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A cluster of highly polymorphic dinucleotide repeats in Intron 17b of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene
Abstract
A cluster of highly polymorphic dinucleotide repeats has been detected in intron 17b of the CFTR gene, 200 bp downstream from the preceding exon. At least 24 alleles, with sizes ranging from 7 to 56 units of a TA repeat, have been identified in a panel of 92 unrelated carriers of cystic fibrosis (CF). The common ones are those with 7, 30, and 31 dinucleotide units, with frequencies of .22, .19, and .12, respectively, among the non-CF chromosomes. Mendelian, codominant segregation of the alleles has been demonstrated in family studies, as expected. A less polymorphic dinucleotide (CA repeat) cluster has also been detected in a region 167 bp downstream from the TA repeat. The length of the CA repeat cluster varies from 11 to 17 dinucleotide units, and it appears to have an inverse relationship to that of the TA repeats. These dinucleotide repeats should be useful in genetic linkage studies, in counseling for CF families with unknown mutations, and in tracing the origins of the various mutant CF alleles.
... comm.). Table 7. Microsatellite markers from chromosome 7 (Zielenski et al., 1991;Weissenbach, 1992). allowing to stand at room temperature for 15 minutes. ...
... Sibling 2 was informative for the CFTR dinucleotide repeat marker in intron 17b of the CFTR gene on chromosome 7q (Zielenski et al., 1991) and for the marker at D7S488 (figure 61). Sibling 2 was uninformative for the markers at D7S493, D7S517 and D7S507 (figures 61 and 62). ...
Angelman syndrome (AS) and Prader-Willi syndrome (PWS) are distinct neurogenetic disorders caused by the loss of function of distinct but closely linked genes on chromosome 15q11-q13, a region subject to genomic imprinting. AS and PWS most commonly arise from a de novo deletion of 15q11-q13. These deletions are maternal in origin in AS and paternal in origin in PWS. AS patients were analysed in order to find atypical deletions, which would define a critical region for the AS gene. One AS individual who had previously been identified with an apparent maternal deletion around the LS6-1 (D15S113) locus and normal DNA methylation imprints, initially appeared deleted for an LS6-1 positive cosmid by fluorescence in situ hybridisation (FISH). Further studies with overlapping cosmid and phage clones, single copy fragments and microsatellite markers, showed that this individual was not deleted for the D15S113 locus and that the original LS6-1 result was due to non-amplification of the maternal allele. A second AS individual was shown by FISH and microsatellite analysis to have an atypical deletion of 15q11-q13 such that the results obtained with PW71B (D15S63) and SNRPN appeared to conflict. A third individual who did not have the characteristic features of AS, but who had a maternally inherited deletion of 15q which included the D15S113 locus was also studied. FISH studies showed that the centromeric breakpoint was contained within a 520 kb yeast artificial chromosome (YAC) which maps between TD3-21 (D15S10) and LS6-1 (D15S113). AS individuals with no apparent maternal deletion of 15q11-q13, aberrant DNA methylation imprints and biparental inheritance of chromosome 15 were studied. Haplotypes were constructed in these individuals and their families, to localise a 'methylation control' locus. These studies were in agreement with a methylation control locus on chromosome 15q11-q13. Prenatal diagnosis was attempted in one family. The fetus had inherited the same critical maternal chromosome 15q11-q13 segment as the two affected siblings for the alleles tested, but had normal DNA methylation imprints. The mother was believed to be mosaic. FISH studies using phage clones obtained from the region on chromosome 15 designated as the 'imprinting centre' were carried out in these AS individuals, but were found to be unsuitable for diagnosing deletions of this region.
... Haplotype a harbors AF508 and carries (GATT)6 and (TA)32 repeat alleles. All CF chromosomes which harbor AF508 carry (GATT)6 (Chehab et al. 1991; Dork et al. 1992), and (TA)32 was present in 22/61 (36%) of AF508 alleles (Zielenski et al. 1991b). Haplotypes b and c both harbor MllOlK and carry (TA)29. ...
... The CFTR allele harboring M1lOlK carries the infrequent IVS17b (TA)29 repeat allele. In Canadian CF families , only 3% of normal alleles and no CF alleles carried (TA)29 (Zielenski et al. 1991b). M1101K appears to be rare outside the Hutterite population, because the mutation has not been found after screening 328 other CF ...
The Hutterite population is a genetic isolate with an increased incidence of cystic fibrosis (CF). Previously we identified three CF haplotypes defined by polymorphisms flanking the CF transmembrane conductance regulator (CFTR) gene. delta F508 was present on one of the haplotypes in only 35% of CF chromosomes. We hypothesized that the other two CF haplotypes, one of which was the most common and the other of which is rare, each harbored different non-delta F508 mutations. Single-strand conformation polymorphism analysis detected a missense mutation, M1101K, in both chromosomes of a Hutterite patient carrying the two non-delta F508 haplotypes. M1101K appears to have originated on an uncommon CFTR allele and to be infrequent outside the Hutterite population. The presence of M1101K on two haplotypes is likely the result of a CFTR intragenic recombination which occurred since the founding, 10-12 generations ago, of the Hutterite population. The crossover was located between exons 14a and 17b, an interval of approximately 15 kbp. delta F508 and M1101K accounted for all of the CF mutations in patients from 16 CF families representing the three subdivisions of the Hutterite population.
... We have typed six intra-and six extragenic RF40LP markers that had been useful in previous studies to characterize the origins of numerous other CFTR mutations (Estivill et al. 1987; Dö rk et al. 1992 Ramsay et al. 1993; Sereth et al. 1993; Cuppens et al. 1994; Morral et al. 1996). In addition, we investigated the three highly informative intragenic CFTR microsatellites that are located in intron 8 (IVS8CA) and intron 17b (IVS17bTA and IVS17bCA) of the CFTR gene (Zielenski et al. 1991; Morral and Estivill 1992; Morral et al. 1993). A common extended 31201GrA–associated haplotype could be derived in each of the four study populations (table 1). ...
... Three of the four single African American patients, however, were compoundFigure 1 Geographic origins of 31201GrA–carrying individuals whose DNA samples were contributed to this study heterozygotes for 31201GrA and for the major CF mutation, DF508. The extensively studied DF508 mutation has been shown to have a single origin in several investigated populations, with a common dimorphic marker haplotype (Kerem et al. 1989; Dö rk et al. 1992; Claustres et al. 1996; Morral et al. 1996) and three major intragenic microsatellite haplotypes—23-31-13, 17-32-13, and 17-31-13—accounting for 185% of DF508 chromosomes (Zielenski et al. 1991; Morral et al. 1993 Morral et al. , 1994 Claustres et al. 1996; Hughes et al. 1996). Under the assumption that the DF508 mutation has occurred only once and has been introduced into the African American population by ethnic admixture, we were able to use the known major DF508 haplotypes to deduce the most likely haplotypes for the 31201GrA allele in the three additional single African American patients. ...
To the Editor:The article by Condit et al. (1998)xDeterminism and mass-media portrayals of genetics. Condit, CM, Ofulue, N, and Sheedy, KM. Am J Hum Genet. 1998; 62: 979–984Abstract | Full Text | Full Text PDF | PubMed | Scopus (66)See all ReferencesCondit et al. (1998) demonstrates some of the limitations of quantitative analysis. The authors select from Reader's Guide articles listed under “heredity” in various time periods. Not surprisingly, such articles consistently attribute characteristics to genes. When the 50 articles selected from the eugenic period attribute human characteristics to heredity at almost the same rate as those selected from the 1990s, the authors conclude that nothing has changed. Predictably, they find that the “degree of determinism” (which they calculate to the fifth decimal) is consistent over 90 years of profound scientific and social change.The paper is an example of the problem of trying to quantitate what is most compellingly understood in qualitative terms. Our study of the gene in popular culture (Nelkin and Lindee 1995xNelkin, D and Lindee, SM. See all ReferencesNelkin and Lindee 1995), a target of Condit et al.'s paper, was not a quantitative study for the precise reason that the counting of such ambiguous and heterogeneous materials provides little insight into the public meaning of science. We focused on qualitative changes in a much broader literature, to suggest that the gene has acquired new powers as a guide to social policy. In the 1990s, the cultural meanings attached to the gene are shaping employment practices, educational policies, and decisions in the courts. The serious issues raised by the high-profile gene deserve more serious analysis.
... IVS17BCA is a CA-repeat in intron 17B with 11±20 repeats and 39% heterozygosity. The IVS17BTA marker is a TA-repeat in intron 17B and has 7±54 repeats with 87% heterozygosity (Zielenski et al., 1991). The extragenic polymorphic markers are also CA-repeats with heterozygosity rates of 80, 77, 87 and 81% respectively (Dreesen et al., 2000). ...
Cystic fibrosis (CF) is an autosomal recessive disease characterized by obstruction and chronic infection of the respiratory tract and pancreatic insufficiency. The first preimplantation genetic diagnosis (PGD) for CF was carried out in 1992. At our centre the first cycle was performed in 1993. However, the number of known CF mutations is >1000, so developing mutation-specific PCR protocols for PGD is unfeasible. This is why a number of marker-based duplex PCRs were developed at the single cell level. A duplex PCR of a mutation and one or two microsatellites is not only a diagnostic tool, but it can also be used as a control for allele drop-out and contamination. During PGD, embryos obtained in vitro are analysed for the presence or absence of a particular genetic disease, after which only embryos shown to be free of this disease are returned to the mother. In total, 22 PGD cycles with duplex PCR (IVS8CA/IVS17BTA, DF508/IVS8CA, DF508/IVS17BTA and D7S486/D7S490) were carried out in 16 couples, which resulted in four ongoing pregnancies and one miscarriage.
... c.3499+200TA(7_56) is a highly polymorphic microsatellite containing TA-repeats located in intron 17b of CFTR gene and have been shown to have at least 24 different alleles with sizes ranging from 7 to 56 repeats (18). D7S523 is a microsatellite containing CA-repeats located 1 cM proximal to the CFTR gene which was reported to show 80% heterozygote frequency in Europe (19). ...
