17^th Workshop of Middle-European Society of Paediatric Endocrinology (MESPE)

November 18–20, 2011

Český Krumlov, Czech Republic 

Abstracts 

Prevalence of metabolic syndrome in Czech children aged 13.0–17.9 years

Aldhoon Hainerová I^1,2, Zamrazilová H¹, Dušátková L¹, Sedláčková B¹, Hlavatý P¹, Kunešová M¹, Hainer V¹

¹Institute of Endocrinology, Prague, Czech Republic; ²Department of Paediatrics, Faculty Hospital Královské Vinohrady and Third Faculty of Medicine, Charles University, Prague, Czech Republic

Introduction. The metabolic syndrome (MS) is already diagnosed in children and adolescents. Our aim was to investigate the prevalence of MS in children/adolescents in general population and in those attending either inpatient or outpatient weight management programmes.

Methods. Two cohorts aged 13.0–17.9 years from the COPAT (Childhood Obesity Prevalence And Treatment) Project were included: 1,533 randomly selected subjects (786 girls; 747 boys) recruited from general paediatricians across the whole Czech Republic and 562 subjects (344 girls; 218 boys) with Body Mass Index (BMI) >90^th percentile recruited from inpatient management centres or outpatient endocrine clinics where they had been treated for overweight. All subjects underwent a detailed examination including anthropometry, hormonal and laboratory investigations. To define the MS, the criteria of International Diabetes Federation (IDF) were used.

Results. Almost 23 % of girls and 25 % of boys of general population (median BMI z-score 0.2) were found to be either overweight or obese. In this cohort, 1.9 % of girls and 5.5 % of boys fulfilled the criteria of MS. In the group of overweight subjects (median BMI z-score 3.7), 12.1 % of girls and 24.7 % of boys were diagnosed with MS.

Conclusions. Almost one fourth of subjects aged 13.0–17.9 years of general population are either overweight or obese. A high prevalence of MS in Czech overweight children and adolescents, esp. in boys, is alarming and requires a prompt appropriate action. 

The study was supported by IGA MZ CR No. NT 12342-5/2011, CZ0123 from Norway through the Norwegian Financial Mechanisms and MSM/7F No. 7F08077.

Endocrine dysfunction after traumatic brain injury in children and adolescents (a single centre prospective study)

Aleksijevic D, Zapletalova J, Krahulik D, Vaverka M, Klaskova E, Wiedermann J, Mihal V

University Hospital, Palacky University, Dept. of Paediatrics and Dept. of Neurosurgery, Olomouc, Czech Republic

Backround. The neuroendocrine dysfunction after traumatic brain injury (TBI) is described in 23–60% of adults and 15-21% of children in retrospective studies.

Objective. To find out the prevalence of the endocrine dysfunction in children after a TBI.

Patients and methods. We evaluated somatic development in 58 patients (29 boys) after TBI. They underwent standard endocrine tests: TSH, fT4, IGF1, PRL, morning cortisol, FSH, LH, testosterone/estradiol in early posttraumatic period (2–14 days, T0) and in 3, 6 and 12 months after the injury (T3, T6, T12). Dynamics tests were performed in patients with abnormalities in clinical examination and/or laboratory results. MRI was made in T12.

Results. Median patients’ age at the time of TBI was 11.3 (0.5–18.7) years. Twenty three patients had GCS <8/15. Diabetes insipidus (DI) occurred in 12 patients and a SIADH in 4 patients respectively, hormonal changes were suggestive of central hypothyroidism in 45% of patients and hypogonadotropic hypogonadism (HH) in 25% of adolescents at T0. Combined pituitary hormone deficiency was found in 2 boys and DI in one patient at T3. Precocious puberty and growth hormone deficiency (GHD) were found in two boys at T6. At T12, a new endocrine dysfunction was diagnosed in five patients (2 GHD, 2 HH and in one patient with a GHD a new central hypothyroidism was confirmed). An empty sella has been found on MRI in two patients. Patients with GCS <8 had hormonal dysfunction more often (6/23) and they had more often DI or SIADH at T0. The occurrence of early endocrine dysfunction significantly correlated with severity of injury (p<0.05), but was not an indicator of development of late hormonal dysfunction (p=0.05).

Conclusions. A hormonal disorder occurred in 15.5% of patients. Risk factors include severity of TBI, abnormalities in the brain-imaging techniques and DI or SIAH in acute posttraumatic phase.  

An ancient founder mutation in PROKR2 impairs human reproduction

Avbelj M^1,2, Jeanpierre M³, Sykiotis GP¹, Young J⁴, Quinton R^5,6, Abreu AP⁷, Plummer L¹, Au MG¹, Balasubramanian R¹, Dwyer AA¹, Florez JC^8,9,10, Cheetham T^5,11, Pearce SH⁵, Purushothaman R¹², Schinzel A¹³, Pugeat M¹⁴, Jacobson-Dickman EE¹², Ten S¹², Latronico AC⁷, Gusella JF^9,15, Dode C³, Crowley Jr WF^1,10, Pitteloud N^1,10

¹Harvard Reproductive Endocrine Sciences Center and the Reproductive Endocrine Unit of the Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts; ²University Children’s Hospital, University Medical Centre Ljubljana, Ljubljana, Slovenia; ³Institut Cochin, Université Paris Descartes, INSERM U1016, Paris, France; ⁴Université Paris-Sud 11 et INSERM U693, Faculté de Médecine Paris Sud, Paris, France; ⁵Institute for Human Genetics, Newcastle University, Newcastle upon Tyne, United Kingdom; ⁶Department of Endocrinology, Newcastle upon Tyne Hospitals, Newcastle upon Tyne, United Kingdom; ⁷Unidade de Endocrinologia do Desenvolvimento, Laboratório de Hormônios e Genética Molecular, LIM/42, Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brasil; ⁸Center for Human Genetic Research and Diabetes Research Center (Diabetes Unit), Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts; ⁹Program in Medical and Population Genetics, Broad Institute, Cambridge, Massachusetts; ¹⁰Department of Medicine, Harvard Medical School, Boston, Massachusetts; ¹¹Department of Paediatrics, Newcastle upon Tyne Hospitals, Newcastle upon Tyne, United Kingdom; ¹²Divisions of Pediatric Endocrinology at Maimonides Infants and Children’s Hospital of Brooklyn and SUNY Downstate Medical Center, Brooklyn, New York; ¹³Institute of Medical Genetics, University of Zürich, Schwerzenbach, Zürich, Switzerland; ¹⁴INSERM U1060, Université Lyon 1, and Fédération d’Endocrinologie, Hospices Civils de Lyon, F-69677 Bron Cedex, France; ¹⁵Center for Human Genetic Research, Massachusetts General Hospital, and Department of Genetics, Harvard Medical School, Boston, Massachusetts

Background. Congenital gonadotropin-releasing hormone (GnRH) deficiency manifests as absent or incomplete sexual maturation and infertility. Although the disease exhibits marked locus and allelic heterogeneity, with the causal mutations being both rare and private, one causal mutation in the prokineticin receptor, PROKR2 L173R, appears unusually prevalent among GnRH-deficient patients of diverse geographic and ethnic origins.

Methods. To track the genetic ancestry of PROKR2 L173R, haplotype mapping was performed in 22 unrelated patients with GnRH deficiency carrying L173R and their 30 first-degree relatives. The mutation’s age was estimated using a haplotype-decay model.

Results. In all informative subjects, the mutation was present on the same ~123 kb haplotype whose population frequency is ≤10%. Thus, PROKR2 L173R represents a founder mutation whose age is estimated at ~9,000 years. Inheritance of PROKR2 L173R-associated GnRH deficiency was complex with highly variable penetrance among carriers, influenced by additional mutations in the other PROKR2 allele (recessive inheritance) or another gene (digenicity).

Conclusion. The paradoxical identification of an ancient founder mutation that impairs reproduction has intriguing implications for the inheritance mechanisms of PROKR2 L173R-associated GnRH deficiency and for the relevant processes of evolutionary selection, including potential selective advantages of mutation carriers in genes affecting reproduction. 

Impact of obesity on blood pressure in children and adolescents

Babinska K¹, Kovacs L¹, Vitariusova E¹, Janko V¹, Dallos T¹, Feber J²

¹2nd Department of Pediatrics, Comenius University Medical School, University Children´s Hospital, Bratislava, Slovakia; ²Division of Nephrology, Department of Pediatrics, Children’s Hospital of Eastern Ontario, Ottawa, Canada

Objective. The goal of our study was to analyze the impact of obesity on the blood pressure in obese children and adolescents. The ambulatory blood pressure monitoring (ABPM) enables a more accurate detection of blood pressure in children with a separate assessment of blood pressures during daytime and nighttime periods.

