Exclusion of ferritins and iron-responsive element (IRE)-binding proteins as candidates for the hemochromatosis gene

We have looked for genes for ferritin and its translational control protein that could account for anomalies in the expression of ferritin (FT) and the transferrin receptor in the duodenum of individuals with hemochromatosis (HC). We show that there are probably only two FTH-like sequences near the HC locus on the short arm of chromosome 6 and no FTL-like sequences. We report the cloning of the previously uncharacterized FTH sequence from 6p (FTHL15) and show that it is probably a processed pseudogene. This gene has been mapped with a panel of radiation hybrid cells to near 6p12. Additionally, we show that there are no sequences on chromosome 6p for a protein that coordinately regulates expression of ferritin and the transferrin receptor.     

Identification of two human ferritin H genes on the short arm of chromosome 6

We have found by analyses of human-hamster hybrid cells that two human ferritin H genes lie near the locus of the iron storage disease idiopathic hemochromatosis on chromosome 6p. One of these genes was isolated and shown to be a processed pseudogene. Comparison of its sequence with those of other ferritin H pseudogenes indicates that they may be derived from a functional H gene other than that on chromosome 11.     

HLA class I and H ferritin gene polymorphisms in normal subjects and patients with haemochromatosis

Summary The gene for idiopathic haemochromatosis is located on the short arm of chromosome 6 within 1 cM of the HLA-A locus. In this region there are many HLA class I genes, and there may also be a gene for the H subunit of ferritin. Both HLA class I and H ferritin genes are therefore candidates for the abnormal gene in idiopathic haemochromatosis. In 15 unrelated patients the frequency of HLA-A3 was 80% compared with 24% for 600 unrelated individuals from South Wales. The most common haplotype involved is probably HLA-A3, B7. DNA was prepared from leucocytes from 12 of these patients and from 85 normal subjects. After digestion with Taq1, electrophoresis, and Southern blotting, class I sequences were detected by hybridisation to an HLA class I probe (pHLA-A). Of the 34 restriction fragments detected, 22 were polymorphic. Particular fragments correlated with the presence of HLA-A antigens A1, 2, 3, 10, 11, w19, and 28, but there was little correlation with B antigens. Restriction fragment patterns specific for haemochromatosis were not found with TaqI or during less extensive studies with other restriction enzymes. No differences in restriction fragment patterns were found between four patients and four normal subjects apparently homozygous for HLA-A3 and B7. Examination of Southern blotting patterns for genomic DNA from patients and normal subjects with a panel of 12 restriction enzymes and a probe for the H ferritin gene (pDBR-2) revealed no polymorphisms associated with either idiopathic haemochromatosis or particular HLA phenotypes. These studies provide no support for either HLA class I genes or the H ferritin gene as candidates for the haemochromatosis gene.     

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.     

Polymorphism in a ferritin H gene from chromosome 6p

Summary This paper addresses the question of whether abnormalities in ferritin expression in the iron storage disease hemochromatosis (HC) involve major deletions or alterations in regions containing the two ferritin H genes that lie near the disease locus on chromosome 6p. We present evidence from analyses of Southern blots that neither gene is deleted in hemochromatosis. We also describe a polymorphism in one of the genes that we have previously shown to be a processed pseudogene. This polymorphism does not correlate with the presence of HC. The PIC value for this polymorphism was calculated as 0.49.     

The haemochromatosis candidate gene HFE (HLA-H) of man and mouse is located in syntenic regions within the histone gene cluster

The HFE (HLA-H) gene is a strong candidate gene for hereditary haemochromatosis and was localized on the short arm of chromosome 6 to 6p21.3-p22. In addition, the sequence of the homologous mouse and rat cDNA and a partial sequence from the mouse gene have been reported recently. In this report, we describe the location of the human and the mouse HFE (HLA-H) gene within the histone gene clusters on the human chromosome 6 and the mouse chromosome 13. Both the human and the murine gene were located on syntenic regions within the histone gene clusters in the vicinity of the histone H1t gene. The genomic sequence of the human HFE (HLA-H) gene and the 3′ portion of the homologous mouse gene were determined. Comparison of the genomic sequences from man and mouse and the cDNA sequence from rat shows significant similarities, also beyond the transcribed region of the mouse gene. J. Cell. Biochem. 69:117–126, 1998. © 1998 Wiley-Liss, Inc.     

Identification of the sex chromosome pair in coho salmon (Oncorhynchus kisutch): lack of conservation of the sex linkage group with chinook salmon (Oncorhynchus tshawytscha)

Fluorescence in situ hybridization (FISH) using a probe to the male-specific GH-Y (growth hormone pseudogene) was used to identify the Y chromosome in coho salmon (Oncorhynchus kisutch). The sex chromosome pair is morphologically similar to chinook salmon (Oncorhynchus tshawytscha) with the GH-Y localized to the small short arm of the largest subtelocentric chromosome pair. FISH experiments with probes containing sex-linked genes in rainbow trout (Oncorhynchus mykiss) (SCAR163) and chinook salmon (Omy7INRA) showed that the coho sex linkage group is different from chinook and rainbow trout and this was confirmed by segregation analysis for the Omy7INRA locus. The telomeric location of the SEX locus, the presence of shared male-specific markers in coho and chinook salmon, and the lack of conservation of sex-linkage groups suggest that transposition of a small male-specific region may have occurred repeatedly in salmonid fishes of the genus Oncorhynchus.     