Cystic fibrosis (CF) is a life-limiting autosomal recessive disorder affecting principally respiratory and digestive system. It is caused by cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation. The aim of this study was to determine the extent of repeat numbers and the degree of heterozygosity for c.3499+200TA(7_56) and D7S523 located in intron 17b and 1 cM proximal to the CFTR gene respectively. Both microsatellites were analyzed by direct electrophoresis of PCR product on 20% polyacrylamide gel in 40 Normal subjects and 40 CF patients originating from North Iran. 9 different alleles were found for D7S523 ranging from 16 to 24 repeats alleles. (CA)20 was the most prevalent allele both in normal individuals and CF patients with 21.3% and 20% frequencies respectively. Heterozygosity frequency of D7S523 in normal individuals and CF patients was 97.5% and 90% respectively. Eighteen different alleles were found for c.3499+200TA(7_56) ranging from 8 to 38 repeats alleles. (TA)9 was the most prevalent allele both in normal individuals and CF patients with 30% and 23.5% frequencies respectively. All normal subjects and 97.5% of CF patients showed heterozyous genotype. The high heterozygosity of the two studied microsatellites witnesses the dynamism of such markers. High degree of heterozygosity of c.3499+200TA(7_56) and D7S523 make these markers, a very useful tool for prenatal diagnosis especially in Iranian population.
... Für die Durchführung eines "indirekten Gentests" stehen eine ganze Reihe intra-und extragenischer, CFTR-gekoppelter Marker zur Verfügung. Die größte Informativität wird durch die Untersuchung des Dinukleotidrepeats IVS17bTA [17] erreicht, doch auch andere Marker haben sich als sehr sinnvoll erwiesen. Die höchste Aussagesicherheit kann erreicht werden, wenn neben den hochinformativen intragenischen Mikrosatelliten auch die flankierenden Restriktionsfragmentlängenpolymorphismen in die Untersuchung einbezogen werden. ...
... Chromosomes bearing the F508del muta-tion and most of the other frequent CFTR mutations are associated with more haplotypes, all of which have been derived by slippage from an ancestor haplotype (see Table 3 in the online Data Supplement). These data confirm the results of previous studies, which analyzed 3 microsatellites and reported a single mutational origin for most CFTR mutations (8,24,25 ). ...
Molecular diagnosis for cystic fibrosis (CF) is based on the direct identification of mutations in the CFTR gene [cystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7)] (detection rate about 90% with scanning procedures) and on segregation analysis of intragenic polymorphisms for carrier and prenatal diagnosis in about 20% of CF families in which 1 or both causal mutations are unknown.
We identified 3 novel intragenic polymorphic repeats (IVS3polyA, IVS4polyA, and IVS10CA repeats) in the CFTR gene and developed and validated a procedure based on the PCR followed by capillary electrophoresis for large-scale analysis of these polymorphisms and the 4 previously identified microsatellites (IVS1CA, IVS8CA, IVS17bTA, and IVS17bCA repeats) in a single run. We validated the procedure for both single- and 2-cell samples (for a possible use in preimplantation diagnosis), and on a large number of CF patients bearing different genotypes and non-CF controls.
The allelic distribution and heterozygosity results suggest that the 3 novel polymorphisms strongly contribute to carrier and prenatal diagnosis of CF in families in which 1 or both causal mutations have not been identified. At least 1 of the 4 previously identified microsatellites was informative in 78 of 100 unrelated CF families; at least 1 of all 7 polymorphisms was informative in 98 of the families. Finally, the analysis of haplotypes for the 7 polymorphisms revealed that most CF mutations are associated with different haplotypes, suggesting multiple slippage events but a single origin for most CFTR mutations.
The analysis of the 7 polymorphisms is a rapid and efficient tool for routine carrier, prenatal, and preimplantation diagnosis of CF.
... The frequent involvement (gain) of 7q32, as observed by Larramendy et al. [14], could not be confirmed in our study: gain of 7q (including 7q32) was found in only a minor portion (6/29, 21%) of the tumors tested. Additionally, Southern blot analysis using a CFTR probe (7q31) [48] showed over-representation in only 2 of the tumors tested (not shown). ...
The C/EBP-homologous transcription factor CHOP (GADD153) is inducible by growth inhibition or DNA damage, and has been shown to be oncogenically activated by the specific (12;16) translocation in human myxoid liposarcoma. We have now found CHOP amplification in two sarcoma cell lines with previously reported amplification of the nearby GLI gene. Among 98 other human sarcomas of various types, CHOP was amplified in a hemangiopericytoma, a liposarcoma, and two osteosarcoma. High constitutive expression levels of CHOP were observed in tumors with gene amplification, but also in some other samples. The nearby MDM2 gene, which codes for a protein that may inactivate wild-type p53, has previously been reported to be frequently amplified in sarcoma. In our sarcoma panel, MDM2 was amplified in 9 cases. MDM2 and CHOP were co-amplified in two of these, whereas the two osteosarcomas had amplified CHOP but not MDM2. CHOP was amplified in both cell lines with GLI amplification, and MDM2 only in one. No mutations in the TP53 gene have been found in samples with amplification of MDM2. In contrast, the cell line in which CHOP but not MDM2 was amplified had mutated TP53, suggesting that selection of this amplicon was not mediated through p53 inactivation.
... One feature that must be built into future statistical modeling of linkage disequilibrium cloning is the relative instability of dinucleotide repeat loci, although this parameter simply broadens the concept of "allele" to include more than one dinucleotide repeat. For example, a TA repeat found in the CF gene shows a "clustering" of alleles (30-32 repeats) in strong linkage disequilibrium with delta-F508, while non-delta-F508 mutations and normal alleles show a much different pattern of allele frequencies over the possible 7-56 repeats (Zielenski et al. 1991). ...
The National Institutes of Health/Department of Energy Human Genome Project has been funding directed research for only 5 years, and it is understandably difficult to cite important research advances directly attributable to the project. However, the project has been constructive in fostering multidisciplinary group research and an inspiring and synergistic "just do it" attitude in both political and scientific circles, domestically and abroad. This collaborative spirit has spawned large-scale genetic and physical mapping projects, with the most impressive and useful results to date being the dense genetic maps produced by the Généthon, a French organization largely supported by the French muscular dystrophy association. With the genetic and physical map reagents now becoming available, disease-gene cloning is proceeding at an increasingly rapid pace. More important than the predictable acceleration of disease-gene mapping are the unpredictable benefits: Will a dense PCR-based dinucleotide-repeat genetic map open novel alternative approaches to disease-gene isolation? Will it become possible to localize disease genes by simply analyzing unrelated, isolated probands rather than the rarer "extended family"? Proband-based "linkage-disequilibrium cloning" may become possible if adequate density, informativeness, and stability of polymorphic loci are obtained. In addition, "genome exclusion cloning" will be added to the established positional, candidate-gene, and functional-disease-gene-cloning experimental approaches. The anticipated exponential expansion of human genetic disease information over the remainder of the 10-year tenure of the Human Genome Project unveils critical yet unresolved issues for medical education and the practice of medicine.(ABSTRACT TRUNCATED AT 250 WORDS)
... There are clear examples of both stable and unstable repeats, and, while mutation rate of dinucleotides is thought to be correlated with the length of the repeat (Weber 1990), this is probably a generalization, and each dinucleotide repeat must be individually evaluated. For example, the prevalence of the AF508 in CF clearly represents founder effect, yet this mutation has been recently found to reside on four different TA repeat haplotypes (Zielenski et al. 1991), and dinucleotide repeats have been successfully used to show apparent founder effect in fragile X (Richards et al. 1992). Clearly, the interpretation of dinucleotide haplotypes and of their relevance to mutation rate and founder effect must be done with caution.Figure 3 Identification of a novel base change in Yugoslavian family I. Left, Detection of two different base changes in the same region (amino acids 1449-1637), by using SSCP analysis. ...
We present a correlation of molecular genetic data (mutations) and genetic data (dinucleotide-repeat polymorphisms) for a cohort of seven hyperkalemic periodic paralysis (HyperPP) and two paramyotonia congenita (PC) families from diverse ethnic backgrounds. We found that each of three previously identified point mutations of the adult skeletal muscle sodium-channel gene occurred on two different dinucleotide-repeat haplotypes. These results indicate that dinucleotide-repeat haplotypes are not predictive of allelic heterogeneity in sodium channelopathies, contrary to previous suggestions. In addition, we identified a HyperPP pedigree in which the dominant disorder was not linked to the sodium-channel gene. Thus, a second locus can give rise to a similar clinical phenotype. Some individuals in this pedigree exhibited a base change causing the nonconservative substitution of an evolutionarily conserved amino acid. Because this change was not present in 240 normal chromosomes and was near another HyperPP mutation, is fulfilled the most commonly used criteria for being a mutation rather than a polymorphism. However, linkage studies using single-strand conformation polymorphism-derived and sequence-derived haplotypes excluded this base change as a causative mutation: these data serve as a cautionary example of potential pitfalls in the delineation of change-of-function point mutations.
β thalassaemia is defined as a group of heterogeneous anaemias in which the β globin peptide synthesis is either absent or reduced by 30% or more. It is the world's most widespread autosomal recessive single gene disorder and represents a major health problem. More than 200 million individuals world-wide carry the β thalassaemia gene with an estimated 75,000 thousands of affected individuals born annually. Because of this β thalassaemia has attracted a great deal of research interest both at the molecular and clinical levels, which has led to the understanding of the complex molecular pathogenesis. Recent clinical management has improved and increased the life expectancy of affected individuals, however, the treatment required is very expensive and unsatisfactory. Therefore, the aim is to control the incidence of the disorder through the provision of carrier detection and prenatal diagnosis. Although prenatal diagnosis has reduced the number of β thalassaemia births, potential risks to the fetus have been associated with the available procedures. Furthermore, pregnancy termination is still unacceptable for many families, and couples at risk are looking for a safer, less emotionally traumatic way to have normal offspring. An alternative is offered by preimplantation genetic diagnosis carried out on the third day after in vitro fertilisation. With the application of nested PCR, amplification of the minute amount of DNA in a single cell has become possible and DNA analysis of single gene disorders for preimplantation diagnosis can be achieved. To enable preimplantation diagnosis of β thalassaemia by direct detection of mutant β globin genes at the single cell level nested PCR and silver stained single stranded conformation polymorphism (SSCP) analysis were employed. However, two main problems with single cell PCR, contamination and allele specific amplification failure in addition to mosaicism can lead to misdiagnosis and the transfer of affected embryos. In this study, the reduction of allele specific amplification failure by the use of SDS/proteinsae K as a lysis buffer, and the application of fluorescence based PCR to reduce the incidence of contamination which becomes apparent when reamplification is done, are reported. Furthermore, this study has also focused on optimising DNA amplification procedures using standard or quantitative multiplex fluorescent PCR in single cells for the detection of mosaicism and age related trisomy 21 along with specific diagnosis of single gene disorder diagnosis and developing mutation analysis techniques such as fluorescent SSCP and automated fluorescent DGGE for either early prenatal diagnosis using transcervical cells or preimplantation diagnosis. Finally, the first step of applying any prevention program for haemoglobinopathies is the identification of mutations within the β globin gene. The molecular characterisation of β thalassaemia has been studied in samples from Jordan and Egypt to facilitate screening and prenatal programmes based on DNA methodology.