Methods. One hundred-nine patients with primary obesity aged 7 to 18 years (mean±SD age = 14.1±3.1) were enrolled in the study. Patients were divided into 3 groups according to BMI Z-scores: Group 1 (n=27): BMI >1.65 and <3.28 SDS; Group 2 (n=55): BMI >3.29 and <4.91 SDS; Group 3 (n=27): BMI >4.92 SDS. Anthropometric, laboratory and ambulatory blood pressure monitoring data were analyzed. Definition and staging of ambulatory hypertension was based on ABPM blood pressure levels and blood pressure load (BPL).

Results. Only 26 patients (24%) had ambulatory normotension, 27 (25%) had ambulatory prehypertension, 3 (3%) had hypertension and 53 (48%) had severe ambulatory hypertension. The severity of hypertension increased significantly with the degree of obesity (Chi square test for trend, p=0.0027). Daytime systolic, diastolic and mean arterial pressures (SBP, DBP, MAP) increased significantly with increased BMI, whereas the nighttime pressure remained elevated regardless of the degree of obesity. Isolated nighttime hypertension was observed in 25% of patients, 38% were classified as non-dippers.

Conclusions. Obesity has a major impact not only on incidence but also on the severity of hypertension. Almost 50% of children with obesity suffer from severe ambulatory hypertension. Significant proportion of them suffers from severe hypertension already at the time of the diagnosis.

Supported by a grant from UK/445/2011. 

Obesity prevalence among 7-years old children in the Czech Republic

Braunerová R¹, Kunešová M¹, Vignerová J², Pařízková J¹, Procházka B³, Riedlová J², Zamrazilová H¹, Šteflová A⁴

¹Obesity Management Centre, Institute of Endocrinology, Prague, Czech Republic; ²National Institute of Public Health, Prague, Czech Republic; ³Pediatric Clinic, Kolín, Czech Republic; ⁴WHO, Prague, Czech Republic

Objective. Childhood obesity is a world-wide health problem. The aim of our study was to evaluate obesity prevalence among 7-years old Czech children and to compare the results with data from 2001 (obtained from 6th National Anthropological Survey – Vignerová, Bláha 2001). The study is a part of the COSI (Childhood Obesity Surveillance Initiative) project, launched by the WHO Regional Office for Europe in 2006.

Methods. The representative sample of Czech 7-year olds included 1704 children. Children were examined during preventive check-ups by their pediatricians. The examination included basic anthropometric measurements, including weight, height, and waist and hip circumference. EpiData Analysis and Statgraphics software were used for statistical analysis.

Results. Obesity prevalence among boys reached 8.2 % in 2008, and 5.4% among girls. Prevalence of obesity and overweight combined reached 15 % among boys and 12.7 % among girls. In comparison with 2001, obesity prevalence among boys increased while obesity prevalence among girls decreased. The percentage of obese and overweight children combined decreased for both genders. Body height remained virtually unchanged in girls but decreased in boys.

Conclusion. The study did not confirm a trend of obesity and overweight prevalence growth among 7 years old children in the Czech Republic.

Supported by grant NS/9832-4 IGA Ministry of Health, Czech Republic. 

Vitamin D and diabetes

Cirmanová V, Čermáková I, Stárka L, Vondra K

Institute of Endocrinology, Prague, Czech Republic

Vitamin D deficiency predisposes individuals to type 1 and type 2 diabetes. It has been shown that vitamin D deficiency impairs insulin synthesis and secretion in humans and in animal models of diabetes, suggesting a role in the development of T2DM. Epidemiological studies suggest a link between vitamin D deficiency in early life and the later onset of T1DM. In some populations, T1DM is associated with certain polymorphisms within the vitamin D receptor gene. In individuals with diabetes mellitus, 25-(OH) vitamin D or 1,25-(OH)₂ vitamin D treatment may increase insulin secretion and improve glucose tolerance.

There is evidence that vitamin D may stimulate pancreatic insulin secretion directly. Vitamin D exerts its effects through nuclear vitamin D receptors, which are found in a wide variety of tissues, including T and B-lymphocytes, skeletal muscle, and the pancreatic islet β-cells. The stimulatory effects of vitamin D on insulin secretion may be manifest only when calcium levels are adequate.

There is some evidence that increased PTH activity is associated with, and possible causes, reduced insulin sensitivity. The prevalence of impaired glucose tolerance and diabetes mellitus is increased in patients with primary hyperparathyroidism. The secondary hyperparathyroidism that often accompanies vitamin D deficiency may thus mediate some of the effects of vitamin D on insulin sensitivity and glucose tolerance.

T1DM is an autoimmune disease and vitamin D is thought to play a role in its pathogenesis by its immunomodulatory actions of reducing lymphocytes proliferation and cytokines production.

Supported by grant No. NS/9831-4.    

GCK-MODY in the Czech Republic: identification of two founder mutations

Dušátková P, Průhová Š, Cinek O, Lebl J

Department of Paediatrics, 2nd Faculty of Medicine, Charles University in Prague and University Hospital Motol, Prague, Czech Republic

Background. Patients with GCK-MODY (glucokinase diabetes or maturity-onset diabetes of the young type 2, MODY2), are clinically characterised with chronic mild hyperglycaemia from birth. GCK-MODY is caused by heterozygous mutations in the glucokinase gene (GCK) which are inherited in autosomal-dominant manner and in most cases are family-specific.

Patients and methods. We have collected 240 Czech families fulfilling the strict clinical criteria of GCK-MODY. We tested GCK gene by direct sequencing. Additionally, after identification of a considerable number of Czech unrelated families with identical GCK mutations (E40K in 21 families, L315H in 15 families, G318R in 13 families, and V33A in 10 families), we tested a possible founder effect using genotypes of 16 markers spanning a 14 Mb region around GCK. Haplotypes were reconstructed using Phase and Haploview programs, and the age of mutations was estimated using DMLE⁺.

Results. GCK-MODY was genetically confirmed in 169 out of 240 families (70%). We found strong evidence that supports ancestral origin of two of the four mutations: E40K was associated with an 11-marker long conserved haplotype, and the L315H was associated with a 7-marker haplotype. The ages of the mutations were estimated to be between 64 and 93 generations old (95% credible sets 45–132).

Conclusion. The study shows a relatively high proportion of GCK mutations among individuals with GCK-like phenotype, confirming the effectiveness of carefully applied clinical criteria prior to genetic testing. We also report the evidence for ancestral origin of two recurrent GCK mutations, implying that GCK-MODY is unlikely to decrease reproductive fitness of its carriers.

The study was supported by the Grant Agency of the Charles University in Prague (GAUK 0840/2010) and the institutional grant from the Czech Ministry of Health (grant MZ NT11402).    

A novel mutation of the parathyroid hormone gene (PTH) in a girl with isolated hypoparathyroidism

Ertl DA¹, Stary S², Streubel B², Raimann A¹, Haeusler G¹

¹Medical University of Vienna, Univ. Clinic of Paediatrics and Adolescent Medicine, Paediatric Pulmology, Allergology and Endocrinology, Vienna, Austria; ²Medical University of Vienna, Institute of Pathology, Genetics, Vienna, Austria

Case report. A 4 months-old female patient presented with severe hypocalcaemia. Treatment with 1,25-(OH)₂-cholecalciferol and calcium was started and serum calcium concentrations were stabilized at the lower normal range. During 6 years of observation, the PTH serum levels were low but detectable, with values between 5.3 and  2.5 pg/ml. Disturbances in the vitamin-D metabolism, autoimmune polyendocrine syndrome (APS), chromosomal anomalies or mutations in the calcium-sensing receptor gene (CaSR) were excluded. Microarray analysis revealed loss of heterozygosity for PTH. Sequencing analysis of PTH showed the presence of c.68C>A homozygous point mutation that causes a premature stop-codon (p.Ser23X), resulting in a non-functional PTH-precursor. The parents and the siblings are heterozygous. A complementary measurement conducted at a specialized institution showed undetectable PTH levels.

Discussion. Isolated hypoparathyroidism is a diagnostic challenge. Besides chromosomal defects (CATCH22, DiGeorge Syndrome), differential diagnosis includes APS and mutations in the CaSR. The detection of measurable PTH serum concentrations in our patient made a defect in the PTH gene rather unlikely – only three PTH mutations have been published so far. However, considering consanguinity, we performed PTH-gene analysis and found a new mutation.

