the complete epidemiological picture. Here we have identified 68 G6PD polymorphisms and analysed 29 of these (i.e. those with a minor allele frequency greater than 1%) in 983 severe malaria cases and controls in Tanzania. We establish, across a number of SNPs including G6PD376, that only female heterozygotes are protected from severe malaria. Haplotype analysis reveals the G6PD locus to be under balancing selection, suggesting a mechanism of protection relying on alleles at modest frequency and avoiding fixation, where protection provided by G6PD deficiency against severe malaria is offset by increased risk of life-threatening complications. Our study also demonstrates that the much-needed large-scale studies of severe malaria and G6PD enzymatic function across African populations require the identification and analysis of the full repertoire of G6PD genetic markers.     

Fragile X syndrome: search for phenotypic manifestations at loci for hypoxanthine phosphoribosyltransferase and glucose-6-phosphate dehydrogenase.

The subjects of this study were individuals with the form of X-linked mental retardation that is associated with the presence of a cytologically variant X chromosome having a secondary constriction or "fragile site" at Xq 27-28 (Fra X). Studies were carried out to test the hypothesis that deletions or modifications at neighboring loci occur as a consequence of events at the fragile site. Skin fibroblasts and peripheral blood lymphocytes from affected males were analyzed with respect to the expression of two X-lined enzymes: glucose-6-phosphate dehydrogenase (G6PD) and hypoxanthine phosphoribosyltransferase (HPRT); loci for these enzymes are known to be located in the region of the fragile site. Although the number of cells resistant to thioguanine (HPRT-deficient) obtained from some cultures from one Fra X male and blood cells of another was greater than expected, the frequency of these cells was not increased in cultures from other Fra X males. Furthermore, our results indicate that the G6PD activity and electrophoretic mobility in Fra X males is similar to that in normal cells, thus providing no evidence for the loss of the long-arm telomere in the fragile X syndrome.     

Neonatal screening of G6PD deficiency in Tunisia

The Glucose-6-phosphate dehydrogenase (G6PI) deficiency is the most common enzymopathy worldwide. WHO had classified Tunisia among countries that are moderately affected by this affection. However, no mass-screening reflecting the real incidence was realized. The aim of this study is to determine the prevalence of this enzymopathy and its molecular basis in Tunisia. A total of 1102 neonates, born in CMNT center of Maternity and of Neonatology of Tunis during the going periods from April, 2005 till May, 2005 and from June, 2006 till September, 2006, have been enclosed in the study. The samplings included 953peripheral venous blood and 149 blood cordon. Among 1102 samplings, only 976 were of use to the screening. In our mass-screening, we consider all newborns that were born in the CMNT during the period of study and were included in the screening. A dosage of the enzymatic activity was realized using spectrophotometric method. G6PD electrophoresis and molecular study by PCR/RFLP were realized for the overdrawn newborn children. Among 976 screening neonates, 43 individuals (4.4%) were found to be G6PD deficient by quantitative enzyme assay. Newborn affected were distributed in 23 boys and 20 girls (sex ratio of 1.15). The electrophoretic mobility and the molecular biology were realized for the affected newborn. Molecular characterization of 30 G6PD deficient neonates revealed that the G6PD A- was the most common and was detected in 20 of 43 individuals (66.7%), followed by G6PD Mediterranean that was detected in 6 (13.3%). At least, 4 other unknown mutations were not able to be determined by PCR/RFLP (n=4). In conclusion G6PD deficiency is frequent in our country, justifying a systematic neonatal screening, to avoid the arisen of grave consequences of this affection. The African variant is the most frequent in our country followed by the Mediterranean one.     

A multipoint linkage map of the distal short arm of the human X chromosome.

