Neonatal and Infant Reference Intervals for FSH, LH, Testosterone, Estradiol, Cortisol and DHEA-S by Electrochemiluminescence in Argentina.

BACKGROUND: Gonadotropin and steroid concentrations obtained in various laboratories cannot often be compared because of methodological differences. AIMS: to determine reference intervals for FSH, LH, T, E2, F and DHEA-S according to age and sex during the first year of life. METHODS: 1236 healthy infants (1-365 days of age) were recruited at Hospital de Niños in Córdoba, Argentina. Serum samples were analyzed using electrochemiluminescence, Cobas e601 analyzer. Reference Intervals and their confidence limits were estimated. RESULTS: Female FSH levels were higher than in males. LH and T levels were higher in males. E2 levels showed a difference between sexes after 60 days of age. F levels showed a wide variation, without differences between sexes. DHEA-S levels were higher at birth and decreased during the first year. CONCLUSION: These reference intervals may help to increase the diagnostic power for the assessment of endocrine disorders during the first year of life.

Molecular analysis of brazilian patients with combined pituitary hormone deficiency and orthotopic posterior pituitary lobe reveals eight different PROP1 alterations with three novel mutations.

BACKGROUND: Mutations in PROP1, HESX1 and LHX3 are associated with combined pituitary hormone deficiency (CPHD) and orthotopic posterior pituitary lobe (OPP). OBJECTIVE: To identify mutations in PROP1, HESX1 and LHX3 in a large cohort of patients with CPHD and OPP (35 Brazilian, two Argentinian). DESIGN AND METHODS: We studied 23 index patients with CPHD and OPP (six familial and 17 sporadic) as well as 14 relatives. PROP1 was sequenced by the Sanger method in all except one sporadic case studied using a candidate gene panel. Multiplex ligation-dependent probe amplification (MLPA) was applied to one familial case in whom PROP1 failed to amplify by PCR. In the 13 patients without PROP1 mutations, HESX1 and LHX3 were sequenced by the Sanger method. RESULTS: We identified PROP1 mutations in 10 index cases. Three mutations were novel: one affecting the initiation codon (c.1A>G) and two affecting splicing sites, c.109+1G>A and c.342+1G>C. The known mutations, c.150delA (p.Arg53Aspfs*112), c.218G>A (p.Arg73His), c.263T>C (p.Phe88Ser) and c.301_302delAG (p.Leu102Cysfs*8), were also detected. MLPA confirmed complete PROP1 deletion in one family. We did not identify HESX1 and LHX3 mutations by Sanger. CONCLUSION: PROP1 mutations are a prevalent cause of congenital CPHD with OPP, and therefore, PROP1 sequencing must be the first step of molecular investigation in patients with CPHD and OPP, especially in populations with a high frequency of PROP1 mutations. In the absence of mutations, massively parallel sequencing is a promising approach. The high prevalence and diversity of PROP1 mutations is associated with the ethnic background of this cohort.

Measurement of Serum 17α-Hydroxyprogesterone in Infants by Radioimmunoassay.

UNLABELLED: Determination of 17α-nyaroxyprogesterone (17OHP) is used for the diagnosis and monitoring of Congenital Adrenal Hyperplasia (CAH). Problems associated with the specificity of antibodies used in direct immunoassays can yield high false results. OBJECTIVES: To analyze serum levels of direct 17OHP (17OHPd) and previous extraction (17OHPe) in the neonatal period, in order to establish reference values. To relate levels of 17OHPd and 17OHPe with other androgens in CAH patients. SUBJECTS AND METHODS: Serum 17OHPd and 17OHPe were measured via RIA-DPC in 400 healthy newborns and infants (aged 2-365 days), and 100 treated CAH patients (aged 1-18 years). The extraction was performed with 3% isopropanol/heptane. The influence of age and gender was assessed by ANOVA. RESULTS: The serum levels of 17OHP were significantly correlated with chronological age, but not with gestational age, sex or birth weight. The difference between 17OHPd and 17OHPe decreased with age. The correlation index between 17OHPd and 17OHPe in CAH patients was 0.93 (p < 0.01). CONCLUSION: The present results provide 17OHP reference values for infants from birth up to one year of life. The extraction method is necessary in the neonatal period up to 6 months of life. Our data might be useful to make an early CAH diagnosis and follow-up newborns with high 17OHP levels without adrenal pathology.