Conclusion. Genetic analysis of the PTH-gene is easy (3 exons) and should be performed in patients with idiopathic isolated hypoparathyroidism and consanguine parents, even before CaSR analysis (6 exons) in order to avoid unnecessary clinical and laboratory screening for autoimmune syndromes. In some laboratories, including ours, the accuracy of PTH serum measurements is poor, thus proving the need of sensitive and standardized assays.     

Cartilage-hair hypoplasia syndrome in a 3-year old girl. Case report

Gécz J¹, Ficek A², Šoltýsová A², Košťálová Ľ¹, Kovács L¹

¹2nd Department of Pediatrics, Comenius University Medical School, University Children´s Hospital, Bratislava, Slovak Republic; ²Department of Molecular Biology, Faculty of Natural Sciences, Comenius University, Bratislava, Slovak Republic

A case of a 3-year old girl with Cartilage-hair hypoplasia syndrome with prenatally diagnosed chondrodysplasia is presented. At age of 1.5 years, megacolon congenitum and anal stricture were diagnosed. Later on, she developed severe anemia with transient pancytopenia and celiac disease. Besides that a combined imunodeficit occurred, with the level of CD4+ lymphocytes ~200/μl. She acquired generalized variccela virus infection with almost fatal end. At age of 3-years she has short stature for her age (-8 SDS), late psychomotoric development with muscle hypotonia, hypopigmentation, fine sparse hair and skeleton deformities. This combination of defects is typical for the Cartilage-hair hypoplasia (CHH) syndrome, a distinct type of metaphyseal dysplasia first described by McKusick in 1965. CHH is inherited in autosomal recessive manner or through the uniparental disomy and caused by a defect in RMRP (RNA component of mitochondrial RNA processing endoribonuclease) gene on chromosome 9p13. When analyzing DNA of our patient we found 3 substitutions in the coding region 156G/C, 177C/T a 222C/T, one substitution in 5´UTR region -6G/A and a duplication of 14 bases 5´aatactactctgtg3´ in 5´UTR region – this was also found in her father´s gene. Based on the results of genetic testing, we may say that this certainly is the first case of CHH in Slovakia. 

Severe inborn deficit of cobalamin hidden behind the picture of methylmalonic aciduria. Case report

Gécz J¹, Virágová L², Košťálová Ľ¹, Kováčik J³, Hlavatá A¹, Kovács L¹

¹2nd Department of Pediatrics, Comenius University Medical School, University Children´s Hospital, Bratislava, Slovak Republic; ²Pediatric Emergency Department, University Children´s Hospital, Bratislava, Slovak Republic; ³Department of Pediatrics, Žilina, Slovak Republic

A case of 6-months old girl with mental and physical retardation and epilepsy is being presented. A severe inborn deficit of cobalamin was diagnosed as it was hidden behind the picture of methylmalonic aciduria. Deficit of cobalamin was caused by its low levels circulating in the mothers blood during intrauterine life. We confirmed that the mother suffers from previously undiagnosed autoimmune pernicious anemia. As we started with cobalamin substitution, all haematological and biochemical parameters normalized, as well as neurological symptoms and MRI findings on the brain. Such a severe case of inborn cobalamin deficiency is very rare in Slovakia. 

15 years of iodine supply monitoring in newborn population of the Czech Republic

Hníková O¹, Kračmar P¹, Vinohradská H², Al Taji E¹, Votava F¹

¹Pediatric Clinic of 3rd Medical Faculty, Charles University, Prague, Czech Republic; ²Neonatal Screening Center, Medical Faculty, Brno, Czech Republic

The history of iodine supplementation in Czechoslovakia started in 1947. The table salt was enriched by potassium iodine at 12 ppm. In 1993–95, an epidemiological study showed unequal iodine deficiency in newborns of 3 regions (median urinary iodines were 28- 45-77 mcg/L). In 1996, the enrichment increased to 27–42 ppm of potassium iodate. Programme with recommendation of iodine tablets –100 mcg/day during pregnancy improved newborns median urinary iodine in all investigated regions to 78 mcg/L in 1997. From 1996, monitoring of iodine supply in newborn population was started, using neonatal TSH (neoTSH). NeoTSH 5.0–20.0 mU/L in no more than 3% of newborns declares normal iodine supply if newborns were sampled at days 5–7. As a result of preventive measures, the nationwide iodine sufficiency in the Czech Republic is evident since 2000 in population aged 5–65 years. However, newborns as the most at-risk group reached iodine suficiency just in 2006, following an extensive education of pregnant mothers. From October 2009, the age at neonatal blood sampling was shifted earlier to 48–72 hours. The neoTSH screening levels presumably could be higher. From our experience for this new situation it will be necessary to change the cut off point from 3% of newborns with neoTSH 5–20 mU/L to at least 5%, as an evidence of suficient iodine supply. Nevertheless the metod, using neoTSH for iodine supply monitoring in newborn population in a long run, proved to be a reliable and unexpensive way, ideal for this purpose.  

Low-dose insulin therapy in patients with cystic fibrosis and early-stage insulinopenia prevents deterioration of lung function

Koloušková S, Zemková D, Bartošová J, Skalická V, Šumník Z, Vávrová V, Lebl J

Department of Pediatrics, Second Faculty of Medicine, Charles University in Prague and University Hospital Prague-Motol, Czech Republic

Objective. Cystic fibrosis related diabetes (CFRD) is primarily an insulinopenic condition. Our prospective study in patients with cystic fibrosis (CFPts) was aimed to an early recognition of insulinopenia and to the evaluation of impact of an early low-dose insulin therapy on the nutritional status and pulmonary function tests (PFTs).

Patients and methods. In 142 CFPts older than 10 years glucose tolerance was investigated once per year using an OGTT. Those with an abnormal result in OGTT (impaired glucose tolerance or diabetes mellitus without fasting hyperglycaemia) underwent a subsequent intravenous glucose tolerance test to asses the stimulated insulin secretion. Controls were sex/age matched to cases.

Results. Twenty eight patients with insulinopenia (IPts) were detected at the age 11.1–20.6 years; median 15.4). At the detection of insulinopenia, body height and body mass index did not differ between both groups except mid-arm ciucumferences (MAC) that was lower in insulinopenic patients (MAC-SDS -1.2±0.9 vs.            -0.8±0.9; p=0.02). PFTs deteriorated in both groups within one year of pre-treatment observation, IPts 81.3+4.3 vs. 73.8+4.3 (p=0.05); controls 76.5+4.1 vs. 71.1+3.8 (p=0.05). During 3 years of insulin treatment PFTs in IPts stabilized (FEV₁ 73.8+4.3 vs. 73.5+4.4). However, in controls PFTs deteriorated during 3 years of observation period (71.1+3.8 vs. 61.0+4.0; p=0.001).

Conclusion. Low-dose insulin administration at an early phase of CFRD stabilizes pulmonary functions. Therefore, we suggest to include a prospective detection of insulinopenia into the routine follow-up of CFpts in order to optimize the timing of start of insulin treatment.

Supported by project MZOFNM2005.  

Gender-specific regulation of retinol binding protein 4 expression and secretion in human adipocytes

Kotnik P^1,2, Tews D¹, Wabitsch M¹, Fischer-Posovszky P¹

 ¹Division of Pediatric Endocrinology and Diabetes, Endocrine Research Laboratory, Department of Pediatrics and Adolescent Medicine, University of Ulm, Ulm, Germany; ²Department of Endocrinology, Diabetes and Metabolism, University Children’s Hospital, UKC Ljubljana, Ljubljana, Slovenia

Background. Retinol binding protein 4 (RBP4) is an adipokine suggested to link obesity and whole body insulin resistance/type 2 diabetes. RBP4 expression is higher in female adipose tissue, whereas RBP4 circulating levels are higher in male.

Aim. To study effects of gender-specific serum factors and the two main sex hormones acting on adipose tissue, estradiol (E) and dihydrotestosterone (DHT), on the expression and secretion of RBP4 in human adipocytes in vitro.

Materials and methods. Human SGBS adipocytes were stimulated with either female or male serum, E (10-¹¹ - 10-⁸ M) or DHT (10-¹² - 10-⁹ M) for 48 hours. Relative gene expression was determined by quantitative real time-PCR. RBP4 protein levels in medium supernatant were measured by ELISA. Results from 3 independent experiments were presented as mean ± SEM. Student t-test or ANOVA and post hoc Dunnett´s test were used for statistical evaluation. P<0.05 was considered statistically significant.

