Genesis of two most prevalent PROP1 gene variants causing combined pituitary hormone deficiency in 21 populations.

Two variants (c.[301_302delAG];[301_302delAG] and c.[150delA];[150delA]) in the PROP1 gene are the most common genetic causes of recessively inherited combined pituitary hormones deficiency (CPHD). Our objective was to analyze in detail the origin of the two most prevalent variants. In the multicentric study were included 237 patients with CPHD and their 15 relatives carrying c.[301_302delAG];[301_302delAG] or c.[150delA];[150delA] or c.[301_302delAG];[ 150delA]. They originated from 21 different countries worldwide. We genotyped 21 single-nucleotide variant markers flanking the 9.6-Mb region around the PROP1 gene that are not in mutual linkage disequilibrium in the general populations--a finding of a common haplotype would be indicative of ancestral origin of the variant. Haplotypes were reconstructed by Phase and Haploview software, and the variant age was estimated using an allelic association method. We demonstrated the ancestral origin of both variants--c.[301_302delAG] was carried on 0.2 Mb-long haplotype in a majority of European patients arising ~101 generations ago (confidence interval 90.1-116.4). Patients from the Iberian Peninsula displayed a different haplotype, which was estimated to have emerged 23.3 (20.1-29.1) generations ago. Subsequently, the data indicated that both the haplotypes were transmitted to Latin American patients ~13.8 (12.2-17.0) and 16.4 (14.4-20.1) generations ago, respectively. The c.[150delA] variant that was carried on a haplotype spanning about 0.3 Mb was estimated to appear 43.7 (38.4-52.7) generations ago. We present strong evidence that the most frequent variants in the PROP1 gene are not a consequence of variant hot spots as previously assumed, but are founder variants.

Newborn Screening in Slovenia.

INTRODUCTION: Newborn screening in whole Slovenia started in 1979 with screening for phenylketonuria (PKU). Congenital hypothyroidism (CH) was added into the programme in 1981. The aim of this study was to analyse the data of neonatal screening in Slovenia from 1993 to 2012 for PKU, and from 1991 to 2012 for CH. METHODS: Blood samples were collected from the heels of newborns between the third and the fifth day after birth. Fluorometric method was used for screening for PKU, CH screening was done by dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA). RESULTS: From 1993 to 2012, from 385,831 newborns 57 were identified with PKU. 184 newborns out of 427,396 screened from 1991 to 2012, were confirmed for CH. Incidences of PKU and CH in the periods stated are 1:6769 and 1:2323, respectively. CONCLUSIONS: Successful implementation of newborn screening for PKU and CH has helped in preventing serious disabilities of the affected children. Adding screening for new metabolic diseases in the future would be beneficial.

Under- and over-reporting of energy intake in slovenian adolescents.

OBJECTIVE: To examine under- and over-reporting of energy intake (EI) among adolescents and to compare relative food and nutrient intakes of under-reporters (UR), over-reporters (OR), and the whole population to acceptable reporters (AR). DESIGN: All adolescents completed food frequency questionnaires at regional health centers, and a subgroup also completed a 3-day weighed dietary protocol at home. SETTING: This study is a part of the first national representative study on dietary habits of Slovenian adolescents. PARTICIPANTS: In total, 2,813 adolescents entering high school (10% of the population); participation rate was 95%. MAIN OUTCOME MEASURES: Absolute EI and relative intakes of food and nutrients. ANALYSIS: Prodi (version 5.2 expert plus, Nutri-Science, Stuttgart, Germany, 2004) software was used to evaluate dietary intakes; t test, analysis of variance, and Mann-Whitney testing were used for differences between means, and chi-square was used for differences between proportions. Level of significance was set at P = .05. RESULTS: The prevalence of UR and OR was 34% and 10% among boys, and 27% and 11% among girls. Under-reporters reported lower energy percentage from carbohydrates, higher energy percentage from fats and proteins, and higher micronutrient densities than AR. CONCLUSIONS AND IMPLICATIONS: Under-reporting and over-reporting are widespread among Slovenian adolescents. Exclusion of UR and OR does not influence mean value of EI when assessing the diet of a group as a whole.