Knockout mice with pituitary malformations help identify human cases of hypopituitarism.

BACKGROUND: Congenital hypopituitarism (CH) and its associated syndromes, septo-optic dysplasia (SOD) and holoprosencephaly (HPE), are midline defects that cause significant morbidity for affected people. Variants in 67 genes are associated with CH, but a vast majority of CH cases lack a genetic diagnosis. Whole exome and whole genome sequencing of CH patients identifies sequence variants in genes known to cause CH, and in new candidate genes, but many of these are variants of uncertain significance (VUS). METHODS: The International Mouse Phenotyping Consortium (IMPC) is an effort to establish gene function by knocking-out all genes in the mouse genome and generating corresponding phenotype data. We used mouse embryonic imaging data generated by the Deciphering Mechanisms of Developmental Disorders (DMDD) project to screen 209 embryonic lethal and sub-viable knockout mouse lines for pituitary malformations. RESULTS: Of the 209 knockout mouse lines, we identified 51 that have embryonic pituitary malformations. These genes not only represent new candidates for CH, but also reveal new molecular pathways not previously associated with pituitary organogenesis. We used this list of candidate genes to mine whole exome sequencing data of a cohort of patients with CH, and we identified variants in two unrelated cases for two genes, MORC2 and SETD5, with CH and other syndromic features. CONCLUSIONS: The screening and analysis of IMPC phenotyping data provide proof-of-principle that recessive lethal mouse mutants generated by the knockout mouse project are an excellent source of candidate genes for congenital hypopituitarism in children.

Exome Sequencing has a high diagnostic rate in sporadic congenital hypopituitarism and reveals novel candidate genes.

CONTEXT: The pituitary gland is key for childhood growth, puberty, and metabolism. Pituitary dysfunction is associated with a spectrum of phenotypes, from mild to severe. Congenital Hypopituitarism (CH) is the most commonly reported pediatric endocrine dysfunction with an incidence of 1:4000, yet low rates of genetic diagnosis have been reported. OBJECTIVE: We aimed to unveil the genetic etiology of CH in a large cohort of patients from Argentina. METHODS: We performed whole exome sequencing of 137 unrelated cases of CH, the largest cohort examined with this method to date. RESULTS: Of the 137 cases, 19.1% and 16% carried pathogenic or likely pathogenic variants in known and new genes, respectively, while 28.2% carried variants of uncertain significance. This high yield was achieved through the integration of broad gene panels (genes described in animal models and/or other disorders), an unbiased candidate gene screen with a new bioinformatics pipeline (including genes high loss of function intolerance), and analysis of copy number variants. Three novel findings emerged. First, the most prevalent affected gene encodes the cell adhesion factor ROBO1. Affected children had a spectrum of phenotypes, consistent with a role beyond pituitary stalk interruption syndrome. Second, we found that CHD7 mutations also produce a phenotypic spectrum, not always associated with full CHARGE syndrome. Third, we add new evidence of pathogenicity in the genes PIBF1 and TBC1D32, and report 13 novel candidate genes associated with CH (e.g. PTPN6, ARID5B). CONCLUSION: Overall, these results provide an unprecedented insight into the diverse genetic etiology of hypopituitarism.

The p.Cys1281Tyr variant in the hinge module/flap region of thyroglobulin causes intracellular transport disorder and congenital hypothyroidism.

