Isolation of additional polymorphic clones from the cystic fibrosis region, using chromosome jumping from D7S8.

The cystic fibrosis (CF) locus has been located, by both linkage analysis and physical mapping, to a 900-kb region of 7q22-31 flanked by D7S8 (J3.11) and D7S23 (XV-2c). Using a 100-kb general jumping library, we isolated two sequential jump clones, J31 and J29, to one side of the D7S8 region and one jump clone, J32, to the other side of D7S8, so that the total region covered is about 300 kb. Three new RFLPs were detected by J29 and J32. Using PFGE mapping and the three jump clones, we found it possible to orient D7S8 on the chromosome and, by linkage analysis, to further narrow the CF region by 100 kb. The orientation of D7S8 will be useful for directing the isolation of other jump clones toward the CF locus. Though the newly described RFLPs are in considerable linkage disequilibrium with D7S8 polymorphisms, they increase the informativeness of genetic markers in the D7S8 region and should be useful in prenatal diagnosis.     

Physical localization of two DNA markers closely linked to the cystic fibrosis locus by pulsed-field gel electrophoresis.

Our previous linkage analysis suggested that the DNA segment D7S122 is located between MET and D7S8, the two genetic markers that are thought to flank the cystic fibrosis locus (CF). Subsequent chromosome walking experiments revealed that D7S122 in within close distance to another randomly isolated DNA marker, D7S340. To determine the physical relationship among D7S122, D7S340, MET, and D7S8, we have constructed a long-range restriction map of the region containing these four DNA segments, by using DNA from a human/hamster somatic hybrid cell line 4AF-KO15 (containing a single human chromosome 7) and a series of rare-cutting restriction enzymes. The combined results of complete, partial, and double digestion analyses confirm that D7S122 and D7S340 are located between MET and D7S8. The order of these markers is MET-D7S340-D7S122-D7S8, with distance intervals of approximately 500, 10, and 980 kbp, respectively. Together with family analysis, this information will be useful for eventual identification of the CF gene.     

Regional assignment of 41 human DNA fragments on chromosome 7 by means of a somatic cell hybrid panel

Summary To detect new restriction fragment length polymorphisms that would cover human chromosome 7 with a network of genetic landmarks, a chromosome 7-specific phage gene library was screened for human single-copy fragments. With use of a somatic cell hybrid panel containing defined regions of human chromosome 7, 41 cloned human single-copy sequences were assigned to five regions of this chromosome. Of special importance are the cell hybrid clones GM1059Rag5 and 7851Rag10-1, derived from human cells with interstitial deletions spanning the bands 7q22-q32, within which the cystic fibrosis gene is located. Twelve new probes are described in 7q22-q32, five of which detect a total of six RFLPs.     

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.     

Characterization and regional mapping of new anonymous chromosome 20-specific DNA markers isolated from a flow-sorted DNA library

Employing the flow-sorted chromosome 20-specific DNA library LL20NS01, we isolated seven novel unique poly- and monomorphic DNA markers specific to human chromosome 20. Initially, 201 phage clones were analyzed regarding insert size and repetitivity. By testing 14 single- and low-copy number clones for their ability to detect RFLPs, three polymorphisms were revealed by two probes, pFMS22-1.4 [D20S22] and pFMS76 [D20S23]. Seven of twenty probes (35%) were assigned to chromosome 20 using a somatic cell hybrid DNA panel. Five of them were regionally mapped by in situ hybridization. Three DNA markers, pFMS51 [D20S29], pFMS76 [D20S23], and pFMS106 [D20S30], were assigned to 20p11.2-p12, and two markers, pFMS22-1.4 [D20S22] and pFMS135 [D20S31], to 20q12-q13.3. Our new chromosome 20-specific DNA markers should be useful for the molecular characterization of this rather underpopulated human chromosome.     

