Genetic association of miRNA-146a with systemic lupus erythematosus in Europeans through decreased expression of the gene.

A recent genome-wide association study revealed a variant (rs2431697) in an intergenic region, between the pituitary tumor-transforming 1 (PTTG1) and microRNA (miR-146a) genes, associated with systemic lupus erythematosus (SLE) susceptibility. Here, we analyzed with a case-control design this variant and other candidate polymorphisms in this region together with expression analysis in order to clarify to which gene this association is related. The single-nucleotide polymorphisms (SNPs) rs2431697, rs2910164 and rs2277920 were genotyped by TaqMan assays in 1324 SLE patients and 1453 healthy controls of European ancestry. Genetic association was statistically analyzed using Unphased. Gene expression of PTTG1, the miRNAs miR-3142 and primary and mature forms of miR-146a in peripheral blood mononuclear cells (PBMCs) were assessed by quantitative real-time PCR. Of the three variants analyzed, only rs2431697 was genetically associated with SLE in Europeans. Gene expression analysis revealed that this SNP was not associated with PTTG1 expression levels, but with the microRNA-146a, where the risk allele correlates with lower expression of the miRNA. We replicated the genetic association of rs2341697 with SLE in a case-control study in Europeans and demonstrated that the risk allele of this SNP correlates with a downregulation of the miRNA 146a, potentially important in SLE etiology.

Opposed independent effects and epistasis in the complex association of IRF5 to SLE.

Genetic variation in the interferon regulatory factor 5 (IRF5) gene affects systemic lupus erythematosus (SLE) susceptibility. However, association is complex and incompletely defined. We obtained fourteen European sample collections with a total of 1383 SLE patients and 1614 controls to better define the role of the different IRF5 variants. Eleven polymorphisms were studied, including nine tag single nucleotide polymorphisms (SNPs) and two extra functional polymorphisms. Two tag SNPs showed independent and opposed associations: susceptibility (rs10488631, P<10(-17)) and protection (rs729302, P<10(-6)). Haplotype analyses showed that the susceptibility haplotype, identified by the minor allele of rs10488631, can be due to epistasis between three IRF5 functional polymorphisms. These polymorphisms determine increased mRNA expression, a splice variant with a different exon 1 and a longer proline-rich region in exon 6. This result is striking as none of the three polymorphisms had an independent effect on their own. Protection was independent of these polymorphisms and seemed to reside in the 5' side of the gene. In conclusion, our results help to understand the role of the IRF5 locus in SLE susceptibility by clearly separating protection from susceptibility as caused by independent polymorphisms. In addition, we have found evidence for epistasis between known functional polymorphisms for the susceptibility effect.

Endothelin-1 gene and endothelial nitric oxide synthase gene polymorphisms in adolescents with juvenile and obesity-associated hypertension.

Hypertension is an increasing public health problem all over the world. Essential hypertension accounts for more than 90% of cases of hypertension. It is a complex genetic, environmental and demographic trait. New method in molecular biology has been proposed a number of candidate genes, but the linkage or association with hypertension has been problematic (lack of gene-gene and gene-environment interaction). It is well known that genetic influences are more important in younger hypertensives, because children are relatively free from the common environmental factors contributing to essential hypertension. The association studies compare genotype ferquencies of the candidate gene between patient groups and the controls, in pathways known to be involved in blood pressure regulation. This study examined three polymorphisms of these factors encoding genes (ET-1 G+5665T (Lys198Asn), endothelial nitric oxide synthase (eNOS) T-786C promoter polymorphism and 27-bp repeat polymorphism in intron 4) in adolescents with juvenile essential and obesity-associated hypertension. Significant differences were found in the G/T genotype of the ET-1 polymorphism in the hypertensive and obese+hypertensive patients (body mass index (BMI) > 30). A strong association was detected between the BMI and the polymorphism of the ET-1 gene. It seems that ET-1 gene polymorphism plays a role in the development of juvenile hypertension associated with obesity. Although no significant differences were seen in the case of the eNOS promoter polymorphism and the eNOS 4th intron 27-bp repeat polymorphism. It seems that eNOS may play a role, but this is not the main factor in the control of blood pressure; it is rather a fine regulator in this process. This study with adolescents facilitates an understanding of the genetic factors promoting juvenile hypertension and obesity.