Congenital hypothyroidism (CH) due to thyroglobulin (TG) variants causes very low serum TG levels with normal or enlarged thyroid glands, depending on the severity of the defect, and with autosomal recessive inheritance. The purpose of this study was to functionally characterize p.Cys1281Tyr variant in the TG gene in order to increase our knowledge of the molecular mechanisms associated with CH. In order to find evidence that support the hypothesis that the p.Cys1281Tyr variant would affect the TG folding were performed amino acid prediction, 3D modeling and transient expression analysis in HEK293T cells. 18 of the 21″in silico" algorithms predict a deleterious effect of the p.Cys1281Tyr variant. The full-length 3D model p.Cys1281Tyr TG showed disulfide bond cleavage between the cysteines at positions 1249 and 1281 and rearrangement of the TG structure, while transient expression analysis indicated that p.Cys1281Tyr causes retention of the protein inside the cell. Consequently, these results show that this pathogenic variant makes it impossible for TG to fulfill its function in the biosynthesis process of thyroid hormones, causing CH. In conclusion, our results confirm the pathophysiological importance of misfolding of TG as a consequence of p.Cys1281Tyr variant located in the hinge module/flap region of TG.

Congenital goitrous hypothyroidism: mutation analysis in the thyroid peroxidase gene.

BACKGROUND: Iodide organification defect (IOD) is characterized by a reduced ability of the thyroid gland to retain iodide resulting in hypothyroidism. Mutations in thyroid peroxidase (TPO) gene appear to be the most common cause of IOD and are commonly inherited in an autosomal recessive fashion. The TPO gene is located on the chromosome 2p25. It comprises 17 exons, covers approximately 150 kb of genomic DNA and codes 933 amino acids. OBJECTIVES: In this study, we characterize the clinical and molecular basis of seven patients from four unrelated families with congenital hypothyroidism (CH) because of IOD. DESIGN AND METHODS: All patients underwent clinical, biochemical and imaging evaluation. The promoter and the complete coding regions of the human TPO along with the flanking intronic regions were analysed by single-strand conformation polymorphism analysis and direct DNA sequencing. Segregation analysis of mutations was carried out, and the effect of the novel missense identified mutations was investigated by 'in silico' studies. RESULTS: All subjects had congenital and persistent primary hypothyroidism. Three novel mutations: c.796C>T [p.Q266X], c.1784G>A [p.R595K] and c.2000G>A [p.G667D] and a previously reported mutation: c.1186_1187insGGCC [p.R396fsX472] have been identified. Four patients were compound heterozygous for p.R396fsX472/p.R595K mutations, two patients were homozygous for p.R595K, and the remaining patient was a compound heterozygous for p.Q266X/p.G667D. CONCLUSIONS: Our findings confirm the genetic heterogeneity of TPO defects and the importance of the implementation of molecular studies to determinate the aetiology of the CH with dyshormonogenesis.

Association of vitamin D receptor gene Cdx2 polymorphism with bone markers in Turner syndrome patients.

BACKGROUND: Turner syndrome (TS) patients usually have low bone mineral density (BMD) and increased risk of osteoporotic fractures. We have previously demonstrated an association of bb (BsmI polymorphic site) and ff (FokI polymorphic site) vitamin D receptor (VDR) genotypes with reduced BMD in TS patients. AIM: To analyze the relationship between VDR-Cdx2 polymorphism and BMD as well as bone metabolic variables in TS patients. METHODS: Fifty-five TS patients and 59 control women were studied. VDR-Cdx2 genotypes were determined using TaqMan probes in a real time thermocycler. Lumbar and femoral BMD were determined by dual-energy X-ray absorptiometry (DEXA) and serum intact parathyroid hormone, osteocalcin and beta3-CrossLaps were determined by electrochemiluminescence. RESULTS: Patients with genotype GG had higher levels of both osteocalcin and beta-CrossLaps as compared to patients with genotype GA (p < 0.01 and p < 0.05, respectively). CONCLUSION: Patients carrying genotype GG have higher levels of bone formation and resorption markers. This indicates a more active bone turnover that could impact on their future bone mineral density.

Silent but Not Harmless: A Synonymous SLC5A5 Gene Variant Leading to Dyshormonogenic Congenital Hypothyroidism.