Members of the human glyceraldehyde-3-phosphate dehydrogenase-related gene family map to dispersed chromosomal locations

Several highly homologous glyceraldehyde-3-phosphate dehydrogenase (GAPD)-related sequences have been identified previously in human DNA by Southern blot analysis. Protein studies have identified only a single expressed locus for this major glycolytic enzyme, and this maps to chromosome 12p13. Sequence analysis of a GAPD muscle cDNA clone and a GAPD-related clone retrieved from an X-chromosome recombinant library showed that the latter was a processed pseudogene that maps to Xp11-p21. In this study, we have determined the chromosomal locations of several of the additional GAPD-related human sequences using a short 3' end sequence from the cDNA to probe DNA from a series of human-rodent somatic cell hybrids on Southern blots. Eight HindIII GAPD-related sequences detected at high stringency have been mapped to 6 different chromosomes. Several of the additional sequences detected at more moderate stringency have been localized to a further 10 chromosomal sites. Together, these sites constitute the known expressed locus, the known X-linked pseudogene, and 15 GAPD-like loci.     

Chromosome loss is responsible for segregation at the HPRT locus in Chinese hamster cell hybrids

The phenomenon of segregation of gene expression has been examined in intraspecific somatic cell hybrids. Specifically, segregation at the hypoxanthine guanine phosphoribosyltransferase (HPRT) locus has been studied in hybrids of Chinese hamster cell lines. The role of chromosome segregation, or other chromosomal events has been assessed by detailed comparison of karyotypes in the 6-thioguanine resistant segregants with those of the parental hybrid lines. The results clearly demonstrate that loss of an entire X chromosome is the primary event responsible for segregation at the HPRT locus, while deletion of a portion of the short arm of an X chromosome was also a frequent event. The results provide the first direct evidence for the assignment of the mapping of this locus to the distal region of the short arm. Analysis of chromosome number distributions in the hybrids and segregants suggests that in selecting chromosomal segregants one may also select for hybrid lines with reduced chromosome stability.     

Structural analysis of the locus containing the human C-reactive protein gene and its related pseudogene

The gene for human C-reactive protein (CRP) is mapped within a 34-kilobase pair genomic DNA segment identified by chromosome walking through overlapping DNA fragments cloned into a lambda phage library. Within 16 kilobase pairs upstream and downstream of the locus for the authentic CRP gene, only one other sequence homologous to that for CRP could be found. Sequencing analysis indicates this sequence to be a pseudogene with 50-80% region-specific homology. Comparison of the authentic CRP gene cloned from genomic DNA libraries independently prepared from three patients indicates no difference in the 5' and 3' flanking region, promoter region, or coding sequence. Only a polymorphism in the length of the poly(GT) stretch located in the intron is observed. There appears to be only one gene locus and copy per haploid chromosome for the authentic CRP gene and its pseudogene.     

A human immunoglobulin kappa orphon without sequence defects may be the product of a pericentric inversion.

The VK gene segments that have been transposed from the kappa locus on the short arm of chromosome 2 at 2p11-12 to other chromosomal sites are called orphons. The 18 VK orphons sequenced up to now carry defects and are to be considered pseudogenes. We now describe the VKI gene segment V108 whose sequence is without any defects and which was localized to the long arm of chromosome 2 at 2q12-14 by in situ hybridization. The V108 region may have been transposed from the short to the long arm of chromosome 2 by a pericentric inversion. Possible reasons for the conservation of its sequence are discussed. In spite of its bona fide sequence V108 is considered to be an unlikely candidate for a VK-JK rearrangement and subsequent functional expression.     

Analysis of complex Y chromosome aberrations using a single DNA probe (Y-367)

A specific cloned DNA sequence (Y-367) detects at least four loci in the euchromatic long arm and in the short arm of the human Y chromosome. Deletion mapping assigns one locus to the distal euchromatic long arm, another to a region close to the centromere on either Yq or Yp, and two additional loci to the Y short arm. Y-367 may thus be used for the rapid screening of even complex Y chromosome aberrations. This is exemplified in a 45,X male with Y chromosome material on the long arm of chromosome 10 by the detection of an inversion of a portion of Yp and by the confirmation of duplications and deletions in two individuals with duplications of part of the Y chromosome.     

