Closing in on the BPES gene on 3q23: mapping of a de Novo reciprocal translocation t(3;4)(q23;p15.2) breakpoint within a 45-kb cosmid and mapping of three candidate genes, RBP1, RBP2, and beta'-COP, distal to the breakpoint

BPES is a genetic disorder presenting with blepharophimosis, ptosis of the eyelids, epicanthus inversus, and telecanthus. BPES type I is associated with female infertility, whereas type II presents without additional symptoms. Hitherto, it remains unknown whether BPES type I results from a defect in a single gene or from a contiguous gene syndrome. Previous cytogenetic and linkage analyses have assigned a BPES locus to 3q23, in a 5-cM interval between D3S1615 and D3S1316. In this report, we describe the molecular and physical characterization of the 3q23 breakpoint in a BPES patient with a t(3;4)(q23;p15.2) translocation. Eight YACs located around and within the D3S1615-D3S1316 interval were mapped relative to the 3q23 breakpoint; 5 YACs spanning the 3q23 breakpoint were identified. Thirteen STSs and ESTs were localized on the YAC map. Subsequent hybridization of 2 YACs spanning the breakpoint to the Human RPCI1 PAC Library and the Human Chromosome 3 LLNL Cosmid Library resulted in the identification of 12 PACs and 50 cosmids respectively, allowing the construction of a detailed PAC and cosmid physical map. A refined position-telomeric to the breakpoint-of 3 candidate genes, cellular retinol-binding proteins 1 and 2 (RBP1, RBP2) and the coatomer beta' subunit (beta'-COP), was obtained on this physical map. Furthermore, a PAC and cosmid contig encompassing the breakpoint was constructed. PAC 169-C 10 and cosmid 11-L 10 crossing the breakpoint have sizes of 110 and 45 kb, respectively. The isolation of coding sequences in these clones and in the rest of the contig will greatly facilitate further efforts toward positional cloning of the gene(s) involved in BPES.     

242 breakpoints in the 200-kb deletion-prone P20 region of the DMD gene are widely spread.

Using whole cosmids as probes, we have mapped 242 DMD/BMD deletion breakpoints located in the major deletion hot spot of the DMD gene. Of these, 113 breakpoints were mapped more precisely to individual restriction enzyme fragments in the distal 80 kb of the 170-kb intron 44. An additional 12 breakpoints are distributed over the entire region, with no significant local variation in frequency. Furthermore, deletion sizes vary and are not influenced by the positions of the breakpoints. This argues against a predominant role of one or a few specific sequences in causing frequent rearrangements. It suggests that structural characteristics or a more widespread recombinogenic sequence makes this region so susceptible to deletion. Our study revealed several RFLPs, one of which is a 300-bp insertion/deletion polymorphism. Abnormally migrating junction fragments are found in 81% of the precisely mapped deletions and are highly valuable in the diagnosis of carrier females.     

Complex arrangement of genes within a 220-kb region of double-duplicated DNA on human 2q37.1

Gene duplication events are followed by divergence of initially identical gene copies, due to the subsequent accumulation of mutations. These mutations tend to be degenerative and may lead to either nonfunctionalization or subfunctionalization of the gene copies. Here we report the molecular characterization of a 220-kb genomic DNA fragment from human 2q37.1, in which a double duplication and a partial triplication event has taken place. As a result, this region contains four copies of alkaline phosphatase (P), four copies of the ECEL1 gene (X), two copies of a newly identified gene (N), and two copies of a cholinergic receptor subunit (R), in the order N-P-X-P-X-P-X-N-P-X-R-R. While three of the four ECEL1 copies, one copy of the phosphatase gene and one copy of the newly identified gene have lost their function, three phosphatase gene copies and the two receptor subunits are still functionally active and thus may provide an example for subfunctionalization of duplicated genes.     

Closing in on a breast cancer gene on chromosome 17q.