The distal portion of the short arm of the human X chromosome (Xp) exhibits many unique and interesting features. Distal Xp contains the pseudoautosomal region, a number of disease loci, and several cell-surface markers. Several genes in this area have also been observed to escape X-chromosomal inactivation. The characterization of new polymorphic loci in this region has permitted the construction of a refined multipoint linkage map extending 15 cM from the Xp telomere. This interval is known to contain the loci for the diseases X-linked ichthyosis, chondrodysplasia punctata, and Kallmann syndrome, as well as the cell-surface markers Xg and 12E7. This region also contains the junction between the pseudoautosomal region and strictly X-linked sequences. The locus MIC2 has been demonstrated by linkage analysis to be indistinguishable from the pseudoautosomal junction. The steroid sulfatase locus has been mapped to an interval adjacent to the DXS278 locus and 6 cM from the pseudoautosomal junction. The polymorphic locus (STS) DXS278 was shown to be informative in all families studied, and linkage analysis reveals that the locus represents a low-copy repeat with at least one copy distal to the STS gene. The generation of a multipoint linkage map of distal Xp will be useful in the genetic dissection of many of the unique features of this region.     

Microsatellite isolation, linkage group identification and determination of recombination frequency in the peach-potato aphid, Myzus persicae (Sulzer) (Hemiptera: Aphididae)

Fifteen polymorphic microsatellite markers were used to establish linkage groups and relative rates of recombination in male and female Myzus persicae (Sulzer) (Hemiptera: Aphididae) (peach-potato aphid). We cloned nine markers from M. persicae and for these we report primer sequences and levels of allelic diversity and heterozygosity in four Australian M. persicae populations. Of the remaining six loci, four loci, also cloned from M. persicae, were obtained from G. Malarky (Natural History Museum, London) and two loci from Sitobion miscanthi were used. Additionally, the primer sequences of locus M77, a locus monomorphic in M. persicae but polymorphic in the closely related Myzus antirrhinii, are presented. Eleven of the 15 polymorphic markers were autosomal and four were X-linked. A linkage analysis was performed on a European pedigree of aphids containing five families with between seven and 11 offspring each. There was no linkage between any loci in females. In males, several pairwise comparisons yielded no recombinant offspring. With the exception of locus M40, these observations were supported in a linkage analysis performed on larger families produced from Australian M. persicae crosses. Locus M40 showed segregation consistent with involvement in a translocation between autosomes 1 and 3 in European samples but not in the Australian samples. From the Australian crosses we report an absence of recombination in males but high recombination rates in females. One X chromosome and four autosomal linkage groups were identified and tentatively assigned to chromosomes. The relevance of achiasmate meiosis to the evolution of sex is discussed.     

Lowe oculocerebrorenal syndrome: DNA-based linkage of the gene to Xq24-q26, using tightly linked flanking markers and the correlation to lens examination in carrier diagnosis.

The Lowe syndrome (LS), or oculocerebrorenal syndrome, has been studied using DNA-based linkage analysis, and the findings have been correlated with the result of a thorough ophthalmologic examination. It was found that the LS gene was linked to markers in the Xq24-q26 region and that the locus DXS42 was the most closely linked marker, giving a LOD score of 3.12 at zero recombination distance. Combined with earlier data, this forms the basis for carrier detection and prenatal diagnosis by using tightly linked flanking markers. A summary of our and other data suggests that the loci DXS17, DXS11, DXS87, and DXS42 are located on the proximal side, and DXS86 and DXS10 on the distal side of the Lowe locus. In isolated cases of LS the question of whether the mother is a carrier of the mutation arises. It was found that a lens examination with slit-lamp illumination and a count of the total number of lenticular opacities is a reliable method of ascertaining the carrier state.     

Linkage studies in Lenz microphthalmia.

Glucose-6-phosphate dehydrogenase (G6PD) phenotype studies were done on a black family with X-linked heredofamilial bilateral microphthalmia (HBM). Three crossovers and three non-crossovers were detected in three informative matings of four generations yielding a recombination value of 0.5. These findings do not provide evidence for linkage between the G6PD and HBM loci, suggesting either that the G6PD and HBM loci are far apart on the X chromosome or that HBM in this family is inherited as an autosomal dominant male sex-limited trait.