Molecular analysis of the human MHC class I region in hereditary haemochromatosis

Summary The unknown allele that predisposes to the development of haemochromatosis in man has been localized to the HLA class I region on the short arm of chromosome 6. We have utilized pulsed-field gel electrophoresis in conjunction with probes that map within, or in the vicinity of, this region to search for structural lesions that may further define the disease locus. Using the enzyme Mlu I, fragments that associated specifically with the HLA-A23, A31 and B8 alleles were identified. However, in members of three pedigrees affected by haemochromatosis, and in six unrelated patients with the disorder, no disease-specific differences were detected in the DNA fragments with four restriction enzymes and eight probes when compared with healthy individuals. These data suggest that the lesion responsible for hereditary haemochromatosis lies beyond the resolution of this technique and does not involve large structural deletions or extensive re-arrangements in this highly polymorphic region of the genome.     

Molecular analysis of the human MHC class I region using yeast artificial chromosome clones

The cloning of large genomic fragments corresponding to the major histocompatibility complex (MHC) class I region provides the necessary framework for a better understanding of its organization and for the localization of new genes involved in MHC-associated disease. Two human genomic libraries constructed in yeast artificial chromosomes (YACs) have been prepared using complete Not I or Mlu I digestion of source DNA. From these libraries three YAC clones with inserts belonging to the MHC class I region have been isolated. They correspond to exact copies of three genomic fragments of 210, 145, and 50 kilobases (kb), respectively and have been precisely located in the restriction map of the region. Detailed rare-cutter restriction maps of the inserts have been generated. Within these clones we have demonstrated the presence of two class I genes, one of which is HLA-E, and of at least three Hpa II tiny fragment (HTF) islands, corresponding to three putative new transcribed sequences. End clones, which are of particular interest in the extension and refinement of the regional map, have been rescued by systematic subcloning of purified YACs.     

Refining the region of branchio-oto-renal syndrome and defining the flanking markers on chromosome 8q by genetic mapping.

Branchio-oto-renal syndrome (BOR) is an autosomal dominant disorder associated with external-, middle-, and inner-ear malformations, branchial cleft sinuses, cervical fistulas, mixed hearing loss, and renal anomalies. The gene for BOR was mapped to the long arm of chromosome 8q. Several polymorphic dinucleotide repeat markers were investigated for linkage in two large BOR families, and the region of localization was refined. Two-point linkage analysis yielded the maximum lod scores of 7.44 at theta = .03 and 6.71 at theta = .04, with markers D8S279 and D8S260, respectively. A multipoint analysis was carried out to position the BOR gene with a defined region using markers D8S165, D8S285, PENK, D8S166, D8S260, D8S279, D8S164, D8S286, D8S84, D8S275, D8S167, D8S273, and D8S271. Haplotype analysis of recombination events at these polymorphic loci was also performed in multigeneration BOR kindreds. The linkage analysis and analysis of recombination events identified markers that clearly flank the BOR locus. The order was determined to be D8S260-BOR-D8S279 at odds > 10(3):1 over the other possible orders. This flanking markers provide a resource for high-resolution mapping toward cloning and characterizing the BOR gene.     

Molecular markers associated with leptinine production are located on chromosome 1 in Solanum chacoense

Leptines of Solanum chacoense are effective natural deterrents against the Colorado potato beetle. Leptines are the acetylated forms of the glycoalkaloids solanine and chaconine and are supposed to be synthesised via hydroxylated derivatives, called leptinines. Inheritance of leptinine production was studied in crosses of closely related S. chacoense genotypes. The segregation data supported a single-gene model for the inheritance of leptinine production. In the segregating F₁ population of a S. chacoense cross, AFLP, RFLP and RAPD markers segregating with the leptinine production have been identified. The locus involved in leptinine synthesis was localised to the short arm of chromosome 1 of the potato where a major QTL for solanidine production, and markers with tight linkage to leptine production, have been mapped before. Our data further support the previous finding that the short arm of chromosome 1 is involved in steroid alkaloid synthesis in potato, and suggest that the genes involved in leptinine and leptine production are tightly linked in S. chacoense. Received: 27 June 2000 / Accepted: 4 August 2000     

Ordering of markers in the pericentromeric region of chromosome 10

Seven polymorphic cosmids previously assigned to 10cen-q11.2 were mapped between D10S34 and RBP3, and ordered by interphase in situ hybridization and yeast artificial chromosome analysis. Some of the presumed unique sequences from the centromeric region have homologies either within the same region or within the centromeric region of other chromosomes.     

