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).

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%.

Lactase haplotype diversity in the Old World.

Lactase persistence, the genetic trait in which intestinal lactase activity persists at childhood levels into adulthood, varies in frequency in different human populations, being most frequent in northern Europeans and certain African and Arabian nomadic tribes, who have a history of drinking fresh milk. Selection is likely to have played an important role in establishing these different frequencies since the development of agricultural pastoralism approximately 9,000 years ago. We have previously shown that the element responsible for the lactase persistence/nonpersistence polymorphism in humans is cis-acting to the lactase gene and that lactase persistence is associated, in Europeans, with the most common 70-kb lactase haplotype, A. We report here a study of the 11-site haplotype in 1,338 chromosomes from 11 populations that differ in lactase persistence frequency. Our data show that haplotype diversity was generated both by point mutations and recombinations. The four globally common haplotypes (A, B, C, and U) are not closely related and have different distributions; the A haplotype is at high frequencies only in northern Europeans, where lactase persistence is common; and the U haplotype is virtually absent from Indo-European populations. Much more diversity is seen in sub-Saharan Africans than in non-Africans, consistent with an "Out of Africa" model for peopling of the Old World. Analysis of recent recombinant haplotypes by allele-specific PCR, along with deduction of the root haplotype from chimpanzee sequence, allowed construction of a haplotype network that assisted in evaluation of the relative roles of drift and selection in establishing the haplotype frequencies in the different populations. We suggest that genetic drift was important in shaping the general pattern of non-African haplotype diversity, with recent directional selection in northern Europeans for the haplotype associated with lactase persistence.

High frequency of alkaptonuria in Slovakia: evidence for the appearance of multiple mutations in HGO involving different mutational hot spots.

Alkaptonuria (AKU) is an autosomal recessive disorder caused by the deficiency of homogentisate 1,2 dioxygenase (HGO) activity. AKU shows a very low prevalence (1:100,000-250,000) in most ethnic groups. One notable exception is in Slovakia, where the incidence of AKU rises to 1:19,000. This high incidence is difficult to explain by a classical founder effect, because as many as 10 different AKU mutations have been identified in this relatively small country. We have determined the allelic associations of 11 HGO intragenic polymorphisms for 44 AKU chromosomes from 20 Slovak pedigrees. These data were compared to the HGO haplotype data available in our laboratory for >80 AKU chromosomes from different European and non-European countries. The results show that common European AKU chromosomes have had only a marginal contribution to the Slovak AKU gene pool. Six of the ten Slovak AKU mutations, including the prevalent G152fs, G161R, G270R, and P370fs mutations, most likely originated in Slovakia. Data available for 17 Slovak AKU pedigrees indicate that most of the AKU chromosomes have their origins in a single very small region in the Carpathian mountains, in the northwestern part of the country. Since all six Slovak AKU mutations are associated with HGO mutational hot spots, we suggest that an increased mutation rate at the HGO gene is responsible for the clustering of AKU mutations in such a small geographical region.

Deep common ancestry of indian and western-Eurasian mitochondrial DNA lineages.

About a fifth of the human gene pool belongs largely either to Indo-European or Dravidic speaking people inhabiting the Indian peninsula. The 'Caucasoid share' in their gene pool is thought to be related predominantly to the Indo-European speakers. A commonly held hypothesis, albeit not the only one, suggests a massive Indo-Aryan invasion to India some 4,000 years ago [1]. Recent limited analysis of maternally inherited mitochondrial DNA (mtDNA) of Indian populations has been interpreted as supporting this concept [2] [3]. Here, this interpretation is questioned. We found an extensive deep late Pleistocene genetic link between contemporary Europeans and Indians, provided by the mtDNA haplogroup U, which encompasses roughly a fifth of mtDNA lineages of both populations. Our estimate for this split is close to the suggested time for the peopling of Asia and the first expansion of anatomically modern humans in Eurasia [4] [5] [6] [7] [8] and likely pre-dates their spread to Europe. Only a small fraction of the 'Caucasoid-specific' mtDNA lineages found in Indian populations can be ascribed to a relatively recent admixture.

Identification of a single ancestral CYP1B1 mutation in Slovak Gypsies (Roms) affected with primary congenital glaucoma.

