Novel and recurrent rearrangements in the CFTR gene: clinical and laboratory implications for cystic fibrosis screening

Because standard techniques used to detect mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene do not detect single or multiple exonic rearrangements, the importance of such rearrangements may be underestimated. Using an in-house developed, single-tube, semi-quantitative fluorescent PCR (SQF PCR) assay, we analyzed 36 DNA samples submitted for extensive CFTR sequencing and identified ten samples with rearrangements. Of 36 patients with classic CF, 10 (28%) harbored various deletions in the CFTR gene, accounting for 14% of CF chromosomes. A deletion encompassing the CFTR promoter and exons 1 and 2 was detected in a sample from one proband, and in the maternal DNA as well. In another family, a deletion of the promoter and exon 1 was detected in three siblings. In both of these cases, the families were African American and the 3120+1G>A splice site mutation was also identified. These promoter deletions have not been previously described. In a third case, a deletion of exons 17a, 17b, and 18 was identified in a Caucasian female and the same mutation was detected in the paternal DNA. In the other seven cases, we identified the following deletions: exons 2 and 3 (n=2); exons 4, 5, and 6a; exons 17a and 17b; exons 22 and 23; and exons 22, 23, and 24 (n=2). In our series, the frequency of CFTR rearrangements in classic CF patients, when only one mutation was identified by extensive DNA sequencing, was >60% (10/16). Screening for exon deletions and duplications in the CFTR gene would be beneficial in classic CF cases, especially when only one mutation is identified by standard methodologies. An erratum to this article can be found at     

ΔF508 frequency and associated haplotypes near the cystic fibrosis locus in the Yugoslav population

Summary Chromosomes from 19 unrelated Southern Yugoslav families in which cystic fibrosis (CF) occurs were analysed for the presence of the ΔF508 mutation, using polymerase chain reaction amplification followed by dot blot and polyacrylamide gel analysis. Of the 38 CF chromosomes, 15 (39.5%) carry the ΔF508 deletion. Restriction fragment length polymorphism haplotypes for KM19/PstI, XV2c/TaqI and J3.11/PstI marker loci were determined and are compared for a total of 34 N and 37 CF chromosomes.     

Use of a non-selective transformation technique to construct a multiply restriction/modification-deficient mutant ofNeisseria gonorrhoeae

A technique that allows for easy identification of transformants ofNeisseria gonorrhoeae in the absence of selective pressure has been developed. A suicide vector that contains a gonococcal DNA uptake sequence was constructed to aid in DNA uptake. In this transformation procedure, a limiting number of cells is incubated with an excess amount of DNA, and the mixture is plated onto a non-selective medium. At least 20% of the resulting colonies contained cells that had been transformed. This strategy was utilized to construct specific deletions of the S.NgoI, II, IV, V and VII restriction-modification (R/M) genes. All five deletions were successfully incorporated into the chromosome of FA19, producing strain JUG029. Strain JUG029 could be transformed with non-methylated plasmid DNA while strain FA19 could not be transformed with such DNA. The development of a simple, non-selective transformation technique, coupled with the construction of a strain that is more permissive for DNA-mediated transformation, will aid in genetic manipulations of the gonococcus.     

Linkage disequilibrium and CF allele segregation analysis in cystic fibrosis families in Northern Ireland

Summary Linkage disequilibrium and cystic fibrosis (CF) allele segregation were analysed in 46 CF families in Northern Ireland. The smaller (+) allele of the KM19/PstI polymorphism and the larger (-) allele of the XV-2c/TaqI polymorphism showed marked linkage disequilibrium with CF. This information can be used to alter the risk of an individual being a carrier of CF away from the expected population risk of 1 in 20. The high-risk genotypes K+K+ or X-X- have a risk of 1 in 10 and the low-risk genotypes K-K- or X+X+ have a risk of 1 in 50. A study of the segregation of CF alleles in the 46 families, using KM19 and Xv-2c, showed preferential inheritance of the paternal (79%), as opposed to the maternal (21%), CF allele by the heterozygous carriers. A mechanism that might explain this observation is discussed.     

