Emőke Endreffy

Haplotype analysis of the apolipoprotein A5 gene in obese pediatric patients

OBJECTIVE Apolipoprotein A5 (APOA5) gene variants have been shown to be associated with elevated TG levels; the T-1131C (rs662799) variant has been reported to confer risk for the metabolic syndrome in adult populations. Little is known about the APOA5 variants in pediatric population, no such information is available for pediatric obesity at all. Here we examined four haplotype-tagging polymorphisms (T-1131C, IVS3 + G476A [rs2072560], T1259C [rs2266788] and C56G [rs3135506]) and studied also the frequency of major naturally occurring haplotypes of APOA5 in obese children. METHODS The polymorphisms were analyzed in 232 obese children, and in 137 healthy, normal weight controls, using PCR-RFLP methods. RESULTS In the pediatric patients we could confirm the already known adult subjects based association of -1131C, IVS3 + 476A and 1259C variants with elevated triglyceride concentrations, both in obese patients and in the controls. The prevalence of the APOA5*2 haplotype (containing the minor allele of T-1131C, IVS3 + G476A and T1259C SNPs together) was 15.5% in obese children, and 5.80% in the controls (p<0.001); multiple logistic regression analysis revealed that this haplotype confers susceptibility for development of obesity (OR=2.87; 95% CI: 1.29-6.37; p≤0.01). By contrast, the APOA5*4 haplotype (with -1131C alone) did not show similar associations. Our findings also suggest that the APOA5*5 haplotype (1259C alone) can be protective against obesity (OR=0.25; 95% CI: 0.07-0.80; p<0.05). CONCLUSIONS While previous studies in adults demonstrated, that the APOA5 -1131C minor allele confers risk for adult metabolic syndrome, here we show, that the susceptibility nature of this SNP restricted to the APOA5*2 haplotype in pediatric obese subjects.

Roles of Paraoxonase and Oxidative Stress in Adolescents with Uraemic, Essential or Obesity-Induced Hypertension

Background/Aims: Paraoxonase 1 (PON1) is associated with high-density lipoproteins in the plasma, and is capable of hydrolysing oxidized lipids and preventing the oxidation of low-density lipoproteins. Oxidative stress and the PON1 (activity and Q192R polymorphism) were analysed in adolescent patients with essential (n = 49) or obesity-induced hypertension (n = 79), uraemic patients (n = 20), and also in obese normotensive patients (n = 60) and age-matched controls (n = 57). Methods: The PON1 activity was measured via paraoxon hydrolysis. The PON1 genotype was determined by real-time PCR. The levels of oxidized and reduced glutathione, the end-products of nitric oxide, cysteine, homocysteine and lipid peroxidation in the plasma were measured and related to the PON1 status. Results: There were no significant differences between the patient groups and the control group in the genotype distributions and the allele frequencies of the Q192R polymorphism. The PON activity was significantly lower (p < 0.001) in the uraemic hypertensive group than in the controls. The MDA concentration was significantly higher in the uraemic hypertensive (p < 0.001) and obese hypertensive (p < 0.05) patients. The plasma NOx concentrations were significantly lower (p < 0.001) and the ratio MDA/NOx were significantly higher in all four patient groups. The GSH levels were significantly lower in the patients with hypertension (p < 0.001) and obesity-induced hypertension (p < 0.05) than in the controls, while the GSSG level (p < 0.01) and the ratio GSSG/GSH (p < 0.05) was significantly higher in the uraemic hypertensive group. The plasma homocysteine level was significantly higher (p < 0.001) in the uraemic hypertensive patients as compared with the controls. Conclusions: We found no significant correlation between the biochemical parameters and neither genotypes nor enzyme activities. The PON1 status and the levels of certain biochemical parameters are independently associated with the hypertension in hypertensive and obese hypertensive patients, and the elevated levels of lipid peroxides and plasma homocysteine may contribute to the increased risk of cardiovascular complications in patients on haemodialysis.

Genetic polymorphisms and the risk of progressive renal failure in elderly Hungarian patients.

The relationship between renal disease progression and genetic polymorphism of enzymes influencing endothelial function remains incompletely understood. We genotyped three cohorts of elderly Hungarian patients: 245 patients with end‐stage renal disease (ESRD) on chronic hemodialysis (HD), 88 patients with mild chronic kidney disease (CKD), and 200 healthy controls. The underlying diagnoses of renal diseases were primary glomerulonephritis, interstitial nephritis, hypertension, diabetic nephropathy, and hereditary diseases. We examined genetic polymorphisms of eight candidate genes associated with endothelial function: endothelial constitutive nitric oxide synthase (ecNOS) T‐786C, endothelin‐1 G5727T, methylenetetrahydrofolate reductase (MTHFR) C677T, paraoxonase‐1 Q192R and M55L, angiotensinogen M235T, angiotensin‐converting enzyme (ACE) I/D and angiotensin II type 1 receptor A1166C gene. Six gene polymorphisms were detected by real‐time polymerase chain reaction with melting‐point analysis, and two via allele‐specific amplification and gel electrophoresis. Control group patients were in Hardy‐Weinberg equilibrium for all tested genotypes. In ESRD patients attributed to hypertension, the endothelin gene G5727T GG genotype occurred significantly less but GT genotype more frequently (P < 0.01 for both). In ESRD patients attributed to primary glomerulonephritis, more ACE DD and less ID genotypes were found (P < 0.02 for both) than in the controls. The underlying diagnosis may modify the association of genetic polymorphism and dialysis‐dependent ESRD.

The effects of vitamin E on tissue oxidation in nephrotoxic (anti-glomerular basement membrane) nephritis

The effects of vitamin E on tissue oxidation, kidney function and morphology were studied in rats with nephrotoxic nephritis (NTN). Thirty-six nephritic animals received no treatment (group 1), while 36 were treated with vitamin E (group 2). Twenty-four hours after the administration of anti-glomerular basement membrane antibody, sulfhydryl-containing renal protein was significantly lower in group 1 than in group 2 (0.70±0.16 and 1.08±0.06 mmol/100 g kidney tissue, respectively), suggesting a free oxygen radical scavenging effect of vitamin E in group 2. The difference was similar on day 14. The creatinine clearance was significantly lower in group 1 than in group 2 on day 1 (40±30 and 204±60 μl/min per 100 g body weight, respectively). The protein excretion was initially high in both groups, but a significant decrease was detected in group 2 relative to group 1 on day 14 (25±18 and 92±38 mg/24 h, respectively). The morphological changes were less severe in group 2. Vitamin E treatment did not alter any of the above values significantly in healthy animals. The release of oxygen free radicals in NTN might play an important role in the pathogenesis, which can be influenced by free radical scavengers through changes in kidney function and morphology.

Efficient Targeted Next Generation Sequencing-Based Workflow for Differential Diagnosis of Alport-Related Disorders

Alport syndrome (AS) is an inherited type IV collagen nephropathies characterized by microscopic hematuria during early childhood, the development of proteinuria and progression to end-stage renal disease. Since choosing the right therapy, even before the onset of proteinuria, can delay the onset of end-stage renal failure and improve life expectancy, the earliest possible differential diagnosis is desired. Practically, this means the identification of mutation(s) in COL4A3-A4-A5 genes. We used an efficient, next generation sequencing based workflow for simultaneous analysis of all three COL4A genes in three individuals and fourteen families involved by AS or showing different level of Alport-related symptoms. We successfully identified mutations in all investigated cases, including 14 unpublished mutations in our Hungarian cohort. We present an easy to use unified clinical/diagnostic terminology and workflow not only for X-linked but for autosomal AS, but also for Alport-related diseases. In families where a diagnosis has been established by molecular genetic analysis, the renal biopsy may be rendered unnecessary.

Early detection of Angelman syndrome resulting from de novo paternal isodisomic 15q UPD and review of comparable cases.

BackgroundAngelman syndrome is a rare neurogenetic disorder that results in intellectual and developmental disturbances, seizures, jerky movements and frequent smiling. Angelman syndrome is caused by two genetic disturbances: either genes on the maternally inherited chromosome 15 are deleted or inactivated or two paternal copies of the corresponding genes are inherited (paternal uniparental disomy). A 16-month-old child was referred with minor facial anomalies, neurodevelopmental delay and speech impairment. The clinical symptoms suggested angelman syndrome. The aim of our study was to elucidate the genetic background of this case.ResultsThis study reports the earliest diagnosed angelman syndrome in a 16-month-old Hungarian child. Cytogenetic results suggested a de novo Robertsonian-like translocation involving both q arms of chromosome 15: 45,XY,der(15;15)(q10;q10). Molecular genetic studies with polymorphic short tandem repeat markers of the fibrillin-1 gene, located in the 15q21.1, revealed that both arms of the translocated chromosome were derived from a single paternal chromosome 15 (isodisomy) and led to the diagnosis of angelman syndrome caused by paternal uniparental disomy.ConclusionsAS resulting from paternal uniparental disomy caused by de novo balanced translocation t(15q;15q) of a single paternal chromosome has been reported by other groups. This paper reviews 19 previously published comparable cases of the literature. Our paper contributes to the deeper understanding of the phenotype-genotype correlation in angelman syndrome for non-deletion subclasses and suggests that patients with uniparental disomy have milder symptoms and higher BMI than the ones with other underlying genetic abnormalities.

Collagen type IV nephropathy: Genetic heterogeneity examinations in affected Hungarian families

The Col4A3, Col4A4 and Col4A5 collagen type IV genes are found to be mutated in Col IV nephropathy. In males with a mutation in the Col4A5 gene (X-linked Alport syndrome: XL-AS), progressive renal disease always develops. Female carriers with a mutation in the Col4A5 gene can develop thin basement membrane nephropathy (TBMN). Males and females who carry 1 Col4A3 or Col4A4 mutation usually manifest TBMN with nonprogressive hematuria. In the event of 2 Col4A3 or Col4A4 gene mutations, the autosomal recessive AS will develop. We examined the cosegregation pattern of hematuria in 20 families. The renal biopsies led to diagnoses of AS in 7 families, and of TBMN in 6 families. In 7 others, the diagnosis of familial hematuria (FHU) was based on the clinical symptoms. Markers of the ColA3/Col4A4 and Col4A5 loci (Col4A3: CA11 and D2S401; Col4A4: HaeIII/RFLP; and Col4A5: DXS456, 2B6 and 2B20) were used to assess their linkage to the clinical symptoms and morphological alterations. Maximum likelihood and the FASTLINK version of the linkage program were applied to compute logarithm of the odds (LOD) scores. A linkage to the Col4A3/Col4A4 genes was identified in 5 families (FHU in 3, AS in 2 families, 25%, LOD score range: 0.20-3.51). The XL-AS pattern of inheritance seemed likely with Col4A5 in 9 families (45%, LOD: 0.43-4.20); we found 4 disease-causative mutations by high-resolution melting curve analysis (LC480) and sequencing in this group. In 2 FHU families, the linkage to chromosomes 2 and X was precluded. Knowledge of the genetic background of Col IV nephropathy is essential to avoid the misdiagnosis of FHU and early AS. The allele frequencies, heterozygosity content and polymorphism information content of the applied STR markers on unrelated Hungarian normal and affected chromosomes 2 and X were also calculated.

Myotubular myopathy. Case report and review of the literature

The first Hungarian report of a case of myotubular myopathy is presented here, which is a recessive congenital disorder linked to X chromosome. The patient presented at birth with severe hypotonia, weak spontaneous movements, arthrogryposis and respiratory insufficiency. The biopsy showed the appearance of myotubular myopathy. The diagnosis was further confirmed by genetic analysis revealing a novel frameshift mutation (1314-1315insT) of the myotubularin-coding MTM1 gene.

Genetic counselling of a male patient with hypohidrotic ectodermal dysplasia

Hypohidrotic ectodermal dysplasias (HED) are characterized by abnormal morphogenesis of epidermis and epidermal appendages. They may be inherited in an autosomal dominant, autosomal recessive or X-linked recessive manner. The most common type shows X-linked inheritance, and males.are usually more severely affected than females. In a male infant who was treated in our department, the diagnosis of HED was made 28 years ago. His mother and grandmother also show typical signs of HED in a milder form, while the other family members are all healthy. This young man wanted to know his risk of having affected children. We examined the segregation of X-chromosomes in ten members of the family with 2 intragenic and one extragenic short tandem repeat (STR) markers of the gene EDA (Xq12-13). According to our results, only family members with clinical signs of HED had the same X-chromosome (the affected son, his mother and grandmother), but none of the healthy family members. Direct mutation analysis of EDA was performed, but no aberration could be detected in this gene. Nevertheless, genetic counselling was possible based on the results of the segregation analysis. In this situation, the male progeny of the patient will not be affected, while female progeny will be carriers.