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Objectives Isolated ACTH deficiency (IAD) is defined as an impaired secretion of ACTH from the pituitary gland without any other anterior pituitary hormonal deficits. The idiopathic form of IAD has been described mainly in adults and is thought to be caused by an autoimmune mechanism. Case presentation Herein, we present an 11-year-old _prepubertal previously healthy boy, who suffered a severe hypoglycemic episode short after the initiation of thyroxine for autoimmune thyroiditis and was finally diagnosed with secondary adrenal failure due to idiopathic IAD, after all other etiologies were excluded, thought an extensive diagnostic work-up. Conclusions Idiopathic IAD is a rare entity of adrenal insufficiency in pediatrics that should be considered as an etiology of secondary adrenal failure in children, when clinical signs of glucocorticoid deficiency are present and other causes are excluded.

Objective The diagnosis of growth hormone deficiency (GHD) in children is not always straightforward because insulin-like growth factor 1 (IGF-I) or GH stimulation tests may not be able to discriminate GHD from constitutional delay of growth and puberty (CDGP) or other causes of short stature. Design Boys and girls (n = 429, 0.7–16 years) who attended our department for short stature participated in this study. They were followed up for an average period of 9 years. At the end of follow-up after reaching the final height, a definitive diagnosis was assigned, and all the components of ternary complex (IGF-I, IGF-binding protein-3 (IGFBP-3), acid-labile subunit (ALS), and IGF-I/IGFBP-3 ratio) were evaluated as biomarkers for the respective diagnosis. Results All the components of the ternary complex were tightly correlated with each other and were positively related to age. IGF-I, IGFBP-3, ALS, and IGF-I/IGFBP-3 ratio differed significantly between GHD and normal groups. IGF-I and ALS levels were lower in GHD compared to children with familial short stature, while IGF-I and IGF-I/IGFBP-3 ratio was significantly lower in GHD compared to children with CDGP. IGF-I and IGF-I/IGFBP-3 receiver operating curve cutoff points were unable to discriminate between GHD and normal groups or between GHD and CDGP groups. Conclusion Despite the tight correlation among all the components of the ternary complex, each one shows a statistically significant diagnosis-dependent alteration. There is a superiority of IGF-I, ALS, and IGF-I/IGFBP-3 ratio in the distinction between GHD and CDGP or between GHD and normal groups but without usable discriminating power, making auxology as the primary criterion for establishing the diagnosis.

Introduction Premature adrenarche (PA) for long time was considered a benign condition but later has been connected to various diseases in childhood and adulthood which remains controversial. Objective To investigate the effect of premature adrenarche on the metabolic phenotype, and correlate the clinical and biochemical data with the metabolic profile of children with PA. Methods Nuclear magnetic resonance (NMR)-based untargeted and targeted metabolomic approach in combination with multivariate and univariate statistical analysis applied to study the metabolic profiles of children with PA. Plasma, serum, and urine samples were collected from fifty-two children with Idiopathic PA and forty-eight age-matched controls from the division of Pediatric Endocrinology of the University Hospital of Patras were enrolled. Results Metabolomic results showed that plasma and serum glucose, myo-inositol, amino acids, a population of unsaturated lipids, and esterified cholesterol were higher and significantly different in PA children. In the metabolic profiles of children with PA and age-matched control group a gradual increase of glucose and myo-inositol levels was observed in serum and plasma, which was positively correlated their body mass index standard deviation score (BMI SDS) values respectively. Urine 1H NMR metabolic fingerprint of PA children showed positive correlation and a clustering-dependent relationship with their BMI and bone age (BA) respectively. Conclusion This study provides evidence that PA driven metabolic changes begin during the childhood and PA may has an inductive role in a BMI–driven increase of specific metabolites. Finally, urine may be considered as the best biofluid for identification of the PA metabolism as it reflects more clearly the PA metabolic fingerprint.

Type 1 diabetes mellitus (T1DM) can cause several complications, among them myopathy, which can appear even in adolescents. This is of importance, since skeletal muscle is the largest of the insulin-sensitive tissues and thus plays a significant role in glucose homeostasis. A prime regulator of skeletal muscle mass is myostatin, a protein which has a negative role in skeletal muscle development but also in glucose homeostasis, causing insulin resistance. Since myopathy is a complication of T1DM and myostatin is a fundamental regulator of skeletal muscle and is also involved in glucose homeostasis, we investigated the serum levels of myostatin in children with T1DM. We determined myostatin serum levels using ELISA in 87 children with T1DM aged 10.62 ± 3.94 years, and in 75 healthy children aged 10.46 ± 3.32 years old. Myοstatin was significantly elevated in T1DM compared to the healthy control children (23.60 ± 7.70 vs 16.74 ± 6.95 ng/ml, p

