Tinka Hovnik

Genetic Polymorphisms in Genes Encoding Antioxidant Enzymes Are Associated With Diabetic Retinopathy in Type 1 Diabetes

OBJECTIVE Oxidative stress plays an important role in the development of microangiopathic complications in type 1 diabetes. We investigated polymorphic markers in genes encoding enzymes regulating production of reactive oxygen species in association with diabetic retinopathy or diabetic nephropathy. RESEARCH DESIGN AND METHODS A total of 124 patients with type 1 diabetes were investigated in this case-control study. All subjects were matched for sex, age, and duration of diabetes. Genotyping was conducted using real-time PCR for p.Val16Ala polymorphism in the MnSOD gene and c.C−262T in the promoter region of the CAT gene. Multiplex PCR method was used for determination of GSTM1 and GSTT1 polymorphic deletions. Fluorescence-labeled PCR amplicons and fragment analysis was used for assessing the number of pentanucleotide (CCTTT)n repeats in inducible nitric oxide synthase. RESULTS A positive association of MnSOD genotype Val/Val (odds ratio [OR] 2.49, 95% CI 1.00–6.16, P = 0.045) and GSTM1–1 genotype (2.63, 1.07–6.47, P = 0.031) with diabetic retinopathy but not with diabetic nephropathy was demonstrated. Additionally, the combination of the two genotypes conveyed an even higher risk (4.24, 1.37–13.40, P = 0.009). No other investigated genetic polymorphisms were associated with either diabetic retinopathy or diabetic nephropathy. CONCLUSIONS Selected polymorphisms in genes encoding MnSOD and GSTM1 could be added to a panel of genetic markers for identification of individuals with type 1 diabetes at an increased risk for developing diabetic retinopathy.

Detection of a complete autoimmune regulator gene deletion and two additional novel mutations in a cohort of patients with atypical phenotypic variants of autoimmune polyglandular syndrome type 1

OBJECTIVE Autoimmune polyglandular syndrome type 1 (APS-1) is characterised by multiple autoimmune diseases. Detection of autoimmune regulator (AIRE) gene mutations facilitates timely and precise diagnosis. DESIGN AIRE mutation detection was performed in a cohort of 11 patients. Two did not meet clinical APS-1 criteria and several started with atypical presentation. METHODS Sequencing and TaqMan genotyping were used to identify AIRE mutations. Complete AIRE deletion was confirmed and framed by real-time PCR, long-range amplification and analysis of the microsatellite markers. RESULTS Seven different mutations were detected, three were novel: c.892G>A in exon 8, silent mutation c.462A>T in exon 3 most likely affecting splicing, and a complete deletion of a single AIRE allele ((?_68)_(1567-14_?)del). Novel (chronic otitis) and rare (systemic juvenile rheumatoid arthritis, autoimmune bronchiolitis, epilepsy) clinical presentations were observed. CONCLUSIONS AIRE mutation detection was valuable in the diagnostics of APS-1 in patients with atypical presentation. Chronic otitis media possibly broadened the cluster of APS-1 manifestations.

Severe progressive obstructive cardiomyopathy and renal tubular dysfunction in Donohue syndrome with decreased insulin receptor autophosphorylation due to a novel INSR mutation

Donohue syndrome (leprechaunism; OMIM *246200) is a rare, recessively inherited disorder of extreme insulin resistance due to mutations in the insulin receptor gene (INSR) causing either defects in insulin binding or receptor autophosphorylation and tyrosine kinase activity. We report a patient with pronounced clinical picture of leprechaunism who developed severe progressive hypertrophic obstructive cardiomyopathy (HOCM) and renal tubular dysfunction which improved on continuous subcutaneous infusion of recombinant human insulin-like growth factor-1 (rhIGF-I). INSR gene molecular analysis and insulin receptor (IR) autophosphorylation on cultured fibroblasts were performed. A novel homozygous missense mutation p.Leu795Pro was found, located in the extracellular portion of the β subunit of the insulin receptor. The post-binding defect of the insulin receptor signaling in cultured fibroblasts demonstrated decreased insulin receptor autophosphorylation. Conclusion: Treatment with rhIGF-I partially reversed severe progressive HOCM and renal tubular dysfunction in a patient with Donohue syndrome associated with a novel p.Leu795Pro INSR gene mutation causing a severe decrease in IR autophosphorylation.

