Four novel mutations of the PKD2 gene in Czech families with autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is a genetically heterogeneous disease caused by mutations in at least three different loci. Mutations in the PKD2 gene are responsible for approximately 15% of the cases of the disease. We have screened 14 Czech families for mutation in the PKD2 gene. Clear evidence against linkage to the PKD1 gene was established by CA-repeat markers in five families. The disease could be linked to both genes according to linkage analysis in nine families but we have chosen these families because of the mild clinical course. An affected member from each family was analyzed by heteroduplex analysis (HA) and single strand conformation polymorphism (SSCP) for all 15 coding regions. Samples exhibiting shifted bands on HA or SSCP gels were sequenced. We detected five mutations (four new, and one which was previously described) and two polymorphisms. The four new mutations include one insertion, one deletion, one substitution (leading to premature translation stop), one amino acid substitution. Our results confirm that different point or small changes distributed throughout the PKD2 gene without clustering are responsible for the disease.

Increased micronuclei frequencies in couples with reproductive failure.

A higher incidence of chromosomal instability in the infertile population is widely recognized. An increased level of micronuclei has been shown to be a marker of chromosome damage. Therefore, micronuclei frequencies were assessed in cytokinesis-blocked lymphocytes of 130 patients (65 couples) with idiopathic infertility or with two or more spontaneous abortions, and 30 healthy fertile donors (15 couples). The frequency of micronucleated cells in the cohort with reproductive failure and healthy controls averaged 14.95+/-6.04 per 1000 and 10.60 +/-2.57 per 1000 (P<0.0001), respectively. When micronuclei frequency sums in particular couples (male + female) were analyzed in the same order, identical statistical significance was reached (P<0.0001). We found no effect of age or sex on micronuclei frequency. In summary, the cytokinesis-blocked micronuclei assay revealed increased micronucleus frequency in couples with infertility or two or more spontaneous abortions, suggesting a possible role of chromosomal instability in reproductive failure.

DNA diagnosis and clinical manifestations of autosomal dominant polycystic kidney disease.

At least 2 genes, detectable by DNA methods, encode autosomal dominant polycystic kidney disease (ADPKD), which remains the most frequent and serious hereditary renal disease. PKD1 gene, localized on chromosome 16, responds for the clinical course in the majority of ADPKD patients, whereas PKD2 gene, localized on chromosome 4, is responsible for less than 10-15% of cases, with presumed milder phenotypic manifestations. To start the clinical and genetic correlation in patients with different genotypes (PKD1 vs. PKD2) in the Czech population, a pilot group of 88 patients with ADPKD was analysed. Families with PKD1 (n = 44) represented 95.6% and families with PKD2 (n = 2) 4.4% of all families investigated (n = 46). Our clinical analysis, yet based only on a limited number of PKD2 subjects, does not definitely support the concept of a milder phenotype and prognosis in PKD2 versus PKD1 patients, in terms of mean age of diagnosis (29 vs. 29 years), mean age at onset of arterial hypertension (33 vs. 33 years), more favourable renal function or ultrasound findings.

[Cleft lip and palate--prenatal diagnosis and counseling]. / Rozstep rtu a patra--prenatální diagnostika a poradenství.

The most frequent congenital developmental defect in the orofacial region are, no doubt, facial clefts which are a serious stress for health professionals and the population. Depending on the type of cleft, the prevalence is between 1 : 1000-2800 births. According to contemporary views in the etiology of orofacial clefts participate genetic as well as environmental factors. That means that specific genetic factors create a certain "sensitivity" for specific factors of the external environment which act as a trigger mechanism and combined they produce the cleft. Cleft lip can be diagnosed already during the 13th week of gestation, while a cleft palate is not necessarily apparent till after the 18th week of gestation as the maxilla is in the process of joining. Presentation of the foetal face and its profile is thus important in particular during the second trimester of gestation and should be part of ultrasonographic screening between the 18th and 20th week of gestation. As more than 8% of facial clefts are associated with chromosomal abnormalities, in all affected foetuses karyotyping is done. The prognosis of satisfactory cosmetic and functional repair in cleft lip and in cleft lip and palate is favourable. In case of associated malformations all depends on the type and severity of these associated defects or on the diagnosis of the syndrome. If median clefts are extensive or associated with cerebral anomalies, the prognosis is as a rule poor. Prenatal diagnosis and management of defects of the orofacial area calls for collaboration of the obstetrician, neonatologist and plastic surgeon already in the stage when the defect is detected to give the expectant mother an opportunity to obtain accurate and unbiased information on possible treatment and prognosis for the foetus.

[Termination of pregnancy in the 2nd trimester using intra-amniotic administration of prostaglandins]. / Ukoncení tehotenství ve II. trimestru gravidity intraamniální aplikací prostaglandinu.