Results. Stimulation of SGBS adipocytes with female serum resulted in higher mRNA RBP4 levels when compared to male serum (.65±.06 vs. .51±.05; ratio of control ± SEM; p<0.05). Both E and DHT increased RBP4 mRNA expression slightly and to comparable levels when compared to the control (E (10-⁹ M) 1.40±.04, DHT (10-⁹ M) 1.34±.05; ratio of control ± SEM; p<0.05). On the other hand DHT decreased RBP4 secretion into medium supernatant in a dose dependant manner (vehicle 18.4±.9 μg/l, DHT(10-¹² M) 15.8±.8 μg/l, DHT(10-¹¹ M) 15.2±1.2 μg/l, DHT(10-¹⁰ M) 13.5±.4 μg/l, DHT(10-⁹ M) 13.3±.6 μg/l; p<0.05) while E had no effect.

Conclusions. Gender-specific serum factors differentially regulate RBP4 mRNA expression in human adipocytes. E and DHT are not directly implicated in the determined differential regulation on mRNA level. On the other hand DHT in a dose dependant manner decreases RBP4 secretion from human adipocytes whereas E has no effect. 

Determination of parameters of insulin pump therapy associated with insulin resistance in adolescents with type 1 diabetes

Kotnik P, Bratina N, Battelino T

Department of Pediatric Endocrinology, Diabetes and Metabolism, University Children’s Hospital, UKC Ljubljana, Ljubljana, Slovenia

Background. Insulin resistance (IR) is involved in the pathogenesis of type 1 diabetes (T1D) complications.

Aim. To determine parameters of insulin pump therapy associated with IR as determined by estimated glucose disposal rate (eGDR).

Subjects and methods. Sixty-four T1D adolescents (15.8±.4 years; 34 girls) on insulin pump therapy were included in this pilot-study. IR was defined as a lower quartile of eGDR (calculated as 24.13 - 12.22 (waist-to-hip ratio) - 3.29 (hypertension status) - 0.57 (A1C)). Data on the last 3 weeks of insulin pump therapy were abstracted by CareLink software (Medtronic).

Data are presented as means ± SEM. Pearson correlation coefficient or ANOVA (with Dunnett’s post-hoc test) were used for statistical analysis when appropriate. P<0.05 was considered statistically significant (*).

Results. eGDR correlated negatively with variability in daily insulin dose, namely SD of daily insulin dose (DD_SD) (r = -.375 p=0.005). As shown in the table, IR subjects significantly differed to non-IR subjects according to A1C and variability in daily insulin dose (DD_SD), but didn’t differ regarding the daily insulin dose (DD), dose used as basal (DDb) or the daily number of boluses. When gender-specific eGDR quartiles were determined, the associations remained significant for boys, but not girls (data not shown).

Conclusions. In T1D adolescents (especially boys) variability in daily insulin dose is associated with IR as determined by eGDR. 

Update on monogenic diabetes: Genetic diagnosis has been clarified in 522 patients from 217 Czech families

 Lebl J, Průhová Š, Dušátková P, Veselá K, Šumník Z, Koloušková S, Štechová K, Obermannová B, Cinek O

Department of Pediatrics, Second Faculty of Medicine, Charles University in Prague and University Hospital Prague-Motol, Czech Republic

Genetic testing for monogenic diabetes was initially offered to Czech diabetic patients in 2000. Currently, we test routinely for GCK, HNF1A, HNF4A, HNF1B, INS, NEUROD1, KCNJ11 and ABCC8 mutations using direct sequencing, with an additional MLPA search for structural derangements. This complimentary genetic service is available for patients with a clinical diagnosis of MODY (autosomal dominant transmission of non-autoimmune diabetes diagnosed before the age of 30 years) or neonatal diabetes (any diabetes diagnosed up to age 6 months).

Currently, the Czech registry of monogenic diabetes includes DNA and clinical data of 1325 subjects from 438 families. Of these, 189 new patient samples from 108 families were gathered within the last year.

GCK diabetes was confirmed in 356 subjects (159 kindreds), HNF1A diabetes in 89 patients (36 kindreds), HNF4A diabetes in 54 patients (17 kindreds), NEUROD1 mutations in 11 patients (2 kindreds), INS mutations in 5 subjects (2 kindreds) and ABCC8 mutations in 7 subjects (1 kindred) with MODY and in one child with neonatal diabetes. No pathogenic gene variants were identified in 82 probands (“MODY X”).

In four children with neonatal diabetes (3 with DEND syndrome), we found KCNJ11 pathogenic variants.

After ten years of educational significance in the Czech medical community, genetic testing is now being employed more frequently in diagnostic work-up of diabetic patients. Over 128 subjects per million of the general population were referred and genetic diagnoses were established in 51 subjects per million. Strict patient selection is a prerequisite of a high rate of positive results.

The project is supported by a research grant MZ ČR NT11402. 

Genetic analysis of the CYP21A2 gene in neonatal dried blood spots from children with transiently elevated 17-hydroxyprogesterone

Malikova J¹, Votava F², Vrzalova Z³, Lebl J¹, Cinek O¹

¹Department of Pediatrics, 2nd Faculty of Medicine, Charles University in Prague and University Hospital Motol, Prague, Czech Republic; ²Department of Pediatrics, 3rd Faculty of Medicine, Charles University in Prague and University Hospital Královské Vinohrady, Prague, Czech Republic; ³Centre of Molecular Biology and Gene Therapy, University Hospital and Brno Masaryk University, Brno, Czech Republic

Background. Neonatal screening for congenital adrenal hyperplasia (CAH) identifies a certain proportion of newborns with transient moderate increase of 17-hydroxyprogesterone (17-OHP) who require a regular follow-up until normalization. We investigated the possibility to reduce the individuals’ recall rates by using genetic methods on their original neonatal dried blood spots.

Patients and methods. We analyzed neonatal dried blood spots from 754 subjects with transiently elevated 17-OHP. The CYP21A2 was sequenced to detect point mutations, and the copy number ratio between CYP21A2 and its pseudogene was tested by two methods utilizing the difference in the sequence of exon 3 (8bp deletion). Performance of the methods was checked using samples from 70 subjects with known CYP21A2 mutations and 191 healthy children.

Results. Among 702 successfully sequenced samples, 670 (95% of the cohort) had no mutations or novel variants in the amplified CYP21A2 gene. We found 22 carriers of known point mutations corresponding with the classic and non-classic forms of CAH, and we further detected eight heterozygous and two homozygous point variants with unknown functional significance. No significant difference was found in the distribution of the gene/pseudogene ratio between the group with transiently elevated 17-OHP and healthy children.

Conclusion. Although CAH due to 21-hydroxylase deficiency could be genetically excluded with a reasonable degree of confidence in 95% of our samples with transiently elevated 17-OHP, the tests performed suboptimally and were time-consuming in comparison with the current practice of repeated measurements of 17-OHP. Their introduction into clinical practice seems to be impractical at this stage.

Osteoporosis in childhood

Marginean O¹, Simedrea I¹, Marcovici T¹, Andor M¹, Maris I¹, Dracea D²

¹Ist Pediatric Clinic of the University of Medicine and Pharmacy, Timisoara, Romania; ²Clinical Children’s Emergency Hospital “Louis Turcanu”, Timisoara, Romania

Aim. To prove the utility of the treatment of osteoporosis during childhood.

Material and methods. Between 2005–2010, we studied the osteoporosis in children admitted to our hospital. The protocol used in order to reach the diagnosis included auxological data, physical examination, calcium/phosphorus metabolism, complementary biological investigations for the additional pathology and imaging (X-ray and DEXA of hip and spine). The treatment protocol involved pamidronat 1 mg/kg at 3 month and, in selected cases, recombinant GH Omnitrope 0.03 mg/kg.

Results and discussions. Eight children (aged 1–14 years) were suffering from osteoporosis of different etiology: osteogenesis imperfecta (4), renal failure (3 cases – one with Prune Belly Syndrome), and osteoporosis secondary to chemotherapy treatment for acute leukemia (1). The main symptoms were severe pain and fractures involving lower back and hip (as a consequence of usual activity). The biologic investigations revealed no specific changes in calcium/phosphorus metabolism. The DEXA exams have shown high risk of fracture in all cases (median T score 4.1). All children were treated with Aredia (pamidronat) 1 mg/kg and have had good evolution (were able to walk alone to school). DEXA exams performed after 1 year revealed a significant improvement of bone density. In cases with short stature and GH treatment, both the statural height and the bone structure improved.

