Pregnancy-associated plasma protein A and proform eosinophilic major basic protein in the detection of different types of coronary artery disease.

Kryptor system was proven to be a rapid, standard method for pregnancy-associated plasma protein A and proform eosinophilic major basic protein (PAPP-A/proMBP) complex detection in coronary artery disease (CAD). No age and/or gender differences in 51 controls and 110 stable coronary artery disease (SCAD) patients were found. SCAD patients did not differ from controls and no difference in PAPP-A/proMBP levels with regards to the number of affected vessels was found. In 21 unstable angina pectoris (UAP), in 35 without and 66 with ST elevation acute myocardial infarctions (NSTEMI, STEMI respectively) patients PAPP-A/proMBP levels were increased (P=0.004 and P<0.0005, respectively). PAPP-A/proMBP levels did not correlate with cardiac troponin I (cTnI) in STEMI and NSTEMI patients. PAPP-A/ proMBP increase was more frequent than cTnI (P=0.036) within the early phase of STEMI. In NSTEMI patients PAPP-A/proMBP positivity was present in 50% of cTnI negative cases. Receiver operating characteristic (ROC) analysis revealed the highest diagnostic accuracy of PAPP-A/proMBP (0.919) in STEMI cTnI positive cases. The highest specificity/sensitivity PAPP-A/proMBP levels for particular acute coronary syndrome (ACS) types were 10.65-14.75 mIU/l. Combination of PAPP-A/proMBP with cTnI increases their diagnostic efficacy within the early phase of ACS. Our results suggest that PAPP-A/proMBP complex is involved in processes preceding vulnerable plaque development in ACS.

[Chromosome Y microdeletions in Czech men with severe reproductive disorders]. / Mikrodelécie chromozómu Y u ceských muzov s t'azkou poruchou reprodukcie.

BACKGROUND: The Y chromosome microdeletions belong to the frequent genetical causes of male infertility. The aim of our study was to introduce reliable molecular genetic diagnosis of Y chromosomal microdeletions and to determine the prevalence of Y chromosomal microdeletions in Czech males with serious reproductive disorders. METHODS AND RESULTS: The Y chromosome microdeletions were screened in 198 Czech men with serious reproductive disorders with decreased sperm count. The Y chromosome microdeletions were disclosed in 8/198 (4.0%) examined males. The AZFc deletion type was revealed in 62.5% (5/8) and the combined AZFc + b microdeletion in 37.5% (3/8) of cases. Neither isolated AZFb nor AZFa microdeletion were found in any subject of the investigated group. CONCLUSIONS: Incidence of individual types of Y chromosomal microdeletions in Czech males with serious reproductive disorders was assessed. The standardised molecular genetic diagnosis of Y chromosomal microdeletions was introduced into the practice.

[Medical genetics in reproductive medicine]. / Vyuzití lékarské genetiky v reprodukcní medicíne.

Reproductive genetics (RG) is another new field of medical genetics, integrated with reproductive medicine, assisted reproduction and developmental genetic. RG is closely linked to the perioconceptional prevention, perinatology, ultrasound and biochemical screening in the end of the first and beginning of the second trimesters. RG is based on the system of specialized genetic counseling, clinical cytogenetics, molecular cytogenetics and molecular genetics to provide prefertilization, preimplantation and classical prenatal diagnosis in the Ist to IIIrd trimesters. Thus, RG is part of the fetal medicine and therapy. The six years experience with RG is summarized. A system of the specialized health care, organized, if possible in one integrated center of RG and reproductive medicine (RM) is presented. Reproductive medicine provides all necessary clinical gynecological and andrological surveillance, with assisted reproduction and further obstetrical ultrasound examinations, including nuchal translucency measurements and 2D, 3D ultrasound, echocardiography examinations, if indicated, as well as the invasive method of prenatal diagnosis and perinatology care. Specialized genetic counseling and cytogenetic analysis, if indicated, should be offered to all partners with reproductive disorders as well as to oocyte donors. Chromosome anomalies are disclosed in 6% of men with abnormal sperm analysis as well as in women with severe reproductive disorders. In males with severe oligo, azoospermia, the sperm aneuploidy analysis by molecular cytogenetic methods is recommended. Advised is also the molecular genetic detection of Y chromosome microdeletions, which is detected in 9% of our azoospermic men with deletions in AZFb region. CFTR gene mutations and intron 8 and 10 polymorphism examination is provided not only in men with obstructive azoospermia (CBAVD), but also if severe oligospermy with less than 1 x 10(6) sperm/ml is detected. Molecular genetic analysis of thrombophilic mutations of factor II., V. (Leiden) and MTHFR gene in unexplained recurrent abortions and in cases with unsuccessful IVF is part of the diagnostic strategy. The population frequencies of carriers of mutations of factor II. (2.3%), factor V.-Leiden (5.7%) and MTHFR gene (38%) were determined. The laser biopsy of the first polar body and of blastomeres was introduced for FISH analysis of chromosome aneuploidies. Quantitative fluorescent PCR (QFPCR) detection is used for testing of the most frequent delta F508 CFTR gene mutation and the most frequent aneuploidies of chromosome 13, 18, 21, X and Y. QFPCR was successfully tested for male fetal sex examination from partially purified fetal cells in the maternal blood. The first trimester ultrasound and biochemical screening is recommended to all successful pregnancies after different IVF methods. If borderline levels of first trimester biochemical screening of PAPP-A protein and beta hCG are detected without pathological ultrasound findings, classical triple test of biochemical screening in 16th week of gestation is recommended. If pathological results of ultrasound and biochemical screening are disclosed, invasive prenatal genetic diagnosis is indicated as well as in pregnancies after ICSL, if there is not any obstetrical contraindication.