Cystic fibrosis (CF) is one of the most common autosomal recessive disorders associated with decreased longevity in the Caucasian population. The disease is classically described as a triad; chronic obstructive pulmonary disease, exocrine pancreatic insufficiency, and elevation of chloride concentration in sweat. The underlying pathophysiology involves abnormal ion transport due to dysfunction of the CF transmembrane conductance regulator (CFTR). Altered salt and water movement across epithelia interferes with the hydration and ionic composition of mucus secretions. Abnormal mucus impairs clearance and defense systems, leading to inflammation and destruction of the lungs, the pancreas, and the developing vas deferens in males. The development of bioavailable small molecules that augment CFTR function is revolutionizing the treatment of CF.
Cystic fibrosis (CF) is one of the most common autosomal recessive disorders associated with decreased longevity in the Caucasian population. The disease is classically described as a triad: chronic obstructive pulmonary disease, exocrine pancreatic insufficiency, and elevation of sodium and chloride concentration in sweat. The underlying pathophysiology involves abnormal ion transport due to dysfunction of the CF transmembrane conductance regulator. Altered salt and water movement across epithelia interferes with the hydration and ionic composition of mucus secretions. Abnormal mucus impairs clearance and defense systems leading to inflammation and destruction of the lungs, the pancreas and the developing vas deferens in males.
The analysis of some extra- and intragenic markers within or closely linked to the cystic fibrosis transmembrane regulator (CFTR) gene is useful as a molecular method in clinical linkage analysis. Indeed, knowing that the molecular basis of cystic fibrosis (CF) is highly heterogeneous in our population, the study of haplotype association with normal and CF chromosomes could be very helpful in cases where one or both mutations remain unidentified. In this study, we analysed with PCR-RFLP and capillary electrophoresis some extra (pJ3.11, KM19 and XV2C) and intragenic (IVS8CA, IVS17bTA and IVS17bCA) polymorphic markers in 50 normal and 10 Tunisian patients carrying the rare E1104X mutation in order to determine the haplotype associated with this mutation. For the extragenic markers, 8 haplotypes were identified. The most frequent of them are the 221 and 112 accounting for 80% of total haplotypes. For the intragenic markers, five haplotypes were present on the E1104X chromosomes. One of them 16-31-13 accounted for 50%. To our knowledge, this is the first work to be interested to the haplotypes linked to the E1104X mutation. This preliminary study of haplotypes could be a helpful method to determine the molecular lesions responsible of this pathology.
delta F508 is the most frequent cystic fibrosis (CF) mutation and accounts for approximately 70% of CF chromosomes worldwide. Three highly polymorphic microsatellite markers have been used to study the origin and evolution of delta F508 chromosomes in Europe. Haplotype data demonstrate that delta F508 occurred more than 52,000 years ago, in a population genetically distinct from any present European group, and spread throughout Europe in chronologically distinct expansions, which are responsible for the different frequencies of delta F508 in Europe.
Three intragenic microsatellites of the CFTR gene, a TA and a CA repeats, namely IVSl7bTA and IVSl7bCA, located in intron 17b and a CA repcat (IVS8CA) located in intron 8 of the CFTR gene, were analyzed in a large sample of Italian cystic fibrosis (CF) and normal chromosomes. Linkage disequilibrium was evaluated between each marker and different CF mutations on a total of 377 CF and 358 normal chromosomes. Our results are consistent with the hypothesis that all AF508 chromosomes derive from a single mutational event. The same hypothesis is valid for mutations G542X, N1303K, 1717-1IG→, which might have been originated more recently than δF508. © 1995 Wiley- Liss, Inc.
Cystic fibrosis (CF) is caused by mutations in the gene for the cystic fibrosis transmembrane conductance regulator (CFTR) that codes for a cAMP-regulated chloride channel. The R347P is a missensemutation located within the first membrane spanning domain (MSD1,) of the CFTR protein. This mutation occurs with an overall worldwide frequency of about 0.2%. The patients, originally described with this mutation were compound heterozygotes with the ΔF508 mutation and had a very mild course of CF, suggesting that R347P, similar to other missense mutations affecting the MSDl domain, causes a mild phenotype. We report here a group of 19 CF patients with the R347P mutation of German, Bulgarian, Czech, and Slovak origin, including two homozygotes. Most patients presented with early disease onset, pancreas insufficiency (PI), and early pulmonary involvement, suggesting that this mutation can lead to a severe course of CF. Most R347P alleles in the group studied share a common polymorphic haplotype. In addition, these analyses gave evidence for recurrence of the mutation in two CF patients of German and Czech origin. © 1995 Wiley-Liss, Inc.
In the cystic fibrosis conductance transmembrane regulator (CFTR) gene a few small deletions and only a large, complex, 50-kb
deletion have been described so far. We report a second large deletion, which had been hypothesized in a patient affected
by cystic fibrosis on the basis of an abnormal pattern of inheritance of the intragenic microsatellites IVS17b/TA and IVS17b/CA.
Southern blot analysis revealed the presence of an anomalous band in the patient and her father, in the region encompassing
exons 13 – 19, approximately 0.6 kb shorten than the one present in normal controls, in addition to the band of the correct
size. Cloning and sequencing the DNA fragments spanning the region of interest demonstrated the presence of a 703-bp deletion
causing complete removal of exon 17b in the paternal cystic fibrosis chromosome. This analysis revealed the presence of two
short direct repeats flanking the breakpoints. The 3′ repeat partially overlapped the IVS17b/CA microsatellite and the number
of CA repeated units present in the paternal cystic fibrosis allele was the shortest ever found among chromosomes so far analyzed.
These data may suggest that the mechanism for the generation of the deletion may have involved a slipped mispairing during
DNA replication, which has not previously been described in the CFTR gene.
We have conducted a comprehensive study of the molecular basis of cystic fibrosis (CF) in 350 German CF patients. A screening approach based on single-strand conformation analysis and direct sequencing of genomic polymerase chain reaction products has allowed us to detect the molecular defects on 95.4% of the CF chromosomes within the coding region and splice sites of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The spectrum of sequence changes comprises 54 different mutations, including 17 missense mutations, 14 nonsense mutations, 11 frameshift mutations, 10 splice site variants and two amino acid deletions. Eleven of these mutations have not previously been described. Our results reflect the marked mutational heterogeneity of CF in a large sample of patients from a non-isolated population.
A cohort of 31 cystic fibrosis patients showing pancreatic sufficiency and bearing an unidentified mutation on at least one chromosome was analyzed through denaturing gradient gel electrophoresis of the whole coding region of the cystic fibrosis transmembrane conductance regulator gene, including intron-exon boundaries. Three new and 19 previously described mutations were detected. The combination of these with known mutations detected by other methods, allowed the characterization of mutations on 56/62 (90.3%) chromosomes. Among those identified, 17 can be considered responsible for pancreatic sufficiency, since they were found in patients carrying a severe mutation on the other chromosome. Among these presumed mild mutations, eight were detected more than once, R352Q being the most frequent in this sample (4.83%). Intragenic microsatellite analysis revealed that the six chromosomes still bearing unidentified mutations are associated with five different haplotypes. This may indicate that these chromosomes bear different mutations, rarely occurring among cystic fibrosis patients, further underlying the molecular heterogeneity of the genetic defects present in patients having pancreatic sufficiency.
The spectra of mutations and polymorphic loci of the gene of cystic fibrosis transmembrane conductance regulator (CFTR) was studied in 60 cystic fibrosis (CF) families from Bashkortostan. Mutations delF508, 394delTT, CFTRdele2,3(21 kb), R334W, and S1196X (33.3, 3.3, 1.7, 0.8, and 0.8%, respectively) were identified. The frequencies of tandem tetranucleotide repeat (TTR) alleles were determined for locus IVS6a-GATT of intron 6 of the CFTR gene and two extragenic loci flanking the CFTR gene, D7S23and MET(probes CS.7 and MetH) in mutant and normal chromosomes. Allelic and haplotypic associations of these loci with the mutations found were estimated. An absolute linkage between the 6TTR allele of locus IVS6a-GATT and the delF508 mutation was ascertained. A considerable linkage disequilibrium between thedelF508mutation and the C2 allele of locus D7S23and between this mutation and the A1 allele of locus MET was found. Most of the other mutant chromosomes carried marker alleles 7TTR, C1, and A2. It was demonstrated that 67% of CF chromosomes carrying delF508 had haplotype 6–2–1 for loci IVS6a-GATT/D7S23/MET, respectively. The frequency distribution of haplotypes in CF chromosomes without delF508had a high variance and did not differ significantly from the distribution in normal chromosomes (2 = 9.415; p > 0.05)).
Simple repeats are abundant in the eukaryotic genome and can markedly affect the expression of genes located in their vicinity.
The mechanism of this effect involves changes in gene transcription and mRNA sensitivity to cell nucleases, frameshift mutations,
etc. The effect depends on the type, length, and location of simple repeats, and is often attributed to their noncanonical
conformation (Z form, triplexes, etc.). An ancient origin is assumed for regulation of gene expression with simple repeats.
We performed prenatal diagnoses for cystic fibrosis in 32 high risk (1:4) couples (including a dizygotic pregnancy). Chorionic villi sampling did not cause abortion or fetal malformation in any case. The preliminary analysis of 9 short tandem repeats always excluded maternal contamination of the DNA extracted from chorionic villi and confirmed paternity. Twenty-two prenatal diagnoses were made by direct analysis of the mutations. In seven cases diagnosis was made by the analysis of intragenic polymorphisms; in three cases, we analyzed two extragenic polymorphisms. The prenatal diagnosis (including genetic counselling) was completed within 24 h from the sampling. Seven prenatal diagnoses revealed an affected fetus; all couples opted for therapeutic abortion. In 17 cases the fetus was heterozygote, and in seven cases it was non carrier of mutated alleles. In the twin pregnancy, mutations were ΔF508/N1303K. Direct analysis of the DNA extracted from the two independent samples of chorionic villi revealed one fetus non carrier of mutated alleles and the other a carrier of the N1303K mutation. Analysis of the HPRT locus predicted both the fetuses as males. Furthermore, the genotype of each fetus was defined after birth. The prenatal diagnosis with chorionic villi sampling plays a key role in the prevention of cystic fibrosis. The laboratories must be equipped for both the direct analysis of mutations and for the analysis of a large number of polymorphisms. The preliminary analysis of short tandem repeats is recommended both to exclude maternal contamination and to confirm parentage.
