Genetic homogeneity of cystic fibrosis.

We studied large Amish/Mennonite/Hutterite kindreds that segregate cystic fibrosis (CF) for linkage between CF and the polymorphic DNA markers pJ3.11 and 7C22 located on chromosome 7. These inbred pedigrees consist of more than 300 members including 30 affected individuals. In these families, linkage between the CF locus and the chromosome 21 marker D21S5 and between CF and the marker at the met oncogene locus on chromosome 7 had been previously indicated. We now report linkage between CF and pJ3.11 (Z = 4.92, theta = 0) and between CF and 7C22 (Z = 3.42, theta = 0). Therefore, CF segregates in these large pedigrees in a manner consistent with data from smaller outbred families with respect to the markers on chromosome 7 closest to CF. These data are consistent with locus homogeneity for the defect causing CF in the populations that have been examined to date.     

Genetic analysis of cystic fibrosis: linkage of DNA and classical markers in multiplex families

Linkage of cystic fibrosis (CF) to DNA and classical markers was studied in 36 families of two or three generations with at least two living affected children. Among the 79 affected children, no recombinants were detected between the disease and the markers MET and pJ3.11, previously shown to be linked to CF. No linkage between the human trypsin gene family (which appears to include at least 10 members) and CF was found, although not all genes of the trypsin family have been screened yet. In one of the CF families, recombination between MET and pJ3.11 was detected in an unaffected sib. Data from our families suggest that the gene order of markers among chromosome 7q is: (7cen;p8.33)collagen(COL1A2);DOCR1-917;paraoxonase+ ++(PON);(MET-cf-J3.11);T-cell receptor beta chain (TCRB);qter. There was no evidence for (or against) either postzygotic selection or meiotic drive to explain the high frequency of CF in Caucasian populations.     

A linkage study of cystic fibrosis in extended multigenerational pedigrees.

The linkage of polymorphic DNA markers on chromosome 7 to cystic fibrosis (CF) was examined in two pedigrees and a number of smaller nuclear families. The pedigrees are multigenerational and together consist of more than 300 members including 30 affected individuals, while the nuclear families each have two generations and either two or three children with CF. Tight linkage was observed between the CF locus and the met oncogene locus theta = 0, zeta = 15.45), pJ3.11 (theta = 0, zeta = 10.07), and 7C22 (theta = 0, zeta = 6.64) in both the pedigrees and nuclear families with no evidence for recombination between CF and any of the DNA markers. Weaker linkage between the CF locus and the locus for the serum enzyme activity marker paraoxonase (PON) was detected, theta = 0.18, zeta = 0.76. The two pedigrees were sufficiently informative to detect significant linkage between CF and each of the three DNA markers previously shown to be tightly linked to the CF locus. These results establish a locus for CF in these pedigrees in the region of chromosome 7 nearest the three DNA markers met, pJ3.11, and 7C22 and are consistent with locus homogeneity for the defect causing CF in these populations and others that have been examined to date.     

Further data supporting linkage between cystic fibrosis and the met oncogene and haplotype analysis with met and pJ3.11

The linkage of cystic fibrosis (CF) and the polymorphic DNA markers pJ3.11, met, 7C22, DOCR1-917, COL1A2, and TCRB have jointly localized the mutation causing CF to chromosome 7q2.1-3.1. We report further linkage data with two polymorphic markers at the met oncogene locus, pmetH and pmetD, which supports the tight linkage found by White et al. between CF and met. One family shows evidence for meiotic recombination between CF and met. Analysis of haplotypes in CF pedigrees collected for linkage studies combined with data from single affected families requesting prenatal diagnosis (Farrall et al., Lancet i:1402-1404, 1986) shows CF and met to be in linkage equilibrium in our population while pJ3.11-CF haplotypes show a deviation from the equilibrium frequencies.     

Chromosome mediated gene transfer of six DNA markers linked to the cystic fibrosis locus on human chromosome seven.

The DNA probes met and pJ3.11 are derived from loci on chromosome seven that are closely linked to, and probably flanking, the gene mutation causing cystic fibrosis (CF). We have shown that mitotic chromosomes from the cell line MNNG-HOS, which contains an activated met oncogene, can induce morphological transformation of mouse NIH-3T3 cells. Southern analysis of isolated transfectant cell lines with cloned dispersed repetitive human DNA sequences as probes demonstrated that several lines of transformed NIH 3T3 cells had stabley incorporated large segments of chromosome seven DNA. Southern blot analysis also demonstrated the presence of met, pJ3.11 and several other single copy sequences that had been previously localised to chromosome 7 within the transgenomes. In this way a further four genetic markers were shown to be physically linked to met, and thus to CF. These probes may prove useful in confirming the order of loci around CF and in the prenatal diagnosis of this common autosomal recessive disease.     

Linkage of DNA markers to cystic fibrosis in 26 families.