Background: Congenital iodide transport defect (ITD) is an uncommon cause of dyshormonogenic congenital hypothyroidism characterized by the absence of active iodide accumulation in the thyroid gland. ITD is an autosomal recessive disorder caused by loss-of-function variants in the sodium/iodide symporter (NIS)-coding SLC5A5 gene. Objective: We aimed to identify, and if so to functionally characterize, novel ITD-causing SLC5A5 gene variants in a cohort of five unrelated pediatric patients diagnosed with dyshormonogenic congenital hypothyroidism with minimal to absent 99mTc-pertechnetate accumulation in the thyroid gland. Methods: The coding region of the SLC5A5 gene was sequenced using Sanger sequencing. In silico analysis and functional in vitro characterization of a novel synonymous variant were performed. Results: Sanger sequencing revealed a novel homozygous synonymous SLC5A5 gene variant (c.1326A>C in exon 11). In silico analysis revealed that the c.1326A>C variant is potentially deleterious for NIS pre-mRNA splicing. The c.1326A>C variant was predicted to lie within a putative exonic splicing enhancer reducing the binding of splicing regulatory trans-acting protein SRSF5. Splicing minigene reporter assay revealed that c.1326A>C causes exon 11 or exon 11 and 12 skipping during NIS pre-mRNA splicing leading to the NIS pathogenic variants p.G415_P443del and p.G415Lfs*32, respectively. Significantly, the frameshift variant p.G415Lfs*32 is predicted to be subjected to degradation by nonsense-mediated decay. Conclusions: We identified the first exonic synonymous SLC5A5 gene variant causing aberrant NIS pre-mRNA splicing, thus expanding the mutational landscape of the SLC5A5 gene leading to dyshormonogenic congenital hypothyroidism.

Mutational screening of the TPO and DUOX2 genes in Argentinian children with congenital hypothyroidism due to thyroid dyshormonogenesis.

PURPOSE: Primary congenital hypothyroidism (CH) is the most common endocrine disease in children and one of the preventable causes of both cognitive and motor deficits. We present a genetic and bioinformatics investigation of rational clinical design in 17 Argentine patients suspected of CH due to thyroid dyshormonogenesis (TDH). METHODS: Next-Generation Sequencing approach was used to identify variants in Thyroid Peroxidase (TPO) and Dual Oxidase 2 (DUOX2) genes. A custom panel targeting 7 genes associated with TDH [(TPO), Iodothyrosine Deiodinase I (IYD), Solute Carrier Family 26 Member 4 (SLC26A4), Thyroglobulin (TG), DUOX2, Dual Oxidase Maturation Factor 2 (DUOXA2), Solute Carrier Family 5 Member 5 (SLC5A5)] and 4 associated with thyroid dysembryogenesis [PAX8, FOXE1, NKX2-1, Thyroid Stimulating Hormone Receptor (TSHR)] has been designed. Additionally, bioinformatic analysis and structural modeling were carried out to predict the disease-causing potential variants. RESULTS: Four novel variants have been identified, two in TPO: c.2749-2 A > C and c.2752_2753delAG, [p.Ser918Cysfs*62] and two variants in DUOX2 gene: c.425 C > G [p.Pro142Arg] and c.2695delC [p.Gln899Serfs*21]. Eighteen identified TPO, DUOX2 and IYD variants were previously described. We identified potentially pahogenic biallelic variants in TPO and DUOX2 in 7 and 2 patients, respectively. We also detected a potentially pathogenic monoallelic variant in TPO and DUOX2 in 7 and 1 patients respectively. CONCLUSIONS: 22 variants have been identified associated with TDH. All described novel mutations occur in domains important for protein structure and function, predicting the TDH phenotype.

Growth hormone treatment in children with idiopathic short stature: correlation of growth response with peripheral thyroid hormone action.