At present automated genotyping in diagnosis involves the detection, digitrzation, and analysis of labeled DNA using computer software. This chapter describes the use of the Applied Biosystems (Foster City, CA) 373 DNA Sequencer and Genescan 672 software for sizing fluorescently labeled PCR products in a diagnostic molecular genetics laboratory. The Applied Biosystems Genotyper software is not covered since this is not used at present in this laboratory. An outline of the steps involved in automated genotyping, from polymerase chain reaction (PCR) to archiving data, is shown in Fig. 1.
Fig. 1.
Overview of the procedure. Labeled PCR products are produced by either incorporation of fluorescent dNTPs or labeled primers. A polyacrylamide gel is cast, scanned, and prerun, and the Genescan collection and analysis files are set up. The PCR products are mixed with a size standard, denatured, and loaded onto the prerun gel. After electrophoresrs the collected data is transferred to another Macintosh for analysis. A results file is generated and the PCR products are scored and checked. The results file is then archived
Cystic fibrosis (CF) is the most common lethal autosomal recessive disorder in Whites, with an incidence of approx 1 m 2500 live births and a carrier frequency of approx 1 in 25. Since the discovery of the cystic fibrosis transmembrane conductance regulator (CFTR) gene m 1989 (1-3), molecular genetics laboratories throughout the world have endeavored to identify the mutations present in their population of CF-bearing chromosomes. Since the entire CFTR gene and its intron-exon boundaries have been sequenced, mutation analysis in CF has become relatively simple, although time consuming. Generally, a number of different methods are applied to mutation analysis, but all involve an imtial step of amplification of part of the gene by polymerase chain reaction (PCR) (4), or a derivative of it, such as amplification refractory mutation system (ARMS) (5).
Generalised atrophic benign epidermolysis bullosa (GABEB) is a form of junctional epidermolysis bullosa with a recessive mode of inheritance. The gene considered likely to be involved in this disease is COL17A1, since in the majority of GABEB patients the product of that gene, the 180-kD bullous pemphigoid antigen (BP180), is undetectable in skin. We have identified an intragenic COL17A1 microsatellite marker for which 83% of randomly selected control individuals are heterozygous. We observed homozygosity for different alleles of this marker in five out of six collagen type XVII-negative GABEB patients of different European descent. Five of the six COL17A1 alleles of three patients originating from the eastern part of The Netherlands were identical, as were the haplotypes including flanking markers. The 2342delG mutation was identified in all these five alleles. This confirms the expectation that due to genetic drift and hidden inbreeding for an autosomal recessive disorder with low gene frequency, such as collagen type XVII-negative GABEB, most disease alleles from a restricted geographical area will be “identical by descent”. Our results demonstrate that involvement of a candidate gene can be confirmed by looking for identity by descent of highly informative intragenic markers.
Apêndice e anexo Orientador: Nelson Augusto Rosário Filho Tese(doutorado) - Universidade Federal do Paraná, Setor de Ciências da Saúde, Programa de Pós-Graduação em Saúde da Criança e do Adolescente. Defesa: Curitiba, 20 de dezembro de 2006. Inclui bibliografia Área de concentração: Doenças respiratórias
The increasing number of laboratories offering molecular genetic analysis of the CFTR gene and the growing use of commercial kits strengthen the need for an update of previous best practice guidelines (published in 2000). The importance of organizing regional or national laboratory networks, to provide both primary and comprehensive CFTR mutation screening, is stressed. Current guidelines focus on strategies for dealing with increasingly complex situations of CFTR testing. Diagnostic flow charts now include testing in CFTR-related disorders and in fetal bowel anomalies. Emphasis is also placed on the need to consider ethnic or geographic origins of patients and individuals, on basic principles of risk calculation and on the importance of providing accurate laboratory reports. Finally, classification of CFTR mutations is reviewed, with regard to their relevance to pathogenicity and to genetic counselling.
Forty-six CF Italian patients and their parents were screened for a highly polymorphic microsatellite consisting of a variable number of CA/GT repeats in intron 8 of the CFTR gene. A strong degree of association was found between alleles 2 and 6 and the CF mutation delta F508. Moreover, considering the haplotypes at the closely linked locus D7S23 and the microsatellite's alleles, a strong linkage disequilibrium was again found for delta F508 and also for non-delta F508 CF chromosomes and the eight commonest haplotypes (B2, B6, C7, A6, A7, B7, D2 and D7). These data, compared with those described in the Spanish population, further support the common origin of the delta F508 mutation in Southern European populations.
q31-q32), the most common to result in cystic fibrosis (CF) is aF508 (allele fre .. quency 70%) (Kerem, 1989). The significance of polymorphisms in introns 6A, 8, and 17B is un .. clear (Gaspirini et al., 1991; Morral et aI., 1991; Zielenski et al., 1991a). Each of these 3 polymor .. phic regions show at least one pattern strongly as .. sociated with presence of a aF508 allele. Intronic polymorph isms may either have no clear func .. tional significance or may be located in regions which affect RNA splicing and potentially CITR function (Chu et al. , 1991; Zielenski et al. , 1991b). We report a sequence variation of human CFrR intron 17B and describe the frequency of its alleles among CF patients. We detected an intron 17B polymorphism by SSCP screening (Dean et al., 1990) of a PCR product which spanned the 3' end of inrron 178, exon 18, and the 5' end' of intron 18 (Fig. 1). Six percent polyacrylamide (29 acrylamide:0.3 bis) nondenaturing SSCP gels were run at 60 W for 4 hr (4°C). PCR amplification was performed on 150 ng of genomic DNA from a CF patient (25 cycles: 94°C 30 sec, 56°C 30 sec, 72°C 1 min) in a 50 J.1.1 reaction mixture containing 50 mM KCI, 10 mM Tris .. HC1, pH 8.8, 1.5 mM MgCI2, 100 J.1.M dNTP, 0.1 ,.,..1 of 32p dCTP (3000 Ci/mmol), 0.5 U
Cystic fibrosis is the most common autosomal disorder in the Caucasian population. Since the description of the major mutation of this disease in 1989, over 150 of additional mutations have been identified in the CFTR gene. This update summarizes the different mutations identified and reported before March 15 by members of the international Cystic Fibrosis Genetic Analysis Consortium. The report includes information on DNA sequence variations found in the gene.
We have analysed the segregation of a TA-repeat polymorphism in intron 17b of the cystic fibrosis transmembrane conductance regulator gene responsible for cystic fibrosis (CF) in 23 French CF families non-informative for the delta F508 mutation (i.e. with at least one parent not carrying delta F508) or closely linked DNA markers. At least 13 different alleles ranging from 7 to 45 repeats were observed and the detected heterozygosity was 89%. Of the 23 families studied, 19 were fully informative for prenatal diagnosis or carrier detection, 3 were partially informative and one was not informative. In 6 families, prenatal diagnosis for CF or carrier detection in siblings of CF cases were performed using this polymorphism.
We have constructed a 2.4-cM resolution genetic linkage map for chromosome 7q that is bounded by centromere and telomere polymorphisms and contains 66 loci (88 polymorphic systems), 38 of which are uniquely placed with odds for order of at least 1000:1. Ten genes are included in the map and 11 markers have heterozygosities of at least 70%. This map is the first to incorporate several highly informative markers derived from a telomere YAC clone HTY146 (locus D7S427), including HTY146c3 (HET 92%). The telomere locus markers span at least 200 kb of the 7q terminus and no crossovers within the physical confines of the locus were observed in approximately 240 jointly informative meioses. The sex-equal map length is 158 cM and the largest genetic interval between uniquely localized markers in this map is 11 cM. The female and male map lengths are 181 and 133 cM, respectively. The map is based on the CEPH reference pedigrees and includes over 4000 new genotypes, our previously reported data plus 29 allele systems from the published CEPH version 5 database, and was constructed using the program package CRI-MAP. This genetic linkage map can be considered a baseline map for 7q, and will be useful for defining the extent of chromosome deletions previously reported for breast and prostate cancers, for developing additional genetic maps such as index marker and 1-cM maps, and ultimately for developing a fully integrated genetic and physical map for this chromosome.
We surveyed the attitudes of a consecutive sample of 306 pregnant Caucasian women toward carrier screening for cystic fibrosis. Of the 214 respondents, 98% said that screening should be offered before pregnancy, and 69% said they would accept carrier screening during pregnancy. Twenty-nine percent of the respondents indicated a willingness to terminate a pregnancy if the fetus were found to have cystic fibrosis. We conclude that carrier screening is of interest to pregnant women, although interest in terminating a pregnancy because of screening results may be limited.
Cystic fibrosis (CF) is the most common severe recessive genetic disorder in the Caucasian population. In 1938, D. H. Anderson provided the first comprehensive description of the disease and also introduced the name “cystic fibrosis of the pancreas.” Patients with CF suffer from excessive mucus accumulation resulting in severe clinical consequences in the respiratory, gastrointestinal, and genitourinary tracts (see Table I). All these symptoms are consistent with defects of exocrine glands, as suggested by S. Farber in 1945; he called the disease “mucoviscidosis,” a name still popular in some parts of continental Europe. CF patients also have elevated electrolyte levels in their sweat, an observation which, first described by di Sant’Agnese et
al. (1953), became the hallmark for CF diagnosis.
Microsatellite analysis of chromosomes carrying particular cystic fibrosis mutations has shown different haplotypes in four cases: R334W, R347P, R1162X, and 3849 + 10kbC-->T. To investigate the possibility of recurrence of these mutations, analysis of intra- and extragenic markers flanking these mutations has been performed. Recurrence is the most plausible explanation, as it becomes necessary to postulate either double recombinations or single recombinations in conjunction with slippage at one or more microsatellite loci, to explain the combination of mutations and microsatellites if the mutations arose only once. Also in support of recurrence, mutations R334W, R347P, R1162X, and 3849 + 10kbC-->T involve CpG dinucleotides, which are known to have an increased mutation rate. Although only 15.7% of point mutations in the coding sequence of CFTR have occurred at CpG dinucleotides, approximately half of these CpG sites have mutated at least once. Specific nucleotide positions of the coding region of CFTR, distinct from CpG sequences, also seem to have a higher mutation rate, and so it is possible that the mutations observed are recurrent. G-->A transitions are the most common change found in those positions involved in more than one mutational event in CFTR.