Two DNA markers, the met oncogene and the anonymous probe, pJ3.11, previously reported to be tightly linked to cystic fibrosis (CF), were used for linkage analysis in 26 families with two or more individuals affected with CF. A new high frequency polymorphism was identified using BanI and the pmetD probe. The results of linkage analysis were as follows: between met and CF, lod score of 18.2 at theta of .009; between pJ3.11 and CF, lod score of 12.1 at theta of 0; and between met and pJ3.11, lod score of 16.7 at theta of 0. These data indicate that most or all of CF is due to an abnormality at a single locus and that the DNA markers are useful for prenatal diagnosis and heterozygote detection within affected families.     

Isolation of a new DNA marker in linkage disequilibrium with cystic fibrosis, situated between J3.11 (D7S8) and IRP.

A cosmid library of recombinants containing nonmethylated CpG sites for rare-cutter restriction enzymes was used previously to isolate the gene IRP and four polymorphic DNA markers (pPT-3, pXV-2c, pCS.7, and pKM.19) which are close to and in linkage disequilibrium with the cystic fibrosis (CF) mutation. We have analyzed several new clones from the same library and have isolated a further cosmid, cNX.6d, which maps approximately 160 kb from CS.7, in the J3.11 direction. A DNA fragment (pMP6d-9) (D7S399) derived from cosmid cNX.6d detects a frequent polymorphism with MspI. Strong linkage disequilibrium between CF and MP6d-9 is found in European populations. Recombinations in two families suggest that CF is between the MspI polymorphic site recognized by pMP6d-9 and the polymorphism recognized by pJ3.11. The new marker is the closest, to date, to CF and will be useful for prenatal diagnosis and carrier testing.     

Cystic fibrosis DNA markers in Alabama blacks

DNA samples from unrelated Alabama blacks with no family history of cystic fibrosis (CF) were analyzed with DNA markers linked to cf. Allelic frequencies of genetic markers detected by probes pmetH, pmetD, XV2c, KM19, and pJ3.11 were compared to those of other populations. Allelic frequencies for pJ3.11, XV2c, and KM19 in Alabama blacks and previously studied Caucasian populations were similar. In contrast, the met locus allelic frequencies in Alabama blacks were markedly different from those in Caucasian populations.     

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.     

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.     

Analysis of 112 haplotypes carrying the cystic fibrosis gene. Applications in genetic counseling

Cystic fibrosis (CF) is always a common lethal genetic disease. The locus is localized to human chromosome 7q22-7q31. Genetic linkage between the CF locus and polymorphic DNA marker is used to realize family studies. We have genotyped 56 families (352 patients) with a CF child. The informativeness with the six markers (Met D/Taq I, Met H/Taq I, Met H/Msp 1, XV2c/Taq 1, km19/pst pJ 3.11/Msp 1) is important (96%). The linkage desequilibrium between alleles detected by XV2 c and Km 19 described by Estivill and al, is also showed in our population. The haplotype B (Km 19 = 6.6 kb, XV2 c = 2.1 kb) is present on 84% of our 112 CF chromosomes. We have established the frequencies of the 10 possible genotypes in the pool of the 112 CF chromosomes and in the pool of the normal chromosome and according to Bayes obtained the predictive positive value to be heterozygote. It is possible to precise the genetic counselling in these families.     

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.     

Cystic fibrosis mutations in the Hutterite Brethren.

The presence or absence of the major cystic fibrosis (CF) mutation, delta F508, in the general patient population was determined by Kerem et al. using allele-specific oligonucleotides for the mutant and normal sequences in the polymerase chain reaction (PCR). delta F508 was identified by Riordan et al., and it is a 3-bp deletion of the phenylalanine codon at position 508. The Hutterite Brethren are an inbred North American population who have three different DNA marker haplotypes of CF chromosomes. Genomic DNA from both a CF child and one parent from each of 10 Hutterite families was analyzed for the presence or absence of the deletion mutation. delta F508 is associated with one of the three CF haplotypes in the Hutterite population, and this is the most common haplotype in a subset of the linkage family data of Kerem et al. The other two Hutterite CF haplotypes are generally rate in Caucasian populations. Since these two CF haplotypes do not carry the deletion mutation, they must carry a different CF mutation(s). The results of the PCR analysis for the deletion mutation lend additional support to our previous conclusion that there were at least three original carriers of CF mutations among the founders of the Hutterite population and that all copies of the same CF haplotype were identical by descent. One Hutterite CF patient has both of the haplotypes which do not carry delta F508. Analysis of this individual's DNA should allow identification of two additional CF mutations in this population.     

Linkage of cystic fibrosis locus and polymorphic DNA markers in 14 families

Linkage relationships between the cystic fibrosis (CF) locus and three polymorphic DNA markers were examined in 14 families, five of which were of Hispanic origin. Tight linkage was found between the CF locus and MET (maximum lod score = 7.16 at theta = .001), and between CF and pJ3.11 (maximum lod score = 3.87 at theta = .001). We observed two recombinations between CF and collagen, yielding a maximum lod score of 0.359 at theta = .125, and one recombination in the cluster CF-MET-pJ3.11. Analysis by the seriation method indicates the order COL-pJ3.11-CF-MET.     

