New Mutations Associated with Rasopathies in a Central European Population and Genotype-Phenotype Correlations.

We performed the genetic analysis of Rasopathy syndromes in patients from Central European by direct sequencing followed by next generation sequencing of genes associated with Rasopathies. All 51 patients harboured the typical features of Rasopathy syndromes. Thirty-five mutations were identified in the examined patients (22 in PTPN11, two in SOS1, one in RIT1, one in SHOC2, two in HRAS, three in BRAF, two in MAP2K1 and two in the NF1 gene). Two of them (p.Gly392Glu in the BRAF gene and p.Gln164Lys in the MAP2K1 gene) were novel with a potentially pathogenic effect on the structure of these proteins. Statistically significant differences in the presence of pulmonary stenosis (63.64% vs. 23.81%, P = 0.013897) and cryptorchidism (76.47% vs. 30%, P = 0.040224) were identified as the result of comparison of the prevalence of phenotypic features in patients with the phenotype of Noonan syndrome and mutation in the PTPN11 gene, with the prevalence of the same features in patients without PTPN11 mutation. Cryptorchidism as a statistically significant feature in our patients with PTPN11 mutation was not reported as significant in other European countries (Germany, Italy and Greece). The majority of mutations were clustered in exons 3 (45.45%), 8 (22.73%), and 13 (22.73%) of the PTPN11 gene.

Rasopathies - dysmorphic syndromes with short stature and risk of malignancy.

OBJECTIVES: The term ´Rasopathies´ represents a group of five neurodevelopmental syndromes (Noonan, LEOPARD, Costello, Cardio-facio-cutaneous, and Neurofibromatose-Noonan syndrome) caused by germline mutation in genes encoding proteins involved in RAS/MAPK (rat sarcoma/mitogen-activated protein kinase) signaling pathway. The RAS/MAPK signaling pathway participates in regulation of cell determination, proliferation, differentiation, migration, and senescence and dysregulation of this pathway can lead to the risk of tumorigenesis. In this review, we aim to summarize the current clinical and molecular genetic knowledge on Rasopathies with special attention for the risk of cancer. We propose also clinical and therapeutic approach for patients with malignancy. METHODS: We are reviewing the clinical and molecular basis of Rasopathies based on recent studies, clinical examination, and molecular diagnostics (mutation analysis of causal genes for Rasopathies) in Slovak pediatric patients. RESULTS: Some clinical features, such as short stature, a specific facial dysmorphology and cardiac abnormalities are common to all of Rasopathy syndromes. However, there are unique signs by which the syndromes can differ from each other, especially multiple lentigo in LEOPARD syndrome, increased risk of malignancy in Costello syndrome, dry hyperkeratotic skin in patients with cardio-facio-cutaneous syndrome, and neurofibromas and cafe-au-lait spots in neurofibromatosis-Noonan syndrome. CONCLUSION: Despite the overlapping clinical features, Rasopathy syndromes exhibit unique fenotypical features and the precise molecular diagnostics may lead to confirmation of each syndrome. The molecular diagnostics may allow the detection of pathogenic mutation associated with tumorigenesis.

Thyroid volume, iodine intake, autoimmune thyroid disorders, inborn factors, and endocrine disruptors: twenty-year studies of multiple effects puzzle in Slovakia.

OBJECTIVE: The aim of this study was to evaluate multiple interrelations between several endogenous and exogenous effects and the thyroid volume and function in large groups of children, adolescents, and adults with a sufficient whole life intake of the iodine. SUBJECTS AND METHODS: The data were obtained either by cross sectioned or longitudinal studies in a total of 4998 children and adolescents (aged 7 to 17 years) and 2501 adults (1071 males and 1430 females aged 20-75 years). Thyroid volume (ThV) was measured by ultrasound, antibodies, and hormones by electrochemiluminiscent immunoassay, and endocrine disruptors (EDs, polychlorinated biphenyls-PCB, dichlorodiethyl-ichloroethylene-DDE, and hexachlorobenzene-HCB) by high resolution gas chromatography/mass spectrometry. RESULTS: 1. In large groups of boys and girls of age 7, 10, 13 or 17 years, the ThV was significantly higher in the 10th decile than in pooled nine lower deciles. Moreover, in 17-year old subjects significantly higher prevalence of hypoechogenicity by ultrasound, positive thyroperoxidase antibodies (TPOab), and increased thyrotropin (TSH) levels were found in the 10th decile. 2. In a small group of children, some individuals revealed consistently higher ThV during the whole 7-year follow-up period irrespective of supplementation with iodine. 3. In 325 sibling pairs of age 10-19 years, born within three years, three groups with different ThV/m2 of body surface were distinguished: Group A (183 pairs having both ThVs small), Group B (103 pairs having both ThVs large); Group C (33 pairs having one ThV small and the other one large). Similar aggregation of ThVs in three groups was observed in 13 pairs of discordant twins and 19 sibling triads in which all the siblings were born within four years. 4. In 42 concordant twins, several pairs had ThV nearly twice as high (in terms of both plain ThV or ThV/m2 of the body surface) as several other pairs of the same age which is assumed to be a result of a genetic background. 5. In large cohorts of males and females, a highly significant positive correlation was found between the ThV and high level of TPOab on one side and EDs on the other side. However, in nearly the same numbers of subjects with low TPOab, negative correlation was seen between ThV and disruptors. These observations may apparently support the synergic effect of the autoimmunity and EDs on the thyroid function. CONCLUSIONS: Several cases of an excessive thyroid growth in the iodine replenished children, adolescents, and adults may apparently result from the autoimmune thyroiditis, probably induced by immunogenic action of iodine in presumably disposed individuals. However, in some cases even simultaneous participation of EDs can not be excluded. Some observations have also suggested that excessive thyroid growth in the iodine replenished adolescent and adult population which was equally exposed to disruptors may also result from other reasons as the unfavorable hereditary background.

