Haplotype mapping of a major quantitative-trait locus for fetal hemoglobin production, on chromosome 6q23.

Fetal hemoglobin (Hb F) and fetal cell (FC) levels in adults show considerable variation and are influenced by several genetic variants; the major determinants appear to be unlinked to the beta-globin gene cluster. Recently, a trans-acting locus controlling Hb F and FC production has been mapped to chromosome 6q23 in an Asian Indian kindred that includes individuals with heterocellular hereditary persistence of Hb F (HPFH) associated with beta thalassemia. We have extended the kindred by 57 members, bringing the total studied to 210, and have saturated the region with 26 additional markers. Linkage analysis showed tight linkage of the quantitative-trait locus (QTL) to the anonymous markers D6S976 (LOD score 11.3; recombination fraction .00) and D6S270 (LOD score 7.4; recombination fraction .00). Key recombination events now place this QTL within a 1-2-cM interval spanning approximately 1.5 Mb between D6S270 and D6S1626. Furthermore, haplotype analysis has led to a reevaluation of the genealogy and to the identification of additional relationships in the kindred.     

Isolation and Chromosomal Localization of a Cornea-Specific Human Keratin 12 Gene and Detection of Four Mutations in Meesmann Corneal Epithelial Dystrophy

Keratin 12 (K12) is an intermediate-filament protein expressed specifically in corneal epithelium. Recently, we isolated K12 cDNA from a human corneal epithelial cDNA library and determined its full sequence. Herein, we present the exon-intron boundary structure and chromosomal localization of human K12. In addition, we report four K12 mutations in Meesmann corneal epithelial dystrophy (MCD), an autosomal dominant disorder characterized by intraepithelial microcysts and corneal epithelial fragility in which mutations in keratin 3 (K3) and K12 have recently been implicated. In the human K12 gene, we identified seven introns, defining eight individual exons that cover the coding sequence. Together the exons and introns span approximately 6 kb of genomic DNA. Using FISH, we found that the K12 gene mapped to 17q12, where a type I keratin cluster exists. In this study, four new K12 mutations (Arg135Gly, Arg135Ile, Tyr429Asp, and Leu140Arg) were identified in three unrelated MCD pedigrees and in one individual with MCD. All mutations were either in the highly conserved alpha-helix-initiation motif of rod domain 1A or in the alpha-helix-termination motif of rod domain 2B. These sites are essential for keratin filament assembly, suggesting that the mutations described above may be causative for MCD. Of particular interest, one of these mutations (Tyr429Asp), detected in both affected individuals in one of our pedigrees, is the first mutation to be identified within the alpha-helix-termination motif in type I keratin.     

Dating the origin of the CCR5-Delta32 AIDS-resistance allele by the coalescence of haplotypes.

The CCR5-Delta32 deletion obliterates the CCR5 chemokine and the human immunodeficiency virus (HIV)-1 coreceptor on lymphoid cells, leading to strong resistance against HIV-1 infection and AIDS. A genotype survey of 4,166 individuals revealed a cline of CCR5-Delta32 allele frequencies of 0%-14% across Eurasia, whereas the variant is absent among native African, American Indian, and East Asian ethnic groups. Haplotype analysis of 192 Caucasian chromosomes revealed strong linkage disequilibrium between CCR5 and two microsatellite loci. By use of coalescence theory to interpret modern haplotype genealogy, we estimate the origin of the CCR5-Delta32-containing ancestral haplotype to be approximately 700 years ago, with an estimated range of 275-1,875 years. The geographic cline of CCR5-Delta32 frequencies and its recent emergence are consistent with a historic strong selective event (e.g. , an epidemic of a pathogen that, like HIV-1, utilizes CCR5), driving its frequency upward in ancestral Caucasian populations.     