Linkage of early-onset familial breast and ovarian cancer to 11 markers on chromosome 17q12-q21 defines an 8-cM region which is very likely to include the disease gene BRCA 1. The most closely linked marker is D17S579, a highly informative CA repeat polymorphism. D17S579 has no recombinants with inherited breast or ovarian cancer in 79 informative meioses in the seven families with early-onset disease (lod score 9.12 at zero recombination). There is no evidence for linkage heterogeneity in the families with early-onset disease. The proportion of older-onset breast cancer attributable to BRCA 1 is not yet determinable, because both inherited and sporadic cases occur in older-onset families.     

Physical mapping of four serpin genes: alpha 1-antitrypsin, alpha 1-antichymotrypsin, corticosteroid-binding globulin, and protein C inhibitor, within a 280-kb region on chromosome I4q32.1.

Alpha 1-antitrypsin (alpha 1AT; protease inhibitor [PI] locus), alpha 1-antichymotrypsin (alpha 1ACT; AACT locus), corticosteroid-binding globulin (CBG; CBG locus), and protein C inhibitor (PCI; PCI locus) are members of the serine protease inhibitor (serpin) superfamily. A noncoding PI-like (PIL) gene has been located 12 kb 3' of the PI gene. The PI, PIL, and AACT loci have been localized to 14q32.1, the CBG locus has been localized to 14q31-14q32.1, and PCI has been mapped to chromosome 14. Genetic linkage analysis suggests tight linkage between PI and AACT. We have used pulsed-field gel electrophoresis to generate a physical map linking these five serpin genes. The order of the genetic loci is AACT/PCI-PI-PIL-CBG, with a maximum distance of about 220 kb between the AACT/PCI and PI genes. These genes form a PI cluster at 14q32.1, similar to that of the homologous genes on murine chromosome l2. The close proximity of these genes has implications for disease-association studies.     

Hyperparathyroidism-jaw tumor syndrome: the HRPT2 locus is within a 0.7-cM region on chromosome 1q.

Hyperparathyroidism-jaw tumor syndrome (HPT-JT) is an autosomal dominant disease characterized by the development of multiple parathyroid adenomas and multiple fibro-osseous tumors of the maxilla and mandible. Some families have had affected members with involvement of the kidneys, variously reported as Wilms tumors, nephroblastomas, and hamartomas. The HPT-JT gene (HRPT2) maps to chromosome 1q25-q31. We describe further investigation of two HPT-JT families (K3304 and K3349) identified through the literature. These two expanded families and two previously reported families were investigated jointly for linkage with 21 new, closely linked markers. Multipoint linkage analysis resulted in a maximum LOD score of 7.83 (at recombination fraction 0) for markers D1S2848-D1S191. Recombination events in these families reduced the HRPT2 region to approximately 14.7 cM. In addition, two of these four study families (i.e., K3304 and K11687) share a 2.2-cM length of their (expanded) affected haplotype, indicating a possible common origin. Combining the linkage data and shared-haplotype data, we propose a 0.7-cM candidate region for HRPT2.     

Large-scale physical mapping within the region 22q12.3-13.1 in meningioma

The lack of physical mapping data strongly restricts the analysis of the meningioma chromosomal region that was assigned to the bands 22q12.3-qter. Recently, we reported a new marker D22S16 for chromosome 22 that was assigned to the region 22q13-qter by in situ hybridization. Utilizing somatic cell hybrids we now sublocalized the marker D22S16 within the band region 22q12-13.1, thus placing it in the vicinity of the gene for the platelet derived growth factor (PDGFB). A physical map was established for the regions surrounding the PDGFB gene and the D22S16 marker. By means of pulsed-field gel electrophoresis (PFGE) D22S16 and PDGFB were found to be physically linked within 900 kb. We also identified two CpG clusters bordering the PDGFB gene. For the enzyme NotI, a variation of the PDGFB restriction pattern was found between different individuals. PFGE analysis of the two loci (PDFGB and D22S16) failed to identify major rearrangements in meningioma.     

Physical mapping of the CC-chemokine gene cluster on the human 17q11. 2 region.

