NOVEL VDR MUTATIONS IN PATIENTS WITH VITAMIN D-DEPENDENT RICKETS TYPE 2A: A MILD DISEASE PHENOTYPE CAUSED BY A NOVEL CANONICAL SPLICE-SITE MUTATION.

Objective: Vitamin D-dependent rickets type 2A (VDDR2A) is a rare autosomal recessive disorder caused by mutations in the vitamin D receptor gene (VDR), leading to end-organ resistance to 1,25-dihydroxyvitamin D3 (1,25[OH]2D3). The objective of this study was to investigate VDR mutations in 11 patients from 8 Turkish-Arab families. Methods: All coding exons and intron-exon boundaries of the VDR gene were amplified by polymerase chain reaction from peripheral leukocyte DNA and sequenced. The effect of splice-site mutations on mRNA splicing was evaluated by a customized VDR mini-gene assay. Results: Homozygous VDR mutations were found in all the patients, including four novel mutations: c.473G>T (p.R158L), c.1-4A>G (IVS3-2A>G), c.755+1G>T, and c.352_356delGACAG (p.D118Sfs*7). The c.1-4A>G mutation was located in the canonical splice acceptor site and 4 base pairs from the original ATG start codon. The mutation resulted in both complete (60% of transcripts) and partial exon 4 skipping (15% of transcripts). The latter was due to activation of a cryptic splice acceptor site and did not disrupt the open reading frame. Both c.755+1G>T and c.352_356delGACAG resulted in frameshifts and a premature stop codon. Clinically, all the patients required continued treatment, except for patient IV-3, who presented with alopecia, hypocalcemia, and increased 1,25(OH)2D3 at 1.5 years of age as a result of the c.1-4A>G mutation. He stopped taking medication at 6 years of age and still maintained normal height and biochemical profile. Conclusion: We have identified four novel VDR mutations. Although canonical splice-site mutations cause premRNA splicing errors that usually lead to a severe disease phenotype, mild disease can also occur due to activation of a cryptic splice site. Abbreviations: 1,25(OH)2D3 = 1,25-dihydroxyvitamin D3 (calcitriol); 25OHD3 = 25-hydroxyvitamin D3; PCR = polymerase chain reaction; PTH = parathyroid hormone; VDDR2A = vitamin D-dependent rickets type 2A; VDR = vitamin D receptor.

A novel DAX-1 mutation presented with precocious puberty and hypogonadotropic hypogonadism in different members of a large pedigree.

Patients with DAX-1 gene mutations on chromosome Xp21 usually present with adrenal hypoplasia congenita and hypogonadotropic hypogonadism. Yet, neither correlation between the type of mutation and the age of onset of the disease nor mechanism of the mutation on puberty is fully understood. Here, we report a novel non-sense p.Gln208X mutation in the amino terminal domain of the DAX-1 gene observed in a large family with three boys presenting with adrenal manifestations at different ages. Furthermore, two boys developed spontaneous puberty that failed to progress at similar ages, whereas the other boy developed precocious puberty at 10 month of age. The unique structure of the DAX-1 gene may explain this phenotypic variability. However, more studies are needed to understand the role of the DAX-1 gene on development of the adrenal gland and hypothalamus-pituitary-gonadal axis.

Clinical and genetic analysis of patients with vitamin D-dependent rickets type 1A.

CONTEXT: Vitamin D-dependent rickets type 1A (VDDR-IA, OMIM 264700) is a rare autosomal recessive disorder and is caused by mutations in the CYP27B1 gene. OBJECTIVES: We aim to investigate CYP27B1 mutation in seven patients from four separate families and characterize the genotype-phenotype correlation. METHODS: The entire coding region of the CYP27B1 gene was sequenced, and genotype-phenotype correlation among patients was assessed. RESULTS: Sequencing analysis identified biallelic CYP27B1 mutations in all patients and monoallelic mutations in their parents. One patient from the first family was compound heterozygous for c.1166G>A (p.Arg389His) and a novel nonsense mutation c.1079 C>A (p.Ser360*). Two patients from the second family were homozygous for a novel splice donor site mutation in intron 1 (c.195 + 2 T>G), causing partial retention of the intron and a shift in the reading frame. Both novel mutations lead to the complete loss of vitamin D1α-hydroxylase activity. Four patients from families 3 and 4 were homozygous for a previously reported duplication mutation in exon 8 (1319-1325dupCCCACCC, Phe443Profs*24). Interestingly, one patient who was presented with severe hypocalcaemia and seizures at 4 months of age as a result of Phe443Profs*24 has improved spontaneously since 11 years of age and does not need regular treatment. Her laboratory tests showed normal serum calcium and 1,25(OH)(2) D after refusing to take medication for 12 months. CONCLUSIONS: There is a good genotype-phenotype correlation in VDDR-IA. However, some patients may recover from the loss of CYP27B1 function, probably due to 1α-hydroxylase activity exerted by a non-CYP27B1 enzyme.

Intrathyroidal ectopic thymic tissue may mimic thyroid cancer: a case report.

Ectopic intrathyroidal thymus tissue that may be present as a thyroid nodule is rarely reported. We present a case of a 4-year-old boy with a solitary thyroid nodule. Real-time thyroid ultrasound showed a calcified nodule in the right lobe. Complete blood count, serum calcitonin, and thyroglobulin concentration were normal and antithyroid antibodies were negative. Fine-needle aspiration (FNA) biopsy was revealed as inadequate for cytological examination. During his follow-up, nodular enlargement was found, and the patient was subjected to surgical total excision of the right lobe of the thyroid gland. Pathological examination showed an ectopic intrathyroidal thymus tissue. In childhood, ectopic intrathyroidal thymus tissue can present as an enlarging microcalcified thyroid nodule that may mimic thyroid cancer and may grow during follow-up.

Variability in the age at diagnosis of diabetes in two unrelated patients with a homozygous glucokinase gene mutation.

Homozygous mutations in the glucokinase gene (GCK) result in a complete deficiency of the GCK enzyme, which leads to permanent neonatal diabetes mellitus. Whilst there has been one report of a patient (with a homozygous p.T168A) who was diagnosed with diabetes at the age of 2 months, all other cases were diagnosed with diabetes within the first 2 weeks of life. We now report a second unrelated patient with the same p.T168A GCK mutation who was diagnosed with diabetes at the age of 9 months. We conclude that the specific GCK mutation, as yet unidentified genetic modifiers, and/or environmental factors might have different effects on pancreatic beta-cell functions, causing variability in the age at diagnosis of diabetes.

The rapid correction of hypercalcemia at presentation of acute lymphoblastic leukemia using high-dose methylprednisolone.

Hypercalcemia is a well-recognized complication of neoplastic disorders. Herein, we report a hypercalcemic pediatric acute lymphoblastic leukemia case at presentation refractory to hydration, furosemide, pamidronate and calcitonin. Normal serum calcium levels were achieved with the initiation of chemotherapy protocol including vincristine, daunomycin and high-dose methylprednisolone. The impact of high-dose methylprednisolone in the correction of severe hypercalcemia in steroid-responsive tumors as an initial treatment approach or for cases refractory to other measures may be life-saving.

Urinary phthalate metabolite concentrations in girls with premature thelarche.

In girls, breast development before eight years of age is called "premature thelarche (PT)". There are few studies in literature that show the interaction between PT and phthalate exposure. The aim of this study was to determine the urinary levels of di-(2-ethylhexyl) phthalate (DEHP) metabolites and other phthalate metabolites in girls with PT. PT group consisted of 29 newly diagnosed subjects. Control group comprised of healthy age-matched girls (n = 25). Urinary phthalate metabolite concentrations were measured by liquid chromatography/tandem mass spectroscopy (LC-MS/MS). The urinary concentrations of mono-(2-ethyl-hexyl)phthalate (MEHP) in the PT group (33.96 ±â€¯6.88 µg/g creatinine) were found to be significantly higher compared to control group (11.54 ±â€¯1.39 µg/g creatinine, p = 0.002). In PT group, %MEHP was also markedly higher vs. control (17.84 ±â€¯3.31 vs. 6.44 ±â€¯1.13, p = 0.001). Our results suggest that DEHP is more efficiently converted to MEHP in girls with PT, the importance of which needs to be further elucidated.

Evaluation of left ventricular functions with two-dimensional speckle-tracking echocardiography (2D-STE) and N-terminal ProBNP in diabetic children.

Simsek A, Turan Ö, Çiftel M, Kardelen F, Durmaz E, Özdem S, Akçurin G, Ertug H. Evaluation of left ventricular functions with twodimensional speckle-tracking echocardiography (2D-STE) and N-terminal ProBNP in diabetic children. Turk J Pediatr 2018; 60: 633-641. The purpose of this study was to examine the existence of subclinical left ventricular dysfunction by using 2D-STE and NT-ProBNP levels in children and adolescent patients with type 1 diabetes mellitus. Furthermore, it was also aimed to investigate the effects of the diabetes duration and the metabolic control of the disease on cardiac functions. The patient group was composed of 63 children who were being followed up for the type 1 diabetes mellitus. The control group was composed of 36 healthy children who were of the similar age. Patients with type 1 diabetes mellitus were divided into groups; according to the duration of the disease; group 1: 3-5 years, group 2: 6-10 years of follow-up. The conventional echocardiography and 2D-STE were applied to all of the patients and control individuals. NT-Pro BNP level was measured in the diabetes group. In the conventional echocardiographic examination; there was no difference between the patient and control groups in terms of left ventricular systolic functions, left ventricular diastolic functions; late-diastolic flow velocity in mitral valve (A) values increased and E-wave/A-wave ratio (E/A ) values decreased in diabetes mellitus patients. According to the 2D-STE results; global longitudinal strain, (-17.28±2.24 vs. -19.49±2.22; p < 0.05) and circumferential strain (-12.86±3.19 vs. -17.71±4.62; p < 0.05) were lower in diabetic patients compared to the parameters of control group individuals. There was no difference between levels of NT-ProBNP of the group 1 and group 2 diabetes mellitus patients. Our study showed that there was a dysfunction on the left ventricular systolic functions of the patients with type 1 diabetes mellitus. NT-Pro BNP levels were not considered as a distinguishing factor for the early stages of diabetes mellitus.

Low zinc levels may contribute to gynecomastia in puberty.

This study aimed to determine whether there were any differences in trace element levels between adolescent boys with gynecomastia and control boys and to determine the correlations between the levels of trace elements and body mass index (BMI) and sex hormones. The pubertal gynecomastia group comprised of 41 patients (mean age=13.2 ±0.9 years), who were admitted to Hacettepe University Ihsan Dogramaci Children's Hospital in Ankara. Control group comprised of 21 healthy male children. Analyses of trace element levels were performed atomic absorption spectrometry. The mean zinc level of control group was 101.33±16.87µg/dL and the mean zinc level of gynecomastia group was 81.36±17,43µg/dL (20% lower in gynecomastia patients, p=0.0001). However, the mean copper and manganese levels of gynecomastia patients were not statistically different than the control group. There were significant positive correlations between plasma zinc and total testosterone levels in gynecomastia group (r=0.592; p<0.05). There was a significant negative correlation between plasma zinc levels and BMI (r=-0.311; p<0.05). These results indicate that zinc deficiency might be one of the underlying factors of gynecomastia, the importance of which needs to be further elucidated.

Hypogonadotropic Hypogonadism due to Novel FGFR1 Mutations.

OBJECTIVE: The underlying genetic etiology of hypogonadotropic hypogonadism (HH) is heterogeneous. Fibroblast growth factor signaling is pivotal in the ontogeny of gonadotropin-releasing hormone neurons. Loss-of-function mutations in FGFR1 gene cause variable HH phenotypes encompassing pubertal delay to idiopathic HH (IHH) or Kallmann syndrome (KS). As FGFR1 mutations are common, recognizing mutations and associated phenotypes may enhance clinical management. METHODS: Using a candidate gene approach, we screened 52 IHH/KS patients. RESULTS: We identified three novel (IVS3-1G>C and p.W2X, p.R209C) FGFR1 gene mutations. Despite predictive null protein function, patients from the novel mutation families had normosmic IHH without non-reproductive phenotype. CONCLUSION: These findings further emphasize the great variability of FGFR1 mutation phenotypes in IHH/KS.

The relation of urinary bisphenol A with kisspeptin in girls diagnosed with central precocious puberty and premature thelarche.

BACKGROUND: Bisphenol A (BPA) is known as an endocrine disruptor and it is supposed to have a role on the development of central precocious puberty (CPP). Kisspeptin, a hypothalamic peptide, is a neuromodulator of gonadotropin releasing hormone and it has an important role on regulation of the onset of puberty. The BPA levels in girls with CPP and premature thelarche (PT) and its relation with kisspeptin levels were investigated. METHODS: Twenty-eight girls with CPP, 28 girls with PT and 22 prepubertal girls as a control group were enrolled to the study. Urinary BPA and serum kisspeptin levels were compared in the groups. Bivariate correlations were performed to evaluate the relations of BPA with kisspeptin and estradiol. RESULTS: There was no statistical difference between groups regarding BPA levels. Serum kisspeptin levels were higher in CPP group than controls [306.56 (interquartile range (IQR), 175.63-504.66) vs. 157.62 (IQR, 55.61-285.00) p: 0.008]. There were no correlations between BPA and kisspeptin levels (r: 0.088, p: 0.391) and between BPA and estradiol (r: -0.171, p: 0.144). CONCLUSIONS: The BPA levels did not differentiate between groups and it seems that the exposed amount of BPA in daily life did not affect kisspeptin levels in girls with CPP and PT.

A Novel Null Mutation in P450 Aromatase Gene (CYP19A1) Associated with Development of Hypoplastic Ovaries in Humans.

OBJECTIVE: The CYP19A1 gene product aromatase is responsible for estrogen synthesis and androgen/estrogen equilibrium in many tissues, particularly in the placenta and gonads. Aromatase deficiency can cause various clinical phenotypes resulting from excessive androgen accumulation and insufficient estrogen synthesis during the pre- and postnatal periods. In this study, our aim was to determine the clinical characteristics and CYP19A1 mutations in three patients from a large Turkish pedigree. METHODS: The cases were the newborns referred to our clinic for clitoromegaly and labial fusion. Virilizing signs such as severe acne formation, voice deepening, and clitoromegaly were noted in the mothers during pregnancy. Preliminary diagnosis was aromatase deficiency. Therefore, direct DNA sequencing of CYP19A1 was performed in samples from parents (n=5) and patients (n=3). RESULTS: In all patients, a novel homozygous insertion mutation in the fifth exon (568insC) was found to cause a frameshift in the open reading frame and to truncate the protein prior to the heme-binding region which is crucial for enzymatic activity. The parents were found to be heterozygous for this mutation. Additionally, all patients had hypoplastic ovaries instead of cystic and enlarged ovaries. CONCLUSION: A novel 568C insertion mutation in CYP19A1 can lead to severe aromatase deficiency. Homozygosity for this mutation is associated with the development of hypoplastic ovaries. This finding provides an important genetic marker for understanding the physiological function of aromatase in fetal ovarian development.

Novel CYP27B1 Gene Mutations in Patients with Vitamin D-Dependent Rickets Type 1A.

The CYP27B1 gene encodes 25-hydroxyvitamin D-1α-hydroxylase. Mutations of this gene cause vitamin D-dependent rickets type 1A (VDDR-IA, OMIM 264700), which is a rare autosomal recessive disorder. To investigate CYP27B1 mutations, we studied 8 patients from 7 unrelated families. All coding exons and intron-exon boundaries of CYP27B1 gene were amplified by PCR from peripheral leukocyte DNA and subsequently sequenced. Homozygous mutations in the CYP27B1 gene were found in all the patients and heterozygous mutations were present in their normal parents. One novel single nucleotide variation (SNV, c.1215 T>C, p.R379R in the last nucleotide of exon 7) and three novel mutations were identified:, a splice donor site mutation (c.1215+2T>A) in intron 7, a 16-bp deletion in exon 6 (c.1022-1037del16), and a 2-bp deletion in exon 5 (c.934_935delAC). Both c.1215 T>C and c.1215+2T>A were present together in homozygous form in two unrelated patients, and caused exon 7 skipping. However, c.1215 T>C alone has no effect on pre-mRNA splicing. The skipping of exon 7 resulted in a shift of downstream reading frame and a premature stop codon 57 amino acids from L380 (p.L380Afs*57). The intra-exon deletions of c.1022-1037del16 and c.934_935delAC also resulted in a frameshift and the creation of premature stop codons at p.T341Rfs*5, and p.T312Rfs*19, respectively, leading to the functional inactivation of the CYP27B1 gene. Clinically, all the patients required continued calcitriol treatment and the clinical presentations were consistent with the complete loss of vitamin D1α-hydroxylase activity. In conclusion, three novel mutations have been identified. All of them caused frameshift and truncated proteins. The silent c.1215 T>C SNV has no effect on pre-mRNA splicing and it is likely a novel SNP. The current study further expands the CYP27B1 mutation spectrum.

Try235Phe homozygous mutation of the steroid 5-a reductase type 2 (SRD5A2) gene in a Turkish patient.

Steroid 5-a reductase type 2 isoenzyme (SRD5A2) deficiency is a male-limited autosomal recessive disorder that results in decreased conversion of testosterone to dihydrotestosterone with various de.gree of incomplete virilization in affected 46, XY infants. No clear genotype-phenotype relationship has been reported till date; moreover, the same mutation can result in considerable heterogeneity in clinical manifestations. Of 6 documented cases with Try235Phe homozygous mutation of the SRD5A2 gene, 3 patients had predominantly female external genitalia whereas the other 3 had predominantly male phenotype. We report Try235Phe homozygous mutation of the SRD5A2 gene in a Turkish patient who was initially assigned as a girl because of the predominantly female appearance of the external genitalia.

Urinary bisphenol a levels in girls with idiopathic central precocious puberty.

OBJECTIVE: Bisphenol A (BPA) is an industrial chemical, particularly used to harden plastics. BPA is thought to have negative health effects on both laboratory animals and humans. Consider ing the decline in age of onset of puberty noted in recent years, particularly among girls, the importance of BPA as an estrogenic endocrine disruptor has increased. In this study, we aimed to determine urinary BPA levels in girls with idiopathic central precocious puberty (ICPP). METHODS: Non-obese girls newly diagnosed with ICPP (n=28, age 4-8 years) constituted the study group. The control group consisted of 25 healthy age-matched girls with no history of ICPP or any other endocrine disorder. Urinary BPA levels were measured by using high-performance liquid chromatography. RESULTS: In the ICPP group, urinary BPA levels were significantly higher compared to the control group [median 8.34 (0.84-67.35) µg/g creatinine and 1.62 (0.3-25.79) µg/g creatinine, respectively (OR=8.68, 95% CI:2.03-32.72, p=0.001)]. There was no marked correlation between urinary BPA levels and body mass index in either group. In the ICPP group, no significant correlations were found between urinary BPA levels and serum luteinizing hormone, follicle-stimulating hormone and estradiol levels. CONCLUSIONS: To our knowledge, this is the first study evaluating the urinary BPA levels in Turkish girls with ICPP. Our results indicate that the estrogenic effects of BPA may be an etiologic factor in ICPP.

Clinical expression of familial Williams-Beuren syndrome in a Turkish family.

Williams-Beuren syndrome (WBS) is a rare genetic disorder characterized by distinctive facial features, intellectual disability with a typical neurobehavioral profile, cardiovascular anomalies, and occasional infantile hypercalcemia. Majority of cases occur sporadically, and only a few cases of familial WBS have been reported. Although pre- and post-natal growth retardation is a common clinical feature of the syndrome, growth hormone deficiency is detected only in a few patients. To our knowledge, there has only been one report about familial Williams-Beuren syndrome in the Turkish population. Here, we report on the three molecular cytogenetically confirmed familial Williams-Beuren syndromes detected in a family with familial short stature. The father, daughter, and son analyzed with clinical and laboratory findings, and reasons of the short stature in Williams-Beuren syndrome are discussed through the literature.

A combination of nifedipine and octreotide treatment in an hyperinsulinemic hypoglycemic infant.

Hyperinsulinemic hypoglycemia (HH) is the commonest cause of persistent hypoglycemia in the neonatal and infancy periods. Mutations in the ABCC8 and KCNJ11 genes, which encode subunits of the ATP-sensitive potassium channel in the pancreatic beta cell, are identified in approximately 50% of these patients. The first-line drug in the treatment of HH is diazoxide. Octreotide and glucagon can be used in patients who show no response to diazoxide. Nifedipine, a calcium-channel blocker, has been shown to be an effective treatment in a small number of patients with diazoxide-unresponsive HH. We report a HH patient with a homozygous ABCC8 mutation (p.W1339X) who underwent a near-total pancreatectomy at 2 months of age due to a lack of response to diazoxide and octreotide treatment. Severe hypoglycemic attacks continued following surgery, while the patient was being treated with octreotide. These attacks resolved when nifedipine was introduced. Whilst our patient responded well to nifedipine, the dosage could not be increased to 0.75 mg/kg/day due to development of hypotension, a reported side effect of this drug. Currently, our patient, now aged 4 years, is receiving a combination of nifedipine and octreotide treatment. He is under good control and shows no side effects. In conclusion, nifedipine treatment can be started in patients with HH who show a poor response to diazoxide and octreotide treatment.

Effect of renal graft on longitudinal growth in prepubertal children.

OBJECTIVES: Linear growth impairment frequently accompanies chronic kidney disease in children. Despite successful renal transplant, growth retardation may persist in renal allograft recipients. MATERIALS AND METHODS: We recorded the longitudinal growth and biochemical data of prepubertal children during the first 2 years after renal transplant in 34 children (18 boys [52.9%]; mean age at renal transplant, 7.3 ± 2.5 y; range, 1.4 to 9.8 y). Height standard deviation scores were calculated. The patients were divided into 2 groups according to the increase in height standard deviation scores over the first 2 years after renal transplant: group 1 (increases in height standard deviation scores < 1) and group 2 (increases in height standard deviation scores > 1). RESULTS: Increases in height standard deviation scores were 0.12 ± 0.34 and 1.62 ± 0.52 for group 1 and group 2 (P < .001). The number of acute rejection episodes was significantly different between groups (P = .04). At renal transplant, increases in height standard deviation scores were negatively correlated with mean age (r: -0.354; P = .04) and height standard deviation scores (r: -0.353; P = .04). In the multivariate model, mean age and height standard deviation scores at renal transplant remained significantly associated with increases in height standard deviation scores (P = .018; ß coefficient: -0.341, 95% CI: -0.17; -0.002; and P = .005; ß coefficient: -0.431, 95% CI: -0.519; -0.101). CONCLUSIONS: Renal transplant improves linear growth by providing moderate or accelerated growth in prepubertal children.

Novel and de novo PHEX mutations in patients with hypophosphatemic rickets.

X-linked hypophosphatemic rickets (XLH) is the most common inherited rickets. XLH is caused by inactivating mutations in the PHEX gene and is transmitted as an X-linked dominant disorder. We investigated PHEX mutation in 10 patients from 6 unrelated Turkish families by PCR-sequence analysis. Six different PHEX mutations were detected in the patients. Four of them were novel: c.1217G>A (p.C406Y) in exon 11, c.2078G>T (p.C693F) in exon 21, a splice donor site mutation in intron 13 (IVS13+1G>T), and a splice acceptor site mutation in intron 13 (IVS13-2A>G). De novo PHEX mutations were found exclusively in female patients from 4 families and inherited mutations were detected from remaining two families. The patients' phenotype was consistent with the loss of PHEX function. Literature review of 78 sporadic cases shows that de novo mutations are present in 83% female patients and female/male ratio is 5 to 1. One patient had biallilic PHEX mutations at c.1735G>A (p.G579R) whereas her mother and two siblings carried a monoallelic mutation. The clinical and laboratory findings of the patient with biallilic PHEX mutation were similar to those with monoallelic mutation. The study shows that PHEX mutation is a common cause of either familial or sporadic hypophosphatemic rickets in Turkish population. Gene dosage effect is not observed. The frequent de novo mutations found in the female patients are likely resulting from mutagenesis of X chromosome in paternal germ cells.