Evolution of Human Polyomavirus JC: Implications for the Population History of Humans

The polyomavirus JC virus (JCV), the etiological agent of progressive multifocal leukoencephalopathy, is ubiquitous in the human population, infecting children asymptomatically, then persisting in the kidney. The main mode of transmission of JCV is from parents to children through long-term cohabitation. Twelve JCV subtypes that occupy unique domains in Europe, Africa, and Asia have been identified. Here, we attempted to elucidate the evolutionary relationships among JCV strains worldwide using the whole-genome approach with which a highly reliable phylogeny of JCV strains can be reconstructed. Sixty-five complete JCV DNA sequences, derived from various geographical regions and belonging to 11 of the 12 known subtypes, were subjected to phylogenetic analysis using three independent methods: the neighbor-joining, maximum parsimony, and maximum likelihood methods. The trees obtained with these methods consistently indicated that ancestral JCVs were divided into three superclusters, designated as Types A, B, and C. A split in Type A generated two subtypes, EU-a and -b, mainly containing European and Mediterranean strains. The first split in Type B generated Af2 (the major African subtype). Subsequent splits in Type B generated B1-c (a minor European subtype) and all seven Asian subtypes (B1-a, -b, -d, B2, MY, CY, and SC). Type C generated a single subtype (Af1), consisting of strains derived from western Africa. While the present findings provided a basis on which to classify JCV into types or subtypes, they have several implications for the divergence and migration of human populations. Received: 4 April 2001 / Accepted: 31 July 2001     

Autosomal Dominant Postaxial Polydactyly, Nail Dystrophy, and Dental Abnormalities Map to Chromosome 4p16, in the Region Containing the Ellis–van Creveld Syndrome Locus

We have studied a four-generation family with features of Weyers acrofacial dysostosis, in which the proband has a more severe phenotype, resembling Ellis-van Creveld syndrome. Weyers acrofacial dysostosis is an autosomal dominant condition with dental anomalies, nail dystrophy, postaxial polydactyly, and mild short stature. Ellis-van Creveld syndrome is a similar condition, with autosomal recessive inheritance and the additional features of disproportionate dwarfism, thoracic dysplasia, and congenital heart disease. Linkage and haplotype analysis determined that the disease locus in this pedigree resides on chromosome 4p16, distal to the genetic marker D4S3007 and within a 17-cM region flanking the genetic locus D4S2366. This region includes the Ellis-van Creveld syndrome locus, which previously was reported to map within a 3-cM region between genetic markers D4S2957 and D4S827. Either the genes for the condition in our family and for Ellis-van Creveld syndrome are near one another or these two conditions are allelic with mutations in the same gene. These data also raise the possibility that Weyers acrofacial dysostosis is the heterozygous expression of a mutation that, in homozygous form, causes the autosomal recessive disorder Ellis-van Creveld syndrome.     

Splicing Defects in the COL3A1 Gene: Marked Preference for 5′ (Donor) Splice-Site Mutations in Patients with Exon-Skipping Mutations and Ehlers-Danlos Syndrome Type IV

Ehlers-Danlos syndrome (EDS) type IV results from mutations in the COL3A1 gene, which encodes the constituent chains of type III procollagen. We have identified, in 33 unrelated individuals or families with EDS type IV, mutations that affect splicing, of which 30 are point mutations at splice junctions and 3 are small deletions that remove splice-junction sequences and partial exon sequences. Except for one point mutation at a donor site, which leads to partial intron inclusion, and a single base-pair substitution at an acceptor site, which gives rise to inclusion of the complete upstream intron into the mature mRNA, all mutations result in deletion of a single exon as the only splice alteration. Of the exon-skipping mutations that are due to single base substitutions, which we have identified in 28 separate individuals, only two affect the splice-acceptor site. The underrepresentation of splice acceptor-site mutations suggests that the favored consequence of 3' mutations is the use of an alternative acceptor site that creates a null allele with a premature-termination codon. The phenotypes of those mutations may differ, with respect to either their severity or their symptomatic range, from the usual presentation of EDS type IV and thus have been excluded from analysis.     