Longevity of the hypopituitary patients from the island Krk: a follow-up study.

Patients with dwarfism on the Krk island in the Adriatic Sea have been known since 1864. Since then 25 related dwarfs (15 males and 10 females) originating from the villages Bascanska Draga and Jurandvor have been recorded. The last patient was born in 1996 and was diagnosed in 2004. In 1988 we were able to prove that the etiology of the hereditary multiple pituitary deficiencies (MPHD) causing the dwarfism is due to a PROP-1 gene mutation, a pituitary transcription factor. During visits in 1988, 1990 and 2007 data on the life span of these patients not treated by growth and sex hormone were collected. We found data for 9 patients (5 males and 4 females). One female died in an accident at age 61; 6 patients (3 males and 3 females) died between ages 68 and 91 due to cardiovascular disease. Two males died at ages 77 and 83, cause unknown. It is concluded that despite the long lasting GH and sex hormone deficiencies and irregular thyroid hormone ingestion, patients with congenital MPHD due to Prop-1 gene defects can live a long life.

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.

Adequate iodine intake of Slovenian adolescents is primarily attributed to excessive salt intake.

In Slovenia, table salt iodization has been applied to combat iodine deficiency. Recently, we found that Slovenian adolescents attained iodine sufficiency (median urinary iodine concentration was 140 microg/L; prevalence of goiter was <1%). National data indicate that salt intake of Slovenian population is too high (150% above the recommended limit); therefore, we hypothesized that sufficient iodine intake in adolescents can be primarily attributed to excessive salt intake. In a cross-sectional study, we investigated iodine and salt intake in Slovenian adolescents as well as the contributions of different foods to their intake. We determined the iodine and salt intake of a national representative sample of 2581 adolescents, aged 14 to 17 years, using the Food Frequency Questionnaire (FFQ). The FFQ covered habitual diets over the past year, and 2485 (96%) adolescents completed a valid FFQ (1370 girls, 1115 boys). The iodine intake was 189.7 +/- 2.6 microg/d (mean +/- standard error of mean), well above the recommended 150 microg/d (P < .001). Table salt was by far the biggest dietary source of iodine and sodium for both sexes. Total salt intake (mean +/- standard error of mean, 10.4 +/- 0.2 g/d) significantly exceeded the upper World Health Organization limit (<5 g/d, P < .001), especially in boys (11.5 +/- 0.3 vs 9.4 +/- 0.2 g/d in girls, P < .001). The main food sources of salt were table salt (33%), bread (24%), salty snack products (10%), meat products (8%), fish products (6%), and milk (4%). Salt intake from foods, excluding table salt, was 6.9 g/d (67% of total salt intake). We conclude that although Slovenian adolescents are iodine sufficient, their salt intake, especially among boys, is too high. Several nutritional interventions are proposed to reduce total salt intake while ensuring adequate iodine intake.

Effects of formula supplementation in breast-fed infants with failure to thrive.

BACKGROUND: The aim of the present study was to assess whether formula supplementation of infants with failure to thrive can improve underweight without jeopardizing breast-feeding. METHODS: In a prospective intervention study 31 term exclusively breast-fed infants were studied, who were admitted to hospital at an age of 28-99 days with failure to thrive (< or =40% expected weight gain for age and/or bodyweight < or =10th percentile for age) without underlying disease. Infant formula was offered ad libitum after each breast-feeding, while continued breast-feeding was supported. RESULTS: Energy intake per day increased from 352 +/- 111 kJ/kg (mean +/- SD) at study start to 587 +/- 115 kJ/kg (P < 0.001, days 1-3 of supplementation) and 501 +/- 99 kJ/kg (days 29-31; P < 0.001 vs study entry). Twenty-five infants continued to be partially (n = 21) or fully (n = 4) breast-fed. Human milk intake decreased from 476 +/- 163 g/day (study days 1-3) to 349 +/- 285 g/day (study days 29-31; P < 0.01). The contribution of breast milk to total milk intake decreased from 100% to 42 +/- 35% (P < 0.001). Supplementation over 31 days led to increased weight (0.98 [0.70], standard deviation scores [SDS]), length (+0.40 [0.41] SDS) and head circumference (+0.59 [0.93] SDS). CONCLUSIONS: One month of formula supplementation successfully improved growth in 72% of infants with failure to thrive on human milk feeding. Breast-feeding was maintained in 81% of infants.