Conclusions. (1) Osteoporosis can be present in childhood as a primary or secondary disorder. (2) The treatment should start as soon as the diagnosis is made. (3) The combination of recombinant GH and pamidronat in selected cases may improve the bone structure.    

Why only monotherapy in congenital hypothyroidism

Muzsnai A, Peter F

Buda Children’s Hospital, Budapest, Hungary

Introduction and objective. It is well-known that major source of triiodothyronine derives from deiodination of thyroxine in extrathyroidal tissues and only a smaller proportion of the daily T3 production is secreted by the thyroid gland. Since the advent of synthetic thyroxine levothyroxine is the drug of choice for treating congenital hypothyroidism. Authors report CH patients replaced with levothyroxine up to age 13–31 years then shifted to combined therapy (T4 plus T3).

Method. CH was confirmed by high TSH and low peripheral hormone levels in infancy. Total or free T4/T3 and TSH were measured regularly to adjust the replacement dose.

Results. Patient 1 was diagnosed and replaced at age 60 days, her TSH had normalized in 4 months. The neglected child presented high TSH, low-normal T4/FT4 and normal T3/FT3 levels time to time which was suspicious for bad compliance. Levothyroxine was supplemented with triiodothyronine when low-normal FT3, normal FT4 and elevated TSH were detected. In a few months both TSH and thyroid hormone levels were normalized. Patient 2 (31 years) and Pt3 (21.5 years) presented elevated TSH despite high FT4 and hyperthyroid signs on high dose levothyroxine. Triiodothyronine was added to monotherapy resulting recovery and normal hormone levels in months. A possible explanation is the decreased activity of deiodinase enzyme in the peripheral tissues.

Conclusion. Combined treatment (T4 plus T3) shows significant advantages after traditional treatment with T4 alone in some CH patients. 

3-M syndrome: Growth pathology behind GH-IGF-I axis. First case in the Czech Republic

Neumann D¹, Hanson D²

¹Dpt. of Paediatrics, University Hospital Hradec Kralove, Czech Republic; ²University of Manchester, Manchester Academic Health Sciences Centre, Paediatric Endocrinology, UK

Case report. Authors present a boy with FGR -5 weeks at 32nd w.g. according to prenatal ultrasound. He was born at 40th w.g. with BW 2090 g, BL 39 cm and OFC 36 cm. He presented with a macrocephaly but a proportionate IUGR. Postnatal adaptation was uneventful and no organ defect was detected. Endocrinological investigations were carried out at age of 15 months. He had normal karyotype 46,XY and the mutation in FGFR3 was excluded. His clinical appearance was as followed: 5.2 kg (-5.5 SD), 62 cm (-6.1 SD), OFC 48.3 cm (60th percentile), walking, speaking and showed a normal intelligence. No major dysmorphic features, except of macrocephaly with the Laron’s syndrome frontal bossing and midfacial dysplasia. Nares were anteverted. Hormonal investigations revealed TFTs in the reference range, as well as biochemistry, blood count and Astrup. The peak of the insulin tolerance test was 27.79 mIU/l, cortisol 938 nmol/l, and 5 days IGF-I generation test levels were 59, 30 and 23 ng/ml (D1, D3 and D5 40th, 8th and 5th percentile, respectively). The X-ray did not show slender long bones and tall vertebrae.

Molecular genetic investigation was done in Manchester AHSC, UK. It resulted in confirmation of the 3-M syndrome and revealed the new CUL7 gene mutation a c.3849 C>T, p.Q1317X.

Conclusion. 3-M syndrome is the pathology behind IGF-I R. Mutation in the CUL7 gene breaks the function of the ubiquitin ligase. The final height is -5 to -6 SD, intelligence normal. Surprisingly, mutations in the CUL7 and OBSL1, genes attributed to 3-M, were recently identified in a consanguineous population of children with Silver-Russell syndrome phenotype. 

Mutations and pituitary morphology in a series of 82 patients with PROP1 gene defects

Obermannová B¹, Pfaeffle R², Zygmunt-Gorska A³, Starzyk J³, Verkauskiene R⁴, Smetanina N⁴, Bezlepkina O⁵, Peterkova V⁵, Frisch H⁶, Cinek O¹, Chris CJ⁷, Blum WF⁸, Lebl J¹

¹Department of Pediatrics, Second Faculty of Medicine, Charles University in Prague, Prague, Czech Republic; ²Department of Pediatrics, University of Leipzig, Leipzig, Germany; ³Department of Pediatric and Adolescent Endocrinology, Polish-American Pediatric Institute, Jagiellonian University, Krakow, Poland; ⁴Lithuanian University of Health Sciences, Medical Academy, Department of Pediatric Endocrinology, Kaunas, Lithuania; ⁵Endocrinology Research Center, Moscow, Russian Federation; ⁶Department of Pediatrics, University of Vienna, Vienna, Austria; ⁷Lilly Research Laboratories, Eli Lilly and Company, Windlesham, UK; ⁸Lilly Research Laboratories, Eli Lilly and Company, Bad Homburg, Germany

Background/Aims. Defects of the PROP1 gene are the most prevalent genetic cause of combined pituitary hormone deficiency. Previous observations in affected patients have shown pituitary size ranging from hypoplasia to overt pituitary mass and evolution of size over the life-span.

Methods. We evaluated pituitary size and morphology in PROP1-mutation carriers who originated from Central and Eastern Europe. We analyzed 112 pituitary MRI scans from 82 patients (42 males) aged 2.5–72.7 years (median 16.6) from 60 kindreds.

Results. Among the 120 independent PROP1 alleles, the most prevalent mutations were delGA301/302 (99 alleles) and delA150 (13 alleles). Median pituitary height at first MRI was 4.7 mm (range 1.0–20.7) and median volume was 127.6 mm³ (range 7.5–3087.0). Pituitary size did not differ between sexes and did not correlate with hormonal phenotype, but significantly decreased with increasing age. However, evaluation of individual values suggested a biphasic mode with increasing volume during childhood, peak in adolescence, and subsequent regression in adulthood.

Conclusion. Although pituitary size was increased in a number of PROP1-deficient patients, none of them suffered permanent damage from pituitary mass; therefore, any proposed surgery should be postponed as long as possible and ultimately may not be necessary due to the self-limiting nature of the pituitary enlargement. 

T1DM risk stratification for the first degree relatives of diabetic patients in the Czech paediatric population

 Petruzelkova L, Vcelakova J, Stechova K, Lebl J, Vyhnankova M, Kolouskova S

Department of Pediatrics, Second Faculty of Medicine, Charles University in Prague and University Hospital Prague-Motol, Prague, Czech Republic

Objectives. The first degree relatives of patients with Type 1 diabetes mellitus (T1DM) are at increased risk of diabetes development. We searched for the strongest predictive combination of type and titer of pancreatic autoantibodies and HLA genotype leading to progression to clinical diabetes.

Methods. This study is a part of the Czech T1DM Prediction Programme where siblings and offspring of T1DM patients are HLA genotyped (DQA1,DQB1 by PCR-SSP) and regularly tested for autoantibodies (GADA, IA2A, IAA by RIA). In our study, 152 children positive for at least one autoantibody and 374 antibody negative children as a control group were enrolled. Median follow up in both groups was 3 years. 23.7% of antibody positive children developed diabetes during their follow-up. The prevalence of GADA and IA2A in antibody positive group was 77.6% and 50.7%, respectively. Seventy-two children from positive group were examined for IAA as well and 22.2% of them were positive. Forty-one antibody positive children underwent an intravenous glucose tolerance test (ivGTT) to assess the first phase insulin release (FPIR).

Results. The strong predictive combination for T1DM development was the positivity of IA2A and presence of the most risky genotype DQA1*05DQB1*0201/DQA1*03DQB1*0302 (p<0.001). Higher titer of IA2A (p<0.01), GADA (p<0.002) or IAA (p<0.012) had an additional progression predictive value. Most predictive for the clinical progression towards T1DM was the number of positive antibodies (p<0.001). Prolonged positivity of antibodies led to slowly decreasing FPIR; however, neither HLA genotype nor the detailed characteristics of antibodies correlated with abnormal FPIR at the ivGTT.

Conclusion. We analyzed the predictive value of selected factors for progression to clinical diabetes. We didn’t observe any association between these factors and the duration of beta cells destruction.

Supported by project MZOFNM2005. 

