Molecular characterization of a ring chromosome 14 showing that the PI locus is centromeric to the D14S1 and IGH loci

Summary Molecular characterization of a ring chromosome 14 was carried out in a patient with the 46,XX,r(14) karyotype. The breakpoints shown by chromosome banding were within bands p11 and q32. Using molecular probes for the immunoglobulin heavy chain (IGH), D14S1 and PI loci located at 14q32, we showed that the IGH and D14S1 loci, located at 14q32.2 and 14q32.2, respectively, were deleted on the ring chromosome 14, but that the PI locus was not. Therefore, the chromosomal break lies between PI and D14S1. These results show that the order of these chromosome 14 markers is cen-PI-D14S1-IGH, in keeping with multipoint linkage data. Further molecular characterization of ring 14 chromosomes should lead to a detailed understanding of the molecular events and clinical consequences of the gene deletion associated with such chromosomal aberrations.     

Distal trisomy 14q

Summary Two cases of chromosome 14 rearrangements with partial duplication which occurred de novo were analyzed by Southern blot analysis using IGH, D14S1 and PI probes. In the first case, with a 46,XX,14p+ karyotype, our study confirms that the additional material on chromosome 14p+ results from a duplication of the 14q region containing the IGH, D14S1 and PI loci. In the second case, our study reveals only one 14q32 locus per chromosome 14 indicating that the extra material does not contain the 14q32 region. Our results demonstrate that molecular probes of the 14q32 region are valuable tools for the characterisation of chromosome 14 abnormalities appearing de novo.     

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.     

Physical mapping of probes within 14q32, a subtelomeric region showing a high recombination frequency

The genetic linkage map of chromosome 14q32 contains 11 loci which span a distance of more than 60 cM. We have assigned 10 of these loci and the AKT1 proto-oncogene to segments of 14q32, using breakpoints derived from four independent chromosomal deletions or rearrangements. The most telomeric breakpoint was found in a proband (HSC 6) carrying a ring-14 chromosome. HSC 6 is monosomic for the distal part of 14q32, which contains the immunoglobulin heavy-chain locus (IGH), and random markers D14S20, D14S19, and D14S23. Two other chromosomal breakpoints, found in probands HSC 121 and HSC 981, could not be distinguished from each other using DNA probes, although the cytogenetic breakpoints appeared to be different at 14q32.32 and 14q32.31, respectively. The region between the breakpoints of HSC 6 and HSC 121 contains AKT1, D14S1, D14S17, and D14S16. The entire telomeric band 14q32 is assumed to contain about 10% of chromosome 14, or approximately 10 Mb. The 8 most telomeric loci, including D14S1, map to 14q32.32-qter, which measures only several megabases. However, these loci span a genetic distance of 23 cM. The high recombination frequency contrasts with the observation that two of the gamma genes in the IGH constant region show a high degree of linkage disequilibrium, though 180 kb apart. This finding suggests that a telomeric localization per se does not lead to a higher recombination frequency and favors the hypothesis that the higher recombination frequency at the telomeres may be due to specific "hot spots" for recombination.     

Molecular characterization of ataxia telangiectasia T cell clones

Summary To delimit the 14q32.1 recurrent breakpoint of ataxia telangiectasia clones, we performed an in situ hybridization study with various probes located on the 14q32 band. We thus mapped this breakpoint between the D14S1 and Pi loci. Furthermore, an interstitial duplication including D14S1 and a part of the IgH locus was demonstrated on a t(14;14) clone.     

Evidence for a second gene for primary microcephaly at MCPH5 on chromosome 1

Primary microcephaly has been mapped to five loci on different chromosomes. We present here the fine mapping of one of the loci, MCPH5, to a region of only 0.58 Mb located at the 1q31.3-1q32.1 junction. A genome scan was performed on five families from the Netherlands and Jordania, with 14 patients affected by microcephaly. A maximum LOD score of 4.78 was found for marker D1S1660 at the MCPH5 locus. Haplotype analysis suggests that the gene causing microcephaly is located between markers D1S3469 and D1S1660, which excludes the previously reported ASPM gene.     

Molecular characterization of ataxia telangiectasia T cell clones

Summary We compared inversions of chromosome 14 in an ataxia telangiectasia clone and in a malignant T cell line (SUPT1). The R-banding chromosome analysis showed a clear difference between the distal breakpoint of the two inversions. Fine mapping of the distal breakpoint in the ataxia telangiectasia inv(14) was performed by in situ hybridization. We conclude that this breakpoint is centromeric to the immunoglobulin heavy chain locus and to the D14S1 anonymous locus. Our results favor the existence of an unknown oncogene in band 14q32.1.     