INTRODUCTION: The method used to terminate pregnancy on medical grounds during the second trimester must be safe, rapid, psychologically feasible and associated with a minimal risk of long-term sequelae. The objective of the present work was a critical analysis of the author's standard protocol of termination of pregnancy during the second trimester. MATERIAL AND METHODS: For induction of abortion during the second trimester the authors used a synthetic prostaglandin analogue (PG) F2 alpha-Dinoprost which was administered in a single dose of 30 mg by the intraamniotic route. At the time of onset of uterine contractions the authors administered peridural anaesthesia. The authors investigated indications, mean period of induction, correlation between the period of induction of abortion and the indications for termination of pregnancy, the week of pregnancy and parity of the mother. They recorded also the type and number of complications. RESULTS: From January 1991 till June 1997 179 pregnancies were terminated by intraamniotic PG administration. After a single intraamniotic PG administration 72% women aborted within 24 hours. In 26% women the intraamniotic administration was repeated twice and in 2% women three times. The mean induction period, i.e. the interval between the administration and abortion of the foetus was 22.6 hours. The interval was significantly longer (28 hours) in foetuses where pregnancy was terminated because of a neural tube defect (p < 0.01). The authors did not detect a correlation between the period of induction and the indication, week of gestation and parity of the mother. COMPLICATIONS: once a general reaction to intraamniotic administration, in three patients a major blood loss replaced by transfusion of erythrocyte mass, no uterine rupture. CONCLUSION: In all instances the therapeutic effect was achieved and there was no need to perform section minor. The disadvantage of the method is the high price of the preparation and need of repeated intraamniotic administration of PG in 29% of the patients.

PIP: In this study F2 alpha-Dinoprost, a synthetic prostaglandin analogue, was tested for use in inducing abortion in second-trimester pregnancy. During the period of January 1991 to June 1997 a total of 179 pregnancies were terminated by intra-amniotic administration of the drug. The indications for the fetus were congenital abnormalities (59 cases or 32%), intrauterine death (41 cases or 24%), and chromosomal aberrations (20 cases or 11%). For the mother the indications were internal disease, psychological reasons (38 cases or 21%), and unsuccessful abortion in the first trimester. The following procedure was carried out: puncture of the amniotic sac under ultrasound control, intra- amniotic administration of a 5 mg of Dinoprost test dose followed by the whole single dose of 30 mg, continuous infusion of 5 mg of Dinoprost at a speed of 10 gtt/min, peridural analgesia, and instrumental curettage. Peridural anesthesia was administered at the time of onset of uterine contractions. Following a single dose 72 women aborted within 24 hours. The intra-amniotic administration of the drug was performed twice in 48 (26%) women and three times in 3 (2%) women. The mean induction period (the interval between dose administration and the expulsion of the fetus) was 22.6 hours. The interval was significantly longer (28 hours) in fetuses for which pregnancy was terminated because of a neural tube defect (p 0.01). Complications included 1 episode of general reaction to intra-amniotic administration; in 3 patients massive blood loss was replaced by the transfusion of 2 x 300 ml erythrocyte mass. Postabortum residue was encountered in 2 cases, cervical rupture in one case, and phlebothrombosis in 1 case. The disadvantage of the method is the high price of the preparation and, to judge by these results, that it requires repeated administration for a substantial minority (29% or 51) of patients.

[The attitude of family members with polycystic kidney disease to the disease and presymptomatic testing]. / Postoje clenu rodin s polycystickou chorobou ledvin k onemocnení a presymptomatickým testum.

BACKGROUND: The application of presymptomatic diagnosis of polycystic kidney disease (PKD) relies on satisfactory collaboration between the affected families and the team of health workers. The objective of the present study was to assess the initial level of knowledge of the disease and its hereditary character, the attitudes of families with PKD to presymptomatic and prenatal testing focused on methods of molecular genetics as well as attitudes to induced abortion if the gene of PKD is detected in the foetus. METHODS AND RESULTS: In a group of 104 subjects who had genetic examinations on account of familial PKD by means of questionnaires knowledge of the diagnosis, clinical and genetic prognosis and attitudes to presymptomatic and prenatal genetic tests were investigated. About 75% of the members of the group had adequate knowledge of the name of the disease and description of the clinical picture, 96.1% were informed on the hereditary character of the disease but only 1.9% knew details about the heredity. The scope of genetic risk was mentioned by some 30%, 51% were unable to express their opinion on the risk. A positive attitude to testing for the presence of the PKD gene was recorded in almost 100% if they themselves were concerned, 94% when their offspring under age was concerned but only 19.2% when the foetus was concerned. Only 5.3% of the respondents would agree with induced abortion in case of positive prenatal evidence of the PKD gene in the foetus and a few more respondents (14%) were opposed to abortion and some 80% were unable or unwilling to express their opinion. CONCLUSIONS: In members of families with PKD an important role is played by knowledge of clinical and genetic aspects of the disease. Genetic counselling must be part of comprehensive care and should always precede indication of molecular genetic methods (DNA analyses).

[Restriction fragment length polymorphisms in the families of patients with Down's syndrome (trisomy 21)]. / Polymorfismy délek restrikcních fragmentu v rodinách pacientu s Downovým syndromem (trisomie 21).

Using the method of polymorphisms of lengths of restriction fragments (RFLP), the authors compare agreement and differences in the normal healthy Czech population and in families with a patient suffering from Down's syndrome. 2-alpha-satellite DNA probes were used which are weighed in the pericentromeric area of heterochromatin of the long arms of chromosomes 13 and 21. These probes contain a number of repetitive sequences, most frequently represented in human heterochromatin of the majority of chromosomes. By hybridization with an alpha-RI-6 probe multiallelic polymorphisms were obtained in families with Down's syndrome in five restrictive endonucleases (Bsp RI, Eco RI, Pst I, Taq I and Xba I). Restrictions with enzymes Bam HI and Hind III were non-polymorphous. Hybridization with the alpha-RI-IB probe revealed polymorphism with restrictive endonuclease Taq I. Enzymes Bam HI, Eco RI, Hind III, Pst I and Xba I (3) were non-polymorphous. The difference of the two probes in the centromeric area of chromosomes 13 and 21 was confirmed by hybridization in situ, using 3H-labelled thymidine triphosphate (TIP) in quantitative experiments on short-term cultures of lymphocytes of healthy subjects (4).

[A genetic registry of pregnant women--SEGRAV]. / Genetický registr gravidních zen--SEGRAV.

The authors describe the structure, contents and use of a genetic register of pregnant women examined at the department of medical genetics. They discuss in detail the investigated data and their classification. The programme makes it possible to use a computer for the longitudinal follow up of the course of pregnancy and the implementation of recommended preventive measures and processing of data for medical statistics. Perspectively the register will be used for evaluation of the validity of genetic prognoses and effectiveness of preventive genetic methods. The register is used in routine practice of genetic clinics since September 1986. The authors give also an account of the experience with the genetic register.

[An alpha-satellite DNA sequence, alpha-RI-6, specific for human chromosomes 13 and 21, detected using the RFLP technic with digoxigenin labelled probes]. / Alfa-satelitní DNA-sekvence, alfa-RI-6, specificka pro lidské chromosomy 13 a 21, detekovaná pomocí RFLP-techniky s digoxigeninovým znacením sondy.

The authors compared two at present most widely used techniques for labelling DNA probes: a) radioactive labelling by means of the radioisotope 32P; non-radioactive labelling using the hapten digoxigenin for the visualization of the hybridization process on nylon membranes. Then sensitivity of the technique of non-radioactive labelling of heterochromatin probes was equivalent to the radioactive method.

[Mutation of the phenylalanine hydroxylase gene in the population of central Bohemia. Relation to the clinical picture of phenylketonuria]. / Mutace genu fenylalaninhydroxylázy ve stredoceské populaci. Vztah ke klinickému obrazu fenylketonurie.

BACKGROUND: Phenylketonuria (PKU) is an autosomal recessive, disease, heterogeneous at the molecular level, caused by mutations in the gene of phenylalanine hydroxylase (PAH). The objective of the present work was to identify mutations and their frequency in the Central Bohemian and Prague population in relation to the clinical phenotype. METHODS AND RESULTS: The authors analyzed a group of 33 patients from 32 unrelated families. The phenotypic manifestations were classified as non-PKU hyperphenylalaninaemia (non-PKU-HPA), mild and classical PKU. Sixty-six mutant alleles of the PAH gene were analyzed by means of the polymerase chain reaction on a Perkin Elmer (480) apparatus and on PHC Techne. A total of eight mutations linked with five haplotypes were detected. R408W mutation linked with 2.4 haplotype was detected on 53% of mutant alleles. No type of mutation was detected by hitherto published procedures in 27% of mutant alleles. CONCLUSIONS: The finding on the distribution and frequency of mutations indicate a genotypic homogeneity of the PKU population in the Central Bohemian area and Prague and are consistent with hitherto published data from the Czech Republic. The revealed data can be used in prenatal and postnatal DNA diagnosis and genotype classification of PKU.

[Autosomal dominant hereditary polycystic kidney disease]. / Autosomálne dominantne dedicná polycystická choroba ledvin.

BACKGROUND: Molecular genetics are a fundamental turning point in the approach to autosomal dominant hereditary renal polycystosis of adults (ADPKD). DNA analysis makes diagnosis possible at in any ontogenic period, i.e. also during the prenatal period. The objective of the present study was to test the presymptomatic DNA diagnosis in a major group of patients with ADPKD and the possibility to detect the disease in the initial stage and influence its development by early treatment and advise the patients on parenthood. METHODS AND RESULTS: In 1990-1994 the authors contacted 157 patients with polycystic kidney disease, adult type (ADPKD). 87 families were examined by Southern's RFLP method (gene PKD1, 16p13.3, standard probe 3'HVR and restrictase Pvu II). Of 493 members of these families only 25 (5.1%) refused to be examined. So far 378 examinations were completed, 90 proceed. In 40 of 132 examined subjects with the risk of ADPKD transmission of the gene was proved. Of four examinations of the foetus with the risk of ADPKD twice transmission of the gene was proved and the pregnancy was terminated. In three families with three or more members suffering from ADPKD in some there was not agreement between the result of linkage analysis of DNA and the clinical finding. The authors analyzed whether the cause is recombination or ADKPD conditioned by mutation of gene PKD2 (4p13-23). Linkage analysis remains in ADPKD the basic examination as the sequence of gene PKD1 is not yet completed. Discovery of gene PKD2 the mutations of which condition as many as 15% of all cases of ADPKD has an impact on the evaluation of linkage analysis. The reliability of prediction rises with the number of examined subjects in the family. The examination revealed that patients are willing to have DNA examinations (as many as 98%); despite this the number of examined subjects is only a small fraction of the anticipated 10,000 people in the Czech Republic suffering from ADPKD: CONCLUSIONS: DNA analysis in patients with autosomal dominant polycystic kidney disease is the most important examination for its diagnosis. It makes the diagnosis possible in all stages of ontogenesis, incl. prenatal diagnosis. It is a highly valid parameter when these patients decide on parenthood. It makes early treatment possible which can influence the development of the disease and its complications.

[Clinical and molecular problems in polycystic kidneys]. / Klinická a molekulární problematika polycystických ledvin.

The authors examined, using nephrological, genealogical and molecular genetic methods, 85 subjects from 19 families with autosomal dominant heredity of polycystic kidney disease. Using the probe 3HVR alpha-globin and restriction endonuclease Pvu II, the authors assessed 95% informative families in the given group and the reliability of the diagnosis was also 95%. The authors tested the homogeneity of the disease in the Czech population and the applicability of the mentioned probe and endonuclease for molecular genetic testing of our population.

[The syndrome of multicystic dysplasia of the kidney, spleen and liver with multiple malformations]. / Syndrom multicystické dysplazie ledvin, slinivky a jater s mnohocetnými malformacemi.

A genealogic analysis carried out in a neonate suffering from multicystic dysplasia of kidneys, pancreas and liver indivated an autosomal recessive type of heredity. Associated malformations of extremities with heterotaxis were present in hitherto undescribed combinations.

Screening of amino acid enzymopathies in pregnancy and possibilities of their prenatal diagnosis.

The report discusses the first results obtained by chromatographic screening of the blood amino acids in pregnant women at the outset of pregnancy. Typical cases of maternal phenylketonuria and maternal hyperphenylalaninaemia found among 2,000 women examined are described in detail, giving a concise metabolic, clinical and genetic picture. The biochemical possibilities of diagnosing congenital disorders of amino acid metabolism before birth are also discussed.

Hyperglycinaemia without ketosis.

When screening mentally backward children we detected a 5-year-old child with elevated urinary glycine excretion and a high blood glycine level. The report discusses the results of a clinical, metabolic and genetic examination of the child and the members of his family with reference to this rare metabolic disorder.

[DNA diagnosis in families with autosomal dominant hereditary polycystic kidney disease]. / Problémy DNA diagnostiky v rodinách s autosomálne dominantne dedicnou polycystickou chorobou ledvin.

In 1990-1992 110 patients with polycystic kidney disease adult type (ADPKD) were contacted. Forty-nine families were examined by Southern's RFLP method (standard probe 3'HVR and Pvu II restrictase). Of 424 members of these families only 25 (5.9%) refused the examination. So far 337 examinations were completed, 62 are under way. In 32 cases of 109 subjects at risk of 50% ADPKD the affection was proved with a probability higher than 95%, in 77 subjects (and 6 subjects with a 25% risk) were eliminated with an equal probability. In two families successful prenatal examinations of the foetus were accomplished. Evaluation of attitudes to DNA diagnostics revealed in our patients a greater willingness to attend postnatal examinations (up to 98%) and a smaller willingness to have prenatal examinations (21%), as compared with data published abroad.