Chemokines are a family of small secreted proteins that are involved in the trafficking of leukocytes by acting on G-protein-coupled receptors. Specific chemokines are also implicated in the regulation of angiogenesis and mobilization of hematopoietic cell precursors. Chemokines are subdivided into four groups on the basis of the relative positions of their conserved cysteines. For the CC-chemokine group, in which the first two (of four) conserved cysteines are adjacent, 22 members have been described so far. In this work, we have analyzed the genomic organization of these genes. We first assigned the genes encoding CC-chemokines to chromosomal regions and organized their relative positioning by using two radiation hybrid panels. Fifteen CC-chemokine genes were shown to be clustered within the 17q11.2 region of the human genome. These genes appeared to be segregated into two subclusters separated by about 2. 25 Mb (9 cR). Contigs of bacterial artificial chromosomes (BAC) covering these two subclusters were subsequently isolated and the localizations of the CC-chemokine genes within these contigs determined. The relative positioning of the BAC clones was determined with the help of fluorescence hybridization on combed genomic DNA. The cluster organization of the various CC-chemokine genes in the genome was found to be grossly consistent with their structural similarities. This map of the CC-chemokine gene cluster should facilitate the determination of the full sequence of the chromosomal region.     

Localization of DNA sequences to a region within Xp11.21 between incontinentia pigmenti (IP1) X-chromosomal translocation breakpoints.

Incontinentia pigmenti (IP) is an X-linked dominant disorder characterized by developmental anomalies of the tissues and organs derived from embryonic ectoderm and neuroectoderm. An IP locus, designated IP1, probably resides in Xp11.21, since five unrelated patients with nonfamilial IP have been identified who possess constitutional de novo reciprocal X;autosome translocations involving Xp11.21. We have used a series of somatic cell hybrids containing the rearranged chromosomes derived from three of the five IP1 patients, along with other hybrid cell lines, to map probes in the vicinity of the IP1 locus. Five anonymous DNA loci--DXS422, DXS14, DXS343, DXS429, and DXS370--have been mapped to a region within Xp11.21, between two IP1 X-chromosomal translocation breakpoints; the IP1 t(X;17) breakpoint is proximal (centromeric) to this region, and the IP1 t(X;13) and t(X;9) X-chromosomal breakpoints lie distal to it. While no IP1 translocation breakpoint has yet been identified by pulsed-field gel electrophoretic (PFGE) analysis, an overlap between three probes--p58-1, 7PSH3.5, and cpX210--has been detected, placing these probes within 125 kb. Four probes--p58-1, 7PSH3.5, cpX210, and 30CE2.8--have been helpful in constructing a 1,250-kb PFGE map of the region between the breakpoints; these results suggest that the IP1 X-chromosomal translocation breakpoints are separated by at least this distance. The combined somatic cell hybrid and PFGE analyses we report here favor the probe order DXS323-(IP1 t(X;13), IP1, t(X;9]-(DXS422, DXS14, DXS343, DXS429, DXS370)-(IP1 t(X;17), DXZ1). These sequences provide a starting point for identifying overlapping genomic sequences that span the IP1 translocation breakpoints; the availability of IP1 translocation breakpoints should now assist the cloning of this locus.     

Fine mapping of the human pentraxin gene region on chromosome 1q23

 The 1q21 to 25 region of human chromosome 1 contains genes which encode proteins with immune- and inflammation-associated functions. These include the pentraxin genes, for C-reactive protein (CRP), serum amyloid P (SAP) protein (APCS), and a CRP pseudogene (CRPP1). The region of chromosome 1 containing this cluster is syntenic with distal mouse chromosome 1. We constructed an approximately 1.4 megabase yeast artificial chromosome (YAC) contig with the pentraxin genes at its core. This four-YAC contig includes other genes with immune functions including the FCER1A gene, which encodes the α-subunit of the IgE high-affinity Fc receptor and the IFI-16 gene, an interferon-γ-induced gene. In addition, it contains the histone H3F2 and H4F2 genes and the gene for erythroid α-spectrin (SPTA1). The gene order is cen.-SPTA1-H4F2-H3F2-IFI-16-CRP-CRPP1-APCS-FCER1A- tel. The contig thus consists of a cluster of genes whose products either have immunological importance, bind DNA, or both. Received: 13 December 1995 / 6 February 1996     

Homozygosity mapping places the acrodermatitis enteropathica gene on chromosomal region 8q24.3

Acrodermatitis enteropathica (AE) is a rare autosomal recessive pediatric disease characterized by dermatitis, diarrhea, alopecia, and growth failure. The disease results from insufficient uptake of zinc by the intestine and can be fatal unless the diet is supplemented with zinc. To map the gene responsible for AE, a genomewide screen was performed on 17 individuals, including 4 affected individuals, in a consanguineous Jordanian family. Three markers-D8S373, D10S212, and D6S1021-had a pattern consistent with tight linkage to a recessive disease: one allele in the affected sibs and multiple alleles in unaffected sibs and parents. Two-point parametric linkage analysis using FASTLINK identified one region, D8S373, with a maximum LOD score >1.5 (1.94 at D8S373: recombination fraction.001). Twelve additional markers flanking D8S373 were used to genotype the extended family, to fine-map the AE gene. All five affected individuals-including one who was not genotyped in the genomewide screen-were found to be homozygous for a common haplotype, spanning approximately 3.5 cM, defined by markers D8S1713 and D8S2334 on chromosomal region 8q24.3. To support these mapping data, seven consanguineous Egyptian families with eight patients with AE were genotyped using these markers, and six patients from five families were found to be homozygous in this region. Multipoint analysis with all consanguineous families, by Mapmaker/Homoz, resulted in a maximum LOD score of 3.89 between D8S1713 and D8S373. Sliding three-point analysis resulted in a maximum LOD score of 5.16 between markers D8S1727 and D8S1744.     

Paint-assisted microdissection-FISH: Rapid and simple mapping of translocation breakpoints in the embryonal rhabdomyosarcoma cell line RD.

BACKGROUND: Spectral karyotyping and multiple fluorophore fluorescence in situ hybridisation (M-FISH) facilitate identification of inter-chromosomal rearrangements, but are of low cytogenetic resolution in mapping translocation breakpoints. Reverse chromosome painting yields increased cytogenetic information but isolation of aberrant chromosomes is technically difficult. We have developed the technique of paint-assisted microdissection FISH (PAM-FISH), which enables microdissection of aberrant chromosomes to be carried out easily and rapidly using relatively simple apparatus. METHODS: A selected chromosome paint is hybridised to abnormal metaphases to label a chromosome of interest, which is then microdissected, amplified, labelled by polymerase chain reaction (PCR), and reverse painted onto extended normal metaphases. RESULTS: PAM-FISH was used to reassess structural chromosomal abnormalities identified by molecular cytogenetics in the rhabdomyosarcoma cell line RD. PAM-FISH improved the analysis of virtually all structural abnormalities, identifying six novel translocations and indicating that seven previously described rearrangements were in fact not present in RD. Accuracy of the breakpoint mapping obtained was confirmed by bacterial artificial chromosome-FISH. CONCLUSIONS: PAM-FISH is ideally suited to analysis of tumour metaphases as it is not affected by poor chromosome morphology. Reagents generated by PAM-FISH are also suitable for other investigations, such as mapping using sequence tagged-site PCR or genomic microarrays. PAM-FISH is technically straightforward and could readily be adopted in a routine cytogenetics laboratory for accurate high-throughput analysis of chromosome breakpoints.     

Nonuniform linkage disequilibrium within a 1,500-kb region of the human immunoglobulin heavy-chain complex.

We have characterized 10 VH polymorphic loci of the VH2, VH3, VH4, and VH5 families. Eight of 10 VH polymorphisms were found to be insertion/deletion polymorphisms, probably the result of nonhomologous recombination over the course of evolution of the current human VH repertoire. The 10 VH polymorphic loci were analyzed in 10 three-generation and 10 two-generation Canadian caucasoid families. Linkage disequilibrium (allelic association) was measured between pairs of VH polymorphic loci, and 12 significant associations were found. The degree of linkage disequilibrium measured between IGH polymorphic loci was then compared with the physical distance separating the loci. The physical distance between IGH polymorphic loci does not entirely determine the degree of linkage disequilibrium between polymorphic loci. Two regions, one in the VH region (between VH3f-2 and VH5-2 and one in the CH region (between C delta and C gamma 3), were found to have linkage disequilibrium values approximately 1/3,000 of that observed in other portions of the IGH region. The previous identification of recombinants in the C delta-to C gamma 3 region indicates that these areas of low linkage disequilibrium are consistent with the presence of recombination hot spots. The observed high amount of recombination in the subtelomeric portion of chromosome 14 therefore appears to be the result of specific hot spots for recombination, rather than a general increase in recombination in this region.     

Genetic and physical fine mapping of a multilocular gene Bjln1 in Brassica juncea to a 208-kb region

Most of the germplasm resources in Brassica juncea produce silique with only two locules, whereas a few varieties can produce silique with three or four locules. The increase in locule number in B. juncea has been shown to cause an increase in the number of seeds per silique, resulting in an increase in the yield per plant. Thus, the development of high-locule-number varieties may be an effective way of improving the yield of B. juncea. Duoshi, a B. juncea landrace originating from the Qinghai–Tibetan plateau, produces silique with 3–4 locules. Genetic analysis has shown that the high-locule-number trait in Duoshi is determined by two recessive genes, tentatively designated as Bjln1 and Bjln2. For fine mapping of the Bjln1 gene, a BC3 population was developed from the cross between Duoshi (multilocular parent) and Xinjie (bilocular parent). Using a combination of amplified fragment length polymorphism (AFLP) and bulked segregant analysis, only two AFLP markers linked to Bjln1 were identified. Preliminary linkage analysis showed that the two AFLP markers were located on the same side of Bjln1. Blast analysis revealed that the sequences of the two AFLP markers had homologues on Scaffold000019 at the bottom of B. rapa A7. Using the results of linkage analysis and BlastN searches, simple sequence repeat (SSR) markers were subsequently developed based on the sequence information from B. rapa A7. Seven SSR markers were eventually identified, of which ln 8 was co-segregated with Bjln1. ln 7 and ln 9, the closest flanking markers, were mapped at 2.0 and 0.4 cM distant from the Bjln1 gene, respectively. The SSR markers were cloned, sequenced and mapped on A7 of B. rapa (corresponding to J7 in the A genome of B. juncea). The two closest flanking markers, ln 7 and ln 9, were mapped within a 208-kb genomic region on B. rapa A7, in which the Bjln1 gene might be included. The present study may facilitate cloning of the Bjln1 gene as well as the selection process for developing multilocular varieties in B. juncea by marker-assisted selection and genetic engineering.     

Novel immunoglobulin superfamily gene cluster,mapping to a region of human chromosome 17q25, linked to psoriasis susceptibility

Chromosome 17q25 harbors a susceptibility locus for psoriasis ( PSORS2). This locus may overlap with loci for atopic dermatitis and rheumatoid arthritis. To further refine the location of PSORS2, we genotyped 242 primarily nuclear families for 15 polymorphic microsatellites mapping to chromosome 17q23-q25. Non-parametric linkage analysis revealed a linkage peak lying close to a novel cluster of genes from the immunoglobulin (Ig) superfamily. This cluster spans >250 kb and harbors five CMRF35-like genes and a sixth inhibitory receptor ( CMRF35H) with three ITIM motifs that is transcribed in the opposite direction from the rest. The Ig domains encoded by these genes are most similar to those of the TREM (triggering receptor expressed selectively in myeloid cells) molecules, NKp44 and the polymeric immunoglobulin receptor. CMRF35-like genes are only expressed in sub-populations of cells of the myeloid lineage. In order to investigate the association of this region with psoriasis, we genotyped the families for 13 novel microsatellites and 19 SNPs from the region of linkage. A maximum NPL of 1.6 ( P=0.05) was obtained within the interval. Two SNP-based haplotypes revealed some evidence for association with psoriasis. One spanned CMRF35H and includes a non-synonymous polymorphism within CMRF35H (R111Q) (TDT P=0.03). The second was a three-locus haplotype lying within the first intron of CMRF35A2 ( TREM5) (TDT P=0.04). The novel markers described here will facilitate additional linkage and association studies between the CMRF35 family and disease.