A common founder effect of the splice site variant c.-23 + 1G > A in GJB2 gene causing autosomal recessive deafness 1A (DFNB1A) in Eurasia.

Mutations in the GJB2 gene are known to be a major cause of autosomal recessive deafness 1A (OMIM 220290). The most common pathogenic variants of the GJB2 gene have a high ethno-geographic specificity in their distribution, being attributed to a founder effect related to the Neolithic migration routes of Homo sapiens. The c.-23 + 1G > A splice site variant is frequently found among deaf patients of both Caucasian and Asian origins. It is currently unknown whether the spread of this mutation across Eurasia is a result of the founder effect or if it could have multiple local centers of origin. To determine the origin of c.-23 + 1G > A, we reconstructed haplotypes by genotyping SNPs on an Illumina OmniExpress 730 K platform of 23 deaf individuals homozygous for this variant from different populations of Eurasia. The analyses revealed the presence of common regions of homozygosity in different individual genomes in the sample. These data support the hypothesis of the common founder effect in the distribution of the c.-23 + 1G > A variant of the GJB2 gene. Based on the published data on the c.-23 + 1G > A prevalence among 16,177 deaf people and the calculation of the TMRCA of the modified f2-haplotypes carrying this variant, we reconstructed the potential migration routes of the carriers of this mutation around the world. This analysis indicates that the c.-23 + 1G > A variant in the GJB2 gene may have originated approximately 6000 years ago in the territory of the Caucasus or the Middle East then spread throughout Europe, South and Central Asia and other regions of the world.

Targeted next-generation sequencing in Slovak cardiomyopathy patients.

OBJECTIVES: For the first time we used targeted next-generation sequencing to detect candidate pathogenic variants in Slovak cardiomyopathy patients. BACKGROUND: Targeted next-generation sequencing is considered to be the best practice in genetic diagnostics of cardiomyopathies. However, in Slovakia, with high cardiomyopathies prevalence of 1/440, the current diagnostic tests are still based on Sanger sequencing of a few genes. Consequently, little is known about the exact contribution of pathogenic variants in known cardiomyopathy genes in Slovak patients. METHODS: We used a panel of 46 known cardiomyopathy-associated genes to detect genetic variants in 16 Slovak cardiomyopathy patients (6 dilated, 8 hypertrophic, 2 non-compaction subtypes). RESULTS: We identified candidate pathogenic variants in 11 of 16 patients (69 %). Genes with higher count of candidate pathogenic variants were MYBPC3, MYH and TTN, each with 3 different variants. Seven variants ACTC1 (c.329C>T), ANKRD1 (c.683G>T), MYH7 (c.1025C>T), PKP2 (c.2003delA), TTN (c.51655C>T, c.84841G>T, c.101874_101881delAGAATTTG) have been detected for the first time and might represent Slovak-specific genetic cause. CONCLUSIONS: We have performed genetic testing of previously untested Slovak cardiomyopathy patients using next-generation sequencing cardiomyopathy gene panel. Given the high percentage of candidate pathogenic variants it should be recommended to implement this method into routine genetic diagnostic practice in Slovakia (Tab. 4, Ref. 39).

Complex phenotypes blur conventional borders between Say-Barber-Biesecker-Young-Simpson syndrome and genitopatellar syndrome.

Say-Barber-Biesecker-Young-Simpson syndrome (SBBYSS) and genitopatellar syndrome (GTPTS) are clinically similar disorders with some overlapping features. Although they are currently considered to be distinct clinical entities, both were found to be caused by de novo truncating sequence variants in the KAT6B (lysine acetyltransferase 6B) gene, strongly suggesting that they are allelic disorders. Herein, we report the clinical and genetic findings in a girl presenting with a serious multiple congenital anomaly syndrome with phenotypic features overlapping both SBBYSS and GTPTS; pointing out that the clinical distinction between these disorders is not exact and there do exist patients, in whom conventional clinical classification is problematic. Genetic analyses revealed a truncating c.4592delA (p.Asn1531Thrfs*18) variant in the last KAT6B exon. Our findings support that phenotypes associated with typical KAT6B disease-causing variants should be referred to as 'KAT6B spectrum disorders' or 'KAT6B related disorders', rather than their current SBBYSS and GTPTS classification.