New markers for the neurofibromatosis-2 region generated by microdissection of chromosome 22

To identify new DNA markers around the neurofibromatosis-2 gene on human chromosome 22, the critical region (22q12-q13.1) was microdissected and microcloned from GTG-banded metaphase chromosomes. Eighteen thousand recombinant clones were obtained. Twenty-seven of 55 clones tested (50%) detected single-copy DNA sequences. Nine of nine clones analyzed in detail were found to map to chromosome 22. Interestingly one clone (EAN04) is part of the leukemia inhibitory factor gene which has previously been mapped to 22q11.2-q13.1. Four clones (EAN01, EAN47, EAN57, and EAN68) detect DNA polymorphisms. These probes were used to compare constitutional and tumor genotypes of 41 patients with acoustic neurinoma. Loss of constitutional heterozygosity was identified in 17 of 31 informative cases (55%). From our data we conclude that the microdissection library is a valuable resource for physical and genetic mapping studies in neurofibromatosis-2.     

Owl monkey gene map: evidence for a homologous human chromosome 7q region near the cystic fibrosis locus

We have demonstrated the assignments of two gene loci (COLIA2, MET) and two noncoding DNA markers (D7S13, D7S8) to owl monkey chromosome 14 (K-VI) by hybridizing DNA probes from the cystic fibrosis (CF) region of human chromosome 7q21-32 to panels of rodent-owl monkey somatic cell hybrids. The assignments are substantiated by in situ chromosome hybridization of markers COLIA2, MET, and D7S13 to the distal long arm of chromosome 14 (K-VI). These results support genomic conservation of the human CF region, at least in the higher primates.     

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.     

Localization of DNA probes with tight linkage to the cystic fibrosis locus by in situ hybridization using fibroblasts with a 7q22 deletion

Summary Five DNA probes known to originate from the region 7q22-q31 were sublocalized by in situ hybridization to metaphase preparations of fibroblasts having besides a normal chromosome 7, a homologue 7 with an apparent interstitial deletion of a large part of band q22. A flow cytometric chromosome analysis confirmed a loss of material from one of the homologues of chromosome 7. Four of the probes, B79a, 7C22,metH, and pJ3.11, have been shown to be closely linked to the cystic fibrosis (CF) locus. We localized probes B79a and 7C22 to the part of 7q22 involved in the deletion, whereasmetH and pJ3.11 could be assigned to band 7q31. Probe pJu28, for which polymorphisms have not yet been described, also appeared to derive from the latter band. Since pJ3.11 andmetH are most tightly linked to the CF locus, this disease locus is indirectly assigned to 7q31. A comparison of our findings with linkage data suggests a discrepancy between genetic and physical distances in the region 7q22-q31.     

Identification of twelve new RFLP-markers on chromosome 22q11-qter

Summary We have previously identified and regionally localized 195 chromosome-22-specific DNA markers. We now report restriction fragment length polymorphisms detected by 9 phage markers mapped to 22q11-q12, two cosmid clones mapped to 22q12-q13 and one plasmid mapped to 22q13-qter. These markers may be useful tools for mapping disease genes such as the NF2 locus, on chromosome 22.     

Identification of sequences of chromosome 7 that are expressed in sweat gland epithelial cells

Summary This paper describes an approach that can be used to identify specifically expressed coding sequences in defined regions of genomic DNA. We developed this method to identify expressed sequences from chromosome 7 located at or near the cystic fibrosis (CF) locus. Radioactively labelled single-stranded cDNAs derived from sweat gland epithelial cells and from fibroblasts were used to screen a genomic library constructed from flow-sorted chromosomes. Differential screening of phage lifts with these two probes yielded 36 different DNA segments. By using somatic cell hybrids containing different portions of chromosome 7, four of the clones were mapped to the 7q31 region in which the CF locus is located. These four clones and two others that gave strong differential epithelial signals but that were not within 7q31 were studied further. Restriction fragment length polymorphisms (RFLPs) were identified for two of the DNA segments within 7q31 and used for linkage analysis using a panel of CF families. One DNA segment was assigned to a location centromeric to the met locus. The other marker did not show recombination with CF but was subsequently excluded from the CF region by physical mapping. Three of the six DNA segments were found to hybridize to various RNAs using the Northern technique and therefore contain portions of genes. One of the clones showed strong differential expression when epithelial tissues were compared to fibroblasts and may represent an epithelium-specific gene.     