The use of automated DNA fragment analysis with the Applied Biosystems 672 Genescanner system was evaluated in a routine diagnostic setting. The aim of the study was to compare automated fragment detection and analysis with conventional methods. For cystic fibrosis analysis the delta F508 mutation in exon 10 of the cystic fibrosis transmembrane regulator (CFTR) gene was multiplexed with two intragenic microsatellites. The analysis of the Prader-Willi/Angelman region of chromosome 15 used a panel of five microsatellites. For dystrophin, seven microsatellites covering the entire dystrophin gene were co-amplified. Automated analysis was faster and more accurate than analysis using radiolabelled products with sequencing gels, although some inconsistencies in the sizing of microsatellite alleles were seen.
The cystic fibrosis transmembrane conductance regulator gene contains three highly informative microsatellites; IVS8CA, IVS17BTA and IVS17BCA. Fluorescent multiplexes of these microsatellites were assayed in 124 CF families carrying 15 different CFTR mutations, from N. Ireland.
We have studied the feasibility of using dinucleotide-repeat microsatellites in the analysis of DNA from ancient bones and teeth. We have used three microsatellites (IVS8CA, IVS17BTA, and IVS17BCA) within the cystic fibrosis transmembrane conductance regulator gene in 28 DNA samples from bones and teeth of up to 5000 years of age. PCR amplification was successful in 71.4% of cases. The repeated analysis of each marker produced different genotypes in 97% of samples, and the same individual genotype was reproduced at least once in 45.5% of cases. Alleles differing from the originals consisted of additions or deletions of 1-39 dinucleotides. The mechanism by which alleles differing from the originals were amplified can be related to the marked degradation of the DNA, with repeat sequences of different length interacting with the partially degraded repeats of the amplified loci. The repeated analysis of each sample allowed us to produce data with some anthropological interest. Among the haptotypes detected in samples from Easter Island, two (16-32-13 and 23-32-13) were found in more than one sample. Similarly, three haplotypes (16-7-17, 16-7-13, and 16-24-13) were detected more than once in samples from the Basque Country. Although haplotypes in the Basque Country are amongst the commonest in European chromosomes, most of those detected in the Easter Island samples are not frequent in Europeans. Thus, the repeated typing of microsatellites allowed us to postulate the genotypes that might be present in the samples but dinucleotide markers do not seem to be reliable enough for genotyping ancient bone and teeth samples.
Molecular diagnosis of cystic fibrosis (CF) in the Italian population, based on the detection of the deltaF508 mutation (51.2% of CF chromosomes), provides full informativity for prenatal diagnosis (PDN) in about 28% of families at risk. Identification of the predominant non-deltaF508 mutations allows the characterization of about 70% of CF chromosomes, making approximately 48% of couples fully informative. In families where at least one chromosome remains uncharacterized, allele segregation is still determined using RFLPs closely linked to the CF gene. The recent identification of three polymorphic clusters of dinucleotide repeats (IVS8/GT, IVS17b/TA and IVS17b/CA) led us to evaluate whether their analysis might improve feasibility studies for prenatal diagnosis or heterozygote identification. One hundred nuclear families with a CF child, reflecting the general Italian deltaF508 mutation distribution, were genotyped for the three microsatellites. In this study microsatellite analysis using IVS8/GT and IVS17b/TA allowed the identification of both parental CF chromosomes in 94% of couples; inclusion in the study of the less polymorphic repeat locus, IVS17b/CA, slightly improved this percentage (97%). Hence, a strategy involving primarily the detection of the deltaF508 mutation and secondarily microsatellite analysis makes possible PDN of CF in virtually all Italian CF families.
In cystic fibrosis (CF), the most common mutation, delta F508 (a three-base-pair deletion) accounts for ca. 70% of mutations in the worldwide population. The majority of other mutations (more than 350 reported so far to the Genetic Analysis Consortium) have been detected in single cases, thus rendering quite cumbersome a molecular diagnostic approach for the identification of CF chromosomes. As an alternative, linkage analysis based on intragenic polymorphism can be useful for prenatal diagnosis and CF-carrier detection, provided that the heterozygosity of the allelic forms is very high. For this purpose, DNA microsatellites, consisting of two to epta nucleotide repeat clusters, displaying a high degree of polymorphism, are being increasingly used as markers in linkage studies. Two main allelic forms, one hexameric (111 bp) and one heptameric (115 bp), of a tetranucleotide (GATT) repeat polymorphism, at the junction of intron IVS6a and exon 6b, have been amplified by PCR technology. These two alleles can be separated in a 10-20% T polyacrylamide gradient gel and detected by ethidium bromide staining. As an alternate procedure, these two fragments are efficiently separated by capillary zone electrophoresis in a viscous solution of 6%T linear polyacrylamide and detected by their intrinsic absorbance at 254 nm.
We have determined the frequency of deletion delta F508 and mutation G542X, a nonsense mutation in exon 11 of the cystic fibrosis (CF) gene, in a sample of 400 Spanish CF families. Mutation G542X represents 8% of the total number of CF mutations in Spain, making it the second most common mutation after the delta F508 deletion, which accounts for 48% of CF chromosomes. G542X has a higher frequency in the Mediterranean coastal area (14%) and in the Canary Islands (25%). About 70% of G542X chromosomes are from Andalucia, Múrcia, Valencia, Catalunya and the Canary Islands. The delta F508 deletion has its highest frequency in the Basque Country (83%). Mutation G542X is associated with the same rare haplotype that is found in association with the delta F508 mutation. The haplotype homogeneity found for G542X, even when intragenic microsatellites (IVS8CA, IVS17BTA and IVS17BCA) are considered, allows us to postulate that this mutation arose from a single mutational event. The geographic distribution of mutations delta F508 and G542X suggests that delta F508 was present in the Iberian Peninsula before the Indo-European invasions, and that G542X was introduced into Spain, via the Mediterranean Sea, probably by the Phoenicians, between 2500 and 3000 years ago.
We describe a fast and reliable method for the nonradioactive analysis of microsatellites. For three dinucleotide repeats within the cystic fibrosis transmembrane conductance regulator (CFTR) gene, the separation of polymerase chain reaction (PCR) products generated with biotinylated primers on a direct blotting electrophoresis system and subsequent chemiluminescence detection is shown. In direct blotting electrophoresis, the separation of DNA fragments depended linearly on size. The reproducible resolution allowed reliable assignment of allele lengths to a given signal. The nonradioactive detection protocol was advantageous compared to radioactive methods: samples could be analyzed within one day due to the fast signal development by 3-(4-methoxyspiro[1,2-dioxetane-3,2'-(5'- chloro)tricyclo[3.3.1.1.3,7]decan]-4-yl)phenylphosphate disodium salt (CSPD). Variation of exposure times enabled differentiation between major bands and byproducts of comparable intensity that are due to the slippage of the Taq polymerase during PCR amplification.
The association of maternal uniparental disomy for chromosome 7 and postnatal growth failure has been reported in four cases and suggests the presence of genomic imprinting of one or more growth related genes on chromosome 7. However, in the reported cases, the possibility of homozygosity for a recessive mutation could not be excluded as the cause of the growth failure as in all cases isodisomy rather than heterodisomy for chromosome 7 was present. We report a case of prenatal and postnatal growth retardation associated with a prenatal diagnosis of mosaicism for trisomy 7 confined to the placenta. DNA typing of polymorphic markers on chromosome 7 has established that the zygote originated as a trisomy 7 with two maternal and one paternal chromosomes 7 with subsequent loss of the paternal chromosome resulting in a disomic child with maternal heterodisomy for chromosome 7. The growth failure seen in this child with heterodisomy 7 lends strong support to the hypothesis of imprinted gene(s) on chromosome 7.
Search for mutations in a cystic fibrosis patient, compound heterozygous for 1717-1G-->A and another uncharacterized molecular defect, revealed the presence of a de novo R1066H mutation on the affected chromosome of paternal origin. Three additional rare mutations (R1066C, R1066S and R1066L), occurring at the CpG dinucleotide at position 3328-3329 of the cystic fibrosis transmembrane conductance regulator gene, have so far been reported. The identification of a R1066H de novo mutation further suggests that this dinucleotide may constitute a mutational hotspot.
Replication errors (RERs) were initially identified in hereditary nonpolyposis colon cancer and other tumors of Lynch syndrome II. Mutations in genes involved in mismatch repair give rise to a mutator phenotype, resulting in RERs. The mutator phenotype is thought to predispose to malignant transformation. Here we show that in the embryonal form of childhood rhabdomyosarcoma, RERs also occur, but in contrast to hereditary nonpolyposis colon cancer, only a subset of the microsatellite loci analyzed show RERs. The occurrence of RERs is strongly correlated with increased fractional allelic loss (P < 0.001), suggesting that the occurrence of RERs is a secondary phenomenon in rhabdomyosarcoma. Coincidental loss of genes involved in mismatch repair, possibly due to their proximity to tumor suppressor genes involved in tumor progression of embryonal form of childhood rhabdomyosarcoma, could explain the observed phenomenon.
We have found records of 1014 Irish cystic fibrosis patients alive by December 1994, belonging to 883 families. Prevalence in the population is 1/3475 and incidence at birth 1/1461, with a gene frequency of 2.6%. Twenty percent of the patients are aged over 20 years, but at present survival rate falls rapidly after that age. We have identified 85% of the mutations on the CFTR gene in a sample of 29% of the families (506 CF chromosomes). Mutation delta F508 is found in 72% of Irish CF chromosomes, G551D in 6.9%, and R117H in 2%. These are the highest frequencies reported for the latter two mutations world wide. Another seven mutations are found in an additional 4% of CF families. We present new microsatellite haplotype data that could be useful for genetic counselling of CF families bearing some of the 15% of CF mutations still unidentified, and comment on possible uses of our database.
The clinical and pathologic features of patients with cystic fibrosis are summarized, and generalized genotype or phenotype correlations are discussed in this article. Incorporation of modern molecular biologic techniques into a rapid, cost-efficient, and specific diagnostic laboratory test is outlined. The protocol for the multiplex polymerase chain reaction detection of five common cystic fibrosis transmembrane conductance regulator (CFTR) mutations by ASO hybridization is detailed. Neonatal screening and issues involved in the genetic counseling of families at risk for cystic fibrosis are presented. Recommendations for molecular diagnostic testing in cystic fibrosis are made.
The gene for Friedreich ataxia (FRDA), an autosomal-recessive neurodegenerative disease, remains elusive. The current candidate region of about 150 kb lies between loci FR2 and F8101 near the D9S15/D9S5 linkage group at 9q13-21.1. Linkage homogeneity between classical FRDA and a milder, slowly progressive Acadian variant (FRDA-Acad) has been demonstrated. An extended D9S15-D9S5 haplotype (C6) predominates in FRDA-Acad chromosomes from Louisiana. We studied 10 Acadian families from New Brunswick, Canada. In eight families, affected individuals conformed to the clinical description of FRDA-Acad; in one, 2 sibs presented with spastic ataxia (SPA-Acad). In the last family, 2 sibs had FRDA-Acad, and one had SPA-Acad. We found that SPA-Acad is linked to the FRDA gene region. The C6 haplotype and a second major haplotype (B7) were identified. The same ataxia-linked haplotypes segregated with both FRDA-Acad and SPA-Acad in two unrelated families. The parental origins of these haplotypes were different. Our observation of different phenotypes associated with the same combination of haplotypes may point to the influence of the parent of origin on gene expression, indicate the effect of modifier genes, or reflect the presence of different mutations on the same haplotypes. Our findings underline the need to investigate families with autosomal-recessive ataxias for linkage to the FRDA region, despite lack of key diagnostic manifestations such as cardiomyopathy or absent deep-tendon reflexes.
The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene contains three highly informative microsatellites: IVS8CA, IVS17bTA, and IVS17bCA. Their analysis improves prenatal/ carrier diagnosis and generates haplotypes from CF chromosomes that are strongly associated with specific mutations. Microsatellite haplotypes were defined for 75 CFTR mutations carried on 437 CF chromosomes (220 for delta F508, 217 for other mutations) from Northern Ireland and three English regions: the North-West, East Anglia, and the South. Fluorescently labelled microsatellites were amplified in a triplex PCR reaction and typed using an ABI 373A fluorescent fragment analyser. These mutations cover all the common and most of the rare CF defects found in the UK, and their corresponding haplotypes and geographic region are tabulated here. Ancient mutations, delta F508, G542X, N1303K, were associated with several related haplotypes due to slippage during replication, whereas other common mutations were associated with the one respective haplotype (e.g., G551D and R560T with 16-7-17, R117H with 16-30-13, 621 + 1G > T with 21-31-13, 3659delC with 16-35-13). This simple, fast, and automated method for fluorescent typing of these haplotypes will help to direct mutation screening for uncharacterised CF chromosomes.
The 3′ flanking region of the interleukin 6 gene is polymorphic due to insertions of different size. Within this region lies
a sequence of approximately 500 base pairs that is AT rich. Based on flanking sequence information we have constructed oligonucleotides
which prime the polymerase chain reaction (PCR) and amplify this AT rich region. The amplification products visualized by
agarose gel electroplioresis gave fragment sizes for both homozygous and heterozygous individuals that were concordant with
those observed by conventional genomic blotting techniques. Alleles that could not be typed by Southern analysis were resolved
with this approach. These results illustrate the value of PCR for the rapid detection of length polymorphisms such as those
due to variable numbers of tandem repeats. In contrast to RFLP analysis this procedure takes less than a day to perform, is
cheaper, avoids the use of radioactivity and requires far less substrate DNA. Three different human alleles were sequenced,
and differences were detected that were due to both large duplications and loss of one or two bases, suggesting that AT rich
regions identify highly polymorphic loci. The same pruners also amplified non-human primate DNA, allowing a comparison of
the human sequence with that of the common chimpanzee and baboon.
CYSTIC fibrosis is the most common lethal inherited disorder in the white population. It is manifested by viscous secretions in the lungs and pancreas and abnormal electrolyte composition of sweat.1 Epithelial cells from patients with cystic fibrosis have abnormal conductance of chloride ions across apical membranes due to defective regulation of a particular chloride channel.2 3 4 Inadequate secretion of chloride is believed to cause the insufficient hydration of mucus in the airways and pancreatic ducts.1 The cystic fibrosis gene has recently been identified, and it is predicted to encode a 1480-amino-acid protein termed the cystic fibrosis transmembrane conductance regulator (CFTR).5 , 6 The . . .
Additional mutations in the cystic fibrosis (CF) gene were identified in the regions corresponding to the two putative nucleotide (ATP)-binding folds (NBFs) of the predicted polypeptide. The patient cohort included 46 Canadian CF families with well-characterized DNA marker haplotypes spanning the disease locus and several other families from Israel. Eleven mutations were found in the first NBF, 2 were found in the second NBF, but none was found in the R-domain. Seven of the mutations were of the missense type affecting some of the highly conserved amino acid residues in the first NBF; 3 were nonsense mutations; 2 would probably affect mRNA splicing; 2 corresponded to small deletions, including another 3-base-pair deletion different from the major mutation (delta F508), which could account for 70% of the CF chromosomes in the population. Nine of these mutations accounted for 12 of the 31 non-delta F508 CF chromosomes in the Canadian families. The highly heterogeneous nature of the remaining CF mutations provides important insights into the structure and function of the protein, but it also suggests that DNA-based genetic screening for CF carrier status will not be straightforward.
The distribution patterns of shared short repetitive motifs in the expansion segments of the large subunit rRNA genes of different
species show that these segments are coevolving as a set and that in two examined vertebrate species the RNA secondary structures
are conserved as a consequence of runs of motifs in one region being compensated by complementary motifs in another. These
unusual processes, involving replication-slippage, have implications for the evolution of ribosomal RNA and for the use of
the rDNA multigene family as a ‘molecular clock’ for assessing relationships between species.
Overlapping complementary DNA clones were isolated from epithelial cell libraries with a genomic DNA segment containing a
portion of the putative cystic fibrosis (CF) locus, which is on chromosome 7. Transcripts, approximately 6500 nucleotides
in size, were detectable in the tissues affected in patients with CF. The predicted protein consists of two similar motifs,
each with (i) a domain having properties consistent with membrane association and (ii) a domain believed to be involved in
ATP (adenosine triphosphate) binding. A deletion of three base pairs that results in the omission of a phenylalanine residue
at the center of the first predicted nucleotide-binding domain was detected in CF patients.
Z-DNA-forming sequences are shown to elicit a biological response in Escherichia coli. Plasmids containing sequences capable of adopting the Z conformation (GC and CA/GT) are shown to be hot spots for spontaneous deletions. All the deletions involve an even number of base pairs. The distribution of the deletion events shows that the process ends when the Z-DNA-forming sequence has been reduced to a size no longer capable of adopting the Z conformation at natural superhelical density.
An understanding of the basic defect in the inherited disorder cystic fibrosis requires cloning of the cystic fibrosis gene
and definition of its protein product. In the absence of direct functional information, chromosomal map position is a guide
for locating the gene. Chromosome walking and jumping and complementary DNA hybridization were used to isolate DNA sequences,
encompassing more than 500,000 base pairs, from the cystic fibrosis region on the long arm of human chromosome 7. Several
transcribed sequences and conserved segments were identified in this cloned region. One of these corresponds to the cystic
fibrosis gene and spans approximately 250,000 base pairs of genomic DNA.
Interspersed DNA elements of the form (dC-dA)n.(dG-dT)n constitute one of the most abundant human repetitive DNA families. We report that specific human (dC-dA)n.(dG-dT)n blocks are polymorphic in length among individuals and therefore represent a vast new pool of potential genetic markers. Comparison of sequences from the literature for (dC-dA)n.(dG-dT)n blocks cloned two or more times revealed length polymorphisms in seven of eight cases. Variations in the lengths of 10 (dC-dA)n.(dG-dT)n blocks were directly demonstrated by amplifying the DNA within and immediately flanking the repeat blocks by using the polymerase chain reaction and then resolving the amplified DNA on polyacrylamide DNA sequencing gels. Use of the polymerase chain reaction to detect DNA polymorphisms offers improved sensitivity and speed compared with standard blotting and hybridization.
We have characterised a highly po1ymorphic region 1.3kb downstream of the human Type II collagen gene. It consists of a highly
AT-rich tandem repetitive region (minisatellite) approximately 650bp long. Two alleles had been observed previously, differing
in size by approximately 300bp. When this region was cloned from four four unrelated individuals carrying the larger allele,
DNA sequence data identified three alleles, suggesting far higher polymorphism than was originally supposed. This minisatellite
was shown to be present in a single copy in the human genome, and to have arisen after the divergence of Old and New World
monkeys.
Analysis of DNA from the beta-globin gene cluster in an Albanian family identified a novel RsaI site approximately 550 base pairs 5' to the beta-globin gene. In this family, two chromosomes carrying otherwise identical beta-globin haplotypes were found to differ at the RsaI site. Population screening demonstrated the presence and absence of the site in DNA from individuals of northern European, Mediterranean, Middle Eastern, Southeast Asian, African, and Asian Indian descent, indicating that this site is a DNA polymorphism common in many ethnic groups. The polymorphism is also present in DNA from individuals carrying different beta-globin alleles. Additional nucleotide sequence changes identified in an RsaI (+) genomic clone in the region immediately 3' to the RsaI site suggest a mechanism for the randomization of the site with respect to haplotype.
A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction. The enzyme, isolated from Thermus aquaticus, greatly simplifies the procedure and, by enabling the amplification reaction to be performed at higher temperatures, significantly improves the specificity, yield, sensitivity, and length of products that can be amplified. Single-copy genomic sequences were amplified by a factor of more than 10 million with very high specificity, and DNA segments up to 2000 base pairs were readily amplified. In addition, the method was used to amplify and detect a target DNA molecule present only once in a sample of 10(5) cells.
Approximately 70 percent of the mutations in cystic fibrosis patients correspond to a specific deletion of three base pairs, which results in the loss of a phenylalanine residue at amino acid position 508 of the putative product of the cystic fibrosis gene. Extended haplotype data based on DNA markers closely linked to the putative disease gene locus suggest that the remainder of the cystic fibrosis mutant gene pool consists of multiple, different mutations. A small set of these latter mutant alleles (about 8 percent) may confer residual pancreatic exocrine function in a subgroup of patients who are pancreatic sufficient. The ability to detect mutations in the cystic fibrosis gene at the DNA level has important implications for genetic diagnosis.
This is a report of the results of a worldwide survey of the most common mutation (deltaF508) that causes cystic fibrosis (CF). The data are listed according to geographical location (in alphabetic order), beginning with the group with the largest number of CF chromosomes. Data consisting of fewer than 10 chromosomes are not included. The overall frequency of deltaF508 in the present mutant CF gene pool is .68, but the study populations are not equally represented; there is marked variation in the proportion of deltaF508 among different geographic populations.
We have determined the frequency of the cystic fibrosis (CF) ΔF508 mutation in a large sample of CF patients originating from
different areas of France, including the greater Paris, Brittany, Alsace, Lorraine and Rhône-Alpes regions. A total of 422
CF chromosomes were studied, and the defect was found to account for 75% of the mutant alleles. In the course of the survey,
a rare nucleotide sequence polymorphism leading to an isoleucine to valine substitution at position 506 of the CF transmembrane
conductance regulator protein has been characterized in an unaffected individual. Our data enable the evaluation of the probabilities
that a chromosome negative for the ΔF508 mutation carriers another CF defect.
