Multipoint linkage analysis in X-linked ocular albinism of the Nettleship-Falls type

Summary The genes that encode the alpha 1 (VI) and alpha 2 (VI) collagen chains, designated COL6A1 and COL6A2, map to human chromosomal band 21q22.3. Using pulsed-field gel electrophoresis and somatic cell hybrids, we found that COL6A1 and COL6A2 form a gene cluster on the most distal part of chromosome 21. Furthermore, we detected several DNA polymorphisms (both restriction site and VNTRs) associated with these loci. These polymorphisms make the COL6A1 and COL6A2 genes among the most informative markers on human chromosome 21.     

Mapping of DNA markers linked to the cystic fibrosis locus on the long arm of chromosome 7.

We have used a panel of eight human/mouse somatic-cell hybrids, each containing various portions of human chromosome 7, and three patient cell lines with interstitial deletions on chromosome 7 for localization of six DNA markers linked to the cystic fibrosis locus. Our data suggest that D7S15 is located in the region 7 cen----q22, that MET is located in 7q22----31, and that D7S8 and 7C22 are located in q22----q32. The hybridization results for COL1A2 and TCRB are consistent with their previous assignment to 7q21----q22 and 7q32, respectively. Given the location of these six markers and their linkage relationships, it is probable that the cystic fibrosis locus is in either the distal region of band q22 or the proximal region of q31. Using the same set of cell lines, we have also examined the location of another chromosome 7 marker PGY1. The data show that PGY1 is located in the region 7cen----q22, a position very different from its previous assignment.     

Molecular Mapping of Uncharacteristically Small 5q Deletions in Two Patients with the 5q- Syndrome: Delineation of the Critical Region on 5q and Identification of a 5q- Breakpoint

Molecular mapping techniques have defined the region of gene loss in two patients with the 5q- syndrome and uncharacteristically small 5q deletions (5q31-q33). The allelic loss of 10 genes localized to 5q23-qter (centromere-CSF2-EGR1-FGFA-GRL-ADRB2-CSF1R-SPARC-GLUH1-NKSF1-FLT4-telomere) was investigated in peripheral blood cell fractions. Gene dosage experiments demonstrated that CSF2, EGR1, NKSF1, and FLT4 were retained on the 5q- chromosome in both patients and that FGFA was retained in one patient, thus placing these genes outside the critical region. GRL, ADRB2, CSF1R, SPARC, and GLUH1 were shown to be deleted in both patients. The proximal breakpoint is localized between EGR1 and FGFA in one patient and between FGFA and ADRB2 in the other, and the distal breakpoint is localized between GLUH1 and NKSF1 in both patients. Pulsed-field gel electrophoresis was used to map the 5q deletion breakpoints, and breakpoint-specific fragments were detected with FGFA in the granulocyte but not the lymphocyte fraction of one patient. This study has established the critical region of gene loss of the 5q- chromosome in the 5q- syndrome, giving the location for a putative tumor-suppressor gene in the 5.6-Mb region between FGFA and NKSF1.     

Mapping of the regulatory subunits RI beta and RII beta of cAMP-dependent protein kinase genes on human chromosome 7.

The genes encoding the regulatory subunits RI beta (locus PRKAR1B) and RII beta (locus PRKAR2B) of human cAMP-dependent protein kinase have been mapped in the basic CEPH (Centre d'Etude du Polymorphisme Humain) family panel of 40 families to chromosome 7p and 7q, respectively, using the enzymes HindIII and BanII recognizing the corresponding restriction fragment length polymorphisms (RFLPs). Previous data from the CEPH database and our present RFLP data were used to construct a six-point local framework map including PRKAR1B and a seven-point framework map including PRKAR2B. The analysis placed PRKAR1B as the most distal of the hitherto mapped 7p marker loci and resulted in an unequivocal order of pter-PRKAR1B-D7S21-D7S108-D7S17-D7S149- D7S62-cen, with a significantly higher rate of male than female recombination between PRKAR1B and D7S21. The 7q regulatory gene locus, PRKAR2B, could also be placed in an unambigous order with regard to the existing CEPH database 7q marker loci, the resulting order being cen-D7S371-(COL1A2,D7S79)-PRKAR2B-MET-D7S87++ +-TCRB-qter. Furthermore, in situ hybridization to metaphase chromosomes physically mapped PRKAR2B to band q22 on chromosome 7.     