Clustering of mutations in the 5' tertile of the NF1 gene in Slovakia patients with optic pathway glioma.

Optic pathway gliomas (OPG) occur in 15% of patients with neurofibromatosis type 1 (NF1; OMIM 162200). Genotype-phenotype correlations in patients with NF1 may help to determine the risk group for developing complications such as OPG in coincidence with other NF1.features. We evaluated 52 patients with NF1 (25 with OPG and 27 without OPG). All subjects underwent a clinical examination focused on neurofibromatosis type 1 and molecular diagnostics of NF1 gene using protocol based on RNA analysis confirming the diagnosis of NF1. In the group with OPG patients, there was a significantly higher incidence of freckling (P=0.017), neurofibromatosis bright objects (NBO) (P=0.0038), compared to the group without OPG. The differences between the groups with respect to Lisch nodules were on the borderline of statistical significance (P=0.088). The frequency of neurofibromas in the group with OPG was not significant (P=0.9). From all patients with the mutation localized in the first tertile of the NF1 gene majority (71%) had optic glioma compared to individuals who didn't have the OPG 29% (P=0.0049). Our results present the clustering of mutations in the 5'tertile of NF1 gene in patients with optic nerve glioma and suggest higher incidence of freckling and neurofibromatosis brain objects in these patients. Molecular analysis of NF1 gene is important part in complex management of NF1 patients and contributes to a better understanding of clinical picture of NF1 patients. .

Prevalence of mutations in thiopurine S-methyltransferase gene among Slovak IBD patients.

BACKGROUND: Thiopurine S-methyltransferase (TPMT) plays an important role in the metabolism of thiopurines. It has been suggested that TPMT genetic polymorphisms lead to dose-related hematopoietic toxicity. Since there are major ethnic differences in the prevalence of particular TPMT variants, it is important for each country to study their own prevalence in order to estimate the role of TPMT variants-related thiopurines toxicity in population suffering from particular inflammatory bowel disease (IBD). AIMS: The aim of this study was to determine the frequency of the four most common allelic variants of TPMT gene in the population of Slovak IBD patients. METHODS: TPMT genetic polymorphisms (TPMT*2, TPMT*3A, TPMT*3B, TPMT*3C) were amplified using PCR and consequently genotyped with genetic analyzer. The allele frequencies of particular allelic variants were calculated and compared with other Caucasian populations reported so far. RESULTS: Three hundred and thirty IBD patients were included; 196/132/2 cases of Crohn´s disease/ulcerative colitis/unclassified colitis; 180 (55 %) males. Ninety-three percent of patients were homozygous for wild-type TPMT variant. Heterozygous genotype of any of the studied polymorphisms was present in 6 % of patients while only one patient was homozygous for TPMT*3A allele (0.3 %). The most prevalent mutant allele was that of TPMT*3A (3.2 %). The distribution of most common allelic variants of TPMT gene among Slovak IBD patients was in accordance with previously reported prevalence in Caucasian populations. CONCLUSION: This study shows the prevalence of TPMT genetic polymorphisms in population of Slovak IBD patients. As in other Caucasian populations, the most common mutant allelic variant is that of TPMT*3A while the prevalence of homozygosity is relatively low (Tab. 3, Ref. 22).

The impact of thiopurine-S-methyltransferase genotype on the adverse drug reactions to azathioprine in patients with inflammatory bowel diseases.