Congenital lipoid adrenal hyperplasia

Pomahačová R¹, Varvařovská J¹, Dort J², Dortová E²

¹Department of Pediatrics, Charles University, Medical Faculty and University Hospital Pilsen, Czech Republic; ²Department of Neonatology, Charles University, Medical Faculty and University Hospital Pilsen, Czech Republic

Background. Congenital lipoid adrenal hyperplasia (lipoid CAH) is a rare form of CAH leading to impaired production of all adrenal and gonadal steroids. Mutations in the gene encoding steroidogenic acute regulatory protein (StAR) or mutations in the gene CYP11A1 encoding the cholesterol side-chain cleavage enzyme (P450scc) cause lipoid CAH. Patients present with primary adrenal insufficiency in early infancy. Defect of sexual steroids leads in male to pseudohermaphroditism.

Case report. Our patient presented early after birth with cardiopulmonary instability, in laboratory with hyponatraemia, hyperkalaemia and metabolic acidosis. Low level of cortisol and high ACTH confirmed primary adrenal insufficiency. The karyotype was 46,XX, clinically no signs of genitalia virilisation. The most frequent enzymatic block of 21-hydroxylase was excluded. Ultrasound of adrenal glands was normal. Hormonal substitution led to normal growth and psychomotor development. At the age of 11 years, high levels of LH and FSH confirmed hypergonadotrophic hypogonadism. This finding reinforced the consideration of etiology of adrenal insufficiency – enzymatic block at the beginning of steroidogenesis. No mutation was identified in the StAR gene. The mutation was confirmed in the CYP11A1 gene. The patient is compound heterozygote for the p.Gly138Arg (c.412G>A) mutation  in exon 2 and the p.Leu170ValfsX30 (c.508_509delCT) mutation in exon 3.

Conclusion. We have to consider the diagnosis of  lipoid CAH in patients with primary adrenal insufficiency and in 46, XY individuals with disorder of sexual development. The knowledge of gene defect allows early diagnosis in other family members and antenatal diagnosis. 

First genetic evidence of Renal Cysts and Diabetes (RCAD) syndrome in the Czech patients with polycystic kidney diseases and renal hypo-dysplasia

Průhová Š, Dušátková P, Malina M, Dušek J, Lebl J, Seeman T, Cinek O

Department of Paediatrics, 2nd Faculty of Medicine, Charles University in Prague and University Hospital Motol, Prague, Czech Republic

Background. The RCAD (Renal Cysts and Diabetes) syndrome (MODY5) is caused by heterozygous mutations in the HNF1B gene leading to developmental kidney damage combined with diabetes mellitus.

Patients and methods. The HNF1B gene was investigated in 104 Czech patients (median age 16 years; interquartile range 11–21 years) with polycystic kidney diseases or kidney hypo-dysplasia by direct sequencing and Multiplex Ligation Probe-dependent Amplification (MLPA).

Results. The HNF1B gene anomalies were detected in 6/104 patients (5.78%): two patients (autosomal dominant polycystic kidney disease) carried heterozygous mutations R165H and IVS1-1G>C; four patients (renal hypo-dysplasia) carried a heterozygous deletion of the whole HNF1B gene. All of the patients had chronic renal insufficiency or end-stage renal failure; two patients were after kidney transplantation at 2 and 5 years of age. Diabetes was detected in only 2/6 patients: 16 years old girl manifested diabetes during the growth hormone (GH) therapy which disappeared after stopping the GH therapy. In a 17 years old boy, glycosuria was randomly found by the preventive investigation. His fasting glycaemia was 14.3 mmol/l and the HbA1c 6.4% (IFCC) at the time of manifestation. He was treated with insulin but after genetic investigation the treatment has been successfully changed to Gliclazide.

Conclusion. Genetic investigation of HNF1B gene is efficient mostly in patients with various developmental kidney diseases, especially cystic kidney diseases. Diabetes is found randomly and patients with RCAD syndrome should be screened for glycaemic control. The HNF1B whole gene deletion prevailed, so the gene-dosage analysis performed by MLPA should precede the direct sequencing.

The study was supported by an institutional grant from the Czech Ministry of Health (grants NT11402 and MZ NT11457). 

Low alkaline phosphatase – a diagnostic key to hypophosphatasia

Raimann A, Ertl D A, Riedl S, Haeusler G

Medical University Vienna, Vienna, Austria

Background. Hypophosphatasia (HPP; OMIM 146300, 241500, 241510) is an inherited disorder characterized by impaired alkaline phosphatase (ALP) activity. Clinical symptoms range from severe disabling bone disease in the infancy and childhood onset form of HPP to isolated teeth mineralization defects in the adult onset form. Recently, enzyme replacement therapy has shown promising results in a phase 2 trial.

Case report. We report the case of a family affected by two different mutations in the ALPL gene. The 11 year old male index-patient presented with short stature, dental abnormalities and valgus deformity of the legs. On radiological examination, rickets-like bone lesions were found. His father complained of chronic bone pain, the only symptom in his sister was enamel defects of the front teeth.

In the index patient, ALP was extremely low (22 U/L, normal range 100–420 U/l) and pyridoxalphosphat levels were 5-fold increased. Analysis of parental ALPL revealed heterosygosity for a mild mutation (E191K) in the mother as well as a dominantly active mutation (T68M) in the father, whereas the index-patient was compound heterozygote (T68M; E191K), confirming the diagnosis of childhood HPP. His symptomatic sister showed a genetic constellation similar to her father.

Discussion. While the maternal mutation in the ALPL gene (E191K) exhibits sufficient residual activity in heterozygotes, the paternal mutation (T68M) diminishes ALP levels to ~5% resulting in a dominant type of HPP. Symptoms in the affected father and sister were unspecific, confirming that mild forms of HPP can easily be missed or misdiagnosed. 

How can GH treatment in Turner Syndrome (TS) become a success?

Ranke MB

University Children`s Hospital, Tübingen, Germany

In most cohorts with TS who were treated with the standard GH dose (<50 µg/kg d) from a starting age of approximately 9 years, an average gain of about 5–10 cm in height has been documented and only about 50% exceeded the 3rd centile of height for adult women. A normal adult height has been reached in a greater proportion only in cohorts treated at an earlier age and with higher GH doses (Dutch study), although at the cost of excessive IGF-I levels. TS girls who were treated very young (<4 years of age) are catching up during childhood, but adult height has not been reported so far.

In normal clinical practice, many TS girls will continue to be presented late; despite of this a fraction will respond well to conventional GH doses. Thus, it is the responsiveness to GH, which can be derived from the growth rate during the first year, determines the outcome. In the case of a poor prospective height gain (total expected gain <5 cm) and poor response (delta height during first year GH <0.5 SDS (norm refs) or <0.3 SDS (TS refs) or a poor responsiveness (SR <-1.0) discontinuation of treatment should be considered, for ethical reasons and costs. Because of safety concerns the presence of excessively high IGF-I blood levels over a longer period of time (>1 year) should to be avoided. 

Severe virilization of a 46,XX newborn and her mother during pregnancy: A novel case of aromatase deficiency

Riedl S¹, Springer A², Huber J³

¹St Anna Children´s Hospital, Medical University of Vienna, Austria; ²Department of Paediatric Surgery, Medical University of Vienna, Austria; ³Department of Obstetrics and Gynaecology, Medical University of Vienna, Austria

Case report. A 2-week-old male-assigned newborn with ambiguous genitalia (Prader 4), 3rd child from Turkish consanguineous parents, presented at our clinic. The child was otherwise healthy without any malformations. From mid-pregnancy on, the mother had developed progressive hirsutism, acne, and voice deepening.

The baby had a 46,XX/SRY-ve karyotype and a normal uterus on sonography. LH and FSH were constantly elevated without measurable estradiol at 3, 5, and 7 weeks. Initial testosterone was 0.37 ng/ml and AMH undetectable. Cortisol rise following 125 µg of synacthen was appropriate. Urinary C21 and C17 metabolites were normal.

The mother´s LH and FSH were suppressed whereas testosterone, androstendione, 17-hydroxyprogesterone, and estradiol were extremely high at 2 weeks, declining rapidly thereafter. Tumor markers were negative. Sonography/MRI revealed markedly enlarged ovaries with PCOS morphology and ruled out an adrenal/ovarian virlilizing tumour.

Genetic testing for a CYP19 defect revealed a homozygous mutation (Glu210Lys) in the baby, inherited from each parent.

Conclusions. This is the 12th case of aromatase deficiency in a 46,XX individual reported so far. Excess of fetal androgens and estrogen deficiency due to a lack of placental aromatization led to virilized external genitalia in the baby (Prader 4) and acne, hirsutism, and voice deepening in the mother. Interestingly, the mother´s ovaries showed PCOS morphology producing high amounts of androgens even some weeks after birth. We propose that increased fetal LHRH, driven by estrogen deficiency, may have crossed the placenta and stimulated maternal gonadotrophins, eventually leading to PCOS associated with transient autonomous overproduction of androgens. 

Similar changes of bone geometry at the radius in Turner syndrome and isolated SHOX deficiency

Soucek O¹, Lebl J¹, Zapletalova J², Novotna D³, Plasilova I⁴, Kolouskova S¹, Zemkova D¹, Rocek M⁵, Hirschfeldova K⁶, Sumnik Z¹

¹Department of Paediatrics, 2nd Faculty of Medicine, Charles University in Prague and University Hospital Motol, Prague, Czech Republic; ²Department of Paediatrics, Faculty of Medicine, Palacky University and University Hospital Olomouc, Olomouc, Czech Republic; ³Department of Paediatrics, Faculty of Medicine, Masaryk University and University Hospital Brno, Brno, Czech Republic; ⁴Department of Paediatrics, Faculty of Medicine, Charles University and University Hospital Hradec Kralove, Hradec Kralove, Czech Republic; ⁵Department of Radiology, 2nd Faculty of Medicine, Charles University in Prague and University Hospital Motol, Prague, Czech Republic; ⁶Institute of Biology and Department of Medical Genetics, 1st Faculty of Medicine, Charles University in Prague and General University Hospital, Prague, Czech Republic

Introduction. Girls with Turner syndrome (TS) have altered bone density and geometry at the radius, which may pose an increased fracture risk in adulthood. The etiology of their low bone strength remains unknown, despite assumptions have been made on the influence of hypogonadism and the haploinsuficiency of the X chromosome. Since short stature homeobox-containing (SHOX) gene plays a major role in long bone development and patients with isolated SHOX deficiency (iSHOX) share some skeletal features with TS, SHOX gene defect could cause specific changes in bone structure in both groups. We aimed to assess bone geometry and volumetric bone density in patients with isolated SHOX deficiency and to compare their results to girls with TS.

Methods. Fourteen patients with isolated SHOX deficiency (median age 11.8 yrs, range 6.9–36.8, 10 children and 4 adults) were examined by peripheral quantitative CT (pQCT) at the non-dominant forearm. Results were expressed as Z-scores using published reference data and then compared to the results of 67 girls with TS (median age 14.3 yrs, range 6.0–19.4). The differences from reference data were tested by one-sample T test and differences between TS and iSHOX were tested by two-sample T test.

Results. Trabecular volumetric bone mineral density (vBMD) was decreased in TS (mean Z-score -0.7±1.3, p<0.001) but normal in iSHOX (Z-score 0.3±1.3, n.s., group difference p<0.01). Cortical vBMD was low in both groups (Z-score -1.6±1.5, p<0.001 and -1.9±2.3, p<0.01, respectively) without significant group difference. Both groups had increased total bone cross-sectional area (CSA) at the diaphysis (Z-score 0.7±1.7, p<0.01 and 2.3±1.6, p<0.001, respectively), but normal cortical bone CSA (0.0±1.0, n.s. and -0.7±2.3, n.s., respectively). Consequently, cortical thickness was decreased in both groups (-0.4±0.5, p<0.001 and -1.1±1.3, p<0.05, resp.).

Conclusions. Patients with Turner syndrome and isolated SHOX deficiency have similar bone geometry at the radius. Our results support the hypothesis that skeletal abnormalities in patients with TS are caused by SHOX deficiency. Low trabecular vBMD in TS is probably a consequence of hypogonadism due to ovarian failure.

Grant support: Charles University Grant Agency (No. 47609), Czech Ministry of Health (No. MZ0FNM2005 64203). 

Clinical and molecular genetics of adrenal tumors associated with Cushing syndrome

Stratakis CA

Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, MD, USA

Molecularly, the overwhelming majority of benign lesions of the adrenal cortex (AC) leading to Cushing syndrome (CS) are linked to one or another abnormality of the cyclic (c) AMP signaling pathway (1). Benign adrenocortical causes of CS include the common and sporadic cortisol-producing adenoma (CPA) and a spectrum of corticotropin (ACTH)-independent, and almost always bilateral, hyperplasias. Macro-hyperplasias are more common among older patients, whereas micro-hyperplasias are frequent among children and young adults. Massive macronodular adrenocortical disease (MMAD) or ACTH-independent macronodular adrenocortical hyperplasia (AIMAH) describes a heterogeneous group of disorders that are associated with aberrant G-protein-coupled receptor (GPCR) expression (E). Abnormal GPCR-E has been found in CPAs; a small number of both MMADs and CPAs harbor somatic GNAS (Gsa) mutations. AIMAH can also be found in the context of McCune-Albright syndrome. Micro-hyperplasias are either pigmented (the classic form being that of primary pigmented nodular adrenocortical disease or PPNAD) or non-pigmented (NP-MAH) and isolated (i) or in the context of other syndromes (Carney complex – CNC). Both CNC and iPPNAD are caused by germline PRKAR1A mutations; somatic mutations of this gene that regulates cAMP-dependent protein kinase (PKA) are also found in 10–20% of all CPAs and abnormalities of PKA are present in most MMADs. NP-MAH forms of adrenal hyperplasia and some CPAs are associated with phopshodiesterase (PDE)-11A and PDE-8B sequence defects. Other PDEs may be involved, too. From all the above, it is clear that increased cAMP signaling leads to tumors in AC. Mouse models of PRKAR1A deficiency also show that increased cAMP signaling leads to tumors in AC and other tissues. But how? Micro-RNA studies identified the Wnt-signaling pathway as a possible culprit (2, 3). We also investigated Prkar1a (+/-) mice when bred within the Rb1 (+/-) or Trp53 (+/-) backgrounds, or treated with a two-step skin carcinogenesis protocol (4). A whole-genome transcriptome profiling of tumors produced by all three models identified Wnt-signaling as the main pathway activated by abnormal cAMP signaling, along with cell cycle abnormalities. siRNA down-regulation of Ctnnb1, E2f1 or Cdk4 inhibited proliferation of human adrenal cells bearing a PRKAR1A-inactivating mutation and Prkar1a (+/-) mouse embryonic fibroblasts. Thus, Prkar1a haploinsufficiency is a relatively weak tumorigenic signal that can act synergistically with other tumor suppressor gene defects or chemicals to induce tumors, mostly through Wnt-signaling activation and cell cycle dysregulation. In a most recent mouse model (5), we further demonstrated that activation of Wnt-signaling is a somewhat generic endpoint of cAMP/PKA activation, even in bone. Micro-RNAs may also activate Wnt in other defects associated with increased cAMP signaling, as in MMAD. We conclude that cAMP signaling aberrations are essential in the pathogenesis of benign cortisol-producing lesions of the AC.

References. (1) Bimpaki et al. Eur J Endocrinol. 2009; 161: 153; (2) Iliopuloulos et al. Cancer Res. 2009; 69: 3278; (3) Bimpaki et al. Clin. Endocrinol (in press); (4) Almeida et al. Hum Mol Genet. 2010; 19: 1387–1398; (5) Tsang et al. Proc Natl Acad Sci USA. 2010; 107(19): 8683–8688.

Sources of support: This work is supported by an intramural grant of the Eunice Kennedy Shriver National Institute of Child Health & Human Development to Dr. C.A. Stratakis (grant# 1 Z01 HD000642-04). 

Low trabecular bone mineral density and sarcopenia, but normal bone strength at the radius in boys with haemophilia

Šumník Z¹, Souček O¹, Komrska V², Lebl J¹

¹Department of Paediatrics and ²Department of Paediatric Haematology and Oncology, University Hospital Motol, 2nd Faculty of Medicine, Charles University in Prague, Czech Republic

Introduction. Although a decreased areal bone mineral density (BMD) has been reported in patients with haemophilia, data are lacking that would reflect the three-dimensional structure of the bone and the muscle-bone relationship.

Aim. We aimed to assess volumetric BMD, bone geometry and muscle-bone phenotype in boys with haemophilia, and to describe the association between clinical characteristics of haemophilia and bone quality and structure.

Methods. A cross sectional study was conducted in 41 boys with haemophilia (mean age 12.4, range 6.6–19.8 years) using peripheral quantitative CT (pQCT) at the non-dominant forearm. Results were transformed into Z-scores using previously published reference data. Significant differences were tested by one sample t-test or sign test. Two-sample t-test and ANOVA were used to compare results between subgroups of patients divided according to the severity of the disease, the fracture history and the number of joint and muscle bleedings.