The human X-linked steroid sulfatase gene and a Y-encoded pseudogene: evidence for an inversion of the Y chromosome during primate evolution

The mammalian X and Y chromosomes are thought to have evolved from a common, nearly homologous chromosome pair. Although there is little sequence similarity between the mouse or the human X and Y, there are several regions in which moderate to extensive sequence homologies have been found, including, but not limited to, the so-called pseudoautosomal segment, in which X-Y pairing and recombination take place. The steroid sulfatase gene is in the pseudoautosomal region of the mouse, but not in man. We have cloned and characterized the human STS X-encoded locus and a pseudogene that is present on the long arm of the Y chromosome. Our data in humans and other primates suggest that there has been a pericentric inversion of the Y chromosome during primate evolution that has disrupted the former pseudoautosomal arrangement of these genes. These results provide additional insight into the evolution of the sex chromosomes and into the nature of this interesting portion of the human genome.     

Exclusion of the HLA locus from a large portion of the long arm of chromosome 6.

HLA antigens were determined in two infants with multiple congenital anomalies and in their healthy parents and one sibling. One infant had a deletion of a major portion of the long arm of chromosome 6. The other child had a translocation of a similar piece of chromosome 6 to the short arm of chromosome 3. The mother and the maternal grandmother showed this translocation in a balanced state. The HLA types of both children and their parents exclude the localization of the major histocompatibility locus from the deleted or translocated portion of the long arm of chromosome 6.     

Molecular cloning of the human methylthioadenosine phosphorylase processed pseudogene and localization to 3q28

Human methylthioadenosine phosphorylase (MTAP) is a purine and methionine metabolic enzyme present ubiquitously in all normal tissues, but often deleted in many types of cancer. The gene for this enzyme maps to chromosome 9 at band p21 where the cyclin-dependent kinase inhibitor genes for p16 and p15 also reside. During our efforts to clone this gene we also isolated a phage clone containing a processed pseudogene of MTAP. The sequence is 92% homologous to the MTAP cDNA, is flanked at its 3′ end by a repetitive element, but does not possess a poly(A) stretch. We localized this processed pseudogene to band 28 on the long arm of chromosome 3 by fluorescence in situ hybridization. All 22 malignant cell lines with deletions at 9p21 screened possessed the pseudogene.     

Sequences homologous to glutamic acid decarboxylase cDNA are present on mouse chromosomes 2 and 10

The chromosomal locations of mouse DNA sequences homologous to a feline cDNA clone encoding glutamic acid decarboxylase (GAD) were determined. Although cats and humans are thought to have only one gene for GAD, GAD cDNA sequences hybridize to two distinct chromosomal loci in the mouse, chromosomes 2 and 10. The chromosomal assignment of sequences homologous to GAD cDNA was determined by Southern hybridization analysis using DNA from mouse-hamster hybrid cells. Mouse genomic sequences homologous to GAD cDNA were isolated and used to determine that GAD is encoded by a locus on mouse chromosome 2 (Gad-1) and that an apparent pseudogene locus is on chromosome 10 (Gad-1ps). An interspecific backcross and recombinant inbred strain sets were used to map these two loci relative to other loci on their respective chromosomes. The Gad-1 locus is part of a conserved homology between mouse chromosome 2 and the long arm of human chromosome 2.     

A resistance-like gene identified by EST mapping and its association with a QTL controlling Fusarium head blight infection on wheat chromosome 3BS.

Fusarium head blight (FHB) is a major disease in the wheat growing regions of the world. A quantitative trait locus (QTL) on the short arm of chromosome 3B controls much of the variation for resistance. The cloning of candidate disease-resistance genes for FHB QTLs on chromosome 3B can provide further elucidation of the mechanisms that control resistance. However, rearrangements and divergence during plant genome evolution often hampers the identification of sequences with similarity to known disease-resistance genes. This study focuses on the use of wheat expressed sequence tags (ESTs) that map to the region on chromosome 3B containing the QTL for FHB resistance and low-stringency BLAST searching to identify sequences with similarity to known disease-resistance genes. One EST rich with leucine repeats and low similarity to a protein kinase domain of the barley Rpg1 gene was identified. Genetic mapping using a Ning894037 x Alondra recombinant inbred (RI) population showed that this EST mapped to the QTL on the short arm of chromosome 3B and may represent a portion of a newly diverged gene contributing to FHB resistance. The EST is a new marker suitable for marker-assisted selection and provides a starting point to begin map-based cloning for chromosome walking and investigate new diverged genes at this locus.     

Localization of mouse parathyroid hormone-like peptide gene (Pthlh) to distal chromosome 6 using interspecific backcross mice and in situ hybridization.

The single copy parathyroid hormone-like peptide gene (Pthlh) was mapped to distal mouse chromosome 6 using genetic linkage analysis with a panel of DNA samples from interspecific backcross mice. In all 114 meiotic events examined, the Pthlh locus cosegregated with the locus for the Kirsten ras-2 gene (Kras-2) which was previously localized to distal mouse chromosome 6. In addition, Pthlh was localized to chromosome 6 band F-G and the mouse parathyroid hormone Pth was localized to chromosome 7 band F, by in situ hybridization. These studies confirm the previous localization of Pthlh to mouse chromosome 6 using somatic cell hybrids and show that the Pthlh/PTHLH locus is a part of a conserved linkage group between distal mouse chromosome 6 and the proximal segment of the short arm of human chromosome 12.