Identification, by homozygosity mapping, of a novel locus for autosomal recessive congenital ichthyosis on chromosome 17p, and evidence for further genetic heterogeneity.

Autosomal recessive congenital ichthyosis (ARCI) comprises a group of severe disorders of keratinization, characterized by variable erythema and skin scaling. It is known for its high degree of genetic and clinical heterogeneity. Mutations in the gene for keratinocyte transglutaminase (TGM1) on chromosome 14q11 were shown in patients with ARCI, and a second locus was described, on chromosome 2q, in families from northern Africa. Three other loci for ARCI, on chromosomes 3p and 19p, were identified recently. We have embarked on a whole-genome scan for further loci for ARCI in four families from Germany, Turkey, and the United Arab Emirates. A novel ARCI locus was identified on chromosome 17p, between the markers at D17S938 and D17S1856, with a maximum LOD score of 3.38, at maximum recombination fraction 0.00, at D17S945, under heterogeneity. This locus is linked to the disease in the Turkish family and in the German family. Extensive genealogical studies revealed that the parents of the German patients with ARCI were eighth cousins. By homozygosity mapping, the localization of the gene could then be refined to the 8.4-cM interval between D17S938 and D17S1879. It could be shown, however, that ARCI in the two Arab families is linked neither to the new locus on chromosome 17p nor to one of the five loci known previously. Our findings give evidence of further genetic heterogeneity that is not linked to distinctive phenotypes.

Assignment of the gene for a new hereditary nail disorder, isolated congenital nail dysplasia, to chromosome 17p13.

Isolated congenital nail dysplasia is an autosomal dominant disorder recently observed in a large family from southern Germany. The disorder is characterized by longitudinal streaks, thinning, and impaired formation of the nail plates leading to increased vulnerability of the free nail margins. In most cases, all fingernails and toenails are similarly involved with some accentuation of the thumb and great toenails. Histologic changes include hypergranulosis of the nail matrix and epithelial outgrowths from the nail bed. Patients do not show any alterations of hair growth and dentition, no malfunction of sweat glands and sensory organs, and no skeletal abnormalities. Isolated congenital nail dysplasia manifests from the first year of life with variable expressivity. In order to localize chromosomally the gene underlying isolated congenital nail dysplasia, linkage to the known keratin gene cluster regions on chromosomes 12q12 and 17q21 was ruled out first. The analysis of 150 microsatellite markers on various chromosomes mapped the isolated congenital nail dysplasia gene to the 6 cM interval between markers at D17S926 and D17S1528 on chromosome 17p13. Markers at D17S849, D17S 1840, and D17S1529 co-segregated completely with the isolated congenital nail dysplasia locus. The maximum two-point LOD score was found for the marker at D17S 1840 (Zmax = 6.72 at Thetamax = 0.00). The identified region harbors no currently known genes involved in skin or nail abnormalities. Isolated congenital nail dysplasia probably represents a novel isolated defect of nail development. The localization of this gene is, therefore, the first step towards the identification of a new factor in nail formation.

Homozygosity mapping in a family with microcephaly, mental retardation, and short stature to a Cohen syndrome region on 8q21.3-8q22.1: redefining a clinical entity.