Peutz-Jeghers Syndrome: Confirmation of Linkage to Chromosome 19p13.3 and Identification of a Potential Second Locus, on 19q13.4

Peutz-Jeghers syndrome (PJS) is an autosomal dominant disease with variable expression and incomplete penetrance, characterized by mucocutaneous pigmentation and hamartomatous polyposis. Patients with PJS have increased frequency of gastrointestinal and extraintestinal malignancies (ovaries, testes, and breast). In order to map the locus (or loci) associated with PJS, we performed a genomewide linkage analysis, using DNA polymorphisms in six families (two from Spain, two from India, one from the United States, and one from Portugal) comprising a total of 93 individuals, including 39 affected and 48 unaffected individuals and 6 individuals with unknown status. During this study, localization of a PJS gene to 19p13.3 (around marker D19S886) had been reported elsewhere. For our families, marker D19S886 yielded a maximum LOD score of 4.74 at a recombination fraction (theta) of .045; multipoint linkage analysis resulted in a LOD score of 7.51 for the interval between D19S886 and 19 pter. However, markers on 19q13.4 also showed significant evidence for linkage. For example, D19S880 resulted in a maximum LOD score of 3.8 at theta = .13. Most of this positive linkage was contributed by a single family, PJS07. These results confirm the mapping of a common PJS locus on 19p13.3 but also suggest the existence, in a minority of families, of a potential second PJS locus, on 19q13.4. Positional cloning and characterization of the PJS mutations will clarify the genetics of the syndrome and the implication of the gene(s) in the predisposition to neoplasias.     

Diversity and Age of the Four Major mtDNA Haplogroups, and Their Implications for the Peopling of the New World

Despite considerable investigation, two main questions on the origin of Native Americans remain the topic of intense debate-namely, the number and time of the migration(s) into the Americas. Using the 720 available Amerindian mtDNA control-region sequences, we reanalyzed the nucleotide diversity found within each of the four major mtDNA haplogroups (A-D) thought to have been present in the colonization of the New World. We first verified whether the within-haplogroup sequence diversity could be used as a measure of the haplogroup's age. The pattern of shared polymorphism, the mismatch distribution, the phylogenetic trees, the value of Tajima's D, and the computer simulations all suggested that the four haplogroups underwent a bottleneck followed by a large population expansion. The four haplogroup diversities were very similar to each other, offering a strong support for their single origin. They suggested that the beginning of the Native Americans' ancestral-population differentiation occurred approximately 30,000-40,000 years before the present (ybp), with a 95%-confidence-interval lower bound of approximately 25,000 ybp. These values are in good agreement with the New World-settlement model that we have presented elsewhere, extending the results initially found for haplogroup A to the three other major groups of mtDNA sequences found in the Americas. These results put the peopling of the Americas clearly in an early, pre-Clovis time frame.     

Human Phenylalanine Hydroxylase Mutations and Hyperphenylalaninemia Phenotypes: A Metanalysis of Genotype-Phenotype Correlations

We analyzed correlations between mutant genotypes at the human phenylalanine hydroxylase locus (gene symbol PAH) and the corresponding hyperphenylalaninemia (HPA) phenotypes (notably, phenylketonuria [OMIM 261600]). We used reports, both published and in the PAH Mutation Analysis Consortium Database, on 365 patients harboring 73 different PAH mutations in 161 different genotypes. HPA phenotypes were classified as phenylketonuria (PKU), variant PKU, and non-PKU HPA. By analysis both of homoallelic mutant genotypes and of "functionally hemizygous" heteroallelic genotypes, we characterized the phenotypic effect of 48 of the 73 different, largely missense mutations. Among those with consistent in vivo expression, 24 caused PKU, 3 caused variant PKU, and 10 caused non-PKU HPA. However, 11 mutations were inconsistent in their effect: 9 appeared in two different phenotype classes, and 2 (I65T and Y414C) appeared in all three classes. Seven mutations were inconsistent in phenotypic effect when in vitro (unit-protein) expression was compared with the corresponding in vivo phenotype (an emergent property). We conclude that the majority of PAH mutations confer a consistent phenotype and that this is concordant with their effects, when known, predicted from in vitro expression analysis. However, significant inconsistencies, both between in vitro and in vivo phenotypes and between different individuals with similar PAH genotypes, reveal that the HPA-phenotype is more complex than that predicted by Mendelian inheritance of alleles at the PAH locus.     

