Ludevit Kadasi

On the physiology and pathophysiology of antimicrobial peptides.

Antimicrobial peptides (AMP) are a heterogeneous group of molecules involved in the nonspecific immune responses of a variety of organisms ranging from prokaryotes to mammals, including humans. AMP have various physical and biological properties, yet the most common feature is their antimicrobial effect. The majority of AMP disrupt the integrity of microbial cells by 1 of 3 known mechanisms—the barrel-stave pore model, the thoroidal pore model, or the carpet model. Results of growing numbers of descriptive and experimental studies show that altered expression of AMP in various tissues is important in the pathogenesis of several gastrointestinal, respiratory, and other diseases. We discuss novel approaches and strategies to further improve the promising future of therapeutic applications of AMP. The spread of antibiotic resistance increases the importance of developing a clinical role for AMP.

Identification of three novel SEDL mutations, including mutation in the rare, non-canonical splice site of exon 4

Spondyloepiphyseal dysplasia tarda (SEDT) is an X‐linked recessive disorder, characterized by disproportionately short stature and degenerative joint disease, which manifests in the early teens. The gene responsible for SED tarda, SEDL, has been identified in Xp22. We report on three novel SEDL mutations. The first mutation is in the rare, non‐canonical 5′ splice site of intron 4 (IVS4+4T>C) in an Italian family. Reverse transcription–polymerase chain reaction (RT–PCR) analysis has revealed that this mutation causes alternative splicing of exon 5, and, as a consequence, inclusion of exon 4b sequence. This gives rise to an altered, truncated SEDL protein. We also describe two new deletions: one is a 4‐bp deletion in exon 6 [333–336del(GAAT)], identified in a Slovak patient with SEDT, and one is a 1.335‐kb deletion (in5/ex6del), found in a Belgian patient. The identification of these novel mutations in SEDL adds to the spectrum of 30 mutations previously identified. A short summary of all currently known SEDL gene mutations is presented.

Twelve novel HGD gene variants identified in 99 alkaptonuria patients: focus on 'black bone disease' in Italy.

Alkaptonuria (AKU) is an autosomal recessive disorder caused by mutations in homogentisate-1,2-dioxygenase (HGD) gene leading to the deficiency of HGD enzyme activity. The DevelopAKUre project is underway to test nitisinone as a specific treatment to counteract this derangement of the phenylalanine-tyrosine catabolic pathway. We analysed DNA of 40 AKU patients enrolled for SONIA1, the first study in DevelopAKUre, and of 59 other AKU patients sent to our laboratory for molecular diagnostics. We identified 12 novel DNA variants: one was identified in patients from Brazil (c.557T>A), Slovakia (c.500C>T) and France (c.440T>C), three in patients from India (c.469+6T>C, c.650–85A>G, c.158G>A), and six in patients from Italy (c.742A>G, c.614G>A, c.1057A>C, c.752G>A, c.119A>C, c.926G>T). Thus, the total number of potential AKU-causing variants found in 380 patients reported in the HGD mutation database is now 129. Using mCSM and DUET, computational approaches based on the protein 3D structure, the novel missense variants are predicted to affect the activity of the enzyme by three mechanisms: decrease of stability of individual protomers, disruption of protomer-protomer interactions or modification of residues in the region of the active site. We also present an overview of AKU in Italy, where so far about 60 AKU cases are known and DNA analysis has been reported for 34 of them. In this rather small group, 26 different HGD variants affecting function were described, indicating rather high heterogeneity. Twelve of these variants seem to be specific for Italy.

Phenylalanine hydroxylase deficiency in the Slovak population: Genotype–phenotype correlations and genotype-based predictions of BH4-responsiveness

We investigated the mutation spectrum of the phenylalanine hydroxylase gene (PAH) in a cohort of patients from 135 Slovak PKU families. Mutational screening of the known coding region, including conventional intron splice sites, was performed using high-resolution melting analysis, with subsequent sequencing analysis of the samples showing deviated melting profiles compared to control samples. The PAH gene was also screened for deletions and duplications using MLPA analysis. Forty-eight different disease causing mutations were identified in our patient group, including 30 missense, 8 splicing, 7 nonsense, 2 large deletions and 1 small deletion with frameshift; giving a detection rate of 97.6%. The most prevalent mutation was the p.R408W, occurring in 47% of all alleles, which concurs with results from neighboring and other Slavic countries. Other frequent mutations were: p.R158Q (5.3%), IVS12+1G>A (5.3%), p.R252W (5.1%), p.R261Q (3.9%) and p.A403V (3.6%). We also identified three novel missense mutations: p.F233I, p.R270I, p.F331S and one novel variant: c.-30A>T in the proximal part of the PAH gene promoter. A spectrum of 84 different genotypes was observed and a genotype based predictions of BH4-responsiveness were assessed. Among all genotypes, 36 were predicted to be BH4-responsive represented by 51 PKU families. In addition, genotype-phenotype correlations were performed.