Toward a long-range map of human chromosomal band 22q11

Human chromosome band 22q11 is involved in numerous chromosomal rearrangements. A long-range molecular map of this region would allow the more precise localization of the various breakpoints of these rearrangements. Toward this goal we have constructed a genomic DNA library that allows the isolation of DNA clones that are directly adjacent to NotI sites. NotI was chosen because it is a restriction enzyme that digests infrequently in the human genome. The genomic DNA used in this library was from a human/hamster hybrid cell line that has a chromosome 22 as the only visible human chromosome. Two clones were isolated and mapped to different regions of 22q11 using a somatic cell hybrid mapping panel. A long-range restriction map flanking the NotI site of each of these two clones was produced using NotI and other infrequently cutting enzymes. Both NotI sites analyzed were located in HTF islands, regions often associated with the 5' end of genes. Thus, the NotI map of 22q11 may also aid in the cloning of undiscovered genes, giving a starting point for the study of duplication/deficiency syndromes of the region.     

Macrorestriction mapping of COL4A1 and COL4A2 collagen genes on human chromosome 13q34

The genes for the alpha-1 and alpha-2 chains of type IV collagen (COL4A1 and COL4A2) map to the same chromosomal band (13q34) and have a high degree of nucleotide homology. We have used pulsed field gel electrophoresis and cloned COL4A1 and COL4A2 DNA fragments as molecular probes to construct a 1200-kb macrorestriction map which encompasses both genes. The two genes are located within a 340-kb region with the 3' end of COL4A2 and the 5' region of COL4A1 separated by at least 100 kb but not more than 160 kb. These genes, therefore, are two members of a gene cluster on chromosome 13q34.     

Refined linkage map of chromosome 7 in the region of the cystic fibrosis gene

The genetic map in the region of human chromosome 7 that harbors the gene for cystic fibrosis (CF) has been refined by multilocus linkage studies in an expanded database including a large set of normal families. Six loci known to be linked to CF were examined: MET, an oncogene; COL1A2, collagen; TCRB, T-cell-receptor beta polypeptide; and three arbitrary loci—D7S8, D7S13, and D7S16—defined by probes pJ3.11, pB79a, and p7C22, respectively. The gene order with greatest statistical support is COL1A2-D7S13-D7S16-MET-D7S8-TCRB. Linkage analysis in families segregating for CF suggested that the most likely location of the CF gene on this map is between MET and D7S8.     

A t(4;22) in a meningioma points to the localization of a putative tumor-suppressor gene.

Cytogenetic analysis of meningioma cells from one particular patient (MN32) displayed the stem-line karyo-type 45, XY, -1, 4p+, 22q-, 22q+, which thus had rearrangements of both chromosomes 22. The 22q+ marker appeared as a dicentric: 22 pter----q11::1p11----qter. The reciprocal product of this translocation has presumably been lost because it lacked a centromere. The 22q- chromosome also appeared to have lost sequences distal to band q11. We assumed that this marker could have been the result of a reciprocal translocation between chromosomes 4 and 22. To investigate the 4p+ and 22q- chromosomes in more detail, human-hamster somatic cell hybrids were constructed that segregated the 22q- and 4p+ chromosomes. Southern blot analysis with DNA from these hybrids showed that sequences from 22q were indeed translocated to 4p+ and that reciprocally sequences from 4p were translocated to 22q-, demonstrating a balanced t(4;22)(p16;q11). On the basis of these results we presume that in this tumor a tumor-suppressor gene is deleted in the case of the 22q+ marker and that the t(4;22) disrupts the second allele of this gene. The latter translocation was mapped between D22S1 and D22S15, a distance of 1 cM on the linkage map of this chromosome. The area in which we have located the translocation is within the region where the gene predisposing to neurofibromatosis 2 has been mapped.     

Refinement of human chromosome 7 map around the pro alpha 2(I)collagen gene by long-range restriction mapping.

The physical proximity of the closely linked pro alpha 2(1)collagen (COL1A2) and erythropoietin (EPO) genes and five loci with no known function was studied by long-range restriction mapping experiments using pulsed-field gel electrophoresis. COL1A2 and D7S64 were found to be within 100 kb of each other, providing a new informative marker for linkage studies with respect to COL1A2. D7S15 and D7S79 were within 350 kb of each other. The physical distance between COL1A2 and EPO was determined to be at least 600 kb. Two CpG rich islands were recognized within 600 kb of COL1A2, suggesting that other genes might lie in the vicinity of COL1A2.     

Molecular cytogenetic assignment of genes to bovine chromosome 5

Seven genes were assigned by molecular cytogenetic methods to bovine chromosome 5. To accomplish this, specific primers were either publicly available or were designed from highly conserved regions of the publicly available mammalian gene sequences. The identity of the amplified segments was verified by sequencing and alignment with the published sequences. The optimized primers that amplified the desired bovine genes were used for screening a bovine bacterial artificial chromosome library. The positive clones were localized to a specific band of bovine chromosome 5 by fluorescence in situ hybridization. The genes HOXC4, SP1 and IGFBP6 were localized to band q21, COL2A1 was localized to bands q21-q23, IGF1 was localized to band q26, MB to band q31 and the gene CYP2D6 was localized to band q35. The cytogenetic assignment of SP1, IGFBP6, COL2A1, IGF1, MB and CYP2D6 is first reported here and the assignment of HOXC4 refines the previous assignment of this gene. The identification and localization of these genes further support the development of the human to bovine comparative map through characterizing the homologous segments conserved in the evolution of these species. This information will be useful for the future localization of genes that affect economically important traits in bovines.     

