Linkage disequilibrium between the fragile X mutation and two closely linked CA repeats suggests that fragile X chromosomes are derived from a small number of founder chromosomes.

In order to investigate the origin of mutations responsible for the fragile X syndrome, two polymorphic CA repeats, one at 10 kb (FRAXAC2) and the other at 150 kb (DXS548) from the mutation target, were analyzed in normal and fragile X chromosomes. Contrary to observations made in myotonic dystrophy, fragile X mutations were not strongly associated with a single allele at the marker loci. However, significant differences in allelic and haplotypic distributions were observed between normal and fragile X chromosomes, indicating that a limited number of primary events may have been at the origin of most present-day fragile X chromosomes in Caucasian populations. We propose a putative scheme with six founder chromosomes from which most of the observed fragile X-linked haplotypes can be derived directly or by a single event at one of the marker loci, either a change of one repeat unit or a recombination between DXS548 and the mutation target. Such founder chromosomes may have carried a number of CGG repeats in an upper-normal range, from which recurrent multistep expansion mutations have arisen.

Identification and regional localization of DNA markers on chromosome 7 for the cloning of the cystic fibrosis gene.

To facilitate mapping of the cystic fibrosis locus (CF) and to isolate the corresponding gene, we have screened a flow-sorted chromosome 7-specific library for additional DNA markers in the 7q31-q32 region. Unique ("single-copy") DNA segments were selected from the library and used in hybridization analysis with a panel of somatic cell hybrids containing various portions of human chromosome 7 and patient cell lines with deletion of this chromosome. A total of 258 chromosome 7-specific single-copy DNA segments were identified, and most of them localized to subregions. Fifty three of these corresponded to DNA sequences in the 7q31-q32 region. Family and physical mapping studies showed that two of the DNA markers, D7S122 and D7S340, are in close linkage with CF. The data also showed that D7S122 and D7S340 map between MET and D7S8, the two genetic markers known to be on opposite sides of CF. The study thus reaffirms the general strategy in approaching a disease locus on the basis of chromosome location.

Progress towards cloning the cystic fibrosis gene.

Genetic linkage analysis with polymorphic DNA markers (restriction fragment length polymorphisms: RFLPS) has allowed the assignment of the cystic fibrosis (CF) locus to the long arm of chromosome 7, within the region of band q31. Two of these markers, MET and D7S8, are tightly linked to the disease locus. Although recent data suggest that they are located on opposite sides of CF, the two can be separated by as much as 5 centimorgans. To obtain a better description of the CF locus and, eventually, to identify the affected gene, additional DNA markers are required to connect MET and D7S8, physically. We have screened the flow-sorted chromosome-7-specific library and thus far isolated 28 new probes from the 7q31 region by DNA hybridization analysis that uses a series of somatic cell hybrids containing various portions of human chromosome 7. Together with the previously identified markers, MET, D7S8, D7S13 and D7S16, these new markers should provide a fine genetic and physical map for the chromosomal region surrounding CF. DNA segments can then be sequentially cloned by chromosome walking from points closest to the CF locus and examined for genes that are preferentially expressed in tissues known to be affected in the disease.

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.

Microdissection and microcloning of the long arm of human chromosome 7.

DNA-fragments from the region of the long arm of human chromosome 7 to which the CF-locus has been mapped recently were isolated by microdissection and microcloning. We developed a new fixation procedure resulting in inserts of 1.0-7.0 kb in length with a mean value of 2.9 kb. Regional mapping of three clones on 7q was carried out by the use of different hybrid cell lines containing fragments of human chromosome 7.

Linkage of cystic fibrosis to the pro alpha 2(I) collagen gene, COL1A2, on chromosome 7.

A linkage has been detected between the locus for cystic fibrosis (CF) and the pro alpha 2(I) collagen gene (COL1A2) which is located in the region q21.3----q22.1 of chromosome 7. Based on the combined linkage data derived from 50 informative two-generation nuclear families collected in Canada and Denmark, the distance between COL1A2 and CF is estimated to be 19 centiMorgans. Close linkage has also been detected between COL1A2 and the DNA marker D7S15 (formerly D0CRI-917) and the serum enzyme activity marker paraoxonase (PON), both of which have previously been found linked to CF. The results of the two-point and three-point linkage analyses indicate that the most probable order of these four genetic loci is COL1A2-D7S15 - PON - CF.