BACKGROUND AND AIMS: The thiopurine drugs, azathioprine (AZA) and 6-mercaptopurine, are established in the treatment of inflammatory bowel diseases (IBD). Polymorphisms in thiopurine S-methyltransferase (TPMT) gene have been associated with adverse drug reactions (ADRs) to AZA. METHODS: The aim of this study was to evaluate TPMT polymorphisms and AZA-related toxicity in a Slovak cohort of 220 IBD patients treated with AZA. In every patient, the dose and duration of AZA therapy, concomitant 5-aminosalicylate (5-ASA) medication, frequency, type, time to onset, dose of ADR and concomitant 5-ASA at the onset of ADR were recorded. Each patient was also genotyped for the presence of variant TPMT alleles (*2,*3A,*3B,*3C). Frequency, type and circumstances of ADRs were compared according to TPMT status. RESULTS: Of the 220 patients, 205 (93.2 %) were wild-type (TPMT*1/*1), one (0.5%) carried a TPMT*1/*3C allele, 13 (5.9 %) carried TPMT *1/*3A allele and one was homozygous for TMPT *3A allele. No TPMT *2 mutation was found. The incidence of adverse drug reactions was 62/205 (30.2 %) in the wild-type group as compared to 13/15 (86.7 %) in the TPMT mutation group, p=2.10-5. Leukopenia (WBC< 3.0*10^9/L) occurred in 21/205 (10.2 %) patients with wild type TPMT versus 11/15 (73.3 %) patients with TPMT mutations, p=0.000001. There was no significant difference between TMPT groups in gastrointestinal or other ADRs. No impact of 5-ASA on the incidence and severity of AZA adverse drug reactions was observed. CONCLUSION: The incidence of leukopenia in TPMT mutant patients was significantly higher and more severe as compared to TPMT wild type patients. We observed no impact of concomitant 5-ASA therapy on AZA induced toxicity (Tab. 4, Fig. 2, Ref. 37).

Prevalence of mutations in thiopurine S-methyltransferase gene among Slovak IBD patients.

BACKGROUND: Thiopurine S-methyltransferase (TPMT) plays an important role in the metabolism of thiopurines. It has been suggested that TPMT genetic polymorphisms lead to dose-related hematopoetic toxicity. Since there are major ethnic differences in the prevalence of particular TPMT variants, it is important for each country to study their own prevalence in order to estimate the role of TPMT variants-related thiopurines toxicity in the particular inflammatory bowel disease (IBD) population. AIMS: The aim of this study was to determine the frequency of the four most common allelic variants of TPMT gene in the population of Slovak IBD patients. METHODS: TPMT genetic polymorphisms (TPMT*2, TPMT*3A, TPMT*3B, TPMT*3C) were amplified using PCR and consequently genotyped on genetic analyzer. The allele frequencies of particular allelic variants were calculated and compared with other Caucasian populations reported so far. RESULTS: Three hundred and thirty IBD patients were included; 196/132/2 Crohn´s disease/ulcerative colitis/unclassified colitis, 180 (55 %) males. Ninety-three percent of patients were homozygous for wild type TPMT variant. Heterozygous genotype of any of the studied polymorphisms was present in 6 % of patients, only one patient was homozygous for TPMT*3A allele (0.3 %). The most prevalent mutant allele was TPMT*3A (3.2 %). The distribution of the most common allelic variants of TPMT gene among Slovak IBD patients were in accordance with previously reported prevalence in Caucasian populations. CONCLUSION: This study shows the prevalence of TPMT genetic polymorphisms in the Slovak IBD patient`s population. As in other Caucasian populations, the most common mutant allelic variant is TPMT*3A, and the prevalence of homozygosity is relatively low (Tab. 3, Ref. 16).

Oral in vivo bactofection in dextran sulfate sodium treated female Wistar rats.