Salmonella-mediated gene therapy in experimental colitis in mice.

Bacterial gene therapy – bactofection is a simple and effective method to deliver plasmid DNA into target tissue. We hypothesize that oral in vivo bactofection can be an interesting approach to influence the course of inflammatory bowel diseases. The aim of this study was to prove the effects of antioxidative and anti-inflammatory bactofection in dextran sulfate sodium (DSS)-treated mice. Attenuated bacteria Salmonella Typhimurium SL7207 carrying plasmids with genes encoding Cu-Zn superoxide dismutase and an N-terminal deletion mutant of monocyte chemoattractant protein-1 were prepared. Male Balb/c mice had ad libitum access to 1% DSS solution in drinking water during 10 days (mild model of colitis). The animals were daily fed with 200 Mio bacteria via gastric gavage during the experiment. Fecal consistency, clinical status, food and water intake were monitored. After 10 days samples were taken and markers of oxidative stress and inflammatory cytokine levels were measured. Colonic tissue was scored histologically by a blinded investigator. DSS treatment significantly increased the levels of inflammatory cytokines and malondialdehyde as a marker of lipoperoxidation in the colon. Anti-inflammatory gene therapy improved the total antioxidative capacity. In comparison with the untreated group, bacterial gene therapy lowered the histological colitis score. Salmonella-mediated antioxidative and anti-inflammatory gene therapy alleviated colitis in mice. The effect seems to be mediated by increased antioxidative status. Further studies will show whether recombinant probiotics expressing therapeutic gene might be used for the therapy of inflammatory bowel diseases.

Effect of unexpected sequence interruptions to conventional PCR and repeat primed PCR in myotonic dystrophy type 1 testing.

Myotonic dystrophy type 1 (DM1) is caused by expansion of the CTG trinucleotide repeat in the DMPK gene. Our study focuses on the effect of recently described unusual sequence interruptions inside the CTG tract on conventional polymerase chain reaction (PCR) and triplet repeat primed PCR (TP-PCR) amplifications, which are the methods now widely used in molecular testing for DM1. For molecular characterization of the CTG repeat tract, we used conventional fluorescent PCR with bidirectional labeling and both forward and reverse direction TP-PCR. Though the results of the methods are still unambiguous for most alleles, mistyping and false results may occur in the typing of some unordinary alleles carrying sequence interruptions. The presence of these interruptions may lead not only to altered TP-PCR profiles, as can be expected, but also to abnormal electrophoretic mobility of complementary strands produced by conventional amplification of such alleles. Our findings suggest that the simultaneous combination of bidirectionally labeled conventional PCR with TP-PCR performed in both directions may be necessary for increasing the reliability and accuracy of the TP-PCR-based assay for DM1 testing.

Rapid detection methods for five HGO gene mutations causing alkaptonuria.

Alkaptonuria (AKU) is an autosomal recessive disorder caused by the deficiency of homogentisate 1,2 dioxygenase (HGO) activity. The disease is characterized by homogentisic aciduria, ochronosis and ochronotic arthritis. AKU shows a very low prevalence (1:250 000), in most ethnic groups. Altogether 43 HGO mutations have been identified in approximately 100 patients. In Slovakia, however, the incidence of this disorder rises up to 1:19 000, and 10 different AKU mutations have been identified in this relatively small country. Here, we report detection methods developed for rapid identification of five HGO mutations. PCR primers were designed enabling detection of mutations IVS5 + 1G→A, R58fs, and V300G by restriction digestion of amplification‐created restriction sites (ACRS). Mutation G152fs is readily identified by heteroduplex analysis, and G161R by amplification refractory mutation system (ARMS) PCR.

Comparison of different DNA binding fluorescent dyes for applications of high-resolution melting analysis.