Mutations of the Fanconi Anemia Group A Gene (FAA) in Italian Patients

Fanconi anemia (FA) is an autosomal recessive disease characterized by progressive pancytopenia, congenital malformations, and predisposition to acute myeloid leukemia. At least five complementation groups (FA-A-FA-E) have been identified. The relative prevalence of FA-A has been estimated at an average of approximately 65% but may widely vary according to ethnic background. In Italy, 11 of 12 patients analyzed by cell-fusion studies were assigned to group FA-A, suggesting an unusually high relative prevalence of this FA subtype in patients of Italian ancestry. We have screened the 43 exons of the FAA gene and their flanking intronic sequences in 38 Italian FA patients, using RNA-SSCP. Ten different mutations were detected: three nonsense and one missense substitutions, four putative splice mutations, an insertion, and a duplication. Most of the mutations are expected to cause a premature termination of the FAA protein at various sites throughout the molecule. Four protein variants were also found, three of which were polymorphisms. The missense mutation D1359Y, not found in chromosomes from healthy unrelated individuals, was responsible for a local alteration of hydrophobicity in the FAA protein, and it was likely to be pathogenic. Thus, the mutations so far encountered in the FAA gene are essentially all different. Since screening based on the analysis of single exons by genomic DNA amplification apparently detects only a minority of the mutations, methods designed to detect alterations in the genomic structure of the gene or in the FAA polypeptide may be helpful in the identification of FAA mutations.     

Mutation rate in human microsatellites: influence of the structure and length of the tandem repeat.

In 10,844 parent/child allelic transfers at nine short-tandem-repeat (STR) loci, 23 isolated STR mismatches were observed. The parenthood in each of these cases was highly validated (probability >99.97%). The event was always repeat related, owing to either a single-step mutation (n=22) or a double-step mutation (n=1). The mutation rate was between 0 and 7 x 10(-3) per locus per gamete per generation. No mutations were observed in three of the nine loci. Mutation events in the male germ line were five to six times more frequent than in the female germ line. A positive exponential correlation between the geometric mean of the number of uninterrupted repeats and the mutation rate was observed. Our data demonstrate that mutation rates of different loci can differ by several orders of magnitude and that different alleles at one locus exhibit different mutation rates.     

The gene for human fibronectin glomerulopathy maps to 1q32, in the region of the regulation of complement activation gene cluster.

Fibronectin glomerulopathy (GFND) is a newly recognized autosomal dominant disease of the kidney that results in albuminuria, microscopic hematuria, hypertension, renal tubular acidosis type IV, and end-stage renal disease in the 2d to 6th decade of life. The disease is characterized histologically by massive deposits of fibronectin (Fn) present in the subendothelial spaces of renal glomerular capillaries. The cause of human GFND is unknown. In order to localize a candidate gene for GFND, we performed linkage analysis of a large, 193-member pedigree containing 13 affected individuals. Since we had previously excluded the genes for Fn and uteroglobin as candidate genes for GFND, a total-genome search for linkage was performed. Examination of 306 microsatellite markers resulted in a maximum two-point LOD score of 4.17 at a recombination fraction of. 00 for marker D1S249, and a maximum multipoint LOD score of 4.41 for neighboring marker D1S2782. By detection of recombination events, a critical genetic interval of 4.1 cM was identified, which was flanked by markers D1S2872 and D1S2891. These findings confirm that GFND is a distinct disease entity among the fibrillary glomerulopathies. Gene identification will provide insights into the molecular interactions of Fn in GFND, as well as in genetically unaltered conditions.     

