Bj Wainwright

Patterns of polymorphism and linkage disequilibrium for cystic fibrosis.

Four polymorphic markers that map within 80 kb of an HTF island which is genetically very close to the cystic fibrosis locus have been identified. We have analyzed the linkage disequilibrium between each of these markers and the cystic fibrosis mutation in 89 families from four European countries, Denmark, Finland, Spain, and Great Britain. Strong linkage disequilibrium between three polymorphic sites and cystic fibrosis was observed. The markers on the J3.11 (D7S8) side of the HTF island show stronger disequilibrium than those on the met side. Linkage disequilibrium between markers and disease alters the probability that a person of a given haplotype is a carrier in some populations and helps to identify regions of a sequence that are most likely to contain the cystic fibrosis mutation.

Isolation of a human gene with protein sequence similarity to human and murine int-1 and the Drosophila segment polarity mutant wingless

An expressed gene sequence which was identified by the isolation of a methylation free CpG island from human chromosome 7 has been cloned from a human lung cDNA library. The deduced protein sequence contains 360 amino acids and has several features of a secreted protein; it is cysteine rich with a signal peptide sequence and two potential asn‐linked glycosylation sites. The protein sequence shows marked similarity with human and murine int‐1 and their Drosophila homolog wingless (Dint‐1). This human int‐1 related protein, int‐1 and Dint‐1 have diverse patterns of expression, but the inferred structural similarities suggest that some of the functional characteristics of these proteins may be shared.

Microdissection of and microcloning from the short arm of human chromosome 2.

A bank of cloned DNA sequences from the distal half of the short arm of human chromosome 2 was generated by using microdissection and microcloning techniques. DNA was purified from 106 chromosomal fragments, manually dissected from peripheral lymphocytes in metaphase, and cloned into the EcoRI site of lambda gt10. A total of 257 putative recombinants were recovered, of which 41% were found to contain human inserts. The mean insert size was 380 base pairs (median size, 83 base pairs), and fewer than 10% of the clones contained highly repetitive sequences. All single-copy sequences examined were shown to map to the short arm of chromosome 2 by using hybrid panels. This technique provides a rapid method of isolating probes specific to a human subchromosomal region to generate linked markers to genetic diseases for which the chromosomal location is known.

Haplotypes in cystic fibrosis patients with or without pancreatic insufficiency from four European populations.

We examined the allele and haplotype frequencies of five polymorphic DNA markers in 355 European cystic fibrosis (CF) patients (from Belgium, the German Democratic Republic, Greece, and Italy) who were divided into two groups according to whether they were or not taking supplementary pancreatic enzymes. The level of linkage disequilibrium between each polymorphism and the CF mutation varied among the different populations; there was no significant association between KM.19 and CF in the Greek population. The distributions of alleles and haplotypes derived from the polymorphisms revealed by probes KM.19 and XV.2c were always different in patients with or without pancreatic insufficiency (PI) in all the populations studied. In particular, among 32 patients without PI, only 9 (or 28%) were homozygous for the KM.19-XV.2c = 2-1 haplotype (which was present in 73% of all the CF chromosomes in our sample) compared to 162 of 252 patients (or 64%) with PI. These findings are consistent with the hypothesis that pancreatic insufficiency or sufficiency may be determined by different mutations at the CF locus.

Chromosome assignment and restriction fragment length polymorphism analysis of the anonymous DNA probe B79a at 7q22 (HMG8 assignment D7S13)

SummaryThe anonymous DNA fragment B79a, which had previously been located to chromosome 7cen-7q22, has been more accurately assigned to 7q22 by dosage analysis in a patient hemizygous for a deletion of that region. By examination of a panel of genomic DNAs from unrelated individuals, digested with a number of different restriction enzymes, we have identified two frequent RFLPs detected by B79a. This probe will be important in the analysis of cystic fibrosis which has been mapped to the region around 7q22.

Cystic fibrosis carrier detection using a linked gene probe.

Cloned DNA markers which are closely linked to the gene defect causing cystic fibrosis have recently been described. These markers are sufficiently informative for carrier detection in 80% of families where there is a living cystic fibrosis child and unaffected sibs. The tightly linked DNA marker pJ3.11 was used in this study to identify carriers in six families and exclude carrier status in two subjects. Risk calculations for recessive diseases using linked DNA probes may be complex, but useful information for counselling can be obtained in this way.

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.

Regional localization of three probes closely linked to the cystic fibrosis locus by deletion analysis

A cell line hemizygous for a deletion of the human chromosome region 7q22----q32 was used for fine mapping three probes closely linked to the cystic fibrosis locus. The three markers, J.3.11, 7c22, and met, were all found to be deleted from the region 7q22----q32. This finding, in combination with previously published mapping data, led to the assignment of J3.11 to 7q22.

A model system for the analysis of gene exclusion: cystic fibrosis and chromosome 19.

We have used multilocus analysis to exclude the cystic fibrosis locus from six polymorphic DNA markers covering most of chromosome 19. A substantial increase in the confidence for exclusion was obtained using the computer programme LINKAGE compared to analysis of pairwise lod scores. A structured approach to the analysis of linkage to autosomal recessive inherited diseases where the biochemical defect is not known is described.

Localisation of a sequence, 7C22, showing close linkage to the cystic fibrosis locus

The DNA sequence 7C22 is known to show close linkage to the cystic fibrosis locus on chromosome 7. We present a regional localisation for this sequence by in situ hybridisation to 7q31.1----q31.2.