Problems of metabolic syndrome diagnostics in children.

OBJECTIVE: Increasing prevalence of exogenous obesity in children appears possibly related to changes in their lipid and carbohydrate metabolism resulting from insulin resistance which, together with obesity and arterial hypertension, are among the components of metabolic syndrome. The aim of this study was to evaluate the age related incidence of obesity complications and the prevalence of metabolic syndrome in children according to the latest criteria. METHODS: A total of 98 obese children were divided in two age groups (5 to 10 and 10 to 16 years). In all patients the BMI was calculated, standard deviation score of BMI (SDS BMI) was estimated according to the data by anthropometric surveys Slovakia and obesity was defined as SDS BMI >2 which is equal to 97th percentile for the appropriate age and gender. Blood pressure >95th percentile for the appropriate gender, age and body was classified as hypertension. Fasting glycemia, total and HDL cholesterol and triglycerides were determined in serum and oral glucose tolerance test was performed. Insulin resistance was classified according to HOMA index. RESULTS: Among 21 children less than 10 years of age lower HOMA values and no impaired glucose tolerance appeared, but hypercholesterolemia was found in 8 cases (38.1 %). Among 77 patients aged 10 to 16 years increased frequency of cases was found with insulin resistance (37.7 %), increased triglycerides (53.3 %), decreased HDL cholesterol (54.4 %) and impaired glucose tolerance (7.8 %). In this group 32.5 % of children showed metabolic syndrome based on modified IDF criteria, while such prevalence rose to 39.0 % if borderline criteria for blood pressure were used. CONCLUSION: The treatment of referred pathological states requires lifestyle changes and follow up at the specialized clinic.

CTLA-4 gene polymorphisms predispose to autoimmune endocrinopathies but not to celiac disease.

OBJECTIVES: The aim of the study was to determine the association of two CTLA-4 gene polymorphisms (CT60, +49 A/G) with Hashimoto thyroiditis (HT), type 1 diabetes mellitus (T1DM) and celiac disease (CD) as well as with the occurrence of multi-organ involvement by autoimmunity in children. METHODS: Genotyping was done by RFLP analysis in Slovak children with HT (n=63) and CD (n=120) and both Slovak and Slovene children with T1DM (n=320) and healthy controls (n=231). RESULTS: We found a significant association of the G allele of the CT60 polymorphism with HT (p<0,0005) in the Slovak population and T1DM in both Slovak (p<0.01) and Slovene populations (p<0.005). The G allele of the +49A/G polymorphism was significantly, though less strongly, associated with T1DM (p<0.05) and HT (p<0.05). Distribution of genotypes of CTLA-4 gene polymorphisms in CD patients did not differ significantly from controls. None of the polymorphisms was associated with multi-organ involvement by autoimmunity. CONCLUSION: The G allele of both examined CTLA-4 gene polymorphisms predisposes to HT and T1DM, but not to CD. No association with multi-organ involvement was found. The GG genotype of the CT60 polymorphism may identify CD patients at an increased risk for concomitant T1DM and HT. Further studies to assess the predictive value of CTLA-4 polymorphisms for the co-occurrence of HT and T1DM in CD patients are needed.

Novel germline mutation in the transmembrane region of RET gene close to Cys634Ser mutation associated with MEN 2A syndrome.

Two mutations on the same allele of RET gene were revealed in a family with predisposition to multiple endocrine neoplasia (MEN) type 2A. The first mutation changes codon 634 from cysteine to serine. The second, a novel mutation in codon 641, changes alanine to serine in the transmembrane domain of the RET protein. Two mutations were present in close proximity in both the patients' germline and tumor DNA and were absent in DNA isolated from healthy family members and control blood donors. All MEN 2A affected family members suffered from medullary thyroid carcinoma and two of ten patients for pheochromocytoma. No parathyroid gland alterations were observed in patients with two RET gene mutations. Analysis of four genetic polymorphisms in the RET gene showed higher incidence of polymorphisms of exons 11 and 15. The observed allelic imbalance in favor of mutated allele in pheochromocytoma corresponded to higher expression of the RET gene. These observations confirm the multifactorial process leading to development of MEN 2A syndrome.