Cardiac damage after treatment of childhood cancer: a long-term follow-up.

BACKGROUND: With improved childhood cancer cure rate, long term sequelae are becoming an important factor of quality of life. Signs of cardiovascular disease are frequently found in long term survivors of cancer. Cardiac damage may be related to irradiation and chemotherapy.We have evaluated simultaneous influence of a series of independent variables on the late cardiac damage in childhood cancer survivors in Slovenia and identified groups at the highest risk. METHODS: 211 long-term survivors of different childhood cancers, at least five years after treatment were included in the study. The evaluation included history, physical examination, electrocardiograpy, exercise testing and echocardiograpy. For analysis of risk factors, beside univariate analysis, multivariate classification tree analysis statistical method was used. RESULTS AND CONCLUSION: Patients treated latest, from 1989-98 are at highest risk for any injury to the heart (73%). Among those treated earlier are at the highest risk those with Hodgkin's disease treated with irradiation above 30 Gy and those treated for sarcoma. Among specific forms of injury, patients treated with radiation to the heart area are at highest risk of injury to the valves. Patients treated with large doses of anthracyclines or concomitantly with anthracyclines and alkylating agents are at highest risk of systolic function defect and enlarged heart chambers. Those treated with anthracyclines are at highest risk of diastolic function defect. The time period of the patient's treatment is emerged as an important risk factor for injury of the heart.

Correlation between AVPR2 mutations and urinary AQP2 excretion in patients with nephrogenic diabetes insipidus.

Activation of the V2 receptor by arginine vasopressin (AVP) results in trafficking of the water channel AQP2 to the luminal plasma membrane and a small amount into the urine. Mutations in the A VPR2 gene, encoding the AVP V2 receptor, result in congenital nephrogenic diabetes insipidus (CNDI). To determine a correlation between A VPR2 mutations and urinary AQP2 excretion, immunobloting was used to detect AQP2 in the urine of patients with CNDI before and after a dehydration test. The patients' genotype was determined using PCR amplification and direct sequencing of the complete A VPR2 gene. Urinary AQP2 excretion was absent in patients with severely debilitating mutations, a novel total deletion of the A VPR2 gene, and a novel nonsense mutation W296X. However, it was detected in siblings with a V88M missense mutation. Urinary AQP2 excretion correlated well with other tested phenotype markers. Urinary AQP2 excretion could be used to evaluate the remaining in vivo integrity of the AVP-V2 receptor-AQP2 cascade in patients with CNDI.

High prevalence of thyroid peroxidase gene mutations in patients with thyroid dyshormonogenesis.

OBJECTIVE: Thyroid dyshormonogenesis is a genetically heterogeneous group of inherited disorders in the enzymatic cascade of thyroid hormone synthesis that result in congenital hypothyroidism (CH). Thyroid peroxidase gene (TPO) mutations are one of the most common causes of thyroid dyshormonogenesis. The aim of this study was to identify TPO gene defects in a cohort of patients with thyroid dyshormonogenesis from Slovenia, Bosnia, and Slovakia. DESIGN AND METHODS: Forty-three patients with permanent CH and orthoptic thyroid glands from 39 unrelated families participated in the study. Mutational analysis of the TPO gene and part of its promoter consisted of single-stranded conformation polymorphism analysis, sequencing, and restriction fragment length polymorphism (RFLP) analysis. RESULTS: TPO gene mutations were identified in 46% of participants. Seven different mutations were identified, four mutations of these being novel, namely 613C > T (R175X), 1519_1539del (A477_N483del), 2089G > A (G667S), and 2669G > A (G860R). Only a single allele mutation was identified in 65% of the TPO mutation carriers. CONCLUSIONS: The results showed a higher prevalence of TPO gene mutations in thyroid dyshormonogenesis when compared with published studies. The high percentage of single allele mutations implied possible intronic or regulatory TPO gene mutations or monoallelic expression.