New polymorphisms and markers in the HLA class I region: relevance to hereditary hemochromatosis (HFE)

Hereditary hemochromatosis (HFE) is an inherited disorder whose gene lies in the proximity of the histocompatability antigen (HLA) class I region, on 6p21.3. Despite efforts in refining the HFE region, a number of informative DNA markers, linked to the disease locus and amenable to use in an assay based on the polymerase chain reaction (PCR) is available. The gene content of this region is high, and the HFE gene has not so far been identified. We have used a strategy based on PCR protocols potentially able to detect both polymorphisms and expressed sequences. This approach has been applied to a 700-kb stretch (approximately) of DNA corresponding to the insert of a Centre d'Etude du Polymorphisme Humain yeast artificial chromosome (225 B1) of the possible candidate region. Five new polymorphisms have been detected among 20 specific fragments isolated. Four of them are tightly linked to the HFE locus. Because of the strong linkage disequilibrium with the disease demonstrated by these markers, they could represent starting points for the identification and characterization of the HFE gene. The remaining non-polymorphic fragments, being amplifiable and in most cases linked to NotI sites, may be useful starting points for the generation of a genomic contig of band 6p21.3 and for gene identification.     

A new highly polymorphic marker in the 5' untranslated region of HLA-F shows strong allelic association with haemochromatosis

The 5 untranslated region of HLA-F contains a polypurine tract comprising repeats of tri- and hexa-nucleotide motifs. We have recently demonstrated that this polypurine tract is highly polymorphic by using the polymerase chain reaction. Here, we demonstrate that some of the alleles can be explained by a deletion of approximately 100 by DNA and show that alleles of this novel, highly polymorphic locus are as strongly associated with haemochromatosis as HLA-A3 or D6S105-8. The observed frequency of heterozygosity at HLA-RF is extremely high (95%) and this locus has been found to be informative in pedigrees that are non-informative at HLA-A and D6S105. We also show an example of replication slippage at HL,A-F in one pedigree.     

High-resolution pachytene chromosome mapping of bacterial artificial chromosomes anchored by genetic markers reveals the centromere location and the distribution of genetic recombination along chromosome 10 of rice

Large-scale physical mapping has been a major challenge for plant geneticists due to the lack of techniques that are widely affordable and can be applied to different species. Here we present a physical map of rice chromosome 10 developed by fluorescence in situ hybridization (FISH) mapping of bacterial artificial chromosome (BAC) clones on meiotic pachytene chromosomes. This physical map is fully integrated with a genetic linkage map of rice chromosome 10 because each BAC clone is anchored by a genetically mapped restriction fragment length polymorphism marker. The pachytene chromosome-based FISH mapping shows a superior resolving power compared to the somatic metaphase chromosome-based methods. The telomere-centromere orientation of DNA clones separated by 40 kb can be resolved on early pachytene chromosomes. Genetic recombination is generally evenly distributed along rice chromosome 10. However, the highly heterochromatic short arm shows a lower recombination frequency than the largely euchromatic long arm. Suppression of recombination was found in the centromeric region, but the affected region is far smaller than those reported in wheat and barley. Our FISH mapping effort also revealed the precise genetic position of the centromere on chromosome 10.     

Mapping cynomolgus monkey MHC class I district on chromosome 6p13 using pooled cDNAs

The cynomolgus monkey (Macaca fascicularis) is a frequently used animal model for studying human diseases, especially immune related ones. For a better understanding of its major histocompatibility complex (MHC) class I district chromosome location, we selected seven cDNA clones as probes for fluorescence in situ hybridization (FISH) from a lymphocyte cell line cDNA library. Expressed sequence tags (ESTs) from these clones were assembled into three clusters and annotated Mafa-A and Mafa-B genes. Further bioinformatics analysis shows that they had multiple duplications spanning approximately 2.8 Mb on the rhesus macaque MHC class I district. Using the FISH technique, we mapped the seven pooled cDNA clones to the short arm of the cynomolgus monkey chromosome 6 on 6p13. To our knowledge, this is the first report of the location of cynomolgus monkey MHC class I district. Using pooled adjacent cDNAs as probes also allows affordable, specific genome region mapping research.     