Genetic richness and diversity in Cryptosporidium hominis and C. parvum reveals major knowledge gaps and a need for the application of “next generation” technologies — Research review

Cryptosporidium species (apicomplexan protists) are a major cause of diarrhoeal disease (= cryptosporidiosis) in humans worldwide. The impact of cryptosporidiosis is also compounded by the spread of HIV/AIDS and a lack of cost-effective anti-cryptosporidial chemotherapeutics or vaccines. Mitigation of the impact of cryptosporidiosis in humans needs to focus on prevention and control strategies, built on a sound understanding of the epidemiology of Cryptosporidium species. Refined epidemiological studies rely on the use of molecular tools employing informative genetic markers. Currently, the 60-kDa glycoprotein gene (gp60) is the most suitable and widely used genetic marker for Cryptosporidium species infecting humans. Here, we undertake an analysis of all publicly-available gp60 sequence data and associated literature for C. hominis and C. parvum, and yield useful insights into the richness, diversity and distribution of genetic variants, and link these variants to human cryptosporidiosis. This global analysis reveals that, despite high genetic richness in Cryptosporidium isolates from humans, there is a surprisingly low diversity. It also highlights limited knowledge about the genetics of cryptosporidiosis in developing nations and in many animals that might act as infection sources. Clearly, there is a major need for more comprehensive studies of Cryptosporidium infecting humans and other animals in Africa and Asia. As molecular technologies improve and become affordable, future studies should utilize “next generation” sequencing and bioinformatic platforms to conduct comparative ‘genome sequence surveys’ to test the validity of current genetic classifications based on gp60 data. Complemented by in vitro and in vivo investigations, these biotechnological advances will also assist significantly in the search for new intervention strategies against human cryptosporidiosis.     

Radioactive labeling techniques for the identification of G antigen in the human Rh blood group system

The G antigen is one of the erythrocyte membrane Rh antigens. The amount of Rh antigen present on the red blood cell is about 10(-15) g and radioactive labeling of membrane proteins is a useful method for its identification and characterization. In this paper, we compare 4 labeling techniques. Using a human monoclonal anti-Rh(G) antibody and an immunofixation technique, we located the G antigen on a polypeptide of an average molecular weight of 28,000 Da.     

A complex of Co(II) with 2-hydroxyphenyl-azo-2'-naphthol (HPAN) is far less cytotoxic than the parent compound on A549-lung carcinoma and peripheral blood mononuclear cells: Reasons for reduction in cytotoxicity

Cytotoxic studies using an azo compound HPAN and its Co(II) complex were carried out on non-small lung epithelium carcinoma (A549) cells and peripheral blood mononuclear (PBM) cells. The results obtained suggest that the Co(II) complex is much less toxic toward both cell lines and the decreased toxicity due to the complex was more pronounced with carcinoma A549 cells. An attempt was made to correlate the findings related to cytotoxicity with the interaction of the compounds with DNA using calf thymus DNA as the target. The study was able to conclude that the complex was a relatively weak binder to calf thymus DNA. This information was used to explain the interaction of azo compounds with DNA in peripheral blood mononuclear cells and A549 lung carcinoma cells. It was concluded that the Co(II) complex interacts with DNA to a much lesser extent than HPAN alone. Cyclic voltammetry experiments carried out with HPAN and the Co(II) complex further showed that the presence of the metal ion in the complex prevents reduction of the azo group to such species that are responsible for inducing cytotoxicity. The overall finding was that complex formation with azo compounds might serve as a possible route to curb their toxicities.     

Functional interaction between purinergic receptors: effect of ligands for A2A and P2Y12 receptors on P2Y1 receptor function

A_2A adenosine receptor (A_2AR), P2Y₁ receptor (P2Y₁R) and P2Y₁₂ receptor (P2Y₁₂R) are predominantly expressed on human platelets. The individual role of each of these receptors in platelet aggregation has been actively reported. Previously, hetero-oligomerization between these three receptors has been shown to occur. Here, we show that Ca²⁺ signaling evoked by the P2Y₁R agonist, 2-methylthioladenosine 5’ diphosphate (2MeSADP) was significantly inhibited by the A_2AR antagonist (ZM241385 and SCH442416) and the P2Y₁₂R antagonist (ARC69931MX) using HEK293T cells expressing the three receptors. It was confirmed that inhibition of P2Y₁R signaling by A_2AR and P2Y₁₂R antagonists was indeed mediated through A_2AR and P2Y₁₂R using 1321N1 human astrocytoma cells which do not express P2Y receptors. We expect that intermolecular signal transduction and specific conformational changes occur among components of hetero-oligomers formed by these three receptors.