Molecular cytogenetics of IGH rearrangements in non-Hodgkin B-cell lymphoma

Rearrangements involving the IGH gene have been identified in about 50% of non-Hodgkin B-cell lymphomas (NHLs) and correlated to clinically relevant subgroups. However, the detection rate largely varied with the technique used. We analyzed the incidence of IGH rearrangements using several fluorescence in situ hybridization (FISH) techniques on metaphases obtained from 96 patients with nodal NHL. An IGH rearrangement was identified in 71 cases (74%). A t(14;18)(q32;q21) was found in 37 of the 42 follicular lymphomas (88.1%) studied and a t(11;14)(q13;q32) in 12 of the 14 mantle cell lymphomas (85.7%). IGH rearrangements were identified in 21 of the 40 diffuse large B-cell lymphomas (52.5%), including seven t(14;18)(q32;q21) and four t(3;14)(q27;q32). Conventional cytogenetics was uninformative in several cases. However, the complemented analysis using 24-color FISH, chromosomal whole paints, telomeric probes and locus specific identifiers enabled us to characterize complex and/or masked IGH translocations in follicular lymphomas and mantle cell lymphomas and to identify all the chromosomal partners involved in IGH rearrangements in diffuse large B-cell lymphomas. This study shows the interest of using metaphase FISH in addition to conventional cytogenetics. Following banding techniques, FISH with the IGH dual color probe can be the first approach in NHL, after which chromosome painting and 24-color FISH can be used to identify the chromosomal partners involved in IGH rearrangements. The identification of these genes is of utmost importance for a better understanding of the molecular mechanisms involved in the genesis of lymphoma.     

Localization of the Krabbe disease gene (GALC) on chromosome 14 by multipoint linkage analysis.

The gene responsible for Krabbe disease, an autosomal recessive disorder caused by deficiency of galactocerebrosidase (GALC), was localized by multipoint linkage analysis on chromosome 14. Eight mapped dinucleotide repeat polymorphisms were tested for linkage to GALC. Two-point linkage analysis demonstrated close linkage of GALC and D14S48, with Z = 13.69 at theta = 0. Multipoint analysis yielded strong support for this finding, with maximum likelihood for GALC located within 1 cM of D14S48. This analysis also identified markers that clearly flank the GALC locus, as the map order of D14S53-GALC-D14S45 is favored by odds greater than 10(6):1. Additional support for close linkage of GALC and D14S48 comes from the apparent linkage disequilibrium between these two loci in a consanguineous Druze community in Israel. These data localize GALC to 14q24.3-q32.1.     

Deletion 14q(q24.3 to q32.1) syndrome: significance of peculiar facial appearance in its diagnosis,and deletion mapping of (α1-antitrypsin)

Summary A 10-month-old Japanese boy who had interstitial deletion of the long arm of chromosome No. 14; 46,XY, del(14)(pter»q24.3::q32.1»qter) is reported. A peculiar facial appearance, including round face, frontal hypertrichosis with thick eyebrows, horizontal narrow palpebral fissures, a short bulbous nose with a flat nasal root, and mild micrognathia, appeared to be common with the two previously reported cases. We stress the significance of this peculiar facial appearance in the diagnosis of 14q-(q24.3 to q32.1) syndrome. The level of ₁-antitrypsin in the patient was only about half of that of his parents and controls, and the Pi locus was tentatively assigned to band 14q32.1.     

The gene for spinal cerebellar ataxia 3 (SCA3) is located in a region of approximately 3 cM on chromosome 14q24.3-q32.2.

SCA3, the gene for spinal cerebellar ataxia 3, was recently mapped to a 15-cM interval between D14S67 and D14S81 on chromosome 14q, by linkage analysis in two families of French ancestry. The SCA3 candidate region has now been refined by linkage analysis with four new microsatellite markers (D14S256, D14S291, D14S280, and AFM343vf1) in the same two families, in which 19 additional individuals were genotyped, and in a third French family. Combined two-point linkage analyses show that the new markers, D14S280 and AFM343vf1, are tightly linked to the SCA3 locus, with maximal lod scores, at recombination fraction, (theta) = .00, of 7.05 and 13.70, respectively. Combined multipoint and recombinant haplotype analyses localize the SCA3 locus to a 3-cM interval flanked by D14S291 and D14S81. The same allele for D14S280 segregates with the disease locus in the three kindreds. This allele is frequent in the French population, however, and linkage disequilibrium is not clearly established. The SCA3 locus remains within the 29-cM region on 14q24.3-q32.2 containing the gene for the Machado-Joseph disease, which is clinically related to the phenotype determined by SCA3, but it cannot yet be concluded that both diseases result from alterations of the same gene.     