Primary congenital glaucoma (PCG) is an autosomal recessive eye disease that occurs at an unusually high frequency in the ethnic isolate of Roms (Gypsies) in Slovakia. Recently, we linked the disease in this population to the GLC3A locus on 2p21. At this locus, mutations in the cytochrome P4501B1 (CYP1B1) gene have been identified as a molecular basis for this condition. Here, we report the results of CYP1B1 mutation screening of 43 PCG patients from 26 Slovak Rom families. A homozygous G-->A transition at nucleotide 1505 in the highly conserved region of exon 3 was detected in all families. This mutation results in the E387K substitution, which affects the conserved K helix region of the cytochrome P450 molecule. Determination of the CYP1B1 polymorphic background showed a common DNA haplotype in all patients, thus indicating that the E387K mutation in Roms has originated from a single ancestral mutational event. The Slovak Roms represent the first population in which PCG is found to result from a single mutation in the CYP1B1 gene, so that a founder effect is the most plausible explanation of its increased incidence. An ARMS-PCR assay has been developed for fast detection of this mutation, thus allowing direct DNA based prenatal diagnosis as well as gene carrier detection in this particular population. Screening of 158 healthy Roms identified 17 (10.8%) mutation carriers, indicating that the frequency of PCG in this population may be even higher than originally estimated.

[Molecular diagnosis of mutations responsible for recurrent and severe forms of primary congenital glaucoma]. / Molekulárna diagnostika mutácie zodpovednej za castú a závaznú formu primárneho kongenitálneho glaukómu.

A PCR-based test has been developed that makes it possible to detect a G to A substitution in the cytochrome P4501B1 gene. This mutation brings about a substitution of glutamic acid to lysine in the cytochrome P4501B1 molecule, and has been shown to be responsible, in homozygous form, for a severe and prognostically unfavourable form of primary congenital glaucoma (PCG). This type of PCG has been previously demonstrated to be extremely frequent in the population of Gypsies (Roms) in Slovakia. In this study, all 33 PCG Gypsy patients examined were found homozygous for this particular mutation, and among 101 unrelated healthy screened subjects from the Gypsy ethnic community, almost 14% of mutation carriers were identified. The test sugesed here makes it possible to perform a direct DNA-based prenatal diagnosis of PCG in the families at risk, as well as to screen for gene carriers.

[2 clinico-genetic types of primary congenital glaucomas in Slovakia]. / Dve klinicko-genetické formy primárneho kongenitálneho glaukómu u nás.

The authors present the results of a 20-year follow-up of a group of 125 patients with primary congenital glaucoma. Based on 10 fundamentally different indicators they provided evidence of two clinical forms of primary congenital glaucoma depending on the ethnic origin and type of heredity. In Slovakia is found: a/autosomal recessive hereditary primary congenital glaucoma in the gypsy population with an identified gene locus GLC3A with a different clinical picture, severe course and unfavourable therapeutic results; b/multifactorial primary congenital glaucoma in the remaining population of Slovakia with different clinical parameters, a milder course and favourable therapeutic results.

Linkage of autosomal recessive primary congenital glaucoma to the GLC3A locus in Roms (Gypsies) from Slovakia.

The autosomal recessive form of primary congenital glaucoma (gene symbol GLC3) has been recently mapped to two different loci, GLC3A (at 2p21), and GLC3B (at 1p36), respectively, on families of Turkish and Saudi Arabian provenance. This disorder is known to occur with an extremely high incidence in Roms (Gypsies) in Slovakia. We performed a standard linkage analysis on a sample of 7 Slovak Gypsy families comprising 18 affected members, and found significant linkage with four STR markers from the chromosomal region of 2p21 (D2S1788, D2S1346, D2S2328, and D2S1356), without heterogeneity. This finding demonstrates that in the Rom population of Slovakia, primary congenital glaucoma is due to the locus GLC3A, and consequently, to the mutation(s) in the cytochrome P4501B1 gene, which has been recently identified as the principal cause of the disease. Roms represent the third population, in which the disorder has been mapped to GLC3A.

PKU in Slovakia: mutation screening and haplotype analysis.

The restriction fragment length polymorphism haplotypes and seven common mutations in the phenylalanine hydroxylase gene were analysed in 49 unrelated Slovak phenylketonuria (PKU) families of Caucasian origin. The predominant mutation in this population sample is R408W, with a frequency of 45.9%. In addition, four other mutations have been identified at relatively high frequencies: IVS12nt1, 10.2%; R158Q, 7.1%; R261Q, 7.1%; R252W, 2.0%. The mutation-haplotype associations correspond to those described in other European populations. The high proportion of mutations (72.4%) amenable to simple rapid detection based on the polymerase chain reaction provides a good basis for direct DNA-diagnosis of PKU in the Slovak population.

