Molecular pathology of haemophilia A in Turkish patients: identification of 36 independent mutations.

Haemophilia A is an X-linked recessive bleeding disorder caused by heterogeneous mutations in the factor VIII gene. In an attempt to reveal the molecular pathology of Turkish haemophilia A patients, the coding sequence of the gene, excluding a large portion of exon 14, was amplified from genomic DNA and subjected to denaturing gradient gel electrophoresis prior to DNA sequencing. Fifty-nine haemophilia A patients were included in the study with severe, moderate and mild phenotypes observed in 24, 15 and 16 patients, respectively. Factor VIII activity and clinical phenotypes were not available for four patients. A total of 36 independent mutations were found, with a mutation detection efficacy of 61%. The mutations that were reported for the first time include 20 point mutations, one 8-bp insertion (TCAAGATA) in exon 4 and one large deletion greater than 2.8 kb involving exon 14. The novel point mutations were composed of three nonsense (Ser681Ter, Cys2021Ter and Gln2113Ter), one splicing error (IVS-1G-->A), 15 missense mutations (Lys48Asn; Leu-98Phe; Thr118Ala; Cys248Tyr; Glu456Lys; Asp560Ala; Tyr664Cys; Phe679Leu; Gly691Trp; Asp1769His; Val1857Leu; Gly2026Gln; Arg2163Pro; Asp2288Ala; and Arg2304Leu) and a T deletion in exon 25 that caused a frameshift followed by a stop codon. All missense mutations except Val1857Leu, which maintained a conserved nonpolar R group, occurred at amino acids conserved among four species and were most probably pathogenic. In addition, two sequence changes (IVS3-9C-->T) and (Leu2230Leu) were also detected in patients carrying Val1857Leu and Phe679Leu missense mutations, respectively. Identification of mutation origins in eight sporadic cases revealed an equal sex ratio of mutations.

Methylation levels at selected CpG sites in the factor VIII and FGFR3 genes, in mature female and male germ cells: implications for male-driven evolution.

Transitional mutations at CpG dinucleotides account for approximately a third of all point mutations. These mutations probably arise through spontaneous deamination of 5-methylcytosine. Studies of CpG mutation rates in disease-linked genes, such as factor VIII and FGFR3, have indicated that they more frequently originate in male than in female germ cells. It has been speculated that these sex-biased mutation rates might be a consequence of sex-specific methylation differences between the female and the male germ lines. Using the bisulfite-based genomic-sequencing method, we investigated the methylation status of the human factor VIII and FGFR3 genes in mature male and female germ cells. With the exception of a single CpG, both genes were found to be equally and highly methylated in oocytes and spermatocytes. Whereas these observations strongly support the notion that DNA methylation is the major determining factor for recurrent CpG germ-line mutations in patients with hemophilia and achondroplasia, the higher mutation rate in the male germ line is apparently not a simple reflection of sex-specific methylation differences.

Comparison between intratracheal and intravenous administration of liposome-DNA complexes for cystic fibrosis lung gene therapy.

Intratracheal (i.t.) and intravenous (i.v.) delivery of DNA-vector formulations are two strategies to obtain gene transfer to the lung, it is still uncertain, however, which of these two modes of delivery will be more effective in the treatment of cystic fibrosis and other lung diseases. In this study, we attempted to optimize formulations of the cationic liposome DODAC:DOPE (dioleoyldimethylammonium-chloride: dioleoylphosphatidylethanolamine) complexed to plasmids encoding chloramphenicol acetyltransferase for i.t. and i.v. injection into CD-2 mice and compared the two methods. Our results showed that both methods conferred reporter gene expression in the lung that was significantly higher relative to injection of plasmid DNA alone. Expression using either mode of administration was maximal 24 h after injection and declined to around 10% of day 1 levels 2 weeks after injection. For i.v. delivery of DODAC. DOPE-DNA complexes multilamellar vesicles were more effective than large unilamellar vesicles in all organs investigated. Recombinant DNA could be detected in the distal lung region following either route of administration. However, i.t. administration predominantly led to DNA deposition in epithelial cells lining the bronchioles, e.g. in clara cells, whereas i.v. administration resulted in DNA deposition in the alveolar region of the lung including type II alveolar epithelial cells.

Characterization of the factor VIII defect in 147 patients with sporadic hemophilia A: family studies indicate a mutation type-dependent sex ratio of mutation frequencies.

The clinical manifestation of hemophilia A is caused by a wide range of different mutations. In this study the factor VIII genes of 147 severe hemophilia A patients--all exclusively from sporadic families--were screened for mutations by use of the complete panel of modern DNA techniques. The pathogenous defect could be characterized in 126 patients (85.7 percent). Fifty-five patients (37.4 percent) showed a F8A-gene inversion, 47 (32.0 percent) a point mutation, 14 (9.5 percent) a small deletion, 8 (5.4 percent) a large deletion, and 2 (1.4 percent) a small insertion. Further, four (2.7 percent) mutations were localized but could not be sequenced yet. No mutation could be identified in 17 patients (11.6 percent). Sixteen (10.9 percent) of the identified mutations occurred in the B domain. Four of these were located in an adenosine nucleotide stretch at codon 1192, indicating a mutation hotspot. Somatic mosaicisms were detected in 3 (3.9 percent) of 76 patients, mothers, comprising 3 of 16 de novo mutations in the patients mothers. Investigation of family relatives allowed detection of a de novo mutation in 16 of 76 two-generation and 28 of 34 three-generation families. On the basis of these data, the male:female ratio of mutation frequencies (k) was estimated as k = 3.6. By use of the quotients of mutation origin in maternal grandfather to patients mother or to maternal grandmother, k was directly estimated as k = 15 and k = 7.5, respectively. Considering each mutation type separately, we revealed a mutation type-specific sex ratio of mutation frequencies. Point mutations showed a 5-to-10-fold-higher and inversions a >10-fold-higher mutation rate in male germ cells, whereas deletions showed a >5-fold-higher mutation rate in female germ cells. Consequently, and in accordance with the data of other diseases like Duchenne muscular dystrophy, our results indicate that at least for X-chromosomal disorders the male:female mutation rate of a disease is determined by its proportion of the different mutation types.

Sequence analysis of the cystic fibrosis gene in patients with disseminated bronchiectatic lung disease. Application in the identification of a cystic fibrosis patient with atypical clinical course.

The diagnosis of classical cystic fibrosis (CF) is easily made by clinical assessment alone, but may be missed or delayed in cases with an atypical clinical course. In a recent major study the age at diagnosis varied between 2 months and 47 years. For diagnostic purposes we have investigated the cystic fibrosis transmembrane regulator (CFTR) gene in 10 adult patients (age 18 to 45 years) with chronic obstructive pulmonary disease since childhood or adolescence and bronchiectases disseminated through both lungs. Only one subject (a 29-year-old male) had exocrine pancreatic insufficiency (PI); all others were pancreatic-sufficient (PS). The first nucleotide (ATP)-binding fold of the CFTR was analyzed by direct sequencing of polymerase chain reaction (PCR)-amplified genomic DNA in these cases. Two patients with different phenotypes (one PI, one PS) were found to be homozygous for the common delta F508 mutation of the CFTR gene, which proved the diagnosis of cystic fibrosis in their cases and allowed genetic counselling. The PS patient had normal sweat tests and had not previously been recognized as having CF. Four other patients were heterozygous for delta F508, with no other mutation in exons 10 or 11 of the gene, and four patients had normal sequences of these exons. Because only about 70% of all CF chromosomes carry delta F508, the unexpectedly high frequency (4/8 = 50%) of heterozygosity for delta F508 among the non-delta F508/delta F508 patients with bronchiectases suggests that some of these might also have unrecognized CF with rare genotypes and mutations in any of the 22 exons not sequenced.(ABSTRACT TRUNCATED AT 250 WORDS)

Prenatal diagnosis of haemophilia B by the use of polymerase chain reaction and direct sequencing.

A second prenatal diagnosis of severe haemophilia B was carried out in a family with no prior history of the disease. The first prenatal diagnosis was based on linkage analysis and showed the male fetus not to be affected because he had inherited the same X-chromosome as his healthy brother. Carrier status in the female at risk could not be assessed by restriction fragment length polymorphisms (RFLPs). She was found to have inherited the same marker constellation as her affected brother. However, due to the fact that a pedigree with no prior history of haemophilia B has been examined diagnosis was impossible. In addition factor IX coagulant and antigen values gave no definitive clue to a haemophilia B carriership. The problems with RFLP analysis in this pedigree were circumvented by polymerase chain reaction (PCR) based direct sequencing of the factor IX gene. A previously unknown mutation could be detected in patient haemophilia B (Kleve) and the carrier status in the female at risk could be confirmed. The second prenatal diagnosis showed that the male fetus had inherited the mutation and will therefore be afflicted with haemophilia B.

Detection and characterisation of two missense mutations at a cleavage site in the factor VIII light chain.

Haemophilia A is an X-linked bleeding disorder caused by a deficiency of factor VIII. As an essential cofactor in the intrinsic clotting cascade, factor VIII is activated and subsequently inactivated by proteolytic cleavages involving factor IIa (thrombin), factor Xa and activated protein C (APC). Investigation of the thrombin cleavage sites at amino acids 372 and 1689 of the factor VIII protein by oligonucleotide screening, DNA amplification and direct sequencing, enabled us to identify two missense mutations in 441 unrelated haemophiliacs. A C-to-T transition, which leads to the substitution of cysteine for arginine at position 1689, was found in a severely affected patient and a previously undescribed G-to-A substitution, causing replacement of arginine1689 with histidine, was found in a patient with mild disease.

Highly variable clinical course in severe alpha 1-antitrypsin deficiency--use of polymerase chain reaction for the detection of rare deficiency alleles.

Among 20 individuals with severe alpha 1-antitrypsin (alpha 1AT) deficiency we observed extremely variable clinical phenotypes ranging from rapidly progressive lung disease fatal at the age of 42 years to an asymptomatic individual with normal lung function at the age of 50 years. Eighteen subjects, including the asymptomatic one, carried the deficient Pi ZZ phenotype as determined by isoelectric focusing (IEF). Their mean alpha 1AT serum level was 36.7 +/- 7.7 mg/dl. DNA restriction analysis showed that all of them had the classical Pi Z-allele-associated DNA haplotype, thus confirming the IEF data. Obviously not all Pi ZZ individuals will have clinical sequelae caused by this genotype. The important differences in clinical course observed could not be explained by smoking habits alone. Probably additional factors are pertinent to the pathogenesis of the lung disease associated with alpha 1AT deficiency (defects in other genes, environmental influences other than smoking). In two patients with very low alpha 1AT serum levels definitive phenotyping by IEF was not possible. Therefore we investigated the molecular basis of their deficiency using polymerase chain reaction (PCR) amplification of the coding exons of their alpha 1AT genes and direct sequencing of the amplification products. Sequence data analysis showed that one of these patients, who had initially been phenotyped as Pi ZZ by IEF, had in fact the genotype Pi QObellinghamZ, thus explaining her low alpha 1AT serum level of 20 mg/dl. The other patient (alpha 1AT serum level 3.7 mg/dl) exhibited the rare genotype Pi MheerlenQOgranite falls. Despite his nearly complete alpha 1AT deficiency, he suffered from only moderately severe pulmonary disease at the age of 42 years.

Microdissection and microcloning of human chromosome 7q22-32 region.

Genetic information contributing to cystic fibrosis in addition to the CF gene is suggested to reside on the long arm of the human chromosome 7. In our attempt to analyze this genomic region in detail, we generated a region-specific DNA probe library by microdissection and microcloning of the midpiece of the chromosome 7q arm. Microdissection was performed in unstained metaphase spreads from a human x mouse hybrid cell line containing chromosome 7 as the only human chromosome. We obtained 593 clones from 75 dissected chromosomal fragments. At least 88% of the microclones were true recombinants; 40% of the clones contained repetitive sequences as determined by plaque hybridization with genomic DNA as probe. The overall mean fragment size of insert fragments was 3.2 kb, the median size was 3.5 kb. Regional mapping of 30 DNA fragments was performed by the aid of hybrid cell lines containing different segments of human chromosome 7; 50% of the microcloned inserts were found to map to 7q22-32.

Regional assignment of 41 human DNA fragments on chromosome 7 by means of a somatic cell hybrid panel.

To detect new restriction fragment length polymorphisms that would cover human chromosome 7 with a network of genetic landmarks, a chromosome 7-specific phage gene library was screened for human single-copy fragments. With use of a somatic cell hybrid panel containing defined regions of human chromosome 7, 41 cloned human single-copy sequences were assigned to five regions of this chromosome. Of special importance are the cell hybrid clones GM1059Rag5 and 7851Rag10-1, derived from human cells with interstitial deletions spanning the bands 7q22-q32, within which the cystic fibrosis gene is located. Twelve new probes are described in 7q22-q32, five of which detect a total of six RFLPs.

[Primary lung cancer from biopsy material of the Pathomorphology Department of the Silesian Medical Academy in Zabrze]. / Pierwotny rak oskrzela w materiale oligobiopsyjnym i Katedry i Zakladu Patomorfologii Slaskiej Akademii Medycznej w Zabrzu.

An analysis was carried out of material biopsied during fiberoptic bronchoscopic examination of 289 patients suspected of lung malignant disorders were present more often in males. In the group of patients older than 40 years the most often diagnosed malignant process was squamous cell carcinoma. A high correlation of clinical suspicion of malignant process and histopathological diagnosis was found. A low rate of diagnosis of operable cases was found.

Prenatal diagnosis of cystic fibrosis.

Cystic fibrosis is the most common autosomal recessive genetic disorder in the Caucasian population (1:2000-1:4000) (Warwick, W. J. (1978) Helv. Paediatr. Acta 33, 117-125). This defect is characterized by chronic obstructive pulmonary disease, pancreatic exocrine insufficiency and abnormally high perspiration electrolytes in most patients (Talamo et al. (1985) In: The metabolic basis of inherited diseases, pp. 1887-1917). The elevated electrolyte level provides the most reliable diagnostic test for cystic fibrosis homozygotes. Although prospects for cystic fibrosis patients have improved, genetically homozygous cystic fibrosis is effectively a lethal disease. Because of the seriousness of the disease, many families with one affected child desire a prenatal diagnosis when a second pregnancy occurs. Despite extensive research, the biochemical basis of cystic fibrosis remains unknown. Secondary effects on microvillar enzymes allow second trimester diagnosis (17-18 weeks of gestation (Brock, D. H. J. (1983) Lancet II, 941-943). First trimester prenatal diagnosis for cystic fibrosis became possible with DNA technology. Application of polymorphic marker loci to problems of prenatal diagnosis and carrier-testing is discussed.

Cystic fibrosis: typing 89 German families with linked DNA probes.

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

Rapid expression of c-fos specific messenger RNA after wounding of a BALB/c-3T3 fibroblast monolayer.

A grid of damaged areas was introduced into serum-starved BALB/c-3T3 fibroblast monolayers. The expression of c-fos specific messenger RNA was examined by the use of the cytoplasmic dot hybridisation technique 15, 30 and 45 min after wounding. The expression of c-fos messenger RNA was highest after 30 min, while microglobulin was constantly expressed. These results show that the unspecific event of wounding induces the specific expression of c-fos messenger RNA, an effect similar to that of growth factors during serum stimulation.

Cystic fibrosis: typing 48 German families with linked DNA probes.

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

Mapping of DNA markers linked to the cystic fibrosis locus on the long arm of chromosome 7.

We have used a panel of eight human/mouse somatic-cell hybrids, each containing various portions of human chromosome 7, and three patient cell lines with interstitial deletions on chromosome 7 for localization of six DNA markers linked to the cystic fibrosis locus. Our data suggest that D7S15 is located in the region 7 cen----q22, that MET is located in 7q22----31, and that D7S8 and 7C22 are located in q22----q32. The hybridization results for COL1A2 and TCRB are consistent with their previous assignment to 7q21----q22 and 7q32, respectively. Given the location of these six markers and their linkage relationships, it is probable that the cystic fibrosis locus is in either the distal region of band q22 or the proximal region of q31. Using the same set of cell lines, we have also examined the location of another chromosome 7 marker PGY1. The data show that PGY1 is located in the region 7cen----q22, a position very different from its previous assignment.

[Possibilities and limits of temporary liver substitution by hemoperfusion with biological material]. / Möglichkeiten und Grenzen des temporären Leberersatzes durch Hämoperfusion mit biologischem Material.

For temporary hepatic assistance we used 200 g porcine liver pieces (5 X 5 X 5 mm3) which were perfused for 6 h with 11 swine blood. ATP and energy reserve values reached their maxima 30 min after starting perfusion, remained unchanged for 120 min, and decreased thereafter. Following 30 min of perfusion energy charge values increased from 0.260 +/- 0.110 mumol/g to 0.560 +/- 0.093 mumol/g (normal value; 0.854 +/- 0.022 mumol/g) and thereafter remained unchanged for 6 h. These results suggest that good energy regulation was maintained in the liver pieces. The small liver cubes showed excellent ammonia and phenol detoxication. However, the liver pieces were not found to be able to conjugate serum bilirubin, which might have been caused by a lack of this anatomic pathway in our model. Levels of hepatic and lytic enzymes in the perfusate increased with the time of perfusion, though they were relatively low as compared to levels in patients with acute hepatic failure. The concentration of free fatty acids in the perfusate, which are known to potentiate hepatic coma, increased slightly. However, methyl mercaptane remained constant during perfusion. Concentrations of nearly all amino acids rose during 6-h perfusion due to damage of hepatic tissues, but the molar ratio of the branched chain amino acid to aromatic amino acid was not changed. These results suggest that liberated substances from the damaged liver would not potentiate hepatic encephalopathy. We feel that hemoperfusion over small liver pieces could be a useful method for hepatic assistance.