Molecular characterization of patients with 18q23 deletions.

The 18q- syndrome is a deletion syndrome that is characterized by mental retardation, hearing loss, midfacial hypoplasia, growth deficiency, and limb anomalies. Most patients with this syndrome have deletions from 18q21-qter. We report on three patients with deletions of 18q23. A mother and daughter with identical deletions of 18q23 have many of the typical features of the 18q- syndrome, including midfacial hypoplasia and hearing loss. In contrast, the third patient has few of the symptoms of the 18q- syndrome. A contig of the 18q23 region was generated to aid in the mapping of the breakpoints. FISH was used to map both breakpoints to the same YAC clone. Furthermore, somatic-cell hybrids from the daughter and the third patient were isolated. The mapping results of sequence-tagged sites relative to the two breakpoints were identical, suggesting that the two deletion breakpoints map very close to one another. The analyses of these patients demonstrate that the critical region for the 18q- syndrome maps to 18q23 but that a deletion of 18q23 does not always lead to the clinical features associated with the syndrome. These patients demonstrate the wide phenotypic variability associated with deletions of 18q.     

No significant effect of monosomy for distal 21q22.3 on the Down syndrome phenotype in “Mirror” duplications of chromosome 21

Three Down syndrome patients for whom karyotypic analysis showed a "mirror" (reverse tandem) duplication of chromosome 21 were studied by phenotypic, cytogenetic, and molecular methods. On high-resolution R-banding analysis performed in two cases, the size of the fusion 21q22.3 band was apparently less than twice the size of the normal 21q22.3, suggesting a partial deletion of distal 21q. The evaluation of eight chromosome 21 single-copy sequences of the 21q22 region--namely, SOD1, D21S15, D21S42, CRYA1, PFKL, CD18, COL6A1, and S100B--by a slot blot method showed in all three cases a partial deletion of 21q22.3 and partial monosomy. The translocation breakpoints were different in each patient, and in two cases the rearranged chromosome was found to be asymmetrical. The molecular definition of the monosomy 21 in each patient was, respectively, COL6A1-S100B, CD18-S100B, and PFKL-S100B. DNA polymorphism analysis indicated in all cases a homozygosity of the duplicated material. The duplicated region was maternal in two patients and paternal in one patient. These data suggest that the reverse tandem chromosomes did not result from a telomeric fusion between chromosomes 21 but from a translocation between sister chromatids. The phenotypes of these patients did not differ significantly from that of individuals with full trisomy 21, except in one case with large ears with an unfolded helix. The fact that monosomy of distal 21q22.3 in these patients resulted in a phenotype very similar to Down syndrome suggests that the duplication of the genes located in this part of chromosome 21 is not necessary for the pathogenesis of the Down syndrome features observed in these patients, including most of the facial and hand features, muscular hypotonia, cardiopathy of the Fallot tetralogy type, and part of the mental retardation.     

Genetic distance of two fibrillar collagen loci, COL3A1 and COL5A2, located on the long arm of human chromosome 2

Two of the human fibrillar collagen genes, proa1(III) (COL3A1) and proa2(V) (COL5A2), map to the same region of the long arm of chromosome 2. To establish the genetic distance between the two loci, we analyzed the segregation of COL3A1 and COL5A2 RFLPs in five families informative for the two loci specific markers. We found that the maximum lod score was 9.33 at a recombination frequency of theta = 0.00. The data therefore provide strong evidence for tight linkage between two evolutionarily related fibrillar collagen genes on the 2q14----2q32 segment of chromosome 2.     

The generation of DNA probes to chromosome 11q23 by Alu PCR on small numbers of flow-sorted 22q- derivative chromosomes

A strategy for the isolation of DNA probes from small numbers of flow-sorted human chromosomes has been developed. A lymphoblastoid cell line carrying the 22q- derivative chromosome product of the constitutional t(11;22) translocation was used as the source of chromosomes. Synthetic oligonucleotide primers, based on the consensus Alu sequence, were used to amplify inter-Alu sequence from 500 flow-sorted 22q- derivative chromosomes. The amplified sequences were cloned into a plasmid vector by blunt-end ligation, yielding clones with inserts in the range of 400 to 1000 bp. Approximately 70% of these clones hybridized to human DNA as single-copy probes. To identify clones derived from chromosome 11, the library was screened with a heterogeneous probe prepared by Alu-PCR amplification from the DNA of a somatic cell hybrid containing one homology of chromosome 11. All the positive clones found were mapped to within the q23-q25 region of chromosome 11 known to be translocated onto the 22q- derivative chromosome. Further mapping studies showed that most of these probes (7/8) lay between the breakpoints for the t(4;11) translocation of acute lymphocytic leukemia and the t(11;22) of Ewing sarcoma. Thus, the use of Alu-PCR on the small derivative chromosome 22q- has provided a greatly enriched source of probes to region 11q23, a part of the genome that is currently of great interest. This approach will be particularly appropriate to small numbers of chromosomes when high specificity rather than total representation is required.     

Fine structure DNA mapping studies of the chromosomal region harboring the genetic defect in neurofibromatosis type 1

To better map the location of the von Recklinghausen neurofibromatosis (NF1) gene, we have characterized a somatic cell hybrid designated 7AE-11. This microcell-mediated, chromosome-transfer construct harbors a centromeric segment and a neo-marked segment from the distal long arm of human chromosome 17. We have identified 269 cosmid clones with human sequences from a 7AE-11 library and, using a panel of somatic cell hybrids with a total of six chromosome 17q breakpoints, have mapped 240 of these clones on chromosome 17q. The panel included a hybrid (NF13) carrying a der(22) chromosome that was isolated from an NF1 patient with a balanced translocation, t(17;22) (q11.2;q11.2). Fifty-three of the cosmids map into a region spanning the NF13 breakpoint, as defined by the two closest flanking breakpoints (17q11.2 and 17q11.2-q12). RFLP clones from a subset of these cosmids have been mapped by linkage analysis in normal reference families, to localize the NF1 gene more precisely and to enhance the potential for genetic diagnosis of this disorder. The cosmids in the NF1 region will be an important resource for testing DNA blots of large-fragment restriction-enzyme digests from NF1 patient cell lines, to detect rearrangements in patients' DNA and to identify the 17;22 NF1 translocation breakpoint.     

11q- syndrome: three cases and a review of the literature.

We report on three children with de novo terminal deletions of the long arm of chromosome 11 (11q-) and breakpoints in 11q23-q24. Eighty-nine other patients with partial monosomy 11q have been reported and were reviewed by us. Salient features of 11q- syndrome are psychomotor retardation, trigonocephaly, telecanthus/hypertelorism, broad depressed nasal bridge, micrognathia, low set abnormal ears, cardiac anomalies and hand/foot anomalies. Renal agenesis and anal atresia are reported first here. Supratentorial white matter abnormality on CT and MRI present in our second patient was reported in three patients. Increased mortality is caused by cardiac anomalies. A third of all patients with partial monosomy 11q had thrombocytopenia or pancytopenia and this seems to be related to the absence of band 11q23-q24. Seventy-six percent of patients have de novo deletions with breakpoints in 11q21-q25. There is no obvious correlation between the length of the deleted segment and the severity of the symptoms. In unbalanced chromosomal patterns with deletions of 11q involving bands 11q23-q24 the typical phenotype of 11q- syndrome remains recognizable. Deletions distal to 11q24.1 do not produce the typical 11q- syndrome.     

A 2-Mb YAC Contig and Physical Map Covering the Chromosome 8q12 Breakpoint Cluster Region in Pleomorphic Adenomas of the Salivary Glands

Pleomorphic adenomas are benign epithelial tumors originating from the major and minor salivary glands. Extensive cytogenetic studies have demonstrated that they frequently show chromosome abnormalities involving chromosome 8, with consistent breakpoints at 8q12. In previous studies, we have shown that these breakpoints are located in a 9-cM interval betweenMOS/D8S285 and D8S260. Here, we describe directional chromosome walking studies starting from D8S260 as well as D8S285. Using the CEPH and ICRF YAC libraries, these studies resulted in the construction of two nonoverlapping YAC contigs of about 2 and 5 Mb, respectively. Initial fluorescencein situhybridization (FISH) analysis suggested that the majority of 8q12 breakpoints clustered within the 2-Mb contig, which was mapped to the centromeric part of chromosome band 8q12. This contig has at least double coverage and consists of 34 overlapping YAC clones. The localization of the YACs was confirmed by FISH analysis. On the basis of mapping data of landmarks with an average spacing of 65 kb as well as restriction enzyme analysis, a long-range physical map was established for the chromosome region spanned by the 2-Mb contig. The relative positions of various known genes and expressed sequence tags within this contig were also determined. Subsequent FISH analyses of pleomorphic adenomas using YACs as well as cosmids revealed that all but two of the 8q12 breakpoints in the primary tumors tested mapped within a 300-kb interval between theMOSproto-oncogene and STS EM156. The target gene affected by the chromosome aberrations mapping within this interval was recently shown to be thePLAG1gene, which encodes a novel zinc finger protein.