Bias in association studies of systemic lupus erythematosus susceptibility due to geographical variation in the frequency of a programmed cell death 1 polymorphism across Europe.

We obtained eight collections of DNA samples from ethnically matched systemic lupus erythematosus (SLE) patients and controls from five European countries totaling 783 patients and 1210 controls. A highly significant cline in the frequency of the PD1.3 A allele was found among controls but not among SLE patients. The frequency of the PD1.3 A allele increased from the Northeast to the Southwest of Europe. The cline was clearly apparent (P=1.2 x 10(-6)) when data from controls of other five SLE susceptibility studies were included in the analysis. This variation has severely biased SLE association studies owing to the lack of parallel changes in SLE patients. As a consequence, the PD1.3 A allele was more common in SLE patients than in controls in the Northeast and Center of Europe, similar to controls in Southeast Europe, and less frequent than in the controls in the Southwest of the Continent. This dissociation in allele frequencies between SLE patients and controls in different subpopulations indicated that programmed cell death 1 variation and disease susceptibility are not independent but the type of relationship is currently unclear. As allele frequency clines are common in other polymorphisms their impact in genetic epidemiology studies should be carefully considered.

Neonatal screening for biotinidase deficiency in Hungary: clinical, biochemical and molecular studies.

From 1989 to 2001, 1,336,145 newborns were screened for biotinidase deficiency in Hungary. Fifty-eight children with the disorder were identified as enzyme-deficient. We have characterized the clinical and biochemical features and mutations of 20 of these children. Eleven children had profound biotinidase deficiency, 7 had partial biotinidase deficiency, and 2 were found to be heterozygous for profound deficiency by mutation analysis. Seventeen different mutations were identified in this population including seven novel mutations. Six of these new mutations are missense, 245C>A, 334G>A, 652G>C, 832C>G, 1253G>C, 1511T>A, and one is a unique allelic double mutation [212T>C;236G>A]. Of five Romanian Gypsies, four were homozygous for the 1595C>T mutation and one was heterozygous for this mutation. Most of the children with profound deficiency have been asymptomatic on therapy; however, four exhibited minimal brain abnormalities, motor delay and abnormal blood chemistries. Compliance with therapy must be questioned in these cases. Of clinical importance, all of the children with partial deficiency exhibited mild symptoms at the time of diagnosis, at several weeks to months of age. These symptoms resolved following biotin therapy. This is in contrast to the experience in the United States, where the children with partial deficiency have been asymptomatic at the time of diagnosis. This finding further indicates that children with partial deficiency should be treated. The incidence of biotinidase deficiency in Hungary is more than twice that observed in a worldwide survey. These results indicate that newborn screening in Hungary is effective and warranted.

Human cytomegalovirus gB genotype 1 is dominant in congenital infections in South Hungary.

On the basis of the sequence variation of the glycoprotein B (gB) gene, human cytomegalovirus (HCMV) can be classified into four gB genotypes. Genotyping of HCMV from congenital infections was carried out on the assumption that the envelope gB may influence the outcome of prenatal infection. Sixty-three pregnant women were included in the study: 40 pregnant women whose fetuses were strongly suspected of having viral infection, and 23 women with normal pregnancies, from whom amniotic fluid was taken for fetal karyotype assessment. The amniotic fluid, fetal blood, blood, and urine of the newborns were examined for HCMV DNA by a nested polymerase chain reaction, and the gB genotype was determined by restriction fragment length polymorphism. HCMV DNA was detected in 12 cases in which the fetuses were suspected of having a viral infection and in 3 of the normal pregnancies. All the HCMV DNA had identical genotype, gB1. These data clearly indicate the dominance of the gB1 genotype in congenital HCMV infections. The clinical outcome of these pregnancies, however, cannot be predicted on the basis of the involvement of this genotype.

Relationship between the occurrence of anti-SSA, anti-SSB autoantibodies and HLA class II alleles from the aspect of in vitro inhibitory effect of glucocorticosteroid on the antibody-dependent cellular cytotoxicity in patients with primary Sjögren's syndrome.

Though at present there is no evidence-based algorithm for the treatment of primary Sjögren's syndrome, it is generally accepted that glucocorticosteroid (GS) therapy must be introduced in cases with severe systemic manifestations. As the side-effects of the GSs are well known, it would be useful to know in advance how the patients will respond to this type of treatment. For this reason we measured the in vitro steroid sensitivity of 29 SS patients using inhibition of antibody dependent cellular cytotoxicity (ADCC) test by methylprednisolone compared to that of 28 controls. SS patients proved to be significantly less sensitive to GSs than controls (inhibition of ADCC reaction: 42.4 vs 53.1%; p < 0.01). This was especially true in SS patients with anti-SSA and/or SSB autoantibody positivity and with HLA-DR2 and/or -DR3 alleles. Comparing the results of the in vitro GS sensitivity and the clinical effectiveness of the previously applied corticosteroid therapy it seems that steroid inhibition of ADCC reaction has a predictive value in determination of in vivo sensitivity to GSs. However, in patients with decreased in vitro GS sensitivity a more expressed in vivo steroid sensitivity cannot be excluded.

HLA-DRB1, -DQA1, and -DQB1 genotypes in patients with nasal polyposis.

OBJECTIVES/HYPOTHESIS: Genetic etiology is suspected in the development of nasal polyposis on the basis of familial aggregation. This study investigated whether there is an association between HLA-DRB1, -DQA1, and -DQB1 alleles and developing nasal polyposis. STUDY DESIGN: Data from 50 polypectomized patients were compared with data from 50 healthy randomly selected controls. Polyp score, possible asthma, aspirin sensitivity, and ASA triad were also recorded. METHODS: Genotyping of HLA-DRB1 alleles was carried out with the Dynal RELI SSO HLA-DRB, a direct DNA probe test that utilizes a polymerase chain reaction (PCR) and nucleic acid hybridization for the differentiation of 70 HLA-DRB alleles and 9 supertypes. For DQA1* genotyping PCR-RFLP (restriction fragment length polymorphism) was used, differentiating eight alleles. The DQB1* typing was carried out using a INNO-LiPA DQB PCR-reverse hybridization kit, allowing the discrimination of 30 alleles. RESULTS: People carrying the HLA-DR7-DQA1*0201, and -DQB1*0202 haplotype were found to have a two to three times higher odds ratios (ORs) for developing the disease, compared with controls. Patients with ASA triad carried the above-mentioned DR7 allele with the linked alleles significantly more often (P < .001). Subjects carrying HLA-DR5 allele and the linked alleles had lower odds ratio values. CONCLUSION: These results underline that allergy is not conditional for the formation of nasal polyps as thought before. Nasal polyposis associated with asthma and aspirin sensitivity is probably a unique form of nasal polyps. The authors plan further investigations in this field.

Analysis of CAG repeat expansion in Huntington's disease gene (IT 15) in a Hungarian population.

Huntington's disease (HD) is a neurodegenerative disorder with autosomal dominant inheritance. The genetic defect is a CAG trinucleotide repeat expansion at the 5' end of the IT 15 gene on chromosome 4. This gene has not been analyzed in the Hungarian population yet. To obtain data DNA from 26 HD patients, 18 members of their families and 70 normal controls was amplified in the involved region by polymerase chain reaction. The CAG repeat numbers varied from 37 to 70 (median: 43) in HD patients and asymptomatic carriers, while individuals of the normal control group had 10-36 CAG repeat numbers (median: 18). The length of CAG repeat expansion in Hungarian HD patients was similar to that reported from other countries. The group of normal controls had the same CAG repeat expansion as populations reported from Western European countries. It is a useful piece of data for population genetics to prove that the population of Hungary is a mélange of different nations that influenced the history of the country in the last 11 centuries. As opposed to this, the only closely related nation, the Finnish, was genetically more isolated during this time, so the frequency of HD (and also the number of CAG repeats in normal individuals) proved to be exceptionally low.

Deletion patterns of dystrophin gene in Hungarian patients with Duchenne/Becker muscular dystrophies.

Deletion pattern analysis of the dystrophin gene was performed in 159 Hungarian patients with Duchenne/Becker muscular dystrophy. In 116 cases (73% of total patients), exon deletions were detected by PCR amplification. In 37 patients (31.9% of patients with a deletion) one exon was deleted, while five or more exons were missing in 40 children (34.4%). With respect to the proximal-distal distribution of the deletions, 90 children (77.6%) had deletions exclusively at the 3' end of the gene, 21 deletions (18.1%) affected only the 5' end, and in five patients (4.3%) large-scale deletions were detected, which affected both regions. Analysis of the breakpoint distribution pattern in the dystrophin gene showed that, similarly to that observed in several Western European populations, intron 44 was involved most frequently (n = 35, 15.1%) as a starting breakpoint. In the Hungarian population introns 50 and 52 were the second (n = 30, 12.9%) and third (n = 29, 12.5%) most frequently observed hot spots at the 3' end; these seem to be characteristic for the Hungarian patients. At the 5' end the breakpoint peak (n = 6, 2.58%) was in intron two. As it was proposed by previous national studies, our findings also suggest that certain intronic sequences, characteristic for a population, probably determine the development of a preferential breakpoint profile in this disease.

Coeliac disease: always something to discover.

The authors present more than 20 years' experience with coeliac disease, with a summary of their published studies. Hair shaft characteristics were determined by scanning electron microscopy. Hair diameter was significantly lower and cuticular erosion scores higher in those who were not on gluten-free diets as compared to controls, showing a tendency towards normal values following start of gluten-free diets. Proton-induced X-ray emission showed significantly lower zinc content of the hair shaft in the group with acute coeliac disease and after a short-term diet, which approached the normal range only after a year-long diet. The serum prolactin levels in healthy controls and in coeliac patients on the diet were within normal limits, whereas in children with coeliac disease taking gluten in their meals, a significant hyperprolactinaemia was found. The erythrocyte glutathione content of coeliac children was elevated, and the glutathione disulfide level was significantly decreased, as compared to values in normal controls. The erythrocyte glutathione disulfide level and glutathione disulfide/erythrocyte glutathione ratio in coeliac children also differed from those in children with iron deficiency. With genotyping, the DQB1*0201/2 (p < 0.00001) and DR3 (p < 0.00001), DR7 (p < 0.01) alleles showed significant positive association with the disease.

Childhood membranous nephropathy, circulating antibodies to the 58-kD TIN antigen, and anti-tubular basement membrane nephritis: an 11-year follow-up.

Childhood membranous nephropathy (MNP) with anti-tubular basement membrane (anti-TBM) nephritis is a rare disorder that may have extrarenal manifestations. This article describes a new case to be added to the 10 previously reported. A renal biopsy specimen from a 1-year-old white boy with nephrotic syndrome, microhematuria, and hypertension showed MNP (granular global IgG, IgA and C3, and segmental IgM and C1q) associated with hypercellularity and granular deposits of IgM and C1q in the mesangium, arteriolar IgA, and linear TBM IgG, IgA, and C3. A biopsy at age 4 years showed MNP (IgG and C3) and linear IgG and C3 along the TBM. Six months later, temporary glucosuria suggested a mild tubular dysfunction. Biopsy at age 8 years showed sclerosing MNP (IgG and C3), linear TBM IgG and C3, and chronic active tubulointerstitial nephritis (TIN). Indirect immunofluorescence showed circulating anti-TBM antibodies, and the enzyme-linked immunosorbent assay (ELISA) approach verified strong reactivity with the 58-kd TIN antigen. Despite trials with steroids, chlorambucil, azathioprine, and cyclosporine, end-stage renal disease developed by the age of 9 years. At age 10 years, the patient received a cadaveric kidney transplant. With the patient now aged 12 years, the graft is still functioning well, without any clinical evidence of disease recurrence. Neurological, ocular, and abdominal symptoms, including nonbacterial diarrhea, were observed during the follow-up period. The pathophysiology of these extrarenal symptoms remains unclear. Serotyping and genotyping of HLA antigens (A2, A10, B12, B41, DR5 [1101, 1103-4, 1106 or 1108-1113], DR6 [1303, 1312, or 1413], DRB3 [*0101 and 0201-2 or 0301], DQA1 [*0501 homozygous], and DQB1 [*0301 homozygous]) did not indicate any HLA association similar to those described previously in childhood MNP with anti-TBM nephritis (HLA-B7 in four patients, HLA-DR8 in two patients). The presented case is the fifth in the literature that displays reactivity with the 58-kd TIN antigen, and for which data on HLA antigens are reported.

Carrier detection by microsatellite analysis of Duchenne/Becker muscular dystrophy in Hungarian families.

Duchenne and Becker muscular dystrophies are among the most severe and frequent inherited disorders. Being still incurable, medical treatment is concentrated on the carrier diagnosis of the members of the affected families. Here we report the results of the studies of 151 members of 41 Hungarian families, obtained with multiplex PCR amplification of 18 exons as well as the muscle specific promoter region, and haplotype analysis of two polymorphic (CA)n repeat microsatellite loci in introns 45 and 49 of the dystrophin gene. The analysis of 15 deletion-type families revealed a frequency of new mutations not differing significantly from that in the other regions of Europe. We also compared the allele distributions of the two microsatellites in randomly selected normal individuals and affected family members. The allele distribution of STRP45 shows interesting differences between the two populations.

Mitochondrial mutation as a probable causative factor in familial progressive tubulointerstitial nephritis.

Renal biopsy of two children and a maternal relative, diagnosed with severe progressive tubulointerstitial nephritis, has shown the presence of distorted mitochondria. Mitochondrial DNA from the blood of these patients was analysed. No major deletions were found, but an A to G mutation was detected in position 5656. It is proposed that this mutation might play a causative role in the renal disease of the patients.

Molecular genetic studies in monogenic and polygenic human diseases.

The main goal of this study was to determine and characterise the types of mutations in two monogenic human disorders: cystic fibrosis (CF) and Duchenne/Becker muscular dystrophy (DMD, BMD) and the susceptibility allele frequency in a polygenic disease: type I insulin-dependent diabetes mellitus (IDDM). After analysing 220 chromosomes for mutations in the CF (Cystic Fibrosis Transmembrane Conductance Regulator = CFTR) gene, delta F508 mutation was most abundant (41%) and out of the non-delta F508 CF mutations 5% was identified as G542X, G551D, R553X, N1303K and W1282X. The CF haplotype analysis by using linked markers to the CFTR gene revealed that the CF "B" haplotype occurred in 66.7% of patients, and this haplotype was 57.2% in patients carrying the delta F508 mutation. Prenatal genetic diagnosis for CF was performed in 10 fetuses: 3 were affected, 6 were carriers, and 1 without any CF mutation. Fifty % of 66 patients with DMB/BMD muscular dystrophy had one or more exon deletions in the dystrophin gene. Eighty-five % of the deletions occurred at the 3' and 15% at the 5' end of the gene. Out of the three prenatal diagnosis in one case DMD was substantiated. Thirty-six % of 50 patients with IDDM possessed four, 44% three and 20% two susceptibility markers in the HLA-DQA1, -DQB1 region. The onset of the disease correlated with the number of susceptibility alleles.

Molecular genetic analysis of HLA-DRB, -DQA, and -DQB polymorphisms in Hungarians and distribution of the DRB1*03 allele in adults and newborns.

We describe here the genetic variability of HLA-DRB1, -DQA, and -DQB in 140 healthy individuals from Hungary, including 95 randomly selected adults and 45 newborns. Allele and haplotype frequencies as well as linkage disequilibria were calculated. It was found that HLA-DRB1*11, -DQA1*0501, and -DQB1*0301 predominate in Hungarians. This information may be helpful in the future for HLA and disease association studies. Simultaneously, we observed that the frequency of the DRB1*03 allele differs between adults and newborns. Since it is well known that bearers of HLA-DR3 (and/or B8) antigens may display significant changes in immune parameters, the lower frequency in adults indicates that children with the DR3 antigen are predisposed to immune diseases in adulthood.