A syndrome of microcephaly, progressive postnatal growth deficiency, and mental retardation was observed in two brothers and their cousin from a multiply consanguineous kindred of Lebanese descent. Hypotonia, chorioretinal dystrophy, and myopia were also identified. The severity of the condition varied among the closely related patients. Because of absence of a distinctive facial appearance, the degree of mental retardation, and short stature, the initially considered clinical diagnosis of Cohen syndrome was withdrawn and a novel genetic entity was assumed. Homozygosity mapping in this family assigned the gene to a 26.8-cM region on the chromosome band 8q21.3 -22.1, between the microsatellites at D8S270 and D8S514. The maximum two-point LOD score was found for marker at D8S267 (Zmax=3.237 at Omax=0.00). Intriguingly enough, the identified gene region overlaps the refined gene region for Cohen syndrome (COH1) [Kolehmainen et al., 1997: Euro J Hum Genet 5:206-213]. This fact encourages the hypothesis that the described kindred segregates for a variant of Cohen syndrome and suggests a redefinition of its phenotype.

Characterization of a novel 21-kb deletion, CFTRdele2,3(21 kb), in the CFTR gene: a cystic fibrosis mutation of Slavic origin common in Central and East Europe.

We report a large genomic deletion of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, viz., a deletion that is frequently observed in Central and Eastern Europe. The mutation, termed CFTRdele2,3(21 kb), deletes 21,080 bp spanning introns 1-3 of the CFTR gene. Transcript analyses have revealed that this deletion results in the loss of exons 2 and 3 in epithelial CFTR mRNA, thereby producing a premature termination signal within exon 4. In order to develop a simple polymerase chain reaction assay for this allele, we defined the end-points of the deletion at the DNA sequence level. We next screened for this mutation in a representative set of European and European-derived populations. Some 197 CF patients, including seven homozygotes, bearing this mutation have been identified during the course of our study. Clinical evaluation of CFTRdele2,3(21 kb) homozygotes and a comparison of compound heterozygotes for deltaF508/CFTRdele2,3(21 kb) with pairwise-matched deltaF508 homozygotes indicate that this deletion represents a severe mutation associated with pancreatic insufficiency and early age at diagnosis. Current data show that the mutation is particularly common in Czech (6.4% of all CF chromosomes), Russian (5.2%), Belorussian (3.3%), Austrian (2.6%), German (1.5%), Polish (1.5%), Slovenian (1.5%), Ukrainian (1.2%), and Slovak patients (1.1%). It has also been found in Lithuania, Latvia, Macedonia and Greece and has sporadically been observed in Canada, USA, France, Spain, Turkey, and UK, but not in CF patients from Bulgaria, Croatia, Romania or Serbia. Haplotype analysis has identified the same extragenic CF-haplotype XV-2c/KM. 19 "A" and the same infrequent intragenic microsatellite haplotype 16-33-13 (IVS8CA-IVS 17bTA-IVS 17bCA) in all examined CFTRdele2,3(21 kb) chromosomes, suggesting a common origin for this deletion. We conclude that the 21-kb deletion is a frequent and severe CF mutation in populations of Eastern- and Western-Slavic descent.

Non-syndromic autosomal dominant progressive non-specific mid-frequency sensorineural hearing impairment with childhood to late adolescence onset (DFNA21).

An autosomal dominant trait of progressive, non-syndromic, non-specific mid-frequency sensorineural hearing impairment was identified in a Dutch family. Many affected family members (n = 21) were identified, among whom seven out of nine relatives aged < 30 years do not show pure mid-frequency hearing impairment, which suggests variable expression. Regression analysis was used to evaluate the age-related hearing threshold data in a cross-sectional analysis in 24 affected patients and in a longitudinal analysis in five of these. At all frequencies, progression in hearing impairment (i.e. the regression coefficient) was significant and fairly similar: the pooled value was about 1.0 dB/y. There was no significant (i.e. not =0 dB) offset threshold (i.e. Y intercept at age 0) found at any frequency. The regression lines could be pooled for the low frequencies (0.25-0.5 kHz) and the mid/high frequencies (1-8 kHz) and this produced apparent onset ages of about 3 and 4 years and annual threshold increases of 0.75 and 1.1 dB/y, respectively. In most patients there is a relatively late onset age (maximum in the range of at least 25-45 years). However, based on the longitudinal analysis of a patient from the age of 4 years onwards in some patients sensorineural hearing impairment might be congenital/prelingual. Oculo-vestibular function was found to be normal. Results from linkage studies tentatively position the underlying gene defect telomeric to the repositioned DFNA13 locus at chromosome 6p21-22.