Identification of expressed bovine class I MHC genes at two loci and demonstration of physical linkage

A cDNA library prepared from lymphocytes of a cow (E98), homozygous at major histocompatibility complex (MHC) loci (BoLA phenotype w10, KN104), was screened with a bovine MHC class I probe. Of the cDNA clones isolated, two, (2.1 and 5.1) were selected and showed divergence at both 5 and 3 termini. E98 DNA was digested with rare-cutter enzymes (Sfi I, Mlu I, Not I, and Cla I) and fragments were size-separated by field inversion gel electrophoresis (FIGE). Hybridization with an entire class I cDNA probe revealed multiple fragments generated by each enzyme. When the 3 untranslated regions (UT) of 2.1 and 5.1 were used as probes, only one fragment was revealed in each digested sample, showing locus specificity of these probes in cattle. Further, DNA of transfected mouse fibroblasts L4 (expressing KN104) and L10 (expressing w10) hybridized to the 3UT regions of clones 2.1 and 5.1, respectively, Northern blot analysis of the mRNA of the L4 and L10 transfected cells provided further evidence that the cDNA clones 2.1 and 5.1 code for the BoLA-KN104 and BoLA-w10 class I molecules respectively, and thus these represent the products of two different genes. A long range physical mapping of the BoLA-w10 and KN104 genes was performed using FIGE analysis of DNA of and homozygous and an heterozygous animal. This analysis revealed that the BoLA-w10 and KN104 genes are separated by not more than 210 kilobases (kb) and that they are components of a multigene family spanning 1550 kb. As the] w10 gene is at the BoLA-A locus we assign the KN104 gene to a B locus.     

Restriction enzyme analysis of mitochondrial DNAs of petite mutants of yeast: Classification of petites, and deletion mapping of mitochondrial genes

Summary We have analyzed the restriction digest patterns of the mitochondrial DNA from 41 cytoplasmic petite strains of Saccharomyces cerevisiae, that have been extensively characterized with respect to genetic markers. Each mitochondrial DNA was digested with seven restriction endonucleases (EcoRI, HpaI, HindIII, BamHI, HhaI, SalI, and PstI) which together make 41 cuts in grande mitochondrial DNA and for which we have derived fragment maps. The petite mitochondrial DNAs were also analyzed with HpaII, HaeIII, and AluI, each of which makes more than 80 cleavages in grande mitochondrial DNA. On the basis of the restriction patterns observed (i.e., only one fragment migrating differently from grande for a single deletion, and more than one for multiple deletions) and by comparing petite and grande mitochondrial DNA restriction maps, the petite clones could be classified into two main groups: (1) petites representing a single deletion of grande mitochondrial DNA and (2) petites containing multiple deletions of the grande mitochondrial DNA resulting in rearranged sequences. Single deletion petites may retain a large portion of the grande mitochondrial genome or may be of low kinetic cimplexity. Many petites which are scored as single continuous deletions by genetic criteria were later demonstrated to be internally deleted by restriction endonuclease analysis. Heterogeneous sequences, manifested by the presence of sub-stoichiometric amounts of some restriction fragments, may accompany the single or multiple deletions. Single deletions with heterogeneous sequences remain useful for mapping if the low concentration sequences represent a subset of the stoichiometric bands. Using a group of petites which retain single continuous regions of the grande mitochondrial DNA, we have physically mapped antibiotic resistance and mit^- markers to regions of the grande restriction map as follows: C (99.3-1.4 map units)-OXI-1 (2.5-15.7)-OXI-2 (18.5-25)-P (28.1-34.2)-OXI-3 (32.2-61.2)-O_II (60-62)-COB (64.6-80.8)-O_I (80.4-85.7)-E (95-98.9).Supported by USPHS Training Grant 5-T01-GM-00090-19.Supported by USPHS Training Grant T32-GM-07197.The Franklin McLean Memorial Research Institute is operated by the University of Chicago for the U.S. Energy Research and Development Administration under Contract EY-76-C-02-0069.     

Electrophoretic analysis of Histoplasma capsulatum chromosomal DNA.