OBJECTIVE: Idiopathic short stature (ISS) describes short children with normal GH secretion. Although GH treatment increases their heights, growth response to the therapy differs among patients. Thyroid hormones (TH) are essential for longitudinal growth acting mainly through TH receptors (TR) α and ß. We have previously reported that GH treatment reduced peripheral TH action in Turner Syndrome by TR down-regulation. The aims of the study were to assess the effect of GH treatment to ISS on peripheral TH action and the correlation between thyroid status and growth response to the therapy. SUBJECTS, DESIGN AND MEASUREMENTS: Eighteen normal (control) and twenty-five ISS children were enrolled and evaluated before and after 12 months of life time (control) or 12 months of GH therapy (ISS). Fasting blood was used for serum biochemical evaluations, peripheral blood mononuclear cells for TR mRNA determination by QRT-PCR and growth parameters by standard methods. RESULTS: GH treatment modified neither TR mRNA levels nor serum markers of TH action in ISS evaluated as a whole group. However, the individual change in TRß mRNA levels correlated to the change in sex hormone-binding globulin (SHBG) levels after GH therapy. The growth response to GH correlated positively with the change in TRα mRNA level and negatively with that in TRß mRNA, TSH and SHBG levels. The change in each TR mRNA isoform after GH treatment correlated negatively with its own basal level. CONCLUSIONS: GH therapy induced individual changes in TR expression in ISS that correlated with their growth response. The basal TR mRNA level could predetermine the change in TR expression and therefore the sensitivity to GH treatment.

Vitamin D receptor genotypes are associated with bone mass in patients with Turner syndrome.

BACKGROUND: Turner syndrome (TS) patients present low bone mineral density (BMD) and increased fracture risk, probably due to a genetic defect aggravated by hormonal deficiency. AIM: To study the relationship between vitamin D receptor (VDR) gene polymorphisms and BMD and bone parameters in TS patients. METHODS: DNA from 65 TS patients and 110 controls was amplified by PCR and digested with FokI, BsmI and ApaI restrictases. Lumbar and femoral BMD were determined by DEXA and serum intact parathyroid hormone, osteocalcin and beta-CrossLaps by electrochemiluminescence. RESULTS: Genotype distribution within the ApaI site was different in both groups: genotype Aa was more abundant in TS (63.8% vs. 41.3%; p<0.01), whereas AA predominated in controls (33.9% vs. 15.5%; p<0.01). Patients carrying genotype bb (BsmI) or ff (FokI) had lower BMD than those with other genotypes (p<0.01 and p<0.05, respectively). CONCLUSION: BsmI and FokI polymorphic sites of VDR could be genetic determinants of BMD in TS patients.

Comparative analysis of clinical, biochemical and genetic aspects associated with bone mineral density in small for gestational age children.

Clinical, biochemical and genetic analysis related to bone mineral density (BMD) were carried out in children born small for gestational age (SGA) that failed to achieve postnatal catch-up growth (CUG), SGA children that completed CUG and adequate for gestational age (AGA) children. Serum IGF-I, IGF-II, IGF binding protein-3 and acid-labile subunit were lower in the SGA-CUG children as compared with the other groups. Frequencies of polymorphic variants of vitamin D receptor, estrogen receptor and collagen genes were similar among groups. The genotype 194-192 of the IGF-I gene was higher in the SGA-CUG and 196-192 was higher in the SGA+CUG group. In the SGA-CUG group, the genotype SS of the COLIA1 gene was associated with lower BMD. Therefore, IGF system and COLIA1 polymorphism distinguish prepubertal SGA-CUG children from the SGA+CUG children of the same age. Furthermore, COLIA1 polymorphism could be useful to predict osteopenia in SGA-CUG children.

A Novel SLC5A5 Variant Reveals the Crucial Role of Kinesin Light Chain 2 in Thyroid Hormonogenesis.

