Alexandra Efthymiadou

Resistance to thyroid hormone caused by a mutation in thyroid hormone receptor (TR)α1 and TRα2: clinical, biochemical, and genetic analyses of three related patients

BACKGROUND 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. METHODS 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. FINDINGS 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. INTERPRETATION 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. FUNDING Wellcome Trust, NIHR Cambridge Biomedical Research Centre, Marie Curie Actions, Foundation for Development of Internal Medicine in Europe.

Fibroblast growth factor 23 and Klotho serum levels in healthy children

Data for fibroblast growth factor 23 (FGF23) and particularly for Klotho in healthy children are limited. We aimed to investigate the relationship between FGF23 and Klotho with age and TmP/GFR and to evaluate parameters that might affect FGF23 and Klotho. In 159 healthy children (82 boys) with a mean±SD age of 8.78±3.47years we measured FGF23 (intact FGF23/iFGF23 and C-terminal FGF23/cFGF23) and soluble aKlotho serum levels by ELISA. Mean±SD value for cFGF23, was 51.14±12.79 RU/ml whereas median (range) values for iFGF23 and Klotho were 35 (8.8, 120) pg/ml and 1945 (372, 5866) pg/ml respectively. Neither FGF23 nor Klotho were significantly associated with age. Pubertal children had higher Klotho than prepubertal (p<0.05), and girls had higher levels of cFGF23 (p<0.05) and Klotho (p<0.001) than boys. Serum phosphate and TmP/GFR were positively associated with cFGF23 (p<0.01 and p<0.001), iFGF23 (p<0.05 and p<0.001) and Klotho (p<0.05 and p<0.01). Klotho was positively correlated with IGF-I (p<0.0001) and 1,25 (OH)2 vitamin D (p<0.05). In this study we provide data on cFGF23, iFGF23, and Klotho measured simultaneously in healthy children. The positive association of serum phosphate and TmP/GFR with FGF23 and Klotho suggests that they have a counterregulatory effect on phosphate homeostasis. The strong association of Klotho with IGF-I could indicate a role of Klotho in linear growth through phosphate regulation, but further studies are required.

Osteoprotegerin, RANKL, ADMA, and Fetuin-A serum levels in children with type I diabetes mellitus

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.

45,X/46,XY mosaicism: a cause of short stature in males.

Abstract45,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.

Hypercalcemia and osteolytic lesions as presenting symptoms of acute lymphoblastic leukemia in childhood. The use of zoledronic acid and review of the literature

Abstract 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.

The effect of GH treatment on serum FGF23 and Klotho in GH-deficient children

BACKGROUND Normal 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. AIM The 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. METHODS We 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. RESULTS After 3 months on GH treatment, the elevation of serum phosphate and TmPO4/GFR (P<0.0001 and P<0.01 respectively) was accompanied by a significant increase in cFGF23 (P<0.01), iFGF23 (P<0.0001), sKlotho (P<0.0001) and IGF1 (P<0.0001). Serum phosphate and TmPO4/GFR were positively associated with iFGF23 (P<0.01 and P<0.05) and IGF1 (P<0.05 and P<0.05). iFGF23 levels were positively correlated with sKlotho (P<0.001), IGF1 (P<0.0001) and height SDS (P<0.0001), whereas sKlotho was positively associated with IGF1 (P<0.0001) and height SDS (P<0.001). CONCLUSION The 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.

Can Kallmann syndrome be occasionally diagnosed during childhood? Genetic diagnosis in a child with associated renal agenesis and mirror movements

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.

A Case of Vitamin-D-Dependent Rickets Type 1A with Normal 1,25-Dihydroxyvitamin D Caused by Two Novel Mutations of the CYP27B1 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.

Anti-Müllerian Hormone in Girls with Premature Adrenarche: The Impact of Polycystic Ovary Syndrome History in their Mothers

Objectives 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. Study design 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. Results 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). Conclusions 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.