Salmonella typhimurium SL7207 carrying Cu-Zn superoxide dismutase and an N-terminal deletion mutant of monocyte chemoattractant protein-1 genes was applied to dextran sodium sulfate treated female Wistar rats. Stool quality, food and water intake were monitored. Markers of oxidative stress, interleukin 1, interleukin 6 and tumor necrosis factor alpha were quantified. No differences were found in body weights, markers of oxidative stress in plasma and inflammatory markers in colon homogenates. Plasma concentrations of I11, I16 were lower in the treatment groups than in the dextran sodium sulfate group. However, dextran sodium sulfate induced inflammation could not be confirmed by plasma levels of I11, I16 and TNFalpha. Although some parameters showed a tendency to improve, the inflammation caused by administration of 4% dextran sodium sulfate during 7 days was low and contradictory to other studies. Results showed the potential synergic effect of combined bacteria-mediated antioxidative and anti-inflammatory gene therapy.

Mutation analysis of the CFTR gene in Slovak cystic fibrosis patients by DHPLC and subsequent sequencing: identification of four novel mutations.

Cystic fibrosis (CF) is the most common lethal autosomal recessive disorder in Caucasians. Its incidence is approximately 1:2500 newborns. CF is caused by mutations in the transmembrane conductance regulator (CFTR) gene, which encodes an important chloride ion channel. The disease affects the respiratory, digestive and reproductive systems. To date more than 1550 mutations and polymorphisms have been identified throughout the CFTR gene, making the DNA diagnosis more difficult. Rapid accurate identification of CFTR gene mutations is important for confirming the clinical diagnosis, for cascade screening in families at risk for CF, for understanding the correlation between genotype and phenotype, and moreover it is also the only means for prenatal diagnosis. Individuals suspect of CF are in Slovakia presently screened for the presence of 30 common mutations, giving mutation detection rate only approximately 48%. To increase the detection rate we applied a gene scanning approach using DHPLC system for analysing specifically all CFTR exons. The fragments showing abnormal elution profiles were subsequently sequenced to characterize the DNA change. We have identified a total of 28 different mutations up to present not found in Slovak CF patients, and 17 different polymorphisms. Four mutations (G437D, H954P, H1375N, and 3120+33G>T) are novel, not yet found in any other CF patient all over the word.

Identification of the deletions in the UGT1A1 gene of the patients with Crigler-Najjar syndrome type I from Slovakia.

Crigler-Najjar syndrome type I (CN I) is a rare autosomal recessive disorder due to hepatic dysfunction of uridine diphospho-glucuronosyltransferase (UGT) activity toward bilirubin. Complete inactivation of this enzyme causing CN I lead to accumulation of unconjugated bilirubin in serum and bile. Here we report the results of the molecular characterization of the uridine diphospho-glucuronosyltransferase 1A1 (UGT1A1) gene in a consanguineous family of Slovak Roms and an unrelated non-Romany family with CN I. Sequence analysis of UGT1A1 gene in all four Romany patients showed mutation in exon 4, a deletion of an A at codon 407 (1220delA), not yet described in homozygous status. All analysed patients were homozygous for 1220delA mutation and their 3 healthy sibs were heterozygous. The non-Romany patient was a compound heterozygote for two different deletions, 1220delA and 717-718delAG at codon 239. In the family of his cousin a son was born affected with CN I, who was homozygote for 717-718delAG mutation. His other niece affected with CN II was heterozygote for mutation 717-718delAG but homozygote for TA insertion and enhancer substitution T-3279G. Haplotype analysis suggests that the 1220delA mutation is identical by descent in both families, though they originate from two ethnically different populations (Slovaks vs. Roms).

Detection of His1069Gln mutation in Wilson disease by bidirectional PCR amplification of specific alleles (BI-PASA) test.