Trisomy 14 (T14) mosaicism is a rare chromosomal condition characterised by various clinical features, including developmental delay, growth impairment, and dysmorphism. Here, we report on a 12-year-old female referred for cytogenetic analysis due to short stature. Standard GTG-banding analysis on the patient’s peripheral blood revealed mosaic Τ14 in the form of an i(14)(q10) in 3% of cells. Furthermore, a small supernumerary marker chromosome (sSMC) had been detected in the first trimester of pregnancy in chorionic villus sampling. A skin biopsy in the patient revealed the presence of a metacentric sSMC in 100% of cells. Cytogenetic and FISH studies showed that it was a de novo metacentric bisatellited sSMC derived from chromosomes 14 or 22. Oligonucleotide array-CGH using skin cells revealed no copy number variations. Studies for uniparental disomy 14 by microsatellite analysis confirmed biparental inheritance. To the best of our knowledge, this is the second report of a patient with 2 abnormal cell lines involving chromosome 14 in different tissues, one with mosaic T14 in the form of i(14)(q10) and one with an sSMC derived from chromosome 14, present in blood and skin, respectively. A rare mechanism of trisomy rescue events is proposed to explain the presence of the different cell lines in the tissues examined. This case highlights the importance of providing the cytogenetics laboratory with adequate clinical data to test for low mosaicism and analyse different tissues if necessary, thus contributing to the suitable clinical management of the patient.

Significance Thyroid hormone deficiencies are the most common preventable causes of intellectual disability. We report that mutations in the thyroid hormone receptor α1 gene (THRA) that result in intellectual disability also reduce brain size. Using human THRA mutation stem cell models, we studied the impact of THRA mutations on human brain development by combining quantitative lineage analysis, gene expression analyses, and novel assays of neuroepithelium formation. We found that THRA regulates the balance between progenitor self-renewal and neurogenesis, and thus overall brain size. Importantly, these in vitro results are consistent with in vivo evidence from magnetic resonance imaging of people with these mutations, advancing our understanding of thyroid hormone action in human brain development., Mutations in the thyroid hormone receptor α 1 gene (THRA) have recently been identified as a cause of intellectual deficit in humans. Patients present with structural abnormalities including microencephaly, reduced cerebellar volume and decreased axonal density. Here, we show that directed differentiation of THRA mutant patient-derived induced pluripotent stem cells to forebrain neural progenitors is markedly reduced, but mutant progenitor cells can generate deep and upper cortical layer neurons and form functional neuronal networks. Quantitative lineage tracing shows that THRA mutation-containing progenitor cells exit the cell cycle prematurely, resulting in reduced clonal output. Using a micropatterned chip assay, we find that spatial self-organization of mutation-containing progenitor cells in vitro is impaired, consistent with down-regulated expression of cell–cell adhesion genes. These results reveal that thyroid hormone receptor α1 is required for normal neural progenitor cell proliferation in human cerebral cortical development. They also exemplify quantitative approaches for studying neurodevelopmental disorders using patient-derived cells in vitro.

Type 1 diabetes mellitus (T1DM) can cause several complications, among them myopathy, which can appear even in adolescents. This is of importance, since skeletal muscle is the largest of the insulin-sensitive tissues and thus plays a significant role in glucose homeostasis. A prime regulator of skeletal muscle mass is myostatin, a protein which has a negative role in skeletal muscle development but also in glucose homeostasis, causing insulin resistance. Since myopathy is a complication of T1DM and myostatin is a fundamental regulator of skeletal muscle and is also involved in glucose homeostasis, we investigated the serum levels of myostatin in children with T1DM.We determined myostatin serum levels using ELISA in 87 children with T1DM aged 10.62 ± 3.94 years, and in 75 healthy children aged 10.46 ± 3.32 years old.Myοstatin was significantly elevated in T1DM compared to the healthy control children (23.60 ± 7.70 vs 16.74 ± 6.95 ng/ml, p 0.0001). Myostatin was not correlated with body mass index (BMI) SD or hemoglobin A1c (HbA1c).Children with T1DM have significantly higher serum levels of myostatin compared to healthy children of the same age and BMI SD. The elevated myostatin in T1DM could reflect impaired muscle function and/or glucose metabolism, or could represent a homeostatic mechanism.

Context Isolated congenital hypoaldosteronism presents in early infancy with symptoms including vomiting, severe dehydration, salt wasting, and failure to thrive. The main causes of this rare autosomal recessive disorder is pathogenic variants of the CYP11B2 gene leading to aldosterone synthase deficiency. Objective To investigate the presence of CYP11B2 pathogenic variants in a cohort of patients with a clinical, biochemical, and hormonal profile suggestive of aldosterone synthase deficiency. Design Clinical and molecular study. Setting Tertiary academic Children’s Hospital, Center for Rare Pediatric Endocrine Diseases. Patients and Methods Sixty-two patients (56 unrelated patients and 6 siblings), with hypoaldosteronism and their parents, underwent CYP11B2 gene sequencing after its selective amplification against the highly homologous CYP11B1 gene. In silico analysis of the identified novel variants was carried out to evaluate protein stability and potential pathogenicity. Results CYP11B2 gene sequencing revealed that 62 patients carried a total of 12 different pathogenic CYP11B2 gene variants, 6 of which are novel. Importantly, 96% of the 56 patients carried the previously reported p.T185I variant either in homozygosity or in compound heterozygosity with another variant. The 6 novel variants detected were: p.M1I, p.V129M, p.R141Q, p.A165T, p.R448C, and the donor splice site variant of intron 8, c.1398 + 1G > A. Conclusion Molecular diagnosis was achieved in 62 patients with aldosterone synthase deficiency, the largest cohort thus far reported. Six novel genetic variants were identified as possibly pathogenic, extending the spectrum of reported molecular defects of the CYP11B2 gene.

Background: Vitamin-D-dependent rickets 1A (VDDR-1A) is caused by mutations of the renal CYP27B1 gene and is a rare form of rickets. Herein, we report a 20-month-old toddler who presented with inability to walk and failure to thrive. The clinical, biochemical and radiological findings were consistent with a diagnosis of rickets, specifically of the genetic type due to increased 25-OH vitamin D stores. Methods and Results: Our patient was a compound heterozygote with 2 novel mutations: c.242G>A(p.Gly81Glu) and c.1144C>G (p.Pro382Ala) in the CYP27B1 gene. Analysis of both mutations with in silico models predicted a deleterious effect on 25-OH vitamin D 1α-hydroxylase function. Interestingly, the levels of 1,25-(OH)2 vitamin D were within normal limits. Our patient was initiated on 1α-hydroxyvitamin D (alfacalcidol) and supplemental calcium. Monitoring of bone metabolism showed a normalization of all bone metabolism serum indices after 3 months of therapy and, thereafter, only alfacalcidol was given at a maintenance dose. The clinical follow-up showed a dramatic improvement in musculoskeletal activity, and the patient regained acceleration in height and weight appropriate for his age. Conclusion: This rare case report of VDDR-1A with normal levels of 1,25-(OH)2 vitamin D enhances our awareness for this type of rickets in clinical practice.

BackgroundNormal phosphate homeostasis is essential for normal linear growth. The phosphaturic fibroblast growth factor 23 (FGF23)/Klotho axis is a major regulator of phosphate homeostasis; therefore, an intact FGF23/Klotho axis is important for normal linear growth. On the other hand, GH/IGF1 axis has opposing effects on phosphate homeostasis, but the underline mechanisms remain unclear.AimThe main objective of this study was to investigate the possible interactions of FGF23 and its co-receptor Klotho, with growth hormone (GH)/IGF1 axis in the regulation of phosphate metabolism in GH-deficient children under GH treatment.MethodsWe studied 23 GH-deficient children, before and 3 months after the onset of GH treatment. Anthropometry and assessment of biochemical parameters were performed, as well as measurement of FGF23 (intact FGF23/iFGF23 and C-terminal FGF23/cFGF23) and soluble α-Klotho (sKlotho) levels.ResultsAfter 3 months on GH treatment, the elevation of serum phosphate and TmPO4/GFR (PPPPPPPPPPPPPPPConclusionThe increase in serum phosphate, which we found in GH-deficient children under GH treatment, is not associated with suppression but rather than with upregulation of the phosphaturic FGF23/Klotho axis.

Introduction Patients with type I diabetes mellitus (T1DM) have increased incidence of atherosclerosis and cardiovascular disease. Although these complications are unusual in children with T1DM, prevention, and early intervention could decrease morbidity and mortality. Osteoprotegerin (OPG), asymmetric dimethylarginine (ADMA), and Fetuin-A have been associated with increased cardiovascular risk (CVR). Increased OPG and ADMA, and decreased or increased Fetuin-A serum levels have been associated with increased CVR. Aim Because patients with T1DM have higher CVR we investigated OPG, ADMA, and Fetuin-A, in children with T1DM. Methods We determined the serum levels of OPG, receptor activator of nuclear factor-κB ligand (RANKL), ADMA, and Fetuin-A by enzyme-linked immunosorbent assay (ELISA) in 56 children with T1DM aged 12.1 ± 3.4 yr and in 46 normal control children, (C) aged 11.3 ± 3.0 yr. Results Serum OPG levels were significantly increased in patients with T1DM (3.352 ± 0.73 pmol/L) compared with C (2.75 ± 0.67 pmol/L, p

To assess whether the serum levels of anti-Müllerian hormone (AMH) are increased in girls with premature adrenarche because they are at a higher risk of developing polycystic ovary syndrome (PCOS) later in life.We measured serum levels of AMH, dehydroepiandrosterone sulfate (DHEAS), testosterone, sex hormone binding globulin, androstenedione, and 17-hyroxyprogesterone in 89 girls with premature adrenarche aged 6.98 ± 1.60 years, and in 55 prepubertal normal girls aged 6.78 ± 1.60 years.AMH was significantly higher in girls with premature adrenarche (2.95 ± 1.20 ng/mL) compared with normal prepubertal girls (2.00 ± 0.95 ng/mL; P .001), whereas their body mass index SD score was similar (P .05). DHEAS, testosterone, and androstenedione were increased in premature adrenarche, whereas sex hormone binding globulin was decreased in girls with premature adrenarche. Among the 89 girls with premature adrenarche, 33 were daughters of mothers with a positive history of PCOS, whereas the mothers of the remaining 56 girls with premature adrenarche had a negative history of PCOS. The girls with a mother with a positive history of PCOS had significantly higher AMH serum levels compared with girls with a mother with a negative history of PCOS (3.37 ± 1.72 ng/mL vs 2.70 ± 1.25 ng/mL; P .05) with no differences in testosterone, DHEAS, androstenedione, and sex hormone binding globulin. The serum concentration of AMH was only positively related to androstenedione (r = 0.538; P .0001).Girls with premature adrenarche, especially those from mothers with a history of PCOS, could have a higher risk of developing PCOS later in life because they have increased serum AMH.

Vitamin-D-dependent rickets 1A (VDDR-1A) is caused by mutations of the renal CYP27B1 gene and is a rare form of rickets. Herein, we report a 20-month-old toddler who presented with inability to walk and failure to thrive. The clinical, biochemical and radiological findings were consistent with a diagnosis of rickets, specifically of the genetic type due to increased 25-OH vitamin D stores.Our patient was a compound heterozygote with 2 novel mutations: c.242GA(p.Gly81Glu) and c.1144CG (p.Pro382Ala) in the CYP27B1 gene. Analysis of both mutations with in silico models predicted a deleterious effect on 25-OH vitamin D 1α-hydroxylase function. Interestingly, the levels of 1,25-(OH)2 vitamin D were within normal limits. Our patient was initiated on 1α-hydroxyvitamin D (alfacalcidol) and supplemental calcium. Monitoring of bone metabolism showed a normalization of all bone metabolism serum indices after 3 months of therapy and, thereafter, only alfacalcidol was given at a maintenance dose. The clinical follow-up showed a dramatic improvement in musculoskeletal activity, and the patient regained acceleration in height and weight appropriate for his age.This rare case report of VDDR-1A with normal levels of 1,25-(OH)2 vitamin D enhances our awareness for this type of rickets in clinical practice.

Patients with type I diabetes mellitus (T1DM) have increased incidence of atherosclerosis and cardiovascular disease. Although these complications are unusual in children with T1DM, prevention, and early intervention could decrease morbidity and mortality. Osteoprotegerin (OPG), asymmetric dimethylarginine (ADMA), and Fetuin-A have been associated with increased cardiovascular risk (CVR). Increased OPG and ADMA, and decreased or increased Fetuin-A serum levels have been associated with increased CVR.Because patients with T1DM have higher CVR we investigated OPG, ADMA, and Fetuin-A, in children with T1DM.We determined the serum levels of OPG, receptor activator of nuclear factor-κB ligand (RANKL), ADMA, and Fetuin-A by enzyme-linked immunosorbent assay (ELISA) in 56 children with T1DM aged 12.1 ± 3.4 yr and in 46 normal control children, (C) aged 11.3 ± 3.0 yr.Serum OPG levels were significantly increased in patients with T1DM (3.352 ± 0.73 pmol/L) compared with C (2.75 ± 0.67 pmol/L, p 0.0001) but RANKL did not change. ADMA was significantly decreased in T1DM compared with C (0.68 ± 0.13 µmol/L versus 0.82 ± 0.18 µmol/L, p 0.0001). Fetuin-A was similar in T1DM (0.551 ± 0.13 g/L) and C (0.540 ± 0.11 g/L) subjects. OPG was positively associated with glycosylated hemoglobin A1c (p 0.001) and negatively associated with BMI (p 0.01). ADMA and Fetuin-A were not associated with A1c and ADMA was only negatively associated with age (p 0.05).OPG is increased, ADMA is decreased, but RANKL and Fetuin-A are unchanged in T1DM children. Whereas increased OPG has been firmly related to increased CVR, more studies, especially longitudinal studies, are needed to delineate the role and clinical significance of decreased ADMA and if Fetuin-A has any role in T1DM.

The increase in genome scanning data, derived from clinical genetics practice, is producing a wealth of information on human sequence variability. The critical issue is to identify if a given nucleotide change results in a benign polymorphism or a disease-causing mutation. We have focused on one specific gene expression step, pre-mRNA processing, where we can functionally define the effect of nucleotide changes and in turn the patient's mutation can shed light on the basic pre mRNA splicing mechanisms. Our results show that several nucleotide changes in CFTR exon 12 induce a variable extent of exon skipping that leads to reduced levels of normal transcripts. This is the case in both natural mutations D565G and G576A (the latter having previously considered a neutral polymorphism) and several site-directed silent substitutions. We demonstrate here that this phenomenon is due to the interference with a new regulatory element that we have named composite exonic regulatory element of splicing (CERES). The effect of single nucleotide substitutions at CERES cannot be predicted by neither SR matrices nor enhancer identification. The recognition and characterization of splicing abnormalities, caused by exon sequence variations at CERES elements, may represent a frequent disease-causing mechanism that also relates to the phenotypic variability. Our results indicate that even the most benign looking polymorphism in an exon cannot be ignored as it may affect the splicing process. Hence, appropriate functional splicing assays should be included in genotype screenings to distinguish between polymorphisms and pathogenic mutations.

Cystic fibrosis (CF) mutation analysis on 437 CF patients, characterized 80 different mutations (20 so far specific to our population) accounting for 91% of CF genes and generating 103 different genotypes. Eight mutations were common [F508del (53.4%), 621+1G>T (5.7%), G542X (3.9%), N1303K (2.6%), 2789+5G>A (1.7%), 2183AA>G (1.4%), E822X (1.4%), R1158X (1%)], 12 showed frequencies between 0.5% and 1%, while the remaining (60) were very rare (1 to 3 alleles). Denaturing gradient gel electrophoresis (DGGE) screening of 12 exons (3, 4, 7, 10, 11, 13, 14b, 16, 17b 20 and 21) detected 85.5% of CF alleles. Haplotypes for eight diallelic and three microsatellite markers have been characterized for the common, a few rare and novel Greek mutations. Results of 165 prenatal diagnoses (including 49 due to bowel hyperechogenicity), testing a total of 41 different parental genotypes, are reported. One hundred and sixteen prenatal tests resulted in 22 affected, 59 heterozygous, 34 normal fetuses and one incomplete diagnosis. Of the 49 echogenic bowel fetuses, 3 were heterozygotes. Carrier screening was initiated, with emphasis on individuals and couples in high-risk groups - with a family history of CF, one partner with CF, and couples with male infertility seeking in vitro fertilization (IVF). Mutation analysis on 672 individuals (120 couples, 91 unaffected CF siblings, 283 CF family relatives and 58 general population subjects), identified a total of 176 heterozygotes and 7 couples where both partners were CF heterozygotes. Prenatal diagnosis was performed in 4 cases and 3 were counseled on the availability of a prenatal test.

Background\ud The thyroid hormone receptor α gene (THRA) transcript is alternatively spliced to generate either thyroid hormone receptor (TR)α1 or a non-hormone-binding variant protein, TRα2, the function of which is unknown. Here, we describe the first patients identified with a mutation in THRA that affects both TRα1 and TRα2, and compare them with patients who have resistance to thyroid hormone owing to a mutation affecting only TRα1, to delineate the relative roles of TRα1 and TRα2.\ud \ud Methods\ud We did clinical, biochemical, and genetic analyses of an index case and her two sons. We assessed physical and radiological features, thyroid function, physiological and biochemical markers of thyroid hormone action, and THRA sequence.\ud \ud Findings\ud The patients presented in childhood with growth failure, developmental delay, and constipation, which improved after treatment with thyroxine, despite normal concentrations of circulating thyroid hormones. They had similar clinical (macrocephaly, broad faces, skin tags, motor dyspraxia, slow speech), biochemical (subnormal ratio of free thyroxine:free tri-iodothyronine [T3], low concentration of total reverse T3, high concentration of creatine kinase, mild anaemia), and radiological (thickened calvarium) features to patients with TRα1-mediated resistance to thyroid hormone, although our patients had a heterozygous mis-sense mutation (Ala263Val) in both TRα1 and TRα2 proteins. The Ala263Val mutant TRα1 inhibited the transcriptional function of normal receptor in a dominant-negative fashion. By contrast, function of Ala263Val mutant TRα2 matched its normal counterpart. In vitro, high concentrations of T3 restored transcriptional activity of Ala263Val mutant TRα1, and reversed the dominant-negative inhibition of its normal counterpart. High concentrations of T3 restored expression of thyroid hormone-responsive target genes in patient-derived blood cells.\ud \ud Interpretation\ud TRα1 seems to be the principal functional product of the THRA gene. Thyroxine treatment alleviates hormone resistance in patients with mutations affecting this gene, possibly ameliorating the phenotype. These findings will help the diagnosis and treatment of other patients with resistance to thyroid hormone resulting from mutations in THRA.\ud \ud Funding\ud Wellcome Trust, NIHR Cambridge Biomedical Research Centre, Marie Curie Actions, Foundation for Development of Internal Medicine in Europe.

The effects of four splicing mutations and one nonsense mutation on cystic fibrosis transmembrane conductance regulator ( CFTR) gene expression were investigated by reverse transcription-polymerase chain reaction analysis of mRNA extracted from nasal epithelial cells harvested from patients harbouring the mutations. We studied four subjects with 621+3A-->G, two with 2751+2T-->A, one with 296+1G-->C, two with 1717-9T-->C-D565G and seven with E822X and compared the results with CFTR mRNA from normal subjects. Our results showed that mutations 621+3A-->G, 2751+2T-->A, and 296+1G-->C, which disrupt the 5' splice donor sites of introns 4, 14a, and 2, respectively, and 1717-9T-->C-D565G, which possibly disrupts the exonic splicing enhancer sequences of exon 12 (owing to the missense mutation in cis), lead to the production of aberrantly spliced mRNA in nasal epithelial cells. Three of the splicing mutations (621+3A-->G, 2751+2T-->A, and 296+1G-->C) result in severe deficiency of normal CFTR mRNA and severe phenotype in the patients. This information is especially useful for mutation 621+3A-->G, which is found in other populations as well, and was initially reported as a polymorphism. The complex allele 1717-9T-->C-D565G results in aberrant splicing of CFTR mRNA with production of transcripts lacking exon 12 (major product), with minor amounts of transcripts revealing joint exon 11 and 12 skipping. Nonsense mutation E822X results in a severe reduction in mRNA levels to about 6% of wild type. Patients with the mutation have a severe clinical phenotype, with both the pancreatic and the pulmonary function affected.

Dear Sir, I am Neoklis A. Georgopoulos, from Department of Obstetrics and Gynaecology, Division of Reproductive Endocrinology, University of Patras Medical School, Greece. We present an early prepubertal diagnosis of Kallmann syndrome (KS) in a child with anosmia, renal agenesis and mirror movements. KS is a congenital defect characterized by the presence of hypogona-dotropic hypogonadism and anosmia or hyposmia 1. Besides hypogonadism and anosmia, the clinical phenotype is characterized by the presence of other associated features, including, among others, mirror movements (synkinesis), cleft palate and renal agenesis/dysgenesis. KS is a genetically heterogeneous disease showing X-linked, dominant or recessive autosomal, or oligogenic inheritance. The gene responsible for an X-linked mode of inheritance was identified in the short arm of the X chromosome (Xp22.3) and named KAL1 2. Mutations within the FGFR1 and the Prok2/ProkR2 genes have been implicated in the autosomal dominant and recessive inheritance forms, respectively. Certain non-reproductive features of KS might be linked to either KAL1 or FGFR1 gene defects. The mirror movements are present in approximately 80% of males with KAL1 mutations, but only in 10% of patients with FGFR1 gene defects. The frequency of KAL1 gene defects is estimated to be around 10% of all sporadic cases, whereas in the presence of associated renal agenesis/dysgenesis the frequency is as high as 89% 3, 4. To date, no case of KS and its associated renal agenesis caused by an FGFR1 mutation has been reported. The diagnosis of hypogonadotropic hypogonadism can be made in a male infant during the first 6 months of life on the basis of activation of the hypothalamic–pituitary–gonadotropic axis 5. Later, in childhood, the axis is suppressed and the definitive diagnosis of KS is therefore delayed until late adolescence, when the lack of pubertal development and the absence of secondary sex characteristics become obvious. The lack of pubertal development can cause considerable psychological damage to the young patient. We report on a child who was admitted to the Paediatric Clinic of Patras Medical School (Rio, Greece) at the age of 12 years for the evaluation of right renal agenesis. His height was 146.6 cm (25th percentile) and his weight was 38.8 kg (50th percentile). On clinical examination by a paediatric endocrinologist, his penis size was found to be normal for a prepubertal boy (length 5 cm, diameter 3.5 cm); his right testis was palpable in the scrotum, which was 2 cm in diameter, but his left testis was not palpable. The associated clinical features included an arched palate, mirror movements and anosmia. He was the only child in the family. As a detailed family history showed no evidence of hypogonadism, anosmia/hyposmia and/or delayed puberty in any relatives, the case was considered sporadic. Serum testosterone level was 8.46 ng dL-1, and there was no response of luteinizing hormone (LH) or follicle-stimulating hormone (FSH) to a gonadotropin-releasing hormone (GnRH) (100 μg) challenge (LH: < 0.01 mIU mL-1, FSH: 0.2 mIU mL-1). A detailed magnetic resonance imaging of the brain showed the absence of the olfactory bulbs and tracts and atrophy of the olfactory gyri. On the basis of the knowledge that KAL1 gene defects are highly probable in the presence of renal agenesis, we decided to sequence the KAL1 gene for a possible early clinical and genetic diagnosis of KS in our patient. We believed that the result of the genetic test would be worth considering, along with the fact that the patient had no clinical signs of hypogonadism. Sequence analysis of the KAL1 gene identified two genetic defects in the patient. The first mutation was a 259G to A transition in exon 1, turning codon 37 encoding glutamic acid into lysine (E37K), and the second an 835A to T transversion in exon 5, turning codon 235 encoding threonine into serine (T235S) (Figure 1). Neither mutation has been described earlier, and they are not identified in the sequence analysis of KAL1 gene in 53 normal men or in the two major public Single Nudeotide Polymorphism (SNP) databases, GenBank and Ensembl. Sequence analysis was carried out for the mother, because the KAL1 gene is located on the X chromosome that is passed on from the mother to the male proband. The mother was a heterozygote for exon 1 with a normal and mutant allele, but she was a wild-type homozygote for exon 5. Therefore, the mother transmitted the mutation in exon 1 to her son. The fact that the second mutation in her genome was absent suggested that the son had a de novo mutation in exon 5 (Figure 1). Figure 1 Electrochromatograms showing the two point mutations. (A) at 259 bp of exon 1 and (B) at 835 bp of exon 5 of KAL1 gene identified in the patient with Kallmann syndrome and renal aplasia and the heterozygotous status of his mother for (C) exon 1 and homozygotous ... The protein encoded by the KAL1 gene, named anosmin-1, is a glycoprotein of the extracellular matrix of unknown function. It has an amino terminus with a cysteine-rich domain, including a whey acidic protein-like four-disulphide core motif (WAP), four fibronectin type III domains (FnIII R1–R4) and a carboxyl-terminal histidine-rich domain. The E37K mutation is localized to the amino-terminal cysteine-rich domain of anosmin-1 and involves the non-conservative replacement of an acidic amino acid by a basic amino acid. The T235S mutation lies within the first R1 FNIII of the protein near a major cell adhesion site, which contains one of the predicted heparin sulphate binding sites of anosmin. A sequence-based prediction of the effects of these amino acid substitutions using the SIFT (Sorting Intolerant From Tolerant) software (http://blocks.fhcrc.org/sift/SIFT.html) revealed that the 259G to A transition (E37K) could affect protein function (with a score of 0.04), whereas the 835A to T transversion (T235S) was predicted to be tolerable. Indeed, with the latter mutation, at the protein level, the amino acid threonine was replaced by serine at position 235. Both amino acids are polar and they differ by only one methyl group (–CH3); therefore, the change was predicted to be tolerable. In contrast, the change from glutamic acid to lysine at position 37 replaces an acidic amino acid with a basic one, which might seriously affect protein function. To our knowledge, a genetic diagnosis of KS has never been reported during childhood. As our case was sporadic, any attempt to identify the genotype would have been extremely costly and time consuming. The KAL1 gene consists of 14 exons, and all reported causative mutations are scattered within the gene without any specific clustering. The finding of a right renal agenesis in a child with coexistent anosmia made sequencing the entire coding region of the KAL1 gene worthwhile, because the possibility of the identification of a causative mutation increased from 9% to 89%. It is therefore worthwhile to apply a genetic test only in the presence of associated non-reproductive clinical phenotypes in KS. Although a normal sequence in the KAL1 gene would not exclude KS, the finding of a causative mutation would further strengthen the diagnosis. Previously, an X-linked mode of inheritance dissociation of anosmia, delayed puberty and hypogonadotropic hypogonadism was reported in members of the same family 4, 6. In this study, the association of the right renal agenesis with anosmia strengthened the clinical diagnosis of KS, and it was further supported by the identification of the causative molecular defect. A normal clinical phenotype has not been reported in a case with KAL1 gene mutation. Through this letter, we wish to emphasise the im- portance of the presence of renal dysgenesis/agenesis and mirror movements as strong indicators for the presence of KAL1 gene mutations in patients with anosmia, even in childhood. Upon a prepubertal genetic diagnosis of the syndrome, the clinician can undertake therapeutic intervention for puberty induction at the age of 14 years, offering the patient a pubertal maturation that is similar to that of his teenage peers. As the correlations between genotypes and phenotypes cannot be predicted with certainty, treatment can be discontinued and the gonadotropic axis can be re-evaluated after puberty is reached. The documentation of the X-linked mode of inheritance could reassure these patients that the disease can only be transmitted as a recessive trait in their female offspring, and prenatal diagnosis could identify the presence of the causative mutation in the KAL1 gene in the male offspring of the next generation.