CONTEXT: Iodide transport defect (ITD) (Online Mendelian Inheritance in Man No. 274400) is an uncommon cause of dyshormonogenic congenital hypothyroidism due to loss-of-function variants in the SLC5A5 gene, which encodes the sodium/iodide symporter (NIS), causing deficient iodide accumulation in thyroid follicular cells. OBJECTIVE: This work aims to determine the molecular basis of a patient's ITD clinical phenotype. METHODS: The propositus was diagnosed with dyshormonogenic congenital hypothyroidism with minimal 99mTc-pertechnetate accumulation in a eutopic thyroid gland. The propositus SLC5A5 gene was sequenced. Functional in vitro characterization of the novel NIS variant was performed. RESULTS: Sanger sequencing revealed a novel homozygous missense p.G561E NIS variant. Mechanistically, the G561E substitution reduces iodide uptake, because targeting of G561E NIS to the plasma membrane is reduced. Biochemical analyses revealed that G561E impairs the recognition of an adjacent tryptophan-acidic motif by the kinesin-1 subunit kinesin light chain 2 (KLC2), interfering with NIS maturation beyond the endoplasmic reticulum, and reducing iodide accumulation. Structural bioinformatic analysis suggests that G561E shifts the equilibrium of the unstructured tryptophan-acidic motif toward a more structured conformation unrecognizable to KLC2. Consistently, knockdown of Klc2 causes defective NIS maturation and consequently decreases iodide accumulation in rat thyroid cells. Morpholino knockdown of klc2 reduces thyroid hormone synthesis in zebrafish larvae leading to a hypothyroid state as revealed by expression profiling of key genes related to the hypothalamic-pituitary-thyroid axis. CONCLUSION: We report a novel NIS pathogenic variant associated with dyshormonogenic congenital hypothyroidism. Detailed molecular characterization of G561E NIS uncovered the significance of KLC2 in thyroid physiology.

Serum levels of adiponectin and leptin in children born small for gestational age: relation to insulin sensitivity parameters.

Children born small for gestational age (SGA) are prone to developing obesity, insulin resistance and type 2 diabetes. Adiponectin and leptin are adipocytokines associated with insulin sensitivity parameters. We aimed to relate serum adiponectin and leptin levels with insulin sensitivity parameters in prepuberal SGA children with and without catch-up growth (SGA+CUG; SGA-CUG, respectively) and to analyze the usefulness of these adipocytokines as early markers of insulin resistance. We analysed adiponectin, proinsulin, leptin, growth factors, insulin, HOMA IR and HOMA beta(cell) in 23 SGA+CUG, 26 SGA-CUG children compared with 48 prepuberal appropiate for gestational age (AGA). SGA children had adiponectin levels comparable to AGA children. Leptin levels were different between sexes, showed to be higher in SGA+CUG group (p=0.040) and these were significantly correlated with insulin sensitivity parameters. These results suggest leptin resistance as an adaptive mechanism to increase energy balance, but an altered functional response of adipocytes cannot be discarded.

Genetic diagnosis of congenital hypopituitarism by a target gene panel: novel pathogenic variants in GLI2, OTX2 and GHRHR.

AIM: Congenital hypopituitarism has an incidence of 1:3500-10,000 births and is defined by the impaired production of pituitary hormones. Early diagnosis has an impact on management and genetic counselling. The clinical and genetic heterogeneity of hypopituitarism poses difficulties to select the order of genes to analyse. The objective of our study is to screen hypopituitarism genes (candidate and previously related genes) simultaneously using a target gene panel in patients with congenital hypopituitarism. METHODS: Screening of 117 subjects with congenital hypopituitarism for pathogenic variants in 26 genes associated with congenital hypopituitarism by massively parallel sequencing using a customized target gene panel. RESULTS: We found three novel pathogenic variants in OTX2 c.295C>T:p.Gln99*, GLI2 c.1681G>T:p.Glu561* and GHRHR c.820_821insC:p.Asp274Alafs*113, and the previously reported variants in GHRHR c.57+1G>A and PROP1 [c.301_302delAG];[c.109+1G>A]. CONCLUSIONS: Our results indicate that a custom-designed panel is an efficient method to screen simultaneously variants of biological and clinical relevance for congenital GH deficiency. A genetic diagnosis was possible in 5 out of 117 (4%) patients of our cohort. We identified three novel pathogenic variants in GHRHR, OTX2 and GLI2 expanding the spectrum of variants associated with congenital hypopituitarism.

Molecular analysis of thyroglobulin mutations found in patients with goiter and hypothyroidism.

Thyroid dyshormonogenesis due to thyroglobulin (TG) gene mutations have an estimated incidence of approximately 1 in 100,000 newborns. The clinical spectrum ranges from euthyroid to mild or severe hypothyroidism. Up to now, one hundred seventeen deleterