Zeynep Atay

Hypogonadotropic Hypogonadism due to a Novel Missense Mutation in the First Extracellular Loop of the Neurokinin B Receptor

CONTEXT The neurokinin B (NKB) receptor, encoded by TACR3, is widely expressed within the central nervous system, including hypothalamic nuclei involved in regulating GnRH release. We have recently reported two mutations in transmembrane segments of the receptor and a missense mutation in NKB in patients with normosmic isolated hypogonadotropic hypogonadism (nIHH). PATIENTS AND METHODS We sequenced the TACR3 gene in a family in which three siblings had nIHH. The novel mutant receptor thus identified was studied in a heterologous expression system using calcium flux as the functional readout. RESULTS All affected siblings were homozygous for the His148Leu mutation, in the first extracellular loop of the NKB receptor. The His148Leu mutant receptor exhibited profoundly impaired signaling in response to NKB (EC(50) = 3 +/- 0.1 nm and >5 microm for wild-type and His148Leu, respectively). The location of the mutation in an extracellular part of the receptor led us also to test whether senktide, a synthetic NKB analog, may retain ability to stimulate the mutant receptor. However, the signaling activity of the His148Leu receptor in response to senktide was also severely impaired (EC(50) = 1 +/- 1 nm for wild-type and no significant response of His148Leu to 10 microm). CONCLUSIONS Homozygosity for the TACR3 His148Leu mutation leads to failure of sexual maturation in humans, whereas signaling by the mutant receptor in vitro in response to either NKB or senktide is severely impaired. These observations further strengthen the link between NKB, the NKB receptor, and regulation of human reproductive function.

Serum alkaline phosphatase levels in healthy children and evaluation of alkaline phosphatase z-scores in different types of rickets.

Objective: Serum alkaline phosphatase (ALP) levels show great variation with age and sex in children and adolescents. Additionally, different buffers used even in the same method cause variable results. This detail is not usually taken into account in the evaluation. We aimed to study pediatric age- and sex-specific reference ranges for ALP by colorimetric assay using p-nitrophenyl phosphate as substrate and diethanolamine as buffer and also to compare the ALP levels in patients with different types of rickets. Methods: 1741 healthy children and adolescents (904 girls) were included in the study for normative data. 77 different ALP measurements from 38 nutritional rickets (NR), 7 vitamin D-dependent rickets (VDDR) and 8 hypophosphatemic rickets (HR) patients were included. Results: Reference values for ALP were constructed. ALP levels demonstrated a tetraphasic course with two peaks at infancy and puberty. There was no difference in ALP levels between boys and girls until puberty. However, higher ALP levels were noted at 10-11 years in girls (p=0.02) and at 12-13, 14-15, 16-17 years in boys (p<0.001). ALP levels start to decline after age 12 and 14 in girls and boys, respectively. Serum ALP levels were highest in the VDDR group and lowest in the HR group (median z-score values in HR, VDDR and NR were 3.6, 10.4 and 6.5, respectively; p<0.001). Similarly, plasma parathormone(PTH) levels ranged from highest to lowest in the VDDR, NR and HR groups (median values: 525, 237 and 98 pg/mL, respectively; p<0.001). Conclusions: This normative data will provide a basis for better evaluation of ALP levels determined by the described method. Furthermore, use of z-scores gives a more precise assessment of changes in ALP levels in rickets and other bone disorders. Conflict of interest:None declared.

Rare Causes of Primary Adrenal Insufficiency: Genetic and Clinical Characterization of a Large Nationwide Cohort

Context: Primary adrenal insufficiency (PAI) is a life-threatening condition that is often due to monogenic causes in children. Although congenital adrenal hyperplasia occurs commonly, several other important molecular causes have been reported, often with overlapping clinical and biochemical features. The relative prevalence of these conditions is not known, but making a specific diagnosis can have important implications for management. Objective: The objective of the study was to investigate the clinical and molecular genetic characteristics of a nationwide cohort of children with PAI of unknown etiology. Design: A structured questionnaire was used to evaluate clinical, biochemical, and imaging data. Genetic analysis was performed using Haloplex capture and next-generation sequencing. Patients with congenital adrenal hyperplasia, adrenoleukodystrophy, autoimmune adrenal insufficiency, or obvious syndromic PAI were excluded. Setting: The study was conducted in 19 tertiary pediatric endocrinology clinics. Patients: Ninety-five children (48 females, aged 0–18 y, eight familial) with PAI of unknown etiology participated in the study. Results: A genetic diagnosis was obtained in 77 patients (81%). The range of etiologies was as follows: MC2R (n = 25), NR0B1 (n = 12), STAR (n = 11), CYP11A1 (n = 9), MRAP (n = 9), NNT (n = 7), ABCD1 (n = 2), NR5A1 (n = 1), and AAAS (n = 1). Recurrent mutations occurred in several genes, such as c.560delT in MC2R, p.R451W in CYP11A1, and c.IVS3ds+1delG in MRAP. Several important clinical and molecular insights emerged. Conclusion: This is the largest nationwide study of the molecular genetics of childhood PAI undertaken. Achieving a molecular diagnosis in more than 80% of children has important translational impact for counseling families, presymptomatic diagnosis, personalized treatment (eg, mineralocorticoid replacement), predicting comorbidities (eg, neurological, puberty/fertility), and targeting clinical genetic testing in the future.

Current Status of Childhood Obesity and its Associated Morbidities in Turkey

As a transitional society, rapid changes have occurred in the social, economic, nutritional and lifestyle aspects of the Turkish population over the last three decades. As a result, the prevalence of overweight and obesity has shown a dramatic increase in the adult Turkish population, reaching figures as high as 30-40%. Although there is no nationwide figure regarding the prevalence of overweight and obesity in Turkish children and adolescents, several local studies performed between 2000 and 2010 in different regions of the country have demonstrated varying prevalence rates of 10.3%-17.6% and 1.9%-7.8% for overweight and obesity, respectively, in children aged 6-16 years. The differences in the figures obtained in these regions are thought to be due to variations in the subject sampling. The figures appear to vary depending on residential (urban vs. rural) and economic conditions. Belonging to a high-income family, living in a large city, having obese parents, being of high birthweight, consuming sugar-sweetened beverages (soft drinks, juice drinks, etc.), and spending time in front of TV and PC were identified as the most common risk factors. Complications and co-morbidities of obesity have also started to appear in our pediatric population. Metabolic syndrome, diagnosed according to the International Diabetes Federation criteria, was found in 2.3% of Turkish schoolchildren aged 10-19 years. This rate was 28% in obese children. Preventive public measures have started to be implemented by the State and other bodies to control the rising trends in obesity. Conflict of interest:None declared.

Puberty and Influencing Factors in Schoolgirls Living in Istanbul: End of the Secular Trend?

OBJECTIVE: To (1) establish the median ages at menarche and pubertal stages and investigate influential factors and (2) assess the secular trend in reaching puberty in a transitional society. MATERIALS AND METHODS: A probit method was used to calculate the median age at menarche and pubertal stages from a cross-sectional study of 4868 healthy schoolgirls (aged 6–18 years) in Istanbul, Turkey. The findings were compared with those from a similar study performed 4 decades earlier. Logistic regression was used to analyze the associations between the odds of attaining puberty stages and putatively influential factors. Simple statistical models were used to test the effects of BMI and consumption of certain foods on the onset of menarche. RESULTS: The median age at menarche is 12.74 years. The median ages at breast stages 2 through 5 are 9.65, 10.10, 11.75, and 14.17 years, respectively, and at pubic-hair stages 2 through 5 are 10.09, 11.19, 12.33, and 14.68 years, respectively. Girls from upper socioeconomic classes are more likely to reach menarche and B4 and B5 stages. Higher BMI seems to be a promoting factor for attaining menarche. Intrauterine growth and gestational age had no effect. The average age at menarche was not associated with the consumption of milk, eggs, chicken, or fish. CONCLUSIONS: The secular trend in puberty is probably about to end in Turkey. Although the median ages at the breast stages show a decreasing trend, the median age at menarche is approximately the same as it was 4 decades ago. Socioeconomic status and BMI are important, and related, factors that affect the age at menarche and pubertal stages.

Clinical and molecular characterization of Turkish patients with familial hypomagnesaemia: novel mutations in TRPM6 and CLDN16 genes

BACKGROUND Recent identification and characterization of novel renal Mg(2+) transporters and ion channels have greatly increased our understanding of the normal physiology of renal magnesium handling. METHODS The present study deals with the clinical and molecular characterization of eight Turkish children (median age 10.6 years, range 3-16.2 years, five boys and three girls) with primary hypomagnesaemia from six families. RESULTS All patients initially presented with tetany and convulsions. Laboratory evaluation yielded severely low serum magnesium levels and low serum calcium levels in all patients. While six patients exhibited inadequately low parathyroid hormone levels, the two remaining patients showed hyperparathyroidism, hypercalciuria and nephrocalcinosis. Genetic studies revealed familial hypomagnesaemia with secondary hypocalcaemia (HSH) due to a TRPM6 mutation in six patients and familial hypomagnesaemia with hypercalciuria and nephrocalcinosis (FHHNC) due to a CLDN16 mutation in one patient. CONCLUSIONS Among recently identified magnesium-wasting disorders, HSH and FHHNC represent two major entities also in the Turkish population. Besides clinical course and laboratory diagnosis of hypomagnesaemia, the detection of renal calcium wasting and parathyroid function are crucial to differentiate between these most prevalent forms of hereditary magnesium deficiency. While TRPM6 mutations underlying HSH almost uniformly lead to a complete loss of function of the TRPM6 protein, the severity of FHHNC phenotype depends on the residual function of the mutated claudin-16 protein.