Identification of crossovers in Wilson disease families as reference points for a genetic localization of the gene

Summary Wilson disease (WD) is an autosomal recessive disorder of copper metabolism. A minimum recombinant analysis using D13S22, ESD, RB1, D13S31, D13S55, D13S26, D13S39, and D13S12, all localized at 13q14-q22, has been carried out in 20WD families of Northwest-European origin. No inconsistencies have been observed with respect to locus order or location of the WD locus (WND) compared with previous linkage studies. D13S31 was mapped as the closest marker proximal to WND, whereas D13S55 and D13S26 were mapped as the closest markers distal to WND. We have identified a crossover between WND and D13S31 in one family and a crossover between WND and D13S55 in another. These crossover sites can be used as reference points for new chromosome 13q14-q21 markers, and are therefore important for a more accurate mapping of the WD locus.     

Mapping of the Gene for Machado-Joseph Disease within a 3.6-cM Interval Flanked by D14S291/D14S280 and D14S81, on the Basis of Studies of Linkage and Linkage Disequilibrium in 24 Japanese Families

The gene locus of Machado-Joseph disease (MJD) has recently been mapped within a 29-cM subregion of 14q chromosome. We did a linkage study of 24 multigenerational MJD Japanese pedigrees, in an attempt to narrow the candidate region of this gene. Pairwise and multipoint linkage analysis, together with haplotype segregation analysis, led to the conclusion that the MJD gene is located at the 6.8-cM interval between D14S256 and D14S81 (Z_max = 24.78, multipoint linkage analysis). D14S291 and D14S280, located at the center of this interval, showed no obligate recombination with the MJD gene (Z_max = 5.93 for D14S291 and 9.99 for D14S280). A weak, but significant, linkage disequilibrium of MJD gene was noted with D14S81 (P < .05) but not with D14S291 or D14S280. These results suggest that a 3.6-cM interval flanked by D14S291/D14S280 and D14S81 is the most likely location of the MJD gene and that it is closest to D14S81.     

Structure of the catfish IGH locus: analysis of the region including the single functional IGHM gene

The catfish IGH locus is large (∼1 Mb) and complex, having undergone multiple internal duplications and transpositions. To define the structure of the locus that contains the single expressed IGHM gene, two overlapping bacterial-artificial-chromosome (BAC) clones spanning the most 3′ end of the channel catfish immunoglobulin heavy (IGH) chain locus have been completely sequenced. The analyses created a contig of 257,153 bp containing 55 VH, 6 D, 12 JH genes and the IGH constant region genes encoding the functional secreted and membrane forms of IgM and the membrane form of IgD. This analysis revealed three major features. First, no C-region genes were found aside from the previously described IGHM1 and IGHD1, with the latter gene being the most 3′ C-region gene of the catfish IGH locus. There was no evidence in the region sequenced for genes that could encode an Ig class similar to the IgZ/IgT described in zebrafish, trout and pufferfish. Second, there are a high number of VH pseudogenes, 28 out of 55 (51%). In contrast, the entire zebrafish IGH locus has 40 functional VH genes and eight pseudogenes (17%). Third, an internal duplication of a 52.4-kb block of VH genes has occurred. These observations suggest that the IGH locus of teleost fish varies significantly from species to species in the diversity of C-region genes as well as the numbers of genes encoding V regions.     

High-density genome scan in Crohn disease shows confirmed linkage to chromosome 14q11-12

Epidemiological studies have shown that genetic factors contribute to the pathogenesis of the idiopathic inflammatory bowel diseases (IBD), Crohn disease (CD) and ulcerative colitis (UC). Recent genome scans and replication studies have identified replicated linkage between CD and a locus on chromosome 16 (the IBD1 locus), replicated linkage between IBD (especially UC) and a locus on chromosome 12q (the IBD2 locus), and replicated linkage between IBD (especially CD) and a locus on chromosome 6p (the IBD3 locus). Since the estimated locus-specific lambdas values for the regions of replicated linkage do not account for the overall lambdas in CD, and since the published genome scans in IBD show at least nominal evidence for linkage to regions on all but two chromosomes, we performed an independent genome scan using 751 microsatellite loci in 127 CD-affected relative pairs from 62 families. Single-point nonparametric linkage analysis using the GENEHUNTER-PLUS program shows evidence for linkage to the adjacent D14S261 and D14S283 loci on chromosome 14q11-12 (LOD = 3.00 and 1.70, respectively), and the maximal multipoint LOD score is observed at D14S261 (LOD = 3.60). In the multipoint analysis, nominal evidence for linkage (P<.05) is observed near D2S117 (LOD = 1.25), near D3S3045 (LOD = 1.31), between D7S40 and D7S648 (LOD = 0.91), and near D18S61 (LOD = 1.15). Our finding of significant linkage to D14S261 and the finding of suggestive linkage to the same locus in an independent study (multipoint LOD = 2.8) satisfies criteria for confirmed linkage, so we propose that the region of interest on chromosome 14q11-12 should be designated the IBD4 locus.     