Thyroid hormone metabolism in pediatric cardiac patients treated by continuous povidone-iodine irrigation for deep sternal wound infection.

OBJECTIVE: The purpose of this study was to assess the influence of povidone-iodine mediastinal irrigation used for the treatment of deep sternal wound infection (DSWI) on thyroid function. METHODS: Thyroid function was studied in 18 pediatric cardiac patients treated with continuous povidone-iodine irrigation for DSWI. The median age of patients was 8 months (18 days-5.3 years). Serum concentrations of total triiodothyronine (TT3), total thyroxine (TT4), free triiodothyronine (FT3), free thyroxine (FT4), thyroid-stimulating hormone (TSH), reverse triiodothyronine (rT3) and thyroxine-binding globulin (TBG) were measured at three time points: (a) prior to mediastinal reexploration (before povidone-iodine exposure); (b) immediately after discontinuation of povidone-iodine irrigation; (c) 2 weeks after discontinuation of mediastinal irrigation. Urinary iodine excretion was examined on the last day of povidone-iodine exposure. RESULTS: Prior to the mediastinal reexploration, the median TT3 and TT4 levels were below the normal range, then increased significantly to concentrations within the normal range. The median serum FT3 levels were within the normal range throughout the observation period, though a significant increase of FT3 levels was observed after discontinuation of irrigation. The median serum FT4 concentrations were within the normal range prior to irrigation and did not change significantly. The median rT3 levels were within the normal range, close to upper normal limit. The median TBG levels were within the normal range throughout the observation period, though a significant increase of TBG levels was observed during the period of mediastinal irrigation. The median TSH level was within the normal range prior to mediastinal irrigation and did not change significantly. Urinary iodine concentrations in infants with povidone-iodine irrigation were significantly higher 6700 microg/l (range, 1600-15000 microg/l) than in the group of 53 healthy infants 200 microg/l (range, 20-780 microg/l, P<0,001). CONCLUSIONS: Our data showed that the use of povidone-iodine irrigation in the patients with DSWI has not lead to any significant alteration in thyroid function within the study period.

Body mass, plasma leptin, glucose, insulin and C-peptide in offspring of diabetic and non-diabetic mothers.

OBJECTIVE: The aim was to investigate the relationship between body mass index (BMI), plasma leptin, glucose, insulin and C-peptide levels in the offspring of diabetic mothers (DM) and non-diabetic healthy mothers (HM). DESIGN: Seventy-two offspring (37 girls and 35 boys, age 4-20 years) of DM were investigated in a prospective study. Those 14-16 years old (Tanner stage II-IV) were compared with age-matched offspring of HM (33 girls and 33 boys). RESULTS: BMI strongly correlated with plasma leptin concentration in the offspring of both DM and HM children. There were higher BMI and plasma leptin and glucose levels in DM than in HM children. There was no difference in plasma insulin or C-peptide levels between HM and age-matched DM children. There was a highly significant positive correlation between plasma leptin and C-peptide in boys of DM. CONCLUSIONS: The higher plasma leptin found in the offspring of DM reflects their higher BMI. A moderately high but still normal glycemia might be a preclinical sign of insulin resistance or other disturbance of glucoregulation.

[Growth and puberty in children after treatment of acute lymphoblastic leukemia]. / Rast a puberta u detí po liecbe akútnej lymfoblastickej leukémie.

BACKGROUND: Growth retardation and other endocrine abnormalities were recognized as sequelae of therapy of acute lymphoblastic leukemia (ALL), especially when chemotherapy was combined with cranial irradiation. The aim of our study was to establish growth and pubertal development in the group of children who had been previously successfully treated for ALL. METHODS AND RESULTS: 58 children (30 F, 28 M, age 8-18 y.) in complete initial remission lasting 4-15 y. (m 8 y.) after standard antileukemic therapy (including cranial irradiation) completed 2-9 y. ago (m 3 including y.) were studied. Standard deviation score (SDS) of standing height (SH) to chronological age (CA) and to genetic target height (GTH), index of body proportionality and timing of puberty were followed up. The final height attained 21 girls and 3 boys. The height of the boys differed neither from the average for our men, nor from their GTH. Girls: SH 148 cm-169 cm, SH of 7 girls > or = -1.5 SDS, 8 girls > or = -1.5 SDS to their GTH. The final height didn't reach yet 23 boys and 9 girls. Their SH to CA as well as SH to GTH didn't substantially differ from average. Index of body proportionality > or = 1.5 SDS in 12/30 girls and 6/28 boys. Menarche was already reached in 25 girls in age ranging from 10-15 y. (mean 11.3 y.), what was less than average for our population (13.3 y.). CONCLUSION: Even the height of prepubertal children under study didn't differ from average, the final height of girls (boys could not be evaluated because of small numbers) was significantly lower as could be expected. We suppose the early ending of puberty as a contributing factor of short stature in girls. Clear tendency to obesity especially among older girls was observed. Children who underwent antileukemic therapy deserve careful endocrinological follow-up.