Physical mapping of the cystic fibrosis region by pulsed-field gel electrophoresis

The gene for cystic fibrosis (CF) is known to be flanked by the closely linked DNA markers met and J3.11 on chromosome 7. Using the technique of pulsed-field gel electrophoresis, we have constructed a complete overlapping restriction map of approximately 3000 kb of DNA in this region. The met and J3.11 probes are found to be between 1300 and 1800 kb apart, which compares well with their genetic distance of 1-2 cM. The CF gene must be located within this interval, and the availability of this physical map should be of considerable utility in mapping additional clones as the search for the gene proceeds.     

Cystic fibrosis: typing 48 German families with linked DNA probes

Summary Two hundred and thirty five subjects from 48 German cystic fibrosis (CF) families were typed for restriction fragment length polymorphisms (RFLPs) detected by the probes pmet H, pmet D, and pJ 3.11, known to be tightly linked to the CF gene. Gene and haplotype frequencies suggest a linkage disequilibrium with the CF locus. The analysis of the predictive value of this typing in individual CF families indicates that the combined use of these probes provides a powerful diagnostic system both for carrier detection and prenatal diagnosis. In 33 out of 48 families carriers and non-carriers could be identified, and in 26 of these 33 families prenatal diagnosis could discriminate between affected and unaffected offspring.     

Cystic fibrosis typing with DNA probes: experience of a screening laboratory

Summary A sample of 125 individuals from 37 British cystic fibrosis (CF) families with at least one living affected child were typed with probes for restriction fragment length polymorphisms (RFLPs) known to be linked to the CF gene. These probes were MetD, MetH, pJ3.11 and 7C22. Using this combination of probes, 30 out of the 37 families were sufficiently informative to enable prenatal diagnosis of the disease. Linkage analysis has also proved to be useful in excluding CF in two cases where diagnosis of the disease was equivocal in the sibling of an affected child.     

Genealogical analysis of cystic fibrosis families and chromosome 7q RFLP haplotypes in the Hutterite Brethren.

In the 100-year period 1880-1980 the Hutterite population increased from about 442 to 23,000 individuals in North America. There are three endogamous subdivisions in this Caucasian genetic isolate. A total of 11 cystic fibrosis (CF) families from Canada and the United States were investigated, including at least two families from each of the three subdivisions, the Dariusleut, Lehrerleut, and Schmiedeleut. A study of RFLPs for the loci D7S8, D7S23, MET, and D7S18 (also called D7S16) in the region of the CF gene in 10 families shows considerable genetic variability. There were three different extended CF gene-region haplotypes on CF chromosomes (CF haplotypes), and there were 13 different extended CF gene-region haplotypes on normal chromosomes (normal haplotypes). The three CF haplotypes have different D7S23 and MET haplotypes. Parents who have the same CF haplotype are, on the average, more closely related than parents who have different haplotypes, but only within the same subdivision. A marriage node graph of 11 families illustrates the complexity of Hutterite genealogies. The frequency distribution of CF haplotypes in the Hutterite sample differs notably from those of larger agglomerates of family data from collaborative studies, with respect to D7S8, MET haplotypes, and D7S23 haplotypes. We propose that there were at least three CF carriers among the founders of the Hutterite population and that copies of a particular CF haplotype in current individuals are identical by descent. The alternative that one or more genetically distinguishable CF haplotypes resulted from recombination since the founding of the population is considered to be less likely.     

The cystic fibrosis locus

The identification of the cystic fibrosis locus (CF) provides a model for the study of single gene defects where the biochemical lesion is not known. Using families each of which has several affected siblings, it was possible to exclude a number of 'candidate genes' which had previously been proposed as possible sites of the CF mutation. Exclusion mapping of the genome using polymorphic protein and DNA markers showed that CF is on the long arm of human chromosome 7. The most closely linked flanking markers were identified, and human chromosome fragments containing them (and therefore the CF locus) were isolated in rodent cell lines by chromosome-mediated gene transfer. The transgenome was then analysed using cosmid contig mapping, pulse-field gel electrophoresis, HTF island identification and linkage disequilibrium. In this way, a candidate coding sequence has been identified which always segregates with CF.     

Cystic fibrosis: typing 89 German families with linked DNA probes

Summary Three hundred and ninety-two subjects from 89 German families were typed for restriction fragment length polymorphisms (RFLPs) detected by the probes pmetH, pmetD, pJ3.11, KM19, and XV2c known to be tightly linked to the cystic fibrosis (CF) gene. The analysis of the predictive value of this typing in individual CF families indicates that the combined use of these probes provides a powerful diagnostic system for both carrier detection and prenatal diagnosis. In 45 families the complete haplotype including all RFLPs was available. Of them 41 (91.1%) were fully informative and 4 were partly informative.