A new method for determining nucleotide sequences in DNA is described. It is similar to the "plus and minus" method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441-448] but makes use of the 2',3'-dideoxy and arabinonucleoside analogues of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase. The technique has been applied to the DNA of bacteriophage varphiX174 and is more rapid and more accurate than either the plus or the minus method.
We describe a polymorphic microsatellite (IVS17BCA) in intron 17B of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. It consists of an 11 to 20 CA/GT dinucleotide repeat, located 424 bp from exon 17B.
DNA 5' to the human myelin basic protein (MBP) gene, mapped to 18q22----qter, is known to manifest multiallelic DNA length variation with heterozygosity of at least 45%. Isolation of genomic DNA containing the MBP gene first exon and its 5' flanking region reveals that this polymorphism arises from a 994-bp region of the diverged tandem repeat (TGGA)249. This sequence is located from 1082 to 2075 bp upstream of the MBP initiator methionine. The repetitive sequence is 18% diverged from (TGGA)249 and from analysis of higher order subsequence reiterations appears to have undergone extensive recombination. The pattern of higher order repetition suggests that multiple crossover and gene conversion events have occurred within a 1.0-kb region. Molecular clones of this sequence represent essentially the longest allelic form of this region seen in Southern transfer analysis. This repetitive DNA is similar to a sequence 5' to the human myoglobin gene.
Cystic fibrosis (CF) is a common recessive lethal genetic disorder, affecting 1 in 1,600 Caucasians. The disease causes defective regulation of chloride-ion transport in exocrine cells. Although in all CF families the disease is linked to a locus on chromosome 7q31, there is clinical heterogeneity in the severity of the disease and the age at which it is diagnosed. CF is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. A three-nucleotide deletion (delta F508) causing the loss of a phenylalanine residue in the tenth exon of the CFTR gene has been found on 70% of CF chromosomes. We have now characterized a CF family in which neither parent of the affected individual carries the common mutation, and identified a two-nucleotide insertion in the CF allele of the mother. The mutation introduces a termination codon in exon 13 of the CFTR gene at residue 821, and is predicted to result in the production of a severely truncated nonfunctional protein.
The gene responsible for cystic fibrosis (CF) has recently been identified and is predicted to encode a protein of 1,480 amino acids called the CF transmembrane conductance regulator (CFTR). Several functional regions are thought to exist in the CFTR protein, including two areas for ATP-binding, termed nucleotide-binding folds (NBFs), a regulatory (R) region that has many possible sites for phosphorylation by protein kinases A and C, and two hydrophobic regions that probably interact with cell membranes. The most common CF gene mutation leads to omission of phenylalanine residue 508 in the putative first NBF, indicating that this region is functionally important. To determine whether other mutations occur in the NBFs of CFTR, we determined the nucleotide sequences of exons 9, 10, 11 and 12 (encoding the first NBF) and exons 20, 21 and 22 (encoding most of the second NBF) from 20 Caucasian and 18 American-black CF patients. One cluster of four mutations was discovered in a 30-base-pair region of exon 11. Three of these mutations cause amino-acid substitutions at residues that are highly conserved among the CFTR protein, the multiple-drug-resistance proteins and ATP-binding membrane-associated transport proteins. The fourth mutation creates a premature termination signal. These mutations reveal a functionally important region in the CFTR protein and provide further evidence that CFTR is a member of the family of ATP-dependent transport proteins.
Five different mutations have been identified in the gene causing cystic fibrosis (CF) through sequencing regions encompassing exons 1-8, including the 5' untranslated leader. Two of these apparent mutations are missense mutations, one in exon 3 (Gly to Glu at position 85; G85E) and another in exon 5 (Gly to Arg at 178; G178R), both causing significant changes in the corresponding amino acids in the encoded protein--cystic fibrosis transmembrane conductance regulator (CFTR). Two others affect the highly conserved RNA splice junction flanking the 3' end of exons 4 and 5 (621 + 1G----T, 711 + 1G----T), resulting in a probable splicing defect. The last mutation is a single-basepair deletion in exon 4, causing a frameshift. These five mutations account for the 9 of 31 non-delta F508 CF chromosomes in our Canadian CF family collection and they are not found in any of the normal chromosomes. Three of the mutations, 621 + 1G----T, 711 + 1G----T, and G85E, are found in the French-Canadian population, with 621 + 1G----T being the most abundant (5/7). There are two other sequence variations in the CFTR gene; one of them (129G----C) is located 4 nucleotides upstream of the proposed translation initiation codon and, although present only on CF chromosomes, it is not clear whether it is a disease-causing mutation; the other (R75Q) is most likely a sequence variation within the coding region.
The gene responsible for cystic fibrosis, the most common severe autosomal recessive disorder, is located on the long arm of human chromosome 7, region q31-q32. The gene has recently been identified and shown to be approximately 250 kb in size. To understand the structure and to provide the basis for a systematic analysis of the disease-causing mutations in the gene, genomic DNA clones spanning different regions of the previously reported cDNA were isolated and used to determine the coding regions and sequences of intron/exon boundaries. A total of 22,708 bp of sequence, accounting for approximately 10% of the entire gene, was obtained. Alignment of the genomic DNA sequence with the cDNA sequence showed perfect colinearity between the two and a total of 27 exons, each flanked by consensus splice signals. A number of repetitive elements, including the Alu and Kpn families and simple repeats, such as (GT)17, (GATT)7, and (TA)14, were detected in close vicinity of some of the intron/exon boundaries. At least three of the simple repeats were found to be polymorphic in the population. Although an internal amino acid sequence homology could be detected between the two halves of the predicted polypeptide, especially in the regions of the two putative nucleotide-binding folds (NBF1 and NBF2), the lack of alignment of the nucleotide sequence as well as the different positions of the exon/intron boundaries does not seem to support the hypothesis of a recent gene duplication event. To facilitate detection of mutations by direct sequence analysis of genomic DNA, 28 sets of oligonucleotide primers were designed and tested for their ability to amplify individual exons and the immediately flanking sequences in the introns.
The gene responsible for cystic fibrosis (CF) has recently been identified, and a three-nucleotide deletion (delta F508 mutation) that results in the loss of a phenylalanine residue in the first putative ATP-binding domain of the predicted protein (CF transmembrane conductance regulator, CFTR) has been found to be the major CF mutation. Although several other mutations have been identified in the CFTR gene, most of them are very rare, making their application to genetic diagnosis difficult. While characterizing the genomic region encompassing the CF locus, we have identified three CA/GT blocks that flank exon 9 of the CF gene. One of the CA/GT blocks exhibits a highly informative variable number of dinucleotide repeats (VNDR) polymorphism. This intragenic VNDR microsatellite should, by itself, provide full information for genetic analysis in approximately 80% of CF families and will help elucidate the associations between DNA polymorphism haplotypes and specific gene mutations. Haplotype analyses of CF chromosomes with and without the delta F508 mutation suggest that the different alleles are generated by slipped-strand mispairing within the dinucleotide repeat during DNA replication, rather than by unequal crossingover within a recombination hot spot.
We have determined the frequency of the cystic fibrosis (CF) delta F508 mutation in a large sample of CF patients originating from different areas of France, including the greater Paris, Brittany, Alsace, Lorraine and Rhône-Alpes regions. A total of 422 CF chromosomes were studied, and the defect was found to account for 75% of the mutant alleles. In the course of the survey, a rare nucleotide sequence polymorphism leading to an isoleucine to valine substitution at position 506 of the CF transmembrane conductance regulator protein has been characterized in an unaffected individual. Our data enable the evaluation of the probabilities that a chromosome negative for the delta F508 mutation carriers another CF defect.
The gene causing cystic fibrosis (CF) has been recently cloned, and the major mutation (delta F508) accounting for approximately 70% of CF chromosomes has been uncovered. We have identified at the 3' end of intron 6 in the CF gene a 4-bp tandem repeat (GATT) that exhibits interesting features. First, PCR screening of 103 normal individuals revealed that the repeat exists only in two polymorphic allelic forms, either as a hexamer or a heptamer. These two alleles are in Hardy-Weinberg equilibrium and predict a heterozygote frequency of 41% (p[seven repeats] = .71; q [six repeats] = .29). Second, the allele with six repeats was found linked to delta F508 on all 76 CF chromosomes investigated, demonstrating strong linkage disequilibrium and suggesting that delta F508 had originated on the gene bearing six repeats. Third, when the repeat alleles are linked to the DNA markers XV2c and KM19, extended haplotypes are generated. These new haplotypes become informative in situations in which prenatal diagnosis cannot be performed solely with XV2c and KM19. Since this repeat marker is located in the CF gene and would be very less likely to recombine with the gene, it can serve as a valuable DNA marker for haplotype analysis. A possible crossover, however, was identified between XV2c and KM19, transferring delta F508 to a different haplotype.
In the cystic fibrosis (CF) gene, recently cloned, a three base pair deletion (delta F508) has been identified in a majority of CF patients. This deletion has been found in 80% of CF chromosomes in families from north west Brittany. In order to identify new mutations we have selected 43 chromosomes negative for the three base pair deletion from these families and directly sequenced exon 11 after DNA amplification by the polymerase chain reaction. We have detected a base change (G----A) at the 3' end of the consensus sequence of intron ten (namely 1717-1). This mutation destroys a splice site in the cystic fibrosis gene which probably produces a mutant allele. This single nucleotide mutation has been reported on two other CF chromosomes.
The cystic fibrosis (CF) gene was recently identified as a gene spanning 250 kilobases (kbp) and coding for a 1480 amino acid protein, cystic fibrosis transmembrane conductance regulator (CFTR). Approximately 70% of CF mutations involve a three-base-pair deletion in CFTR exon 10, resulting in the loss of a phenylalanine at position 508 in the gene product (delta F508). In order to screen for other molecular defects, we have used a strategy based on denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified gene segments. This method, which permits rapid detection of any sequence change in a given DNA stretch, was used successfully to analyse 61 non-delta F508 CF chromosomes from French CF patients. A study of CFTR exons 10, 11, 14a, 15 and 20 detected three mutations located in exons 14a, 15 and 20, along with several nucleotide sequence polymorphisms. These nucleotide changes were identified by direct sequencing of PCR fragments displaying altered electrophoretic behaviour, together with some of the polymorphisms and mutations previously characterized by others. The strategy presented here constitutes a valuable tool for the development of carrier testing for individuals or couples with a family history of cystic fibrosis, and will contribute to deciphering the functionally important regions of the CFTR gene.
The human genome contains approximately 50,000 copies of an interspersed repeat with the sequence (dT.dG/dA.dC)n, where n = approximately 10-60. We and others have found that several of these repeats have variable lengths in different individuals, with allelic fragments varying in size by multiples of 2 bp. These "microsatellite" variable number of tandem repeats (VNTRs) may be scored by PCR, using unique flanking primers to amplify the repeat-containing regions and resolving the products on DNA sequencing gels. Since few VNTRs have been found on the X chromosome, we screened a flow-sorted X chromosome-specific genomic library for microsatellites. Approximately 25% of the phage clones hybridized to a poly (dT-dG).poly(dA-dC) probe. Of seven X-linked microsatellites present in positive phages, five are polymorphic and three have both eight or more alleles and heterozygosities exceeding 75%. Using PCR to amplify genomic DNAs from hybrid cell panels, we confirmed the X localization of these VNTRs and regionally mapped four of them. The fifth VNTR was regionally mapped by virtue of its tight linkage to DXS87 in Centre du Polymorphisme Humain families. We conclude that whatever factors limit the occurrence of "classical" VNTRs and RFLPs on the X chromosome do not appear to operate in the case of microsatellite VNTRs.
To identify DNA polymorphisms that are abundant in the human genome and are detectable by polymerase chain reaction amplification of genomic DNA, we tested the hypothesis that the polydeoxyadenylate tract of the Alu family of repetitive elements is polymorphic among human chromosomes. We analyzed the 3' ends of three specific Alu sequences and found that two (in the adenosine deaminase gene and the beta-globin pseudogene) were polymorphic. This novel class of polymorphisms, termed AluVpA [Alu variable poly(A)] may represent one of the most useful and informative group of DNA markers in the human genome.
We have identified three different point mutations in the coding region of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Each mutation segregates with the disease in two- or three-generation pedigrees and is not found on the normal chromosome of any documented cystic fibrosis carrier. One of the mutations is found in two independent families that contain at least one individual with a mild course of disease. All of these alterations replace charged amino acids with less polar residues and are found in the putative transmembrane sections of the molecule. The mutated amino acids are found to be conserved in both rodents and amphibians and lie in a region of CFTR that is believed to form a channel in the membrane. Although these alterations are rare, they provide important clues to functionally important regions of the molecule.
A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction. The enzyme,
isolated from Thermus aquaticus, greatly simplifies the procedure and, by enabling the amplification reaction to be performed
at higher temperatures, significantly improves the specificity, yield, sensitivity, and length of products that can be amplified.
Single-copy genomic sequences were amplified by a factor of more than 10 million with very high specificity, and DNA segments
up to 2000 base pairs were readily amplified. In addition, the method was used to amplify and detect a target DNA molecule
present only once in a sample of 10(5) cells.
The human genome contains approximately 50,000 copies of an interspersed repeat with the sequence (dT-dG)n, where n = approximately 10-60. In humans, (TG)n repeats have been found in several sequenced regions. Since minisatellite regions with larger repeat elements often display extensive length polymorphisms, we suspected that (TG)n repeats ("microsatellites") might also be polymorphic. Using the polymerase chain reaction to amplify a (TG)n microsatellite in the human cardiac actin gene, we detected 12 different allelic fragments in 37 unrelated individuals, 32 of whom were heterozygous. Codominant Mendelian inheritance of fragments was observed in three families with a total of 24 children. Because of the widespread distribution of (TG)n microsatellites, polymorphisms of this type may be generally abundant and present in regions where minisatellites are rare, making such microsatellite loci very useful for linkage studies in humans.
Approximately 70 percent of the mutations in cystic fibrosis patients correspond to a specific deletion of three base pairs, which results in the loss of a phenylalanine residue at amino acid position 508 of the putative product of the cystic fibrosis gene. Extended haplotype data based on DNA markers closely linked to the putative disease gene locus suggest that the remainder of the cystic fibrosis mutant gene pool consists of multiple, different mutations. A small set of these latter mutant alleles (about 8 percent) may confer residual pancreatic exocrine function in a subgroup of patients who are pancreatic sufficient. The ability to detect mutations in the cystic fibrosis gene at the DNA level has important implications for genetic diagnosis.
Short simple sequence stretches occur as highly repetitive elements in all eukaryotic genomes and partially also in prokaryotes
and eubacteria (1–6). They are thought to arise by slippage like events working on randomly occuring internally repetitive
sequence stretches (3, 7, 8). This predicts that they should be generally hypervariable in length. I have used the polymerase
chain reaction (PCR) process to show that several randomly chosen simple sequence loci with different nucleotide composition
and from different species show extensive length polymorphisms. These simple sequence length polymorphisms (SSLP) may be usefully
exploited for identity testing, population studies, linkage analysis and genome mapping.
The usual advice given in standard texts is to combine classes when expected numbers are too small. It is suggested that an alternative would be to use the exact values of teh mean and variance of χ2, for which algebraic expressions are provided. The reliability of the test can be improved by using √χ2 instead of χ2 as the test criterion.
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Cystic fibrosis (CF) is the most common autosomal recessive disorder affecting approximately 1 out of 2000 live births in the Caucasian population (Talamo et al. 1983). The major clinical manifestations of CF include chronic obstructive pulmonary disease, pancreatic enzyme insufficiency, and elevated sweat electrolyte levels, all of which are suggestive of an inborn error of metabolism involving exocrine glands. If untreated, CF individuals usually die at an early age due to severe lung infection, but, because of the advances in disease treatment, many patients now survive into adulthood. On the other hand, despite extensive research efforts, the basic defect of this disease remains unknown. Although a defective regulation of chloride ion transport seems to be the most consistent measurable parameter in CF (Knowles et al. 1983; Quinton 1983; Quinton and Bijman 1983; Sato 1984; Widdicombe et al. 1985; Yankaskas et al. 1985), it is still not clear whether this abnormality...
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The discovery of specific restriction endonucleases (Smith and Wilcox 1970) made possible the isolation of discrete molecular fragments of naturally occurring DNA for the first time. This capability was crucial to the development of molecular cloning (Cohen et al. 1973); and the combination of molecular cloning and endonuclease restriction allowed the synthesis and isolation of any naturally occurring DNA sequence that could be cloned into a useful vector and, on the basis of flanking restriction sites, excised from it. The availability of a large variety of restriction enzymes (Roberts 1985) has significantly extended the utility of these methods.
The de novo organic synthesis of oligonucleotides and the development of methods for their assembly into long double-stranded DNA molecules (Davies and Gassen 1983) have removed, at least theoretically, the minor limitations imposed by the availability of natural sequences with fortuitously unique flanking restriction sites. However, de novo synthesis, even with automated...
By Southern blotting and hybridization analysis using 32P-labeled poly(dT-dG) . poly(dC-dA) as a probe, we have found, in eukaryotic genomes, a huge number of stretches of dT-dG alternating sequence, a sequence that has been shown to adopt the Z-DNA conformation under some conditions. This sequence was found in all eukaryotic genomes examined from yeast to human, indicating extraordinary evolutionary conservation. The number of the sequence ranged from about 100 in yeast to tens of thousands in higher eukaryotes. Comparison of nucleotide sequences of dT-dG alternating regions and its flanking regions in several cloned genes showed that the repeated element [the Z(T-G) element]] consists only of dT-dG alternating sequence with variable length. The presence of another purine-pyrimidine alternating sequence was also surveyed in eukaryotic genomes by Southern blot hybridization using 32P-labeled poly(dG-dC) . poly(dG-dC) as the probe. The stretches of dC-dG alternating sequence [the Z(C-G) element] were found to be moderately repetitive in human, mouse, and salmon genomes. However, a few and no copies of the Z(C-G) element were found in yeast and calf genomes, respectively. These results provide evidence for the abundance of potential Z-DNA-forming sequences in nature.
Mutation analysis for heterozygote detection and the prenatal diagnosis of the cystic fibrosis
- A Kornberg
- Dna Replication
- San Francisco Wh Freeman
- Wk Lemna
- Gl Feldman
- B Kerem
- Sd Fernbach
- Ep Zevkovich
- O Brien
- We Collins
Kornberg A (1980) DNA replication. WH Freeman, San
Francisco
Lemna WK, Feldman GL, Kerem B, Fernbach SD, Zevkovich EP, O'Brien WE, Collins FS, et al (1990) Mutation
analysis for heterozygote detection and the prenatal diagnosis of the cystic fibrosis. N Engl J Med 322:291-296
Two cystic fibrosis patients with mild pulmonary disease and nonsense mutations in each CFTR gene
- G R Cutting
- L M Kash
- B J Rosenstein
- L-C Tsui
- H H Kazazian
- S E Antonarakis
Cutting GR, Kash LM, Rosenstein BJ, Tsui L-C, Kazazian
HH Jr, Antonarakis SE (1990a) Two cystic fibrosis patients with mild pulmonary disease and nonsense mutations in each CFTR gene. N EngI J Med 323:1685-1689
A cluster of cystic fibrosis mutations in the first nucleotide-binding fold of the cystic fibrosis conductance regulator protein
- G R Cutting
- L M Kash
- B J Rosenstein
- Zielenskij
- L-C Tsui
- S E Antonarakis
- H H Kazazian
Cutting GR, Kash LM, Rosenstein BJ, ZielenskiJ, Tsui L-C,
Antonarakis SE, Kazazian HH Jr (1990b) A cluster of
cystic fibrosis mutations in the first nucleotide-binding
fold of the cystic fibrosis conductance regulator protein.
Nature 346:366-369
Worldwide survey ofthe AF508 mutation-report from the Cystic Fibrosis Genetic Analysis Consortium
Cystic Fibrosis Genetic Analysis Consortium (1990) Worldwide survey ofthe AF508 mutation-report from the Cystic Fibrosis Genetic Analysis Consortium. Am J Hum
Genet 47:354-359
The polydeoxyadenylate tract of Alu repetitive elements is polymorphic in the human genome
- E P Economou
- A W Bergen
- A C Warren
- S E Antonarakis
Economou EP, Bergen AW, Warren AC, Antonarakis SE
(1990) The polydeoxyadenylate tract of Alu repetitive
elements is polymorphic in the human genome. Proc Natl
Acad Sci USA 87:2951-2954
Five polymorphic microsatellite VNTRs on the human X chromosome
- J A Luty
- Z Gou
- H F Willard
- D H Ledbetter
- S Ledbetter
- M Litt
Luty JA, Gou Z, Willard HF, Ledbetter DH, Ledbetter S,
Litt M (1990) Five polymorphic microsatellite VNTRs on
the human X chromosome. Am J Hum Genet 46:776-783