Endothelial Nitric Oxide Synthase T(–786)C Polymorphism in Children and Adolescents with Type 1 Diabetes and Impaired Endothelium-Dependent Dilatation

Background/Aims: This study aimed to investigate the association of endothelial nitric oxide synthase (eNOS) polymorphisms with impaired endothelium-dependent dilatation (EDD) in a cohort of children and adolescents with type 1 diabetes. Methods: Ninety-seven children and adolescents with type 1 diabetes underwent ultrasound assessment of EDD. The association of various factors with EDD was analyzed with multivariate linear regression analysis. Genotypes were determined for the eNOS T(–786)C and 4ab polymorphisms, and their association with EDD was tested with logistic regression analysis. Results: Thirty-three percent of children had impaired EDD. EDD was independently associated with A1c (p = 0.0005) and inversely correlated with A1c (p = 0.0037, OR = 2.14) using logistic regression analysis. The presence of any C allele at eNOS (–786) was significantly more frequent in patients with impaired EDD (OR = 2.97, 95% CI = 1.08–8.87, p = 0.03). Logistic regression analysis revealed OR of 3.09 for impaired EDD for patients with any C allele as compared to TT patients when controlling for all other covariates (p = 0.048). Conclusion:A third of children and adolescents with type 1 diabetes had impaired EDD independently associated with A1c. The presence of any C at (–786) of the eNOS gene conveyed a significantly increased independent risk for impaired EDD.

The association of folate pathway and DNA repair polymorphisms with susceptibility to childhood acute lymphoblastic leukemia.

Genetic factors may play an important role in susceptibility to childhood acute lymphoblastic leukemia (ALL). The aim of our study was to evaluate the associations of genetic polymorphisms in folate pathway and DNA repair genes with susceptibility to ALL. In total, 121 children with ALL and 184 unrelated healthy controls of Slovenian origin were genotyped for 14 polymorphisms in seven genes of folate pathway, base excision repair and homologous recombination repair (TYMS, MTHFR, OGG1, XRCC1, NBN, RAD51, and XRCC3). In addition, the exon 6 of NBN was screened for the presence of mutations using denaturing high performance liquid chromatography. Twelve polymorphisms were in Hardy-Weinberg equilibrium in controls and their genotype frequencies were in agreement with those reported in other Caucasian populations. Among the investigated polymorphisms and mutations, NBN Glu185Gln significantly decreased susceptibility to B-cell ALL (p=0.037), while TYMS 3R allele decreased susceptibility to T-cell ALL (p=0.011). Moreover, significantly decreased susceptibility to ALL was observed for MTHFR TA (p=0.030) and RAD51 GTT haplotypes (p=0.016). Susceptibility to ALL increased with the increasing number of risk alleles (ptrend=0.007). We also observed significant influence of hOGG-RAD51 and NBN-RAD51 interactions on susceptibility to ALL. Our results suggest that combination of several polymorphisms in DNA repair and folate pathways may significantly affect susceptibility to childhood ALL.

GPR143 Gene Mutation Analysis In Pediatric Patients With Albinism

Background: X-linked ocular albinism type 1 is difficult to differentiate clinically from other forms of albinism in young patients. X-linked ocular albinism type 1 is caused by mutations in the GPR143 gene, encoding melanosome specific G-protein coupled receptor. Patients typically present with moderately to severely reduced visual acuity, nystagmus, strabismus, photophobia, iris translucency, hypopigmentation of the retina, foveal hypoplasia and misrouting of optic nerve fibers at the chiasm. Materials and Methods: Following clinical ophthalmological evaluation, GPR143 gene mutational analyses were performed in a cohort of 15 pediatric male patients with clinical signs of albinism. Results: Three different mutations in the GPR143 gene were identified in four patients, including a novel c.886G>A (p.Gly296Arg) mutation occurring “de novo” and a novel intronic c.360 + 5G>A mutation, identified in two related boys. Conclusions: Four patients with X-linked ocular albinism type 1 were identified from a cohort of 15 boys with clinical signs of albinism using mutation detection methods. Genetic analysis offers the possibility of early definitive diagnosis of ocular albinism type 1 in a significant portion of boys with clinical signs of albinism.