OBJECTIVES Different applications of high-resolution melting (HRM) analysis have been adopted for a wide range of research and clinical applications. This study compares the performance of selected DNA binding fluorescent dyes for their possible application in HRM. DESIGN AND METHODS We compared twelve dyes with basic properties considered relevant for PCR amplification and melting curve analysis. These included PCR inhibition, fluorescence intensity, the ability to generate melting curves and their effect on melting temperature (Tm). Seven of these dyes with promising properties were then evaluated for possible use in basic HRM applications; such as small amplicon genotyping, genotyping of a 1 kb insertion/deletion polymorphism, probe-based genotyping and mutation screening. RESULTS Five dyes failed to exhibit promising properties during the first part of the study, and these were excluded from further testing. Of the remaining dyes, SYTO11, SYTO13 and SYTO16 showed better PCR inhibitory and Tm affecting properties compared to commercial HRM dyes LCGreen Plus, EvaGreen and ResoLight. Although the SYTO dyes generally exhibited good discrimination powers in HRM applications, SYTO11 and SYTO14 gave low signal intensity and lower quality results. CONCLUSIONS Our results suggest that the best performing dyes for HRM are those commercially offered for HRM analyses. However, the performance of SYTO16 and SYTO13 was comparable to the HRM dyes in the majority of our assays, thus demonstrating that they are also quite suitable for both real-time PCR and HRM applications.

High-resolution melting analysis for genotyping of the myotonic dystrophy type 1 associated Alu insertion/deletion polymorphism

Since its introduction, high-resolution melting (HRM) analysis has been used for genotyping of various types of sequence alterations. In this study, we report the use of HRM for genotyping of the 1-kb insertion/deletion polymorphism, involving a problematic region of five consecutive Alu elements, that is associated with myotonic dystrophy type 1. We combined a three-primer polymerase chain reaction (PCR) amplification approach with HRM using two primer sets. Analyses based on curve shapes are sensitive enough to differentiate between genotypes with both primer sets. In addition, the newly designed insertion-specific primer from the second primer set equalizes the allele-specific amplicon lengths, thereby reducing the possibility of preferential amplification of shorter fragments.

GJB2 gene mutations in Slovak hearing-impaired patients of Caucasian origin: spectrum, frequencies and SNP analysis.

To the Editor: In developed countries, due to genetic factors, more than 60% of patients suffer from prelingual, moderate to profound hearing impairment, and more than 70% of hereditary hearing impairment cases are non-syndromic (nonsyndromic hearing impairment, NSHI). Of these, approximately 80% are estimated to be autosomal recessive, with 22 recessive genes identified so far (http://webhost.ua.ac.be/hhh/). Among them, most causative mutations are in the gene GJB2 (1). This gene is located at 13q12 and belongs to the connexin gene family. Connexin genes are simple in organization, composed of two exons. The first exon encodes the 50-untranslated region, while the second exon encodes the complete open reading frame and the 30-untranslated region. This makes connexin genes relatively easy targets for screening of mutations by direct sequencing. Thus far, more than 100 different NSHIcausing mutations and 24 polymorphic variants have been identified worldwide within the GJB2 gene (http://davinci.crg.es/deafness/). Although the majority of mutations are recessive, some of them are dominant [W44C (2); R75W (3)]. As it is the case in many recessive disorders, the mutational spectra often vary at this locus between different populations, with a single mutation being sometimes prevalent and thus specific for a particular population. In Caucasians, the 35delG mutation accounts for up to 85% of the GJB2 mutant alleles (4–6). However, it is less frequent in populations of non-European origin, where other GJB2 mutations prevail. Thus, the mutation 235delC is prevalent in Koreans (7), Japanese (8), Chinese (9) and Taiwanese (10); 167delT prevails in Ashkenazi Jews (11), R143W in Africans (12, 13) and W24X in NSHI patients of Indian origin (14–16). The exact frequency of congenital hearing impairment or deafness in the Slovak population is unknown, but it is estimated to be approximately 3–4 in 1000 newborns with impaired hearing and 1 in 1000 with deafness (personal communication with Dr Zuzana Kabatova, First Department of Otolaryngology, Comenius University Faculty of Medicine). This study is aimed to determine the spectrum and frequencies of GBJ2 gene (coding region) mutations in Slovak NSHI patients of Caucasian origin.