Wilson disease (WD) is an autosomal recessive disorder of hepatic copper metabolism caused by mutations in a gene encoding a copper-transporting P-type ATPase, ATP7B. The majority of known mutations affecting this gene are frequent in different populations, which may help to introduce rapid diagnostic procedures based on direct DNA analysis into routine clinical practise. The His1069Gln mutation in exon 14 is the most frequent one, accounting for 30-60% of all mutations in Caucasian patients. The aim of the present work was to introduce DNA-based direct analysis into routine molecular screening for the above mutation in Slovak WD patients and to assess its frequency in patients as well as in a control population. Twenty seven clinicaly diagnosed patients from twenty five families, twenty relatives of index patients and three hundred and six control DNA samples were tested using two different DNA-based methods: the earlier described amplification created restriction site (ACRS) for Alw21I in combination with nested PCR and the amplification refractory mutation system (ARMS). In 18 of 25 unrelated patients (72%), the mentioned genetic defect was present in at least one copy. In ten of them (40%), the above mutation was detected in homozygous and in eight individuals (32%) in heterozygous state. In seven WD patients (28%), this mutation was not detected. The allele frequency of His1069Gln in Slovak patients with WD was 56%, which was higher as reported in other populations. In a control group of 306 random DNA samples (612 alleles), the His1069Gln mutation was observed in 3 samples (carrier frequency 1%; allele frequency 0.49%). These frequencies correspond to figures observed in different population of European origin. Taken together, we have provided further evidence that the His1069Gln mutation is the prevalent ATP7B mutation in central-european WD patients. Although both methods used in this study worked in our hands reliably, there are in every-day use some drawbacks and limitations inherent to them (PCR reactions in two tubes, possibility of star activity or not complet digestion by restriction endonuclease, etc.). Therefore we developed a simpler, cost effective and rapid DNA diagnostic test based on bidirectional amplification of specific alleles (BI-PASA), which enables detection of homozygotes (wild and mutant) and heterozygotes, respectivelly, in one PCR reaction. The test was highly sensitive and specific, yielding no false-positive or false-negative results. Its reliability and discriminating power was tested on samples of 27 WD patients and 120 random control DNA's, previously genotyped by above mentioned methods. Comparing results of BI-PASA with ACRS and ARMS tests showed 100% concordance.

TaqI digestion of PCR product increases the informativity of St14 VNTR for the diagnosis of hemophilia A.

Recently, a pair of PCR primers have been described that make it possible to amplify a highly polymorphic VNTR locus DX552 (St14). PCR products range in size from approximately 650 to 3000 bp. Ninety X chromosomes from unrelated Caucasian subjects were investigated. Digestion of the PCR products with TaqI revealed the presence of a polymorphic TaqI restriction site within the product 200 bp from the end. This restriction site is present on 60% and absent on 40% of all alleles, but the absence is confined solely to the alleles 1690 bp (39%) and 2100 bp (1%). Thus, there is a strong allelic association between the most frequent 1690 bp allele and the absence of the TaqI restriction site. Determination of this polymorphisms within the St14 VNTR region increases the expected heterozygosity at the DXS52 locus from 72% to 80%. This increases the fraction of hemophilia A families where this marker is informative for indirect prenatal diagnosis and carrier identification.

Long distance PCR in detection of inversion mutations of F8C gene in hemophilia A patients.

In the present paper, the experience with detection of intron 22 inversion of F8C gene in severe hemophilia A patients using a recently described long-distance PCR (LD-PCR) method was reported. To test the sensitivity and the specifity of the LD-PCR, analysis of 46 DNA samples of patients and their family members, previously tested by Southern hybridization, was performed. In addition, 16 DNA samples of severe hemophilia A patients in which causative mutation was unknown, were included in analysis. Four-primers, P, Q, A&B, which are able to differentiate between the affected males with or without the inversion, and in female carriers, were used in LD-PCR. Two primers, P&Q, are located within the F8C gene flanking int22h1. Two further primers, A&B, flank int22h2 and int22h3, extragene homologs of int22h1. Nine combinations of four primers were used to identify the optimal one. Four-primers (P, Q, A&B), three-primers (P,Q & B;P, A & B; A, B & Q;P, Q & A) and two-primers (A & B; P & Q; A & Q; P & B) PCR amplifications were performed in the hemophilia A patients as well as in obligate carriers DNA samples. Successful amplification required introduction of some modifications of the original protocol. The most reproducible and uniform results were obtained using two-primers PCR, performed in four single reactions. Thus, a total of 46 DNA samples, 22 were hemizygous for inversion, 6 without the inversion, 14 carriers and 4 non-carriers of inversion. Perfect correlation between genotypes determined using Southern hybridization and LD-PCR was achieved. The optimalized two-primers LD-PCR protocol was used for analysis of 16 DNA samples of severe hemophilia A patients with unknown mutation. Ten cases of inversions and six cases without them were detected. Thus in additional 10 severe hemophilic patients DNA diagnosis was completed. The most successful and reproducible results were obtained performing four single LD-PCR reactions with combinations of two-primers A & B; P & Q; A&Q, and P&B in each DNA sample and this approach is recommended for routine using in clinical practice.