Lack of association of common allelic variants in the thyroglobulin gene with Hashimoto's thyroiditis in young subjects with type 1 diabetes.

Background/Aim: Four SNPs (E10SNP24, E10SNP158, E12SNP, E33SNP) in the Tg gene are suspected to be involved in the development of autoimmune thyroid diseases. The aim of the study was to determine whether these variants play a role in the development of Hashimoto’s thyroiditis in young subjects with type 1 diabetes, in whom autoimmune thyroid diseases are significantly more common than in the general population. Subjects and Methods: Seventy-six subjects with type 1 diabetes and Hashimoto’s thyroiditis and 110 subjects with only type 1 diabetes were studied. Hashimoto’s thyroiditis was determined according to the clinical, biochemical and ultrasonographic criteria. SNPs were determined by the TaqMan SNP method. Results: In young subjects with type 1 diabetes, no association between any of the tested SNPs or their combinations and Hashimoto‘s thyroiditis was found. Conclusions: This is the first study to investigate the association of SNPs located inside the coding region of the Tg gene with the development of Hashimoto’s thyroiditis in subjects with type 1 diabetes. The lack of an association is in concordance with a study where marker Tgms2, located inside intron 27, was not associated with joint susceptibility for autoimmune thyroid disease and type 1 diabetes.

Association of Average Telomere Length with Body-Mass Index and Vitamin D Status in Juvenile Population with Type 1 Diabetes / Povezava Povprečnih Dolžin Telomerov Z Indeksom Telesne Teže in Vitaminom D Pri Mladostnikih S Sladkorno Boleznijo Tipa 1

Abstract Background. Type 1 diabetes (T1D) is an autoimmune chronic disease where hyperglycemia, increased risk of oxidative stress, advanced glycation end-products and other genetic and environmental factors lead to T1D complications. Shorter telomeres are associated with hyperglycemic levels and lower serum vitamin D levels. Methods. Average telomere length (ATL) in whole blood DNA samples was assessed with qPCR method in 53 Slovenian T1D children/adolescents (median age 8.7 years, 1:1.3 male/female ratio). Body mass index standard deviation score (BMI-SDS), glycated haemoglobin and serum level of vitamin D metabolite (25-(OH)-D3) and the age at the onset of T1D were collected from the available medical documentation. Results. Results indicate shorter ATL in subjects with higher BMI-SDS when compared to those with longer ATL (0.455 ± 0.438, -0.63 ± 0.295; p=0.049). Subjects with higher BMI-SDS had lower serum vitamin D levels when compared to those with lower BMI-SDS (40.66 ± 3.07 vs. 52.86 ± 4.85 nmol/L; p=0.045). Vitamin D serum levels did not significantly differ between subjects with longer/shorter ATL. Conclusion. T1D children/adolescents with shorter ATL tend to have higher BMI-SDS. Lower serum vitamin D levels were associated with higher BMI-SDS, while associations between vitamin D serum levels, age at the onset of T1D, glycated haemoglobin and ATL were not observed. Additional studies with more participants are required to clarify the role of the telomere dynamics in T1D aetiology and development of complications. Izvleček Izhodišče. Sladkorna bolezen tipa 1 (SBT1) je kronična avtoimunska bolezen, pri kateri hiperglikemija ter zvišana raven oksidativnega stresa in končnih produktov glikacije skupaj z genetskimi in okoljskimi dejavniki privedeta do nastanka diabetičnih zapletov. Krajše dolžine telomerov so povezane s hiperglikemičnimi epizodami in nižjimi serumskimi vrednostmi vitamina D. Metode. Z metodo qPCR smo iz vzorcev DNK periferne krvi določili povprečne dolžine telomerov 53 slovenskim bolnikom s SBT1 (povprečna starost 8,7 leta, razmerje med dečki in deklicami 1:1,3). Indeks standardnega odklona indeksa telesne teže (BMI-SDS), vrednosti serumskega metabolita vitamina D - 25-hidroksikalcifediola (25-(OH)-D3), glikiran hemoglobin in starost preiskovancev ob izbruhu bolezni smo pridobili iz razpoložljive medicinske dokumentacije. Rezultati. Rezultati nakazujejo krajše dolžine telomerov pri bolnikih z višjimi vrednostmi BMI-SDS (0,455 ± 0,438, -0,63± 0,295; p=0,049). Preiskovanci z višjimi vrednostmi BMI-SDS so imeli nižje vrednosti 25-(OH)-D3 kot preiskovanci z nižjimi vrednostmi BMI-SDS (40,66 ± 3,07 proti 52,86 ± 4,85 nmol/L; p=0,045). Vrednosti 25-(OH)-D3 niso statistično značilno različne pri preiskovancih z višjimi oziroma nižjimi povprečnimi dolžinami telomerov. Zaključki. Otroci in mladostniki s SBT1 s krajšimi dolžinami telomerov imajo nekoliko višje vrednosti BMI-SDS. Nižje vrednosti 25-(OH)-D3 so povezane z višjim BMI-SDS. Povezav med serumskimi vrednostmi 25-(OH)-D3, starostjo bolnikov ob izbruhu bolezni, glikiranim hemoglobinom in povprečnimi dolžinami telomerov nismo zaznali. Za razjasnitev vloge telomerov v etiologiji, patogenezi in nastanku zapletov SBT1 bodo potrebne nadaljnje raziskave z večjim številom preiskovancev.

Association of Glycemic Control and Cell Stress With Telomere Attrition in Type 1 Diabetes

Association of Glycemic Control and Cell Stress With Telomere Attrition in Type 1 Diabetes Hyperglycemia and abnormal blood glucose levels induce oxidative stress, promote the development of microvascular and macrovascular complications,1 and may increase telomere loss in patients with type 1 diabetes.2,3 All studies of adults with type 1 diabetes, except one, report a shorter telomere length associated with age, duration of type 1 diabetes, and higher glycated hemoglobin A1c (HbA1c) levels.4 To expand these data in a pediatric population at the onset of type 1 diabetes by assessing the association between telomere length and age or HbA1c level,5 we conducted a retrospective longitudinal study evaluating the association of glycemic control and associated cell stress with telomere dynamics during a 7-year follow-up.

Metabolic control, ApoE genotypes, and dyslipidemia in children, adolescents and young adults with type 1 diabetes

BACKGROUND AND AIMS Limited data is available on the factors influencing the lipid profiles and the prevalence of dyslipidemia in children, adolescents and young adults with type 1 diabetes. We aimed at assessing the influences of metabolic control and ApoE genotypes on lipid profiles and the prevalence of dyslipidemia in children, adolescents and young adults with type 1 diabetes. METHODS Children, adolescents and young adults with type 1 diabetes from our nationwide cohort attending the annual check-up were prospectively included. Data on metabolic control and expanded lipid profiles were collected, and ApoE genotyping performed. Test for homoscedasticity of continuous variables was followed by ANOVA and Welchs ANOVA tests, and Chi-square test was used for categorical variables with Kruskal-Wallis test as a control. RESULTS 467 patients were included in the data analysis: 226 female (48.4%), mean age 14.71 ± 5.09 years and diabetes duration 6.74 ± 4.54 years. Mean HbA1c was 61 ± 5 mmoL/mol (7.71 ± 1.22%), with no gender-related differences. Females had higher mean total cholesterol (p < 0.001), LDL-C (p = 0.005), HDL-C (p < 0.001), non-HDL-C (p = 0.003), and ApoB levels (p < 0.001). 26.3% of participants had LDL-C levels above the type 1 diabetes LDL-C-goal of 2.6 mmoL/L, and 19.5% had elevated/borderline-elevated lipoprotein(a) (Lp(a)). HbA1c levels were positively related to higher levels of LDL-C (p = 0.0070) and Lp(a) (p = 0.0020). Participants with ApoE4(e3/e4) allele had higher levels of LDL-C (p = 0.010), independently of HbA1c. CONCLUSIONS Females and subjects with suboptimal metabolic control had more adverse lipid profiles. ApoE4(e3/e4) alleles were associated with significantly higher LDL-C levels, independently of HbA1c.