Fine mapping of the Autosomal Dominant Split Hand/Split Foot Locus on Chromosome 7, Band q21.3-q22.1

Split hand/split foot (SHFD) is a human developmental defect characterized by missing digits, fusion of remaining digits, and a deep median cleft in the hands and feet. Cytogenetic studies of deletions and translocations associated with this disorder have indicated that an autosomal dominant split hand/split foot locus (gene SHFD1) maps to 7q21-q22. To characterize the SHFD1 locus, somatic cell hybrid lines were constructed from cytogenetically abnormal individuals with SHFD. Molecular analysis resulted in the localization of 93 DNA markers to one of 10 intervals surrounding the SHFD1 locus. The translocation breakpoints in four SHFD patients were encompassed by the smallest region of overlap among the SHFD-associated deletions. The order of DNA markers in the SHFD1 critical region has been defined as PON–D7S812–SHFD1–D7S811–ASNS. One DNA marker, D7S811, detected altered restriction enzyme fragments in three patients with translocations when examined by pulsed-field gel electro-phoresis (PFGE). These data map SHFD1, a gene that is crucial for human limb differentiation, to a small interval in the q21.3-q22.1 region of human chromosome 7.     

A new polymorphic locus, D7S411, isolated by cloning from preparative pulse-field gels is close to the mutation causing cystic fibrosis

The mutation causing cystic fibrosis (CF) has been localized to the DNA sequence of 700 kb bounded by the loci identified by the markers pMP6d-9 (D7S399) and pJ3.11 (D7S8). A 560-kb fragment obtained after SacII digestion of DNA from a cell line containing this region of human chromosome 7 in a mouse background was separated using pulse-field gel electrophoresis and isolated from the gel. The DNA was digested with BamHI prior to cloning into lambda EMBL3. Approximately 0.1% of the resulting clones contained human repetitive sequences, and 24 such recombinants were studied. Of these, 23 are on chromosome 7; 8 clones were duplicated, and of the 15 different recombinants, 7 are between MET and INT1L1, and a further 7 are between INT1L1 and pMP6d-9, leaving a single marker, pG2, which is between pMP6d-9 and pJ3.11. pG2 recognizes an RFLP with XbaI. A cosmid walk from pG2 has generated a further marker, H80, which recognizes an RFLP with PstI. This new locus (D7S411) divides the remaining region between the CF flanking markers, thereby making it more accessible to fine pulse-field mapping and allowing the precise localization of further clones to this region. Although it is not possible to position the CF locus unequivocally with respect to D7S411, both polymorphic markers at this locus exhibit low but significant linkage disequilibrium with CF, placing the emphasis for the search for the gene on the D7S399 to D7S411 interval of 250 kb.     

High-resolution genetic map around the spinal muscular atrophy (SMA) locus on chromosome 5.

Although autosomal recessive spinal muscular atrophy (SMA) has been mapped to chromosome 5q12-q13, there is for this region no genetic map based on highly informative markers. In this study we present the mapping of two previously reported microsatellite markers in 40 CEPH and 31 SMA pedigrees. We also describe the isolation of a new microsatellite marker at the D5S112 locus. The most likely order of markers (with recombination fractions given in parentheses) is 5cen-D5S6-(.02)-D5S125-(.04)-(JK53CA1/2,D5S11 2)-(.04)-D5S39-qter. The relative order of D5S6, D5S112, and D5S39 was confirmed by in situ hybridization. Multipoint linkage analysis in 31 SMA families indicates that the SMA locus lies in the 6-cM interval between D5S6 and JK53CA1/2, D5S112.     

A Common Region of Deletion on Chromosome 17q in Both Sporadic and Familial Epithelial Ovarian Tumors Distal to BRCA1

