RNA-based mutation analysis identifies an unusual MSH6 splicing defect and circumvents PMS2 pseudogene interference.

Heterozygous germline mutations in one of the mismatch repair (MMR) genes MLH1, MSH2, MSH6, and PMS2 cause hereditary nonpolyposis colorectal cancer (HNPCC) or Lynch syndrome, a dominantly inherited cancer susceptibility syndrome. Recent reports provide evidence for a novel recessively inherited cancer syndrome with constitutive MMR deficiency due to biallelic germline mutations in one of the MMR genes. MMR-deficiency (MMR-D) syndrome is characterized by childhood brain tumors, hematological and/or gastrointestinal malignancies, and signs of neurofibromatosis type 1 (NF1). We established an RNA-based mutation detection assay for the four MMR genes, since 1) a number of splicing defects may escape detection by the analysis of genomic DNA, and 2) DNA-based mutation detection in the PMS2 gene is severely hampered by the presence of multiple highly similar pseudogenes, including PMS2CL. Using this assay, which is based on direct cDNA sequencing of RT-PCR products, we investigated two families with children suspected to suffer from MMR-D syndrome. We identified a homozygous complex MSH6 splicing alteration in the index patients of the first family and a novel homozygous PMS2 mutation (c.182delA) in the index patient of the second family. Furthermore, we demonstrate, by the analysis of a PMS2/PMS2CL "hybrid" allele carrier, that RNA-based PMS2 testing effectively avoids the caveats of genomic DNA amplification approaches; i.e., pseudogene coamplification as well as allelic dropout, and will, thus, allow more sensitive mutation analysis in MMR deficiency and in HNPCC patients with PMS2 defects.

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.

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%.

Glutathione S-transferase polymorphisms influence the level of oxidative DNA damage and antioxidant protection in humans.

Glutathione S-transferase genotypes GSTT1, GSTM1, GSTP1 were characterised in 155 middle-aged men and compared with parameters of oxidative stress at the level of DNA and lipids, with antioxidant enzymes, and with plasma antioxidants in smokers and non-smokers. Smokers had on average significantly lower levels of Vitamin C, beta-carotene and beta-cryptoxanthin and higher amounts of oxidised purines and pyrimidines in lymphocyte DNA. The GSTM1 null genotype was associated with elevated glutathione as well as with higher Vitamin C concentration in plasma. Vitamin C was higher in GSTT1+ compared with GSTT1 null--as was glucose-6-phosphate dehydrogenase activity. The homozygous GSTP1 a/a genotype was associated with significantly higher levels of GST activity measured in lymphocytes, in comparison with the b/b genotype. Using multifactorial statistical analysis we found significant associations between smoking, GSTP1 genotype, plasma Vitamin C, and purine base damage in lymphocyte DNA. The difference in Vitamin C plasma levels between smokers and non-smokers was seen only with the GSTP1 b/b genotype. This group accounted also for most of the increase in purine oxidation in smokers. In contrast, the link between smoking and oxidised pyrimidines in DNA was seen only in the GSTT1 null group. It seems that polymorphisms in the phase II metabolising enzyme glutathione S-transferase may be important determinants of commonly measured biomarkers.