The prevalence and risk factors of premature thelarche and pubarche in 4- to 8-year-old girls

Aim:  To assess the prevalence of premature thelarche (PT) and pubarche in healthy 4‐ to 8‐year‐old girls and to investigate factors associated with early pubertal development.

An atypical case of familial glucocorticoid deficiency without pigmentation caused by coexistent homozygous mutations in MC2R (T152K) and MC1R (R160W).

Context: Familial glucocorticoid deficiency (FGD) is a rare autosomal recessive disorder characterized by isolated cortisol deficiency. Mutations in the gene encoding the ACTH receptor (MC2R) account for 25% of cases. One significant feature is generalized skin hyperpigmentation, which is thought to be due to elevated ACTH acting on the melanocortin 1 receptor (MC1R). Objective: The aim of the study was to determine the cause of a nonhyperpigmented case of FGD. Patients: The patient presented at 4 yr of age with hypoglycemia after prolonged fasting during a respiratory tract infection. She had further hypoglycemic attacks and was diagnosed with isolated glucocorticoid deficiency at 6 yr of age. Her parents were consanguineous, and she had two unaffected sisters. Her physical examination was normal, except that her height and weight were greater than the 97th centile for a sex- and age-matched reference population. Interestingly, she had no hyperpigmentation despite very high ACTH levels. Results: Nucleotide sequence analysis revealed homozygous mutations c.478C>T in MC1R and c.455C>A in MC2R leading to R160W and T152K changes in the amino acid sequences, respectively. The R160W MC1R change has previously been implicated in a red hair/pale skin phenotype, and MC2R -T152K is trafficking defective. Both parents and two unaffected sisters were heterozygous for the MC1R mutation; additionally, one unaffected sister was heterozygous for the MC2R mutation, and the other was wild-type. Conclusion: We report an unusual case of FGD without hyperpigmentation due to coexistent MC1R/MC2R mutations. This case is important because it demonstrates for the first time that the assumption that the action of ACTH on MC1R causes skin hyperpigmentation is correct.

Effect of BCG vaccine on tuberculin skin tests in 7-11-year-old children.

Aim: To determine the effect of Bacillus Calmette Guerin (BCG) vaccination on tuberculin skin test responses in 7–11‐year‐old children, and also to clarify whether the number of vaccinations and the time interval between vaccination and tuberculin skin test have an effect on the test responses. Method: 1200 primary school children were evaluated for the presence and number of BCG scars. They were then given 5 TU PPD‐S intra‐dermally. Seventy‐two hours after the application of tests, PPD indurations were measured. Results: Mean indurations were 3.7 ± 3.9, 6.5 ± 5.4 and 9.2 ± 7.1 mm in children with no scar, one scar and two scars, respectively. No statistical difference was found between mean induration of children with one scar and those with two scars.

Evidence of hormone resistance in a pseudo-pseudohypoparathyroidism patient with a novel paternal mutation in GNAS

CONTEXT Loss-of-function GNAS mutations lead to hormone resistance and Albrights hereditary osteodystrophy (AHO) when maternally inherited, i.e. pseudohypoparathyroidism-Ia (PHPIa), but cause AHO alone when located on the paternal allele, i.e. pseudoPHP (PPHP). OBJECTIVE We aimed to establish the molecular diagnosis in a patient with AHO and evidence of hormone resistance. CASE The patient is a female who presented at the age of 13.5years with short stature and multiple AHO features. No evidence for TSH or gonadotropin-resistance was present. Serum calcium and vitamin D levels were normal. However, serum PTH was elevated on multiple occasions (64-178pg/mL, normal: 9-52) and growth hormone response to clonidine or L-DOPA was blunted, suggesting hormone resistance and PHP-Ia. The patient had diminished erythrocyte Gsα activity and a novel heterozygous GNAS mutation (c.328 G>C; p.A109P). The mother lacked the mutation, and the fathers DNA was not available. Hence, a diagnosis of PPHP also appeared possible, supported by low birth weight and a lack of AHO features associated predominantly with PHP-Ia, i.e. obesity and cognitive impairment. To determine the parental origin of the mutation, we amplified the paternally expressed A/B and biallelically expressed Gsα transcripts from the patients peripheral blood RNA. While both wild-type and mutant nucleotides were detected in the Gsα amplicon, only the mutant nucleotide was present in the A/B amplicon, indicating that the mutation was paternal. CONCLUSION These findings suggest that PTH and other hormone resistance may not be an exclusive feature of PHP-Ia and could also be observed in patients with PPHP.