Linkage analysis in familial breast and ovarian cancer and studies of allelic deletion in sporadic ovarian tumors have identified a region on chromosome 17q containing a candidate tumor-suppressor gene (referred to as BRCA1) of likely importance in ovarian carcinogenesis. We have examined normal and tumor DNA samples from 32 patients with sporadic and 8 patients with familial forms of the disease, for loss of heterozygosity (LOH) at 21 loci on chromosome 17 (7 on 17p and 14 on 17q). LOH on 17p was 55% (22/40) for informative 17pl3.1 and 17pl3.3 markers. When six polymorphic markers flanking the familial breast/ovarian cancer susceptibility locus on 17ql2-q21 were used, LOH was 58% (23/40), with one tumor showing telomeric retention. Evaluation of a set of markers positioned telomeric to BRCA1 resulted in the highest degree of LOH, 73% (29/40), indicating that a candidate locus involved in ovarian cancer may reside distal to BRCA1. Five of the tumors demonstrating allelic loss for 17q markers were from individuals with a strong family history of breast and ovarian cancer. More important, two of these tumors (unique patient number [UPN] 57 and UPN 79) retained heterozygosity for all informative markers spanning the BRCA1 locus but showed LOH at loci distal to but not including the anonymous markers CMM86 (D17S74) and 42D6 (D17S588), respectively. Deletion mapping of seven cases (two familial and five sporadic) showing limited LOH on 17q revealed a common region of deletion, distal to GH and proximal to D17S4, that spans −25 cM. These results suggest that a potential tumor-suppressor gene involved in both sporadic and familial ovarian cancer may reside on the distal portion of chromosome 17q and is distinct from the BRCA1 gene.     

A sequence-ready 840-kb PAC contig spanning the candidate tumor suppressor locus DBC1 on human chromosome 9q32-q33.

A putative tumor suppressor locus involved in bladder cancer has been mapped to human chromosome 9q32-q33 and designated DBC1. Our previous microsatellite-based deletion mapping study indicated that DBC1 was localized between D9S1848 and AFMA239XA9. We have constructed an 840-kb sequence-ready contig composed of bacteriophage P1-derived artificial chromosomes (PACs), which encompasses DBC1. Clones were initially identified by screening a PAC library with markers localized to the region by physical mapping, and subsequently PAC end probes were used to complete the contig. This contig contains a minimum tiling path of six PAC clones between D9S1848 and AFMA239XA9. Three expressed sequence tags (ESTs) were mapped to the DBC1 region by screening 24 ESTs mapped to the surrounding area by radiation hybrids. One represented the gene for DBCCR1, a known candidate for DBC1, and the other two were novel. This contig and preliminary expression map form the basis for the identification of the bladder cancer tumor suppressor gene in this region.     

Linkage of DNA probe B79a (D7S13) to cystic fibrosis

We have conducted, in 64 affected families, a study of linkage between the anonymous DNA segment pB79a (D7S13) and the locus for cystic fibrosis (CF) on chromosome 7q. The maximum lod score was 12.60 at theta = .08 (confidence bounds .045-.135). Although D7S13 is not sufficiently close to CF for routine use in DNA-based prenatal diagnosis, it will be helpful in certain families when other nearby markers are uninformative. D7S13 will also be useful for refining the linkage map of the CF region.     

Isolation of chromosome-21-specific DNA probes and their use in the analysis of nondisjunction in Down syndrome

Summary Thirteen single-copy, chromosome-21-specific DNA probes were isolated from a recombinant library made from flow-sorted chromosome 21 DNA and regionally mapped using a panel of somatic cell hybrids. Five probes mapped in the 21q21-q22.1 region, six to the 21q22.1-qter region, and one to each of the regions 21q22.1-q22.2 and 21q22.3. Two of these probes, one of which maps in the critical region for Down syndrome, have recently been shown to be expressed at high levels in Down syndrome brain tissue (Stefani et al. 1988). Following preliminary screening for restriction fragment lenght polymorphisms (RFLPs), five polymorphisms were discovered with four of the chromosome 21 DNA probes. A frequent MspI polymorphism detected by one of the probes was used in conjunction with four previously described polymorphic chromosome 21 probes to analyse the origin of nondisjunction in 33 families with a child or fetus with trisomy 21. The parental origin of the additional chromosome 21 was determined in 12 cases: in 9 (75%) of these it was derived from the mother and in the other 3 cases (25%) it was of paternal origin. Cytogenetic analysis of Q-banding heteromorphisms was informative in three of five families tested, and in each case the RFLP results were confirmed. The meiotic stage of nondisjunction was defined with confidence in five families, the results being obtained with pericentromeric RFLP or cytogenetic markers. Recombination between two nondisjoined chromosomes was demonstrated in one family and is consistent with the view that a lack of recombination between chromosome 21 homologues or failure of their conjunction is not the invariable cause of trisomy 21.