Hypercalcemia is rare in children with etiology different from adults. We report an 11.5-year-old boy with developmental delay that presented with difficulty in walking and vomiting. Laboratory investigations revealed hypercalcemia (Ca 13.8 mg/dL) with low serum parathormone, normal full blood count and no circulating blasts. Osteolytic lesions were discovered on radiological survey. Bone marrow examination was consistent with the diagnosis of common B acute lymphoblastic leukemia (ALL). Hypercalcemia was successfully treated with zoledronic acid. Hypercalcemia is a rare but well-recognized complication of ALL, usually present at the time of initial diagnosis. We reviewed the literature and analyzed the clinical and laboratory data in 36 cases of childhood ALL presenting with hypercalcemia. In conclusion, hypercalcemia alone or combined with osteolytic lesions can be the only presenting symptom of ALL in children.

45,X/46,XY mosaicism is associated with a broad spectrum of phenotypes ranging from apparently normal male development to individuals with incomplete sexual differentiation and clinical signs of Turner syndrome in both males and females. The most common presentation among individuals with a 45,X/46,XY karyotype is sexual ambiguity, accounting for approximately 60% of cases, while the least common category of 45,X/46,XY patients consists of those with bilaterally descended testes, found in 11–12%. We report on two patients with an apparently normal male phenotype and 45,X/46,XY mosaicism who were diagnosed postnatally because of short stature. Both of these boys presented at the age of 15 years with short stature, minor Turner-like stigmata, normal male external genitalia and spontaneous pubertal development. One of them had coarctaction of the aorta with bicuspid aortic valve, an uncommon clinical feature in boys with mosaicism. The same patient underwent a trial of GH replacement therapy with poor response and his sperm analysis revealed azoospermia. Like our patients, most mosaic 45,X/46,XY children with bilateral scrotal testes go unrecognised at birth and throughout childhood unless they have somatic features of Turner syndrome or significant growth retardation. We recommend that boys with otherwise unexplained short stature, being short for their families, should be karyotyped routinely as is recommended in short-stature girls. In addition, boys with 45,X/46,XY mosaicism require a thorough clinical evaluation similar to that performed in girls with Turner syndrome and must be routinely followed up for their potential to respond favorably to GH treatment and for late onset abnormalities, such as infertility and gonadal tumors.

In order to investigate the incidence of cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations and unclassified variants in chronic pulmonary disease in children and adults, we studied 20 patients with asthma, 19 with disseminated bronchiectasis (DB) of unknown aetiology, and 12 patients with chronic obstructive pulmonary disease (COPD), and compared the results to 52 subjects from the general Greek population. Analysis of the whole coding region of the CFTR gene and its flanking intronic regions revealed that the proportion of CFTR mutations was 45% in asthma (P0.05), 26.3% in DB (P0.05), 16.7% in COPD (P0.05), compared to 15.4% in the general population. Seventeen different molecular defects involved in disease predisposition were identified in 16 patients. Three potentially disease-causing mutations, T388 M, M1R and V11I, are novel, found so far only in three asthma patients. The hyperactive M470 allele was found more frequently in COPD patients (frequency 70.8%, P0.01) than in the controls. The study of the TGmTnM470 V polyvariant CFTR allele revealed the presence of CFTR function-modulating haplotypes TG13/T5/M470, TG11/T5/M470, TG12/T5/V470 and TG12/T7, combined with M470 or V470, in six asthma patients, four DB patients (P0.01), and two COPD patients (P0.05). These results confirm the involvement of the CFTR gene in asthma, DB and possibly in COPD.

The intracellular localization of cystic fibrosis transmembrane conductance regulator (CFTR) in native tissues is a major issue in the study of mutation, processing, and trafficking effects in CFTR and in the evaluation of therapeutic strategies in cystic fibrosis (CF). This work evaluated the applicability of ten different antibodies (Abs) under various fixation techniques for CFTR localization in fresh-brushed nasal epithelial cells collected from CF patients homozygous for F508del and control individuals. In parallel, the same Ab panel was also tested on BHK cell lines overexpressing wild-type or F508del CFTR. The Abs MATG1061, 169, Lis1, MP-CT1, CC24-R, MAB25031, and MAB1660 gave the best detection of CFTR in the apical region (AR) of nasal tall columnar epithelial (TCE) cells. The labeling pattern of these Abs was consistent with the postulated processing defect of F508del CFTR because only a minority of CF TCE cells present CFTR in the AR. In contrast, M3A7, MM13–4, and L12B4 weakly react with the AR and stain almost exclusively a cis-Golgi-like structure in the majority of CF and non-CF airway cells. In BHK cells, all the Abs enabled distinction between wild-type CFTR localization in cell membrane from F508del CFTR, which in these cells is exclusively located in the endoplasmic reticulum.