Relationship between the duration of the breast-feeding period and the lipoprotein profile of children at the age of 13 years.

Selected parameters of lipid metabolism were studied in a group of 76 children aged 12-13 years. The children were divided into 4 subgroups according to the duration of neonatal nutrition (no breast feeding, breast feeding for 3, 6 or more than 6 months). We studied the concentration of total serum cholesterol, its distribution into lipoprotein fractions, the concentration of serum triacylglycerols and apolipoproteins A1 (Apo A1) and B (Apo B). Atherogenic indexes were calculated from the data obtained. The highest cholesterol levels (5.20+/-0.15 mmol x l(-1)) were found in children who had been breast-fed for more than 6 months, while the highest concentrations of Apo B (0.80+/-0.07 g x l(-1)) and Apo A1 (1.76+/-0.06 g x l(-1)) and the highest Apo B/Apo A1 ratio (0.45+/-0.04) were found in children with the shortest period of breast-feeding. No significant sex-related differences in total, VLDL, LDL, HDL cholesterol, triacylglycerols and apolipoproteins were observed.

Presence of atriopeptin-like immunoreactivity in human and rat milk.

Maternal milk is a significant source of hormones and other bioactive substances. They might be involved either in the control of mammary gland function or in the regulation of growth and development of the neonate. Atriopeptin (atrial natriuretic factor, ANF) is a peptide with strong diuretic, natriuretic and vasorelaxant actions, and it has been suggested to play an important role in the circulatory adaptation to extrauterine life. The aim of this study was to determine whether ANF is present in maternal milk, using radioimmunological analysis. The levels of ANF-like substance in human milk were found to be in the range of 0.3-3.0 pg/ml, those in rat milk between 37-117 pg/ml. The measured concentrations of ANF were proportional to the volume of the extracted milk. Serial dilutions of the extracts yielded curves which were not totally parallel to the human alpha-ANF standard curve. Our data indicate that, during the first days after delivery, ANF levels in human milk are higher than those in later periods of lactation. This pilot study provides the first description of the presence of atriopeptin in milk. Though a detailed characterization of milk ANF-like immunoreactivity is needed, a biological significance of present findings seems possible.

Early diagnosis of medullary thyroid carcinoma in a 13 years old girl.

Early stage of medullary thyroid carcinoma was diagnosed in a 13 years old girl from the family with incidence of MEN IIa. High level of calcitonin after pentagastrin stimulation was crucial for the diagnosis. Pentagastrin test as a regular screening for medullary thyroid carcinoma for all children over 3 years of families with MEN IIa is recommended.

Search for optimal age for weaning. Ten-year prospective study.

The effect of infant nutrition on some clinical and biochemical parameters was studied from birth in a longitudinal prospective study. The data covering the period up to 7 years were reported previously. At the age 10 years 80 children were examined. Two children have been regularly followed up because of high blood pressure. Both had been breast-fed for less than one week and are obese at present. Surprisingly serum total cholesterol was the highest in the group breast-fed for more than 6 months. There were no pronounced differences in the levels of lipoprotein cholesterol carriers. The incidence of a high (over 6.0) atherogenic index (total chol - HDL chol) x APOB/APOA1 x HDL chol) and cholesterolemia over 4.8 mmol/l was significantly (chi 2 test) higher in the group breast-fed for over 6 months. Thyroid function parameters were not affected by the duration of breast-feeding. We conclude that besides undoubtedly favorable effects of breast-feeding, some functions might be also adversely affected if it lasts for more than 6 months. For justified recommendation of optimal duration of breast-feeding all aspects should be considered and more data collected.

Our experience with childhood histiocytosis X.

A retrospective analysis of the course of histiocytosis X in 16 children treated at our hospital over the past 15 years is presented. Nearly all of the patients were at generalized stages of the disease. In this study the staging according to Greenberg was used. The disease had a favorable course at Stages I and II, 1 patient at Stage III and 2 at Stage IV died. All the children were treated by chemotherapy, in some of them combined with radiotherapy. Individual patients differed from one another with respect to chemotherapeutic regimens. The actuarial survival rate was 78% for the whole group. Better results were obtained after a less intense but long-term treatment than after an intense but interrupted chemotherapy.