Autoimmune regulator-1 messenger ribonucleic acid analysis in a novel intronic mutation and two additional novel AIRE gene mutations in a cohort of autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy patients.

CONTEXT: Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a rare autosomal recessive disease associated with mutations in the AIRE gene. OBJECTIVE: Our objective was to investigate clinical and mutational characteristics of 12 Slovenian patients from 10 families with APECED. METHODS: Direct sequencing, restriction fragment length polymorphism, and amplification refractory mutation system analyses were used to identify AIRE gene mutations. Autoimmune regulator (AIRE)-1 mRNA analysis was used to confirm pathogenicity of the intronic mutation. RESULTS: The prevalence of APECED in Slovenian population was estimated to be 1 in 43,000, which is significantly higher compared with the neighboring populations. Three novel mutations were identified among six different mutations detected in the AIRE gene. The first novel mutation was an intronic mutation (653-7_-5delCTC) affecting proper splicing by using a nearby new acceptor splice site as demonstrated by AIRE-1 mRNA analyses. The second and third novel mutations were frame-shift mutations located in exon 5 (540delG) and exon 9 (1064-1068dupCCCGG), both leading to premature truncation of the AIRE protein. The Finnish R257X mutation was the most frequent AIRE gene mutation in Slovenian patients with APECED (16 of 24 alleles). CONCLUSIONS: Three novel AIRE gene mutations were identified. For the first time, a novel intronic mutation was investigated on the mRNA level in APECED. This could be particularly important for APECED patients where no or only heterozygous mutation on the genomic DNA level is detected.

Altered expression of COX-1, COX-2, and mPGES in rats with nephrogenic and central diabetes insipidus.

Prostaglandins have an important role in renal salt and water reabsorption. PGE2 is the main kidney prostaglandin and is thought to be mainly produced in the kidney inner medulla (IM). There are indications that PGE2 synthesis in nephrogenic (NDI) and central (CDI) diabetes insipidus is altered. We hypothesize that the expression of the major PGE2 synthesis enzymes cyclooxygenases 1 and 2 (COX-1, COX-2) and membrane-associated PGE2 synthase (mPGES) is altered in the kidneys of rats with NDI and CDI. Wistar rats treated with lithium for 4 wk were used as the NDI model. One-half of the NDI model rats were additionally dehydrated for 48 h. Brattleboro (BB) rats that lack endogenous antidiuretic hormone were used as the CDI model. Expression and localization of COX-1, COX-2, and mPGES in IM, inner stripe of outer medulla (ISOM), and cortex were determined by immunoblotting and immunohistochemistry. In lithium-induced NDI, expression of COX-1, COX-2, and mPGES was markedly decreased in IM. In ISOM and cortex, COX-1 expression was marginally reduced and mPGES expression was unaltered. COX-2 expression was undetected in ISOM and marginally increased in cortex. Consistent with this, the density of COX-2-expressing cells in macula densa was significantly increased, indicating differential regulation of COX-2 in IM and cortex. Dehydration of NDI rats resulted in a marked increase in COX-2 immunolabeling in IM interstitial cells, and there was no significant change in COX-1 and mPGES expression in any kidney zone. Treatment of DDAVP in BB rats for 6 days resulted in a markedly increased expression of COX-1, COX-2, and mPGES in IM. In the cortex, there were no changes in the expression of COX-1 and mPGES, whereas COX-2 expression was decreased. These results identify markedly reduced expression of COX-1, COX-2, and mPGES in IM in lithium-induced NDI. Furthermore, there were major changes in the expression of COX-1, COX-2, and mPGES in rats with CDI.