High frequency of GJB2 mutation W24X among Slovak Romany (Gypsy) patients with non-syndromic hearing loss (NSHL).

Mutations in the GJB2 gene (connexin 26) represent a major cause of autosomal recessive non-syndromic hearing loss (NSHL) worldwide. In most Caucasian populations, the 35delG mutation in this gene was found to account for up to 50% of cases of the genetic non-syndromic childhood deafness. In populations of non-European ethnic background, other GJB2 gene mutations are occasionally common, e.g. 167delT in Ashkenazi Jews, R143W in Africaans and 235delC in Koreans. In this work, DNA samples from 54 unrelated NSHL patients from endogamous and inbred population of Slovak Roms (Gypsies) from Eastern Slovakia were screened for GJB2 mutations. The coding region of the GJB2 gene of patients was sequenced and mutations W24X, R127H, V153I, L90P and V37I were found. In Slovak Romany population, mutation W24X accounts for 23.2%, R127H for 19.4%, 35delG for 8.3%, V153I for 3.7%, L90P for 3.7% and V37I for 0.9% of screened chromosomes. As the W24X mutation was previously found in India and Pakistan, were from the European Romanies originate, it was brought by the European Romnanies from their Indian homeland. The carrier frequency of 35delG was estimated for Slovak non-Romany population to be 3.3%, and for Slovak Romany population to 0.88%. The carrier frequency of W24X varied in different Slovak Romany subpopulations from 0.0% up to 26.1%.

A new extremely large allele at the D1S80 (MCT118) locus.

A new extremely large allele at locus D1S80, segregating in a three-generation family is described. The length of PCR-generated allele is approximately 1000 bp. Restriction analysis indicates that this increase is due to an increased number of basic core sequence. The assessed number of tandem repeats is in range 52-55, corresponding to 979-1027 bp exact length of the PCR-generated fragment.

[The yield and quality of DNA extracted from blood samples stored under various conditions]. / Výt'azky a kvalita DNA extrahovanej z krvných vzoriek pri rozlicných spôsoboch ich uskladnenia.

The effect of different conditions of blood sample storage on the yield and quality of the extracted DNA was studied. Samples of whole umbilical blood were divided into eight groups containing 10 samples each and stored as follows: A--no storage, DNA extracted immediately; B--24 hours at 4 degrees C; C--24 hours at room temperature; D--7 days at 4 degrees C; E--3 months at -18 degrees C; F--6 months at -70 degrees C; G--14 days at -70 degrees C, the samples were thawed one time and frozen again; H--14 days at -70 degrees C, the samples were 3 times thawed and frozen. DNA was isolated and yields quantified by spectrophotometry at 260 and 280 nm. The average DNA yield in the individual groups ranged from 574.5 to 1,075 micrograms per 10 ml of whole blood. Although variations in the yield of DNA were observed both within and among groups, there were no significant changes with respect to different storage conditions. However group H yielded significantly more DNA (900 micrograms/10 ml, p less than 0.05) compared to the control group A. After digestion with restriction endonuclease and electrophoresis in agarose gel, all DNA preparations were found to be of high molecular weight and in digestible condition. The results can be used to advantage in storing DNA for purposes of molecular diagnosis of genetic diseases.

[Analysis of inversion in intron 22 of the factor F VIII:C gene in patients with hemophilia A in the Slovak population]. / Analýza inverzií v 22. intróne génu pre faktor F VIII:C u pacientov s hemofíliou A v Slovenskej populácii.

Hemophilia A is the result of Factor F VIIIC (F8C) gene mutations. Predominating mutation is inversion, occurring in about 50% of patients with severe form of the disease. Inversion is the result of homologous recombination between gene A located on the 22. introne of the F8C gene and one of its telomeric copies located about 500 kb from 5'end of the factor F VIIIC gene. This study presents the results of this mutation screening in 84 nonrelated patients with hemophilia A. Inversion was identified in 22 (50%) of 44 patients with severe form and in 1 (from 13) with moderate form of the disease. Distal type of inversion was more frequent (82.6%) than proximal one. The identification of iversions enabled direct DNA diagnosis in 50% of patients with severe form of the disease and will be successfully used in the prenatal diagnosis and carrier testing, mainly in families with sporadic occurrence of the disease. (Tab. 1, Fig. 2, Ref. 18.)

[Use of DNA analysis in the diagnosis and prevention of hemophilia A]. / Uplatnenie analýzy DNA v diagnostike a prevencii hemofílie A.

Hemophilia A belongs to the monogenically determined diseases. The methods of molecular genetics (recombinant DNA) have greatly contributed both to the elucidation of the genetic basis of these diseases and to the elaboration of effective preventive approaches either by prenatal genetic diagnosis or by detection of heterozygous transmitters. The authors present a short survey of the results in the field of genetic research of hemophilia A at molecular level achieved over the last years and they report on their own results of DNA analysis in 15 families with the occurrence of the disease. Of 17 potential subjects transmission was excluded in 10 and confirmed in 7 cases. The importance of early and complete detection of families with the occurrence of hemophilia A is emphasized particularly in the light of effective prevention.

[Molecular genetic analysis of deletions in the Duchenne and Becker types of progressive muscular dystrophy]. / Molekulárno-genetická analýza delécií pri progresívych svalových dystrofiách typu Duchenne a Becker.

Methods of molecular genetics (Southern's hybridization and DNA amplification by the PCR method) were used to search the DNA of patients suffering from the Duchenne (DMD) and the Becker (BMD) type of progressive muscular dystrophy for deletions in the dystrophin gene. The series consisted of 29 patients with DMD and 2 patients with BMD. As hybridization probes cloned cDNA sections were used designated as CF56a, CF56b, 1-2a, 2b-3, 4-5a, 5b-7 and 8. With the PCR methods means for exons 8, 19, 45 and 48 were used. No deletion was found in either of the BMD patients. In 13 (44.8%) of the 29 DMD patients deletion with at least one cDNA probe was found. Most deletions were detected with the probes 8 (46.2%) and 1-2a (30.8%). The high proportion of deletions in the etiology of DMD/BMD has both a high differential diagnostic value and allows to make direct prenatal diagnosis as well as to determine transmission in these families with subsequent elimination of the risk of diagnostic error resulting from recombination in DNA diagnosis by means of binding. (Tab. 1, Fig. 2, Ref. 20.)

[Identification of a de novo mutation in a factor FVIII:C gene in a family requesting prenatal diagnosis of hemophilia A]. / Identifikácia de novo mutácie v géne pre faktor FVIII:C v rodine pozadujúcej prenatálnu diagnostiku hemofílie A.

Hemophilia is caused by wide spectrum of different mutations in the F8C gene which made the direct DNA diagnosis of the diseases not the case of choice. Indirect DNA diagnosis by means of linked restriction fragment length polymorphisms (RFLPs) provides the alternative. Using this method authors identified de novo mutation in a family requiring prenatal diagnosis of hemophilia A. This de novo mutation arose during the spermatogenesis of the proband's father. Attempts to characterize the mutation on the molecular level are presented. (Ref. 15, Fig. 1.).