[DNA analysis in classic phenylketonuria--screening for mutations and haplotype analysis in Slovak families]. / DNA-analýza pri klasickej fenylketonúrii--skríning mutácií a haplotypová analýza v slovenských rodinách.

Authors in this contribution present the results of screening for mutations in PAH gene responsible for classical phenylketonuria (PKU), and that of haplotype analysis, based on DNA analysis in 49 Caucasian families with at least one affected child from Slovak Republic. The clearly predominant PKU mutation in this population was the R408W with proportion of 45.9% among all PKU mutations. In addition four other mutations have been identified: IVS12nt1-10.2%, R158Q-7.1%, R261Q-7.1%, and R252W-2.0%. the overall proportion of identified PKU mutations equals 72.4%. Considering the fact, that these mutations are amenable to rapid and rather simple detection using PCR, the DNA analysis is recommended as a method of direct diagnosis in clinical practice as well as in prevention.

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.

Deletion delta F508 and haplotype analysis of CFTR gene region in Slovak CF patients.

Analysis of a sample of 50 unrelated cystic fibrosis (CF) patients and 46 nuclear families from Slovakia (Czechoslovakia) by the polymerase chain reaction and Southern hybridization revealed that the proportion of the delta F508 mutation was 58% in this population, and that the frequency of the B (i.e., KM19/XV2c [1-2]) haplotype was increased in both delta F508 and non-delta F508 CF chromosomes (98% and 46%, respectively). These results support the view that the trans-European gradient of the delta F508 frequency is of a geographical rather than of an ethnic origin, and that in Slavonic populations, there exists an as yet unidentified but frequent CF mutation other than delta F508, associated with the B haplotype.

[The delta F508 mutation which causes cystic fibrosis and its association with closely linked DNA polymorphisms in the Slovak population]. / Mutácia deltaF508 zaprícinujúca cystickú fibrózu a jej asociácia s tesne viazanými polymorfizmami DNA v slovenskej populácii.

Linkage relationships between DNA polymorphism metH/TaqI as well as KM19/PstI and the mutation causing cystic fibrosis (CF) were analyzed in 48 families from Slovakia with th occurrence of CF. The polymorphism metH/TaqI did not show linkage disequilibrium with CF mutation. A pronounced allelic association was however found between CF mutation and KM19/PstI polymorphism. Of the 83 CF chromosomes analyzed, the given mutation was associated with the 6.6 kb allele in 82% of cases, while the rate of this allele in chromosomes without the mutation amounts only to 24%. The value of the standardized disequilibrium coefficient SCD = 0.58. Delta F508 deletion was addressly studied in 25 patients (i.e. 50 CF chromosomes). Of the 50 CF mutations, the given deletion was in 64% (32), while the remaining 36% (18) of mutations were of other, closely not identified types. Delta F508 deletion is in marked allelic association with the 6.6 kb allele of KM19/PstI polymorphism (SCD = 0.68). Between the given allele of KM19/PstI polymorphism and CF mutation no other allelic association was found but with delta F508. (Tab. 6, Fig. 1, Ref. 18).

[Rapid prenatal diagnosis of cystic fibrosis using the polymerase chain reaction: results of the first 5 cases]. / Rýchla prenatálna diagnostika cystickej fibrózy metódou polymerázovej ret'azovej reakcie (PCR): výsledky prvých piatich vysetrení.

Cystic fibrosis (CF) is an autosomal recessive lethal disease with an incidence in Slovakia of 1 affected in 1800 newborns. Within a year the incidence amounts to about 50 cases. Though the responsible gene has already been cloned, the only effective approach to prevention is prenatal diagnosis in the first and second trimester of pregnancy. The paper presents the results of the first five cases of prenatal diagnosis of CF established by the new rapid method of DNA analysis, polymerase chain reaction (PCR). Delta F508 deletion mutation and closely linked DNA polymorphism KM19/PstI were assessed. In two of the five cases studied the fetuses were found to be affected and pregnancy termination was indicated. To exclude the possibility of fetal DNA contamination with maternal DNA, the hypervariable DNA polymorphism VNTR apoB was determined simultaneously. The advantages of this approach are demonstrated on cases of prenatal diagnosis performed in two families where contamination of fetal DNA could be excluded. The value of the PCR method is being compared with that of Southern's hybridization method. (Tab. 2, Fig. 4, Ref. 27.).

[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.