Seven chromosome-sized DNA molecules in the Downs strain of Histoplasma capsulatum were resolved by using chromosome-specific DNA probes in blot hybridizations of contour-clamped homogeneous electric field (CHEF) and field-inversion gel electrophoresis (FIGE) agarose gels. The sizes of the chromosomal DNA bands extended from that of the largest Saccharomyces cerevisiae chromosome to beyond that of the Schizosaccharomyces pombe chromosomes. Under our experimental conditions, the order of the five largest DNA bands was inverted in the FIGE gel relative to the CHEF gel, demonstrating a characteristic of FIGE whereby large DNA molecules may have greater rather than lesser mobility with increasing size. Comparison of the Downs strain with other H. capsulatum strains by CHEF and FIGE analysis revealed considerable variability in band mobility. The resolution of seven chromosome-sized DNA molecules in the Downs strain provides a minimum estimate of the chromosome number.     

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

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

Pulsed field gel electrophoresis of representatives of Mycobacterium tuberculosis and Mycobacterium bovis BCG strains

Abstract Using field inversion gel electrophoresis (FIGE), different Mycobacterium tuberculosis strains, such as phage prototypes, exhibit different DNA restriction patterns which are easy to compare. Virulent and avirulent variants of M. tuberculosis H37, as well as daughter strains of M. bovis BCG, display characteristic DNA profiles. BCG strains isolated from suppurative adenitis following vaccination of French patients showed patterns identical to the BCG Pasteur strain used for vaccination. These results demonstrate that FIGE of DNA restriction fragments generated by Dra I represents a suitable technique for the analysis of mycobacteria at a genomic level. The Dra I profiles allow the differentiation and precise identification of the BCG Pasteur, Glaxo, Russian and Japanese strains.     

Separation of Different Conformations of Plant Mitochondrial DNA Molecules by Field Inversion Gel Electrophoresis

Mitochondrial (mt) DNA structure in higher plants is still unclear as to the circularity or linearity of the genome. We have developed a system to electrophoretically separate distinct populations of mtDNA, with some populations enriched for networked linear and circular DNA molecules. Using field inversion gel electrophoresis (FIGE) and electron microscopy (EM), we have identified four distinct populations of mtDNA from two Brassica species. Using FIGE, two slow migrating mtDNA populations ran faster than a 66 kbp Escherichia coli circular plasmid marker, while these same populations comigrated in the compression zone in contour-clamped homogeneous electrophoretic field (CHEF) gels. A fast-migrating mtDNA population was also resolved by FIGE as a diffuse band between 20 to 70 kbp when compared with linear lambda () markers. FIGE resolved the 66 kbp circular marker into several multimers, while CHEF resolved only open-circular monomers and linears. In agreement with FIGE results, EM analysis indicated the two slow migrating mtDNA populations contained circular (both supercoiled and relaxed circles) and free linear molecules of 10-60 kbp, and networked linear molecules of 45–140 kbp total size that may represent recombination intermediates. The fast migrating population consisted of 10–50 kbp linear molecules. Well-bound mtDNA showed only long linear molecules of 40–150 kbp with no detection of circles or complex/rosette molecules. This report shows that FIGE has clear advantages over CHEF for separating large DNA molecules with different conformations, and may be very useful for studies to characterize genome structure in complex systems such as plant mitochondria.     

Frequency of the F508 deletion in cystic fibrosis patients from the European part of the USSR

Summary The frequency of the F508 deletion (F508) has been analyzed in 189 cystic fibrosis (CF) patients from the European part of the USSR, viz. 127 nothern Slavonians (Leningrad region), 30 southern Slavonians (the Ukraine), 10 central Slavonians (Moscow region), 14 Moldavians (Kishenev region) and 8 Lithuanians (Vilnius region). The distribution of CF⁺ chromosomes with and without F508 varied significantly in the different ethnic groups studied and correlated with the clinical manifestation of CF. The overall frequency of F508 in Slavonian patients is equal to 62.5%, approximately 90% of them being heterozygous or homozygous for this mutation. The frequency of the deletion among 99 Slavonian patients with severe disease manifestation (pancreatic insufficiency, PI) is equal to 67.5%, only 12 patients having pancreatic sufficiency (PS, 17.5%). The highest value of F508 (77.4%) is registered in PI/CF patients of the southern Slavonian group; it is much less frequent (about 57%) in relevant groups of Slavonians from the northern and central parts of the country. Unusually low frequencies (24% and 26%) of F508 are detected in a few samples of Lithuanian and Moldavian CF patients, respectively. All F508⁺CF-chromosomes of Slavonian origin are associated with haplotypes 2.2.2. defined by the restriction fragment length polymorphism sites KM19/PstI, CS.7/Hin6I and MP6d-9/MspI, although a high proportion (about 25%) of unknown mutations is associated with the same haplotype. Haplotype B (allele 1XV2c/TaqI; allele 2 KM19/PstI) accounts for 91% of F508⁺CF chromosomes. Our data are consistent with the hypothesis of a single origin and subsequent diffusion of this major CF mutation; however, its interpopulational dissemination in Eastern Europe does not follow the suggested south-east to north-west gradient in Western Europe. The significance of these data for prenatal diagnosis and carrier screening of CF mutations is briefly discussed.     

A new type of IS1-mediated deletion

Summary Genetical tests and DNA sequence analysis revealed that the mechanism of formation of IS1-induced type I and type II deletions differs.IS1-mediated type II deletions occur at the termini of the integrated element and do not remove the element. This process is independent of the cellular recA system and does not involve DNA sequence homology.Conversely, the formation of IS1-induced type I deletions differs substantially. They require recA gene product, small DNA sequence duplications and a topological arrangement of the DNA molecule to allow alignment of duplications.     

Marker haplotype association with growth in German cystic fibrosis patients

Summary In 84 families with 101 children with cystic fibrosis (CF) and 103 unaffected siblings, the haplotype of CF chromosomes was determined with six restriction fragment length polymorphism (RFLP) markers that span the CF gene locus. Patient groups with different genotypes in the more distant flanking marker loci MET D, MET H, and D7S8 differed significantly from each other with respect to percentile height and weight, and percentage of weight for height. Patients homozygous 1-1 in met D (TaqI) and met H (TaqI) were thin and tall when homozygous 1-1 in J3.11 (MspI), and small when homozygous 2-2 in J3.11. Heterozygosity in 3.11 and met H and homozygosity 1-1 in met D segregated with the most severe growth retardation. In contrast, growth was normal in patients who were heterozygous in met D and/or had an uncommon KM.19/XV-2c haplotype. Most patients with pancreatic sufficiency and/or borderline sweat test values were carrying rare haplotypes on their CF chromosomes. Adult patients clustered in genotype groups with normal height percentile distributions. This association between haplotype and clinical severity of CF in the German population provides evidence for genetic microheterogeneity of the CF locus, either because of the existence of multiple alleles of the CF gene itself and/or because of the existence of closely linked polymorphic genes that control growth and development and hence modulate the clinical course and prognosis of CF.     

EVOLUTION AND MITOCHONDRIAL DNA IN NEUROSPORA CRASSA

We studied mitochondrial DNA variability in 19 natural Neurospora crassa isolates and one wild-type isolate to examine evolution of these fungi and their mitochondrial DNA (mtDNA). We combined restriction endonuclease analysis of natural isolate mtDNA with DNA-DNA hybridization to cloned EcoR I fragments of a wild-type genome to discriminate between length mutations and site changes due to nucleotide substitution. Most variability was due to length mutations (insertions and deletions); genome size could vary 25% between pairs of isolates. Length-mutation distribution was not random, nor simply explained by the presence of coding versus noncoding regions. Restriction-site changes were few; the estimated amount of nucleotide substitution per nucleotide between the most divergent pair of isolates was 0.78%. Evolutionary relationships among isolates based on both types of mutations were compatible, and suggest that geographically distinct populations of mitochondrial DNA exist in the biological species, N. crassa. In contrast, no such correlation was shown by the previously determined distribution of nuclear heterokaryon incompatibility genes in the same isolates (Mylyk, 1975, 1976).     

Use of a non-selective transformation technique to construct a multiply restriction/modification-deficient mutant ofNeisseria gonorrhoeae

A technique that allows for easy identification of transformants ofNeisseria gonorrhoeae in the absence of selective pressure has been developed. A suicide vector that contains a gonococcal DNA uptake sequence was constructed to aid in DNA uptake. In this transformation procedure, a limiting number of cells is incubated with an excess amount of DNA, and the mixture is plated onto a non-selective medium. At least 20% of the resulting colonies contained cells that had been transformed. This strategy was utilized to construct specific deletions of the S.NgoI, II, IV, V and VII restriction-modification (R/M) genes. All five deletions were successfully incorporated into the chromosome of FA19, producing strain JUG029. Strain JUG029 could be transformed with non-methylated plasmid DNA while strain FA19 could not be transformed with such DNA. The development of a simple, non-selective transformation technique, coupled with the construction of a strain that is more permissive for DNA-mediated transformation, will aid in genetic manipulations of the gonococcus.     

First analysis of the F508 deletion in cystic fibrosis patients from the GDR

Summary Cystic fibrosis (CF) patients (n = 157) from the GDR were analysed for the occurrence of the recently discovered 3bp deletion causing CF. About 50% of all investigated patients were homozygotes and about 30% heterozygotes for this deletion. Of the analysed CF chromosomes from these patients, 62% carry the deletion, which is in strong linkage disequilibrium with the KM19 restriction fragment length polymorphism allele 2 and the 1/2 XV2c/KM19 haplotype.     

The mutation spectrum of the CFTR gene in mucoviscidosis patients from Bashkortostan

Mutations of CFTR were studied in patients with cystic fibrosis (CF) from Bashkortostan. In total, 15 mutations were observed and 51% of all mutant alleles identified. The most diagnostically significant mutations were delF508 (33.8%), 394delTT (3.52%), CFTRdele2.3(21 kb) (1.41%), R334W (1.41%), 3849+ 10 kbC-->T (1.41%), and N1303K (1.41%). Mutations G542X, 2184insA, S1196X, and W1282X were each found in less than 1% patients. Five new mutations and two neutral substitutions were revealed. These were I488M (exon 10), 1811 + 12A-->C (intron 11), T663S (exon 13), I1226R (exon 19), 4005 + 9A-->C (intron 20), 2097A-->C (A655A, exon 13), and 3996G-->C (V1288V, exon 20). Bashkortostan was shown to differ in CFTR mutation spectrum from other regions of Russia. The results will allow direct DNA diagnostics of CF in far more families. Molecular screening of probands' relatives will contribute to identification and medical genetic counseling of heterozygous carriers, which is essential for CF prevention.     

Complex Mutations & Subpopulations of Deletions at Exon 19 of EGFR in NSCLC Revealed by Next Generation Sequencing: Potential Clinical Implications

Microdeletions at exon 19 are the most frequent genetic alterations affecting the Epidermal Growth Factor Receptor (EGFR) gene in non-small cell lung cancer (NSCLC) and they are strongly associated with response to treatment with tyrosine kinase inhibitors. A series of 116 NSCLC DNA samples investigated by Sanger Sequencing (SS), including 106 samples carrying exon 19 EGFR deletions and 10 without deletions (control samples), were subjected to deep next generation sequencing (NGS). All samples with deletions at SS showed deletions with NGS. No deletions were seen in control cases. In 93 (88%) cases, deletions detected by NGS were exactly corresponding to those identified by SS. In 13 cases (12%) NGS resolved deletions not accurately characterized by SS. In 21 (20%) cases the NGS showed presence of complex (double/multiple) frameshift deletions producing a net in-frame change. In 5 of these cases the SS could not define the exact sequence of mutant alleles, in the other 16 cases the results obtained by SS were conventionally considered as deletions plus insertions. Different interpretative hypotheses for complex mutations are discussed. In 46 (43%) tumors deep NGS showed, for the first time to our knowledge, subpopulations of DNA molecules carrying EGFR deletions different from the main one. Each of these subpopulations accounted for 0.1% to 17% of the genomic DNA in the different tumors investigated. Our findings suggest that a region in exon 19 is highly unstable in a large proportion of patients carrying EGFR deletions. As a corollary to this study, NGS data were compared with those obtained by immunohistochemistry using the 6B6 anti-mutant EGFR antibody. The immunoreaction was E746-A750del specific. In conclusion, NGS analysis of EGFR exon 19 in NSCLCs allowed us to formulate a new interpretative hypothesis for complex mutations and revealed the presence of subpopulations of deletions with potential pathogenetic and clinical impact.