The use of continuous subcutaneous insulin infusion (CSII) as the treatment of choice in children and adolescents with type 1 diabetes.

Continuous subcutaneous insulin infusion (CSII) by external pumps is shown in several clinical trials to be a safe and effective treatment modality for patients with T1D. The present prospective observational study evaluated the efficacy and safety of CSII in children and adolescents treated in a routine clinical setting. 186 patients using CSII from 3 to 30 months were included in the analysis. A significant decrease of GlyHbA1c was observed after 3 months (p < 0.005) and was sustained throughout the study with a mean all over reduction of 0.7%. Events of ketoacidosis and severe hypoglycemia amounted to 0.027 and 0.014 per patient-year, respectively. Routine use of CSII in children and adolescents with T1D is effective in improving metabolic control and associated with a very low incidence of ketoacidosis or severe hypoglycemia.

A novel L94Q mutation in the CDKN2A gene in a melanoma kindred.

About 10% of melanoma cases have clinical factors indicative of hereditary cancer. CDKN2A is a major melanoma susceptibility gene in familial malignant melanoma. In this study a novel L94Q missense mutation of the CDKN2A gene is described in a melanoma kindred with two affected second-degree family members. To detect the mutation, polymerase chain reaction (PCR) amplification methods and direct sequencing were used. The presence of the mutation was confirmed by restriction fragment length polymorphism analysis after digestion of the PCR amplicons with the restriction endonuclease BspMI. The penetrance of the novel mutation was shown to be incomplete. Functional importance of the mutation was assumed from the protein p16 structure.

Frequencies of Q188R and N314D mutations and IVS5-24g>A intron variation in the galactose-1-phosphate uridyl transferase (GALT) gene in the Slovenian population.

Numerous mutations in the galactose-1-phosphate uridyl transferase (GALT) gene have been found to impair GALT activity to different extent, causing galactosemia. This disorder exhibits considerable allelic heterogeneity in different populations and ethnic groups. The Q188R mutation accounts for 60-70% of classical galactosemia alleles in the Caucasian population. Individuals homoallelic for Q188R have a severe phenotype with complete loss of enzyme activity. Another form of GALT deficiency is Duarte galactosemia with N314D mutation associated alleles (Duarte-2). Although heterozygotes for classical galactosemia are asymptomatic at birth and Duarte galactosemia appears to be quite benign, there are some indications that these disorders can increase the risk of developing certain diseases later in life. The aim of our study was to analyze a healthy Slovenian population for the frequencies of Q188R and N314D mutations, and for the Duarte-2 indicative intronic variation IVS5-24G>A. DNA samples from 174 healthy subjects were analyzed for all three mutations by polymerase chain reaction and digestion with restriction enzymes. Allele frequencies for Q188R and N314D mutations and IVS5-24G>A intron variation were found to be 0.29%, 8.0% and 5.7%, respectively. These results correlate well with those reported for most other healthy Caucasian populations.

TGF-ß activates genes identified by differential mRNA display in pancreatic rudiments.

The effect of TGF-ß on gene activation in embryonic pancreatic rudiments was investigated using differential mRNA display. Several cDNA bands were augmented and some were suppressed in the presence of TGF-ß. Differentially expressed cDNAs were re-amplified, sequenced, and sequences compared to the GeneBank database. Glucagone and brain α-tropomyosin cDNAs were identified from the group of augmented cDNAs, and B-carboxypeptidase form the group of suppressed cDNAs. PCR experiments were confirmed with Northern blots. Obtained results are in accordance with immunohistochemical findings and render differential mRNA display a useful technique in identifying differentially expressed genes in embryonic pancreatic rudiments. Several unknown differentially expressed cDNA sequences obtained in our experiments remain to be identified.