Results. Boys with haemophilia had a decreased trabecular volumetric BMD (mean Z-score -0.5, p<0.01), while their cortical volumetric BMD was increased (mean Z-score 0.4, p<0.05). The volumetric bone mineral content and the bone geometry at the radial diaphysis were normal when adjusted for patients’ shorter body height. Muscle area was decreased (mean Z-score -1.0, p<0.001), irrespective of age. No association was observed of bone quality parameters and bone geometry with the disease severity, fracture history or number of bleedings.

Conclusion. Bone strength measured at the diaphysis of the radius is not impaired in boys with haemophilia. The finding of the decreased trabecular bone density can be most likely attributed to their sarcopenia.

Grant support: GAUK 47609, MZ0FNM2005 64203. 

Ectopic thyroid tissue presenting as a midline neck mass

Tar A¹, Szécsényi Nagy I², Hegedűs E²

¹Heim Pál Hospital Saint John and ²Buda Children’s Hospital, Budapest, Hungary

Midline neck masses are not uncommon in the pediatric population. The differential diagnosis of a midline neck mass includes thyroglossal duct cysts, dermoid cysts, sebaceous cysts, ectopic thyroid tissue, thyroid nodules, lymph nodes lipomas and neoplasms. It is important to distinguish between ectopic thyroid tissue and other causes of anterior neck swelling. It is crucial to know if this is the only functioning thyroid tissue. Ectopic thyroid tissue may also lie along the anterior neck, anywhere from the foramen cecum to the anterior trachea where the thyroid gland normally rests. Rarely is ectopic thyroid tissue found in the mediastinum, larynx or esophagus.

We present the case of a fifteen year old female where the ectopic thyroid tissue with normal vascularization together with the normal gland were present as identified by ultrasound, a good noninvasive study helpful in distinguishing solid from cystic lesions. ^99mTechnetium labeled pertechnetate radionuclide thyroid scanning showed that the ectopic thyroid tissue dominated the thyroid gland in normal position. Fine needle aspiration cytology showed colloidal and follicular structure with sign of regression of the ectopic thyroid mass along with the ductus thyreoglossus. Thyroid function tests performed at presentation were as follows: TSH: 7.06 mIU/ml, FT3: 5.18 pmol/l, FT4: 13.19 pmol/l, Anti-TPO: <10 IU/ml, Anti-Thyreoglobulin: <20 IU/ml. Treatment with 100 μg L-Thyroxin qd was started. Repeated ultrasound examination after one year therapy showed volume reduction in the ectopic. 

Peripheral blood karyotype poorly represents tissue mosaicism determined by FISH and QF-PCR – A case report

Török D¹, Haltrich I¹, Nagy B², Tóth Zs¹, Jórász³, Luczay A¹

¹Semmelweis University, 2nd Dept. of Pediatrics, Budapest, Hungary; ²Semmelweis University, 1st Dept. of Obstetrics and Gynecology, Budapest, Hungary; ³Bethesda Children’s Hospital, Budapest, Hungary

Background. Cytogenetic analysis in disorders of sexual differentiation is routinely performed on peripheral blood lymphocytes. The presence of Y chromosome material in the gonads is of major interest because of the risk of gonadoblastoma development. However, there is a lack of evidence whether the gonadal karyotype is congruent with lymphocyte karyotype.

Patient and methods. A three-week-old boy with normal male genitals presented with left inguinal hernia and cryptorchidism. The inguinal hernia surgery revealed an unusual anatomic situation. His free testosterone was normal (23.21 pg/ml). Peripheral blood karyotype was mos 45,X[25]/46,X,i(Y)(p10).ish idic(Y)(q11)(DYZ3++)Y[6]. An SRY duplication was detected by FISH. Histological examination found an immature testis on the left, a dysgenetic gonad on the right side and a uterus-like organ. Based on the different degree of differentiation of the two gonads and the normal hormone production we hypothesized that the mosaicism – namely the ratio of Y chromosome – may vary from loci to loci, moreover in some tissues might be significantly different from that of the peripheral lymphocytes. To test this hypothesis blood, gonad and subcutaneous fibroblast samples were analyzed by FISH and QF-PCR.

Results. The X:Y ratio was normal in the testis and nearly 2:1 in the blood, fibroblast and a dysgenetic gonad samples by QF-PCR. However, FISH analysis in the testis have found one X centromere in 60% and presence of different mosaic SRY+ cell lines (with single or double SRY signals) in 40% of cells (nuc ish(DXZ1+)(SRY-)[201]/(DXZ1+)(SRY+)[88]/ (DXZ1+)(SRY++)[52]/ (DXZ1++)(SRY+)[12]).

In addition, the father’s karyotype was also analyzed and found to be a normal male without SRY duplication.

Conclusions. Our case underlines that peripheral blood lymphocyte karyotype might not provide a reliable representation of specific tissue karyotypes. Future studies may prove whether Y chromosome material present in the gonads would be completely absent or undetected in lymphocytes. Peripheral blood is an easy and reliable source of material for cytogenetic analysis, however, the interpretation of test results might require caution.  

Newborn screening in Czechia: Overview of the last 9 years, with emphasis on congenital adrenal hyperplasia (dedicated to anniversary of Prof. Olga Hníková)

Votava F¹, Kračmar P¹, Dejmek P¹, Chrastina P², Kožich V², Pešková K², Šťastná S², Adam T³, Friedecký D³, Hlídková E³, Vinohradská H⁴, Krulišová V⁵, Holubová A⁵, Balaščáková M⁵, Piskáčkova T⁵, Macek M Jr⁵, Gaillyová R⁶, Truellová I⁷, Švagera Z⁸

¹Dept. of Pediatrics, Charles University, 3rd Faculty of Medicine and University Hospital Kralovske Vinohrady, Prague; ²Institute of Inherited Metabolic Disorders, Charles University, 1st Faculty of Medicine and General University Hospital, Prague; ³Laboratory of Inherited Metabolic Disorders, Palacky University, Faculty of Medicine and University Hospital, Olomouc; ⁴Dept. of Clinical Biochemistry and Hematology, Children Hospital, Masaryk University, Faculty of Medicine and University Hospital, Brno; ⁵Institute of Biology and Medical Genetics, Charles University, 2nd Faculty of Medicine and University Hospital Motol, Prague; ⁶Dept. of Medical Genetics, Masaryk University, Faculty of Medicine and University Hospital, Brno; ⁷Dept. of Health Services, Ministry of Health of the Czech Republic, Prague; ⁸Institute of Clinical Biochemistry, University Hospital, Ostrava, Czech Republic

Background. The nationwide newborn screening (NS) was started in 1975 for phenylketonuria (PKU); in 1985 for congenital hypothyreosis (CH); in 2006 for congenital adrenal hyperplasia (CAH) and since October 2009 for cystic fibrosis (CF) and 9 expanded inborn metabolic disorders (MSUD, GA I, IVA, MCADD, LCHADD, VLCADD, CPTD I and II, CACTD). The aim is to summarize results from January 2002 through December 2010.

Methods. A total of 961,887 newborns were screened for PKU and CH; phenylalanine was measured by bacterial inhibition method, chromatography, enzymoimmunoassay, later by tandem mass spectrometry (MS/MS); thyreotropin was determined by fluoroimmunoassay (FIA). The 17-hydroxyprogesterone was assayed in 535,673 newborns by FIA. The level of immunoreactive trypsinogen (IRT) was measured in 138,780 newborns by FIA and the CFTR gene was analysed in 1,485 of them. The spectrum of metabolites was analysed in 145,404 newborns by MS/MS.

Results. PKU was detected in 120 newborns (prevalence 1:8,015); CH was confirmed in 248 (1:3,878); CAH in 47 (1:11,397); CF in 27 (1:5,140); 14 cases of MCADD (1:10,386) were detected; two probands with LCHADD (1:72,702); two cases of GA I (1:72,702); one and one proband with MSUD and VLCADD was caught (1:145,404). Cumulative detection rate was increased from 1:2,701 in 2002–2005 over 1:2,072 in 2007–2008 to 1:1,085 in 2010. False positive rate (FPR) in CAH reached 0.51%.

Conclusion. NS is effective for presymptomatic detection of serious rare diseases. FPR in NS for CAH should be improved.

Supported by IGA 9986/3, 9981/3, MSM0021620814 and VFN MZ0VFN2005.