Linkage disequilibrium between haplotypes and allelic incompatibilities detected with p49f,a probe located on the Y chromosome

We have analyzed linkage disequilibrium between haplotypes and allelic incompatibilities betweenTaqI RELPs concerning the probe 49f, located on the Y chromosome. For most (but one) observed haplotypes, discrepancies between observed and expected frequencies can be easily explained allowing a small number of incompatibilities beetween alleles.     

Multiple genetic mechanisms have contributed to the generation of the HLA-A2/A28 family of class I MHC molecules

The genetic events that produce diversity in class I MHC genes and proteins has been investigated by using a family of closely related HLA-A alleles. Five genes coding for HLA-A2.2Y, HLA-A2.3, and HLA-Aw68.2 have been isolated. Exon sequences are compared with the known sequences for HLA-A2.1, HLA-A2.2F, HLA-A2.4, HLA-Aw68.1, and HLA-Aw69. Pairwise comparison of the eight unique sequences shows that point mutation, reciprocal recombination, and gene conversion have all contributed significantly to the diversification of this family of alleles. These results are compared with those of other studies that have emphasized the role of gene conversion. A predominance of coding substitutions in the alpha 1 and alpha 2 domains is found, consistent with positive selection for polymorphism being a major factor in the fixation of these alleles. In the three cases examined, genes for phenotypically identical proteins gave identical nucleotide sequences, indicating that most, if not all, of the class I polymorphism is detectable by immunological methods. The apparent stability of the sequences suggests that the events generating some of the alleles occurred before the origin of modern Homo sapiens.     

湖羊6号染色体微卫星标记多样性与产羔数的关系

选择位于绵羊6号染色体上FecB基因紧密连锁的6个微卫星标记, 即LSCV043、BMS2508、GC101、300U、Bulge5和471U, 分析其在湖羊中的遗传多样性以及和产羔数的关系。结果表明, 6个微卫星位点均属多态性位点, 共检测到34个等位基因、53种基因型。LSCV043、BMS2508和300U属高度多态位点, 其多态信息含量分别为0.6674、0.6035和0.5615。对不同基因型群体的产羔率进行统计分析, LSCV043基因型为107 bp/123 bp所对应的总体产羔数明显高于110 bp/123 bp基因型群体所对应的产羔数(P<0.05); BMS2508基因型为154 bp/154 bp、154 bp/170 bp和154 bp/200 bp所对应的群体第一胎产羔数明显高于170 bp/170 bp所对应的产羔数(P<0.05), 基因型170 bp/170 bp的群体总体产羔数均低于该位点的其他基因型(P<0.05); 其他位点各基因型之间产羔数均无显著差异。     

On the distribution of some genetic markers in libya.

On a sample of N equals 169 nonrelated blood donors from Libya several blood group, serum protein group and enzyme frequencies are within the range of those of other Caucasoid populations. However, the frequencies of cDe, Jk-a, Fy-c, Hp-1, Gc-1, aP-b and AK-1 show some greater differences and seem to indicate certain Negroid influence on the genetic composition of our Libyan sample. This assumption is conceivable considering the racial history of Libya.     

The T cell receptor beta chain genes are located on chromosome 6 in mice and chromosome 7 in humans

Homologous clones that encode the beta chain of the T cell antigen receptor have been isolated recently from both murine and human cDNA libraries. These cDNA clones have been used in connection with interspecies hybrid cell lines to determine that the murine T cell receptor gene is located on chromosome 6 and the human gene on chromosome 7. In situ hybridization confirms these data and further localizes these genes to band B of chromosome 6 in the mouse and bands 7p13-21 in the human genome. The organization of the T cell antigen receptor J beta gene segments and C beta genes appears to be conserved, since very few intraspecies polymorphisms of restriction fragment length have been detected in either mouse or human DNA.