[From pedigree analysis to gene defects].
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Finland↗
Biomedical subjects
J Kere
Publications and source records attributed to J Kere.
Fine mapping of the EDA gene: a translocation breakpoint is associated with a CpG island that is transcribed.
In order to identify the gene for human X-linked anhidrotic ectodermal dysplasia (EDA), a translocation breakpoint in a female with t(X;1)(q13.1;p36.3) and EDA (patient AK) was finely mapped. The EDA region contains five groups of rare-cutter restriction sites that define CpG islands. The two more centromeric of these islands are associated with transcripts of 3.5 kb and 1.8 kb. The third CpG island maps within <1 kb of the translocation breakpoint in patient AK, as indicated by a genomic rearrangement, and approximately 100 kb centromeric from another previously mapped translocation breakpoint (patient AnLy). Northern analysis with a probe from this CpG island detected an approximately 6-kb mRNA in several fetal tissues tested. An extended YAC contig of 1,200 kb with an average of fivefold coverage was constructed. The two most telomeric CpG islands map 350 kb telomeric of the two translocations. Taken together, the results suggest that the CpG island just proximal of the AK translocation breakpoint lies at the 5' end of a candidate gene for EDA.
Base Sequence↗
Patterns of matrix metalloproteinase and TIMP-1 expression in chronic and normally healing human cutaneous wounds.
The present study was carried out to characterize the patterns of expression of matrix metalloproteinases or their tissue inhibitor (TIMP-1) in normally healing, acute vs. chronic, skin wounds. In situ hybridization was performed to localize collagenase, stromelysin-1, stromelysin-2, matrilysin, urokinase plasminogen activator (uPA) and TIMP-1 mRNAs in 14 chronic venous ulcers and 10 normally healing wounds, representing different time points after wounding. Surgical wounds, made in piglets harvested at several time points, were studied as controls. Collagenase, stromelysin-1 and -2, as well as uPa, were expressed in keratinocytes in both acute and chronic wounds, while epithelial TIMP-1 mRNA was not detected in any chronic wound biopsies studied. However, TIMP-1 was expressed at the epithelial edges of both acute human and pig wounds. Our results suggest that the balance between metalloenzymes and their inhibitor TIMP-1, is disturbed, in poorly healing wounds.
Acute Disease↗
A 6-Mb YAC contig in Xp22.1-p22.2 spanning the DXS69E, XE59, GLRA2, PIGA, GRPR, CALB3, and PHKA2 genes.
We report the generation of an approximately 6-Mb contig of 70 overlapping yeast artificial chromosomes (YAC) covering the interval between DXS16 and DXS1229 in Xp22.1-p22.2. Within this region lie the genes for calbindin (CALB3), gastrin-releasing peptide receptor (GRPR), phosphatidyl-inositol glycan-class A protein (PIGA), glycine receptor alpha-2 (GLRA2), phosphorylase kinase alpha (PHKA2), XE59 (a gene escaping X chromosome inactivation), and DXS69E (71-7A). YACs were isolated initially from four libraries either by hybridization or using sequence tagged sites (STSs) for DXS16, DXS9, GLRA2, DXS207, DXS43, DXS1416, DXS1317, DXS1195, and DXS418. Additional STSs were obtained from the end fragments of the original YACs studied, thus allowing us to cover the contig with a series of 73 STSs, approximately 1 per 100 kb. YAC contig construction allowed the following locus order to be established: Xpter-DXS16-DXS69E-DXS414-XE59 - DXS9 - (GLRA2, DXS987) - (PIGA, DXS207) - DXS1053-DXS197-(GRPR,DXS43)-CALB3-DXS14 16- DXS1317 - DXS1195 - DXS418 - DXS257 - (PHKA2, DXS999)-DXS443-DXS1229-Xcen. Restriction mapping of the DXS16-DXS43 interval predicted the existence of several CpG islands, suggesting the presence of other genes in the region. This work provides a starting point for further mapping and positional cloning of several X-linked disease genes.
Base Sequence↗
Fine mapping of the congenital chloride diarrhea gene by linkage disequilibrium.
Congenital chloride diarrhea is a recessively inherited intestinal disorder affecting electrolyte transportation. The clinical presentation is a life-threatening watery diarrhea with a high chloride content. Recently, the congenital chloride diarrhea gene (CLD) was assigned to chromosome 7 by linkage in eight Finnish families. In the present study, refined mapping of CLD was performed by studying linkage and linkage disequilibrium in 24 Finnish and 4 Swedish families. Recombination mapping assigned CLD to an approximately 10-cM region flanked by D7S515 and D7S799. Linkage disequilibrium was detected over this large genetic region, with the strongest allelic association at D7S496. Application of the Luria and Delbrück-derived analysis allowed for a further narrowing of the CLD region to approximately 0.37 cM from the marker D7S496. Haplotype analysis placed CLD unequivocally between D7S501 and D7S692, very close to D7S496 and most likely on the distal side of D7S496. This combined analytical approach allowed highly accurate mapping of CLD, each component adding complementary and consistent mapping information.
Child↗
Characterization of four human YAC libraries for clone size, chimerism and X chromosome sequence representation.
Four collections of human X-specific YACs, derived from human cells containing supernumerary X chromosomes or from somatic cell hybrids containing only X human DNA were characterized. In each collection, 80-85% of YAC strains contained a single X YAC. Five thousand YACs from the various libraries were sized, and cocloning was assessed in subsets by the fraction of YAC insert-ends with non-X sequences. Cocloning was substantial, ranging up to 50% for different collections; and in agreement with previous indications, in all libraries the larger the YACs, the higher the level of cocloning. In libraries made from human-hamster hybrid cells, expected numbers of clones were recovered by STS-based screening; but unexpectedly, the two collections from cells with 4 or 5 X chromosomes yielded numbers of YACs corresponding to an apparent content of only about two X equivalents. Thus it is possible that the DNA of inactive X chromosomes is poorly cloned into YACs, speculatively perhaps because of its specialized chromatin structure.
Animals↗
Structure of the human CCG1 gene: relationship between the exons/introns and functional domain/modules of the protein.
The human CCG1 gene, encoding CCG1/TAFII250/p250, was isolated by complementing tsBN462, a mutant BHK21 cell line that shows cell-cycle arrest at high temperature. Using the cDNA as a probe, the locations of exon-intron junctions were determined in the genomic DNA. Thirty-eight exons ranging from 68 to 219 bp in size were found. All the exon-intron junctions followed the GT-AG rule. Using a newly developed method, we performed a module analysis of the CCG1 protein. The functional domain previously predicted in CCG1 was further confirmed to be encoded in a single predicted module that is the minimal functional unit in the protein. The boundaries of the predicted modules show a close correlation to the intron/exon junction of CCG1. The entire gene, at least 110 kb long, has been recovered in a YAC, which provides a route to the further study of module function.
Base Sequence↗
Cystic fibrosis in a low-incidence population: two major mutations in Finland.
The incidence of cystic fibrosis (CF) in Finland, 1:25,000 newborn, is one of the lowest in Caucasian populations. The delta F508 mutation accounts for 18/40 (45%) of CF chromosomes in Finland. Other mutations were therefore sought among the remaining 55%. Twelve out of 40 chromosomes (30%) were found to carry 394delTT, whereas G542X and 3732delA were each detected in one chromosome. Eight mutations remained unidentified using a testing panel for 26 mutations. Mutation 394delTT was associated exclusively with haplotype 23-36-13. Five unknown mutations were associated with different haplotypes for microsatellite markers, whereas three shared the same haplotype. Most delta F508 mutations and all unidentified mutations originated from regions of old and dense settlement in the coastal regions, whereas 394delTT was geographically clustered and enriched in a rural location, consistent with a local founder effect. The remote location of Finland and her population history give a plausible explanation for the rarity of CF in Finland.
Adult↗
Reactive oxygen species of neutrophils from patients with monosomy 7 in the bone marrow: contradictory chemiluminescence activity by whole blood or by purified cells.
Monosomy 7, a deletion of the long arm of chromosome 7, was shown in the neutrophils of peripheral blood in 5 of 6 patients with a myelodysplastic syndrome or leukemia who had monosomy 7 in their bone marrow cells. In a chemiluminescence assay the production of reactive oxygen species by neutrophils from patients was increased in whole blood and decreased in purified cells, which suggests that neutrophils with monosomy 7 tolerate poorly the cell purification procedures used. The in vitro migration of purified neutrophils obtained from patients with monosomy 7 was impaired. It is known that patients with monosomy 7 have an increased susceptibility to infections. It is possible that neutrophils with monosomy 7 are too easily triggered to a full-scale respiratory burst and thereby the cells exhaust their ability to eliminate invading microbes efficiently.
Adult↗
Paternal isodisomy for chromosome 7 is compatible with normal growth and development in a patient with congenital chloride diarrhea.
Uniparental disomy for maternal chromosome 7 has been described in three patients with recessive disorders. Short stature in each of these patients has been explained by the effect of imprinting of growth-related genes on maternal chromosome 7. Alternatively, although less likely, all these patients may be homozygous for a rare recessive mutation. Here we report both paternal isodisomy for chromosome 7 and normal growth in a patient with a recessive disorder, congenital chloride diarrhea. She had inherited only paternal alleles at 10 loci and was homozygous for another 10 chromosome 7 loci studied. Her physical status and laboratory tests were normal except for a mild high-frequency sensorineural hearing loss. As the patient has normal stature, it is likely that the paternal chromosome 7 lacks the suggested maternal imprinting effect on growth. Paternal isodisomy for human chromosome 7 may have no phenotypic effect on growth.
Adult↗
[Linkage and linkage disequilibrium in the Finnish disease heritage].
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Finland↗
The gene for congenital chloride diarrhea maps close to but is distinct from the gene for cystic fibrosis transmembrane conductance regulator.
Congenital chloride diarrhea (CLD) is characterized by watery stools with high chloride content beginning prenatally and is inherited as an autosomal recessive trait. Perfusion studies have established a basic defect in ileal and colonic Cl-/HCO3- transport, resulting in defective chloride absorption. The protein and its gene defects have, however, remained uncharacterized. We attempted to exclude candidate genes by considering linkage disequilibrium as well as genetic linkage in a small number of Finnish families. Initial results were suggestive of linkage between CLD and the cystic fibrosis transmembrane regulator gene (CFTR). Extended analysis in eight families confirmed close linkage to chromosome 7 markers proximal of CFTR, with maximum logarithm of odds scores of 5.11 and 5.06 for D7S501 and D7S496, respectively, at zero recombination. Allelic associations were observed that were striking between CLD and D7S496 and weaker between CLD and D7S501. Multipoint analyses mapped CLD unequivocally at D7S496 with a maximum logarithm of odds score of 9.33. We conclude that the CLD gene maps close to, but is distinct from, CFTR.
Chlorides↗
Anhidrotic ectodermal dysplasia gene region cloned in yeast artificial chromosomes.
Anhidrotic ectodermal dysplasia (EDA), an X-chromosomal recessive disorder, is expressed in a few females with chromosomal translocations involving bands Xq12-q13. Using available DNA markers from the region and somatic cell hybrids we mapped the X-chromosomal breakpoints in two such translocations. The breakpoints were further mapped within a yeast artificial chromosome contig constructed by chromosome walking techniques. Genomic DNA markers that map between the two translocation breakpoints were recovered representing putative portions of the EDA gene.
Base Sequence↗
Ordered shotgun sequencing, a strategy for integrated mapping and sequencing of YAC clones.
Ordered shotgun sequencing proposes to organize the mapping and sequencing of YACs with a hierarchical strategy that incorporates a feedback loop. Building on current protocols, a YAC is subcloned into plasmids, plasmid insert ends are sequenced, and the sequences are overlapped to create a partial map. Complete sequencing then starts with plasmids whose end-sequence tracts have overlapped, but to a minimal extent. The next plasmids to be sequenced are again selected for least overlap, striking out progressively to span the YAC with minimal directed gap-filling. Simulations support its feasibility and indicate that during the generation of the complete sequence, the approach facilitates the early choice of regions for selective sequencing, for example, for coding units. The sequencing of plasmids would also require less redundancy, and discriminate repetitive sequences more easily, than random sequencing across larger clones. The overall effort scales with YAC size and can be further reduced by additional mapping information.
Chromosome Mapping↗
A rare reciprocal translocation (12;21) segregating for nine generations.
An autosomal reciprocal translocation (12;21) was found in five seemingly unrelated families in Finland. Three families had had multiple spontaneous abortions and two families had a child with Down's syndrome. The genealogies of the five families were traced using population registries, and four families were found to have a common ancestor born in 1752. Kinship to the fifth family could not be established but its ancestors were traced back to the same rural parishes as those of the four other families. The translocation segregated at the same frequency as normal chromosomes. A statistically insignificant increase in spontaneous abortions was detected when the matings of translocation carriers were compared with non-carrier matings. The increase may however be clinically significant. These results permit more accurate counselling in these and similar translocation families.
Abortion, Habitual↗
Haplotype analysis to determine the position of a mutation among closely linked DNA markers.
Positional cloning involves first finding linkage between an inherited phenotype (such as a disease) and a DNA marker, followed by the use of a variety of physical and genetic mapping techniques to move from linkage to mutation. If there is a founder effect within a population, crossovers are often rare between the mutation causing the phenotype and closely situated markers and increasing disequilibrium may be observed as the site of the mutation is approached. Standard coefficients of disequilibrium may, however, be insensitive to the relative position of close markers and the mutation, because they depend upon allele frequencies in the normal population compared to those of the founder chromosome. Using cystic fibrosis in European populations as a model system, alternative methods for determining the position of a mutation are discussed. These include haplotype parsimony and three-way interval likelihood analysis. Both methods predict the location of the major CF mutation accurately from a real set of more than 600 European CF chromosomes.
Alleles↗
[Current status of the human genome project].
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Chromosome Mapping↗
Evidence that the MCC-APC gene region in 5q21 is not the site for susceptibility to hereditary nonpolyposis colorectal carcinoma.
Hereditary nonpolyposis colorectal carcinoma (HNPCC) is the most common form of hereditary colon cancer. Autosomal dominant inheritance is evident from pedigrees but the genetic basis of the disorder is otherwise unknown. Recently, two genes in 5q21 involved in colon carcinogenesis, APC and MCC, were identified, and APC was shown to be the gene predisposing to familial adenomatous polyposis. To determine if these genes also confer susceptibility to HNPCC we performed linkage analyses in nine affected families. The MCC-APC region could be formally excluded as the locus for HNPCC in seven families. In one family the results were suggestive of exclusion, although they were not conclusive. The remaining family was uninformative. We used two alternative definitions of affected status. Based on haplotypes for MCC and APC the added pairwise logarithm-of-odds score for all nine families was -22.57 at the recombination fraction of 0.00 using more stringent criteria for the HNPCC phenotype and -22.67 for less stringent criteria. In addition to blood DNA samples from living family members, DNA from formaldehyde-fixed archival pathology specimens from decreased individuals contributed to these linkage results.
Base Sequence↗