Localization of the restriction fragment length polymorphism D14S1 (pAW-101) to chromosome 14q32.1 leads to 32.2 by in situ hybridization

The restriction fragment length polymorphism D14S1 is delineated by the cloned, single-copy DNA fragment pAW-101. This cloned fragment can therefore serve as a useful marker for gene linkage studies, and the exact location on the gene map is of great interest. pAW-101 was 3H-labeled and hybridized in situ to normal, prometaphase chromosome preparations. Analysis of the grain distribution shows this fragment to be localized to the long arm of chromosome 14 at band q32. Using lymphoid cell lines with 8;14 reciprocal translocations (q24.1;q32.3) from patients with Burkitt lymphoma, we found that the DNA fragment hybridizes to the rearranged chromosome 14 proximal to the breakpoint. These results localize D14S1 to the region 14q32.1 leads to 32.2 This is consistent with localization of this fragment utilizing somatic cell hybrids and family studies.     

A locus for autosomal dominant accessory auricular anomaly maps to 14q11.2–q12

Accessory auricular anomaly is a small excrescence of skin that contains elastic cartilage on different regions of the helix and the face. Previous work has shown that the genetic trait of some patients with the isolated symptom of accessory auricular anomaly is autosomal dominant. To map the gene for autosomal dominant accessory auricular anomaly (ADAAA), we investigated a Chinese family with 11 affected individuals. We performed linkage analysis with microsatellite markers spanning the whole human-genome in the family. The inheritance pattern of the ADAAA family was autosomal dominant with complete penetrance. Two-point linkage analysis revealed significant maximum LOD scores of 4.20(D14S990 and D14S264, sita = 0) in the family. Haplotype construction and multipoint linkage analysis also confirmed the locus and defined the isolated ADAAA locus to a 9.84 cM interval between the markers D14S283 and D14S297. Our study assigned an isolated ADAAA locus to 14q11.2–q12. This is the first ADAAA locus reported to date.Y. Yang and J. Guo contribute to this work equally.     

Localization and genetic linkage of the human immunoglobulin heavy chain genes and the creatine kinase brain (CKB) gene: Identification of a hot spot for recombination

The immunoglobulin heavy chain (IGH) gene cluster and the gene coding for the brain form of the enzyme creatine kinase (CKB) have previously been localized to chromosome 14, at 14q32.3 and 14q32, respectively. Here we report more precise regional localization of these genes by dosage studies using DNA from a child hemizygous for the region from 14q32.32 to 14qter. CKB and IGH are present in a single dose in the proband. Dosage studies in a second patient with a similar but smaller deletion due to a ring chromosome 14 show that CKB is proximal to the IGH cluster. An EcoRI restriction site polymorphism was found with probes for the CKB gene. Linkage analysis of family data indicates that CKB is closely linked to IGH. Linkage analysis also revealed unusually high recombination (beta = 3.2%) between the C delta and C gamma 3 genes of the IGH constant region, which are only 60 kb apart. This finding, in combination with a previous observation of linkage equilibrium in the region, suggests that the C delta-C gamma 3 region contains a recombination hot spot.     

Localization of a DNA segment encompassing four tRNA genes to human chromosome 14q11 and its use as an anchor locus for linkage analysis

The chromosomal location of an 8.2-kb genomic fragment encompassing a cluster of four human tRNA genes has been determined by three complementary methods including Southern analysis of human/rodent somatic cell hybrids, in situ hybridization, and genetic linkage analysis. This tRNA cluster (TRP1, TRP2, and TRL1) is located near the T-cell receptor alpha (TCRA) locus at 14q11, and several RFLPs were detected at this site. These RFLPs and those at the TCRA and MYH7 (cardiac beta-MHC gene) loci have been used to type all informative members of the CEPH pedigrees. This has permitted ordering of these three gene loci and two anonymous probes (D14S26 and D14S25) in a 20-cM interval just below the centromere of chromosome 14. Based upon the chromosomal location and the polymorphisms at this site, one or more members of this gene cluster could serve as a useful anchor locus on chromosome 14.