[Cystic fibrosis--a disease of adolescents and adults?]. / Cystická fibróza--nemoc dospívajících a dospelých?

BACKGROUND: Cystic fibrosis (CF) is no longer a childhood disease. Since the identification of the gene in 1989 research has made advances and changed views on the pathogenesis, diagnosis and treatment. The objective of the present work is to make doctors treating adult patients familiar with modern therapeutic methods and their value. METHODS AND RESULTS: In the CF Centre of the Faculty Hospital in Prague Motol 349 patients are followed up on a long-term basis, incl. 95 who died since 1985. Hundred and twenty six (36.1%) patients survived to the age of 18 years, of those 41 died and 85 patients live. Comparison of semilongitudinal data of a group of 83 patients born before 1975 whose treatment during childhood and puberty was inadequate and 196 patients born in 1976-90 treated by modern methods proved the great effect of treatment on the course and prognosis of the disease. The median age at death increased during from 12.2 years in 1985-90 to 18.8 years in 1991-1998 (p = 0.004). The nutritional status of adult patients is satisfactory in 40.4%, poor in 33.3% and marginal in 26.3%. A normal pulmonary function was recorded in 17.5%, 22.8% are severely affected, the majority of patients (59.7%) has values within 40 to 80% of normal levels. CONCLUSIONS: Modern intensive treatment improved the prognosis and quality of live in patients with CF. Critical deterioration of the clinical condition shifted to the threshold of adult age. It is therefore essential that doctors treating such patients should be familiar with this issue.

[Treatment of infertility in nonobstructive azoospermia using the TESE method (testicular sperm extraction)--clinical results]. / Lécba neplodnosti u neobstrukcní azoospermie metodou TESE (Testicular Sperm Extraction)--klinické výsledky.

Sperm was found in 56.6% of TESE cycles in 27 men with non-obstructive azoospermia. Using testicular sperm, 30% oocytes were fertilized. Embryos were transferred in 11 cycles. Twins were delivered in the 34th week of pregnancy. Two further pregnancies are ongoing. The pregnancy rate was 27% per ET. Testicular biopsy in all azoospermic men is recommended in special IVF centers not only for histological examination but also for ICSI procedure with testicular sperm which could be provided at the same time.

[Treatment of infertility in non-obstructive azoospermia using the TESE (testicular sperm extraction) method--clinical study]. / Lécba neplodnosti u neobstrukcní azoospermie metodou TESE--klinická vysetrení.

Testicular sperm extraction (TESE) was performed in 27 men in 30 cycles. All men were examined for genetics, serum hormonal status, biochemical status of semen samples. All men were examined by an urologist. No prognostic evaluation able to provide information about the prognosis of TESE procedure was found. Even a high FSH level, testicular hypotrophy or previous histological examination cannot exclude any patient from testicular biopsy.

Genotype/phenotype correlation in autosomal recessive lamellar ichthyosis.

Autosomal recessive lamellar ichthyosis is a severe congenital disorder of keratinization, characterized by variable erythema of the whole body surface and by different scaling patterns. Recently, mutations have been identified in patients with lamellar ichthyosis in the TGM1 gene coding for keratinocyte transglutaminase, and a second locus has been mapped to chromosome 2. We have now analyzed the genotype/phenotype correlation in a total of 14 families with lamellar ichthyosis. Linkage analyses using microsatellites in the region of the TGM1 gene confirmed genetic heterogeneity. In patients not linked to the TGM1 gene, the second region identified on chromosome 2 and a further candidate region on chromosome 20 were excluded, confirming as well the existence of at least three loci for lamellar ichthyosis. Sequence analyses of the TGM1 gene in families compatible with linkage to this locus revealed seven different missense mutations, five of these unpublished so far, and one splice mutation. No genotype/phenotype correlation for mutations in the TGM1 gene was found in this group of patients, which included two unrelated patients homozygous for the same mutation. Similarly, no clear difference in the clinical picture was seen between patients with TGM1 mutations and those unlinked to the TGM1 locus. Comparison of genetic and clinical classifications for patients with lamellar ichthyosis shows no consistency and thus indicates that clinical criteria currently in use cannot discriminate between the molecularly different forms of the disease.

SCA6 is caused by moderate CAG expansion in the alpha1A-voltage-dependent calcium channel gene.

Recently, moderate (CAG)>20 repeat expansions in the alpha1A-voltage-dependent calcium channel gene (CACNL1A4) have been identified in a previously unmapped type of SCA which has been named SCA6. We investigated the (CAG)n repeat length of the CACNL1A4 gene in 733 patients with sporadic ataxia and in 46 German families with dominantly inherited SCA which do not harbor the SCA1, SCA2, or MJD1/SCA3 mutation, respectively. The SCA6 (CAG)n expansion was identified in 32 patients most frequently with late manifestation of the disease. The (CAG)n stretch of the affected allele varied between 22 and 28 trinucleotide units and is therefore the shortest trinucleotide repeat expansion causing spinocerebellar ataxia. The (CAG)n repeat length is inversely correlated with the age at onset. In 11 parental transmissions of the expanded allele no repeat instability has been observed. Repeat instability was also not found for the normal allele investigating 431 meioses in the CEPH families. Analyzing 248 apparently healthy octogenerians revealed one allele of 18 repeats which is the longest normal CAG repeat in the CACNL1A4 gene reported. The SCA6 mutation causes the disease in approximately 10% of autosomal dominant SCA in Germany. Most importantly, the trinucleotide expansion was observed in four ataxia patients without obvious family history of the disease which necessitates a search for the SCA6 (CAG)n expansion even in sporadic patients.

SCA2 trinucleotide expansion in German SCA patients.

Autosomal dominant spinocerebellar ataxias (SCA) are a group of clinically and genetically heterogeneous neurodegenerative disorders which lead to progressive cerebellar ataxia. A gene responsible for SCA type 2 has been mapped to human chromosome 12 and the disease causing mutation has been identified as an unstable and expanded (CAG)n trinucleotide repeat. We investigated the (CAG)n repeat length of the SCA2 gene in 842 patients with sporadic ataxia and in 96 German families with dominantly inherited SCA which do not harbor the SCA1 or MJD1/SCA3 mutation, respectively. The SCA2 (CAG)n expansion was identified in 71 patients from 54 families. The (CAG)n stretch of the affected allele varied between 36 and 64 trinucleotide units. Significant repeat expansions occurred most commonly during paternal transmission. Analysis of the (CAG)n repeat lengths with the age of onset in 41 patients revealed an inverse correlation. Two hundred and forty-one apparently healthy octogenerians carried alleles between 16 and 31 repeats. One 50-year old, healthy individual had 34 repeats; she had transmitted an expanded allele to her child. The small difference between 'normal' and disease alleles makes it necessary to define the extreme values of their ranges. With one exception, the trinucleotide expansion was not observed in 842 ataxia patients without a family history of the disease. The SCA2 mutation causes the disease in nearly 14% of autosomal dominant SCA in Germany.

Possible association of the allele status of the CS.7/HhaI polymorphism 5' of the CFTR gene with postnatal female survival.

Cystic fibrosis (CF) patients show a high degree of linkage disequilibrium between the CF transmembrane conductance regulator (CFTR) gene and polymorphisms 5' of that gene. To determine whether the region 5' of CFTR contains biologically important sequences, the allele frequencies of six CFTR-linked polymorphisms (metH/MspI, XV-2c/TaqI, CS.7/HhaI, KM19/PstI, MP6d9/MspI, J44/XbaI) were determined in 417 randomly selected elderly individuals (over 75 years of age) from the Czech population. The elderly individuals were considered "escapees" of strong selective pressures that had operated during their lifetime, prior to the introduction of modern health care since 1950. The pooled allele frequencies of the analyzed marker polymorphisms in the elderly did not significantly differ from published data. However, when analyzed by ex, the allele frequencies of markers CS.7/HhaI and KM19/PstI differed significantly (P < 0.05) between elderly females and males. The allele frequencies of the six polymorphisms were then determined in 646 newborns and 345 young adults of reproductive age; these individuals were selected in a similar manner and drawn from the same population. In these control groups, the studied marker polymorphisms exhibited no statistically significant differences between sexes and/or between individuals of the same sex, only between different age groups. A gradual relative increase in the frequency of allele "2" of marker CS.7/HhaI was observed from newborn females to elderly women, the overall difference in allele frequencies of this marker polymorphism between newborn females and elderly women reaching statistical significance (P < 0.05). Interestingly, allele "2" is the major constituent of the extended "B-haplotype", which is in strong linkage disequilibrium with common CF alleles. Taken together, our data suggest that the region spanning markers CS.7 and KM19 is associated with a genetic factor that influences postnatal female survival, providing a possible mechanism for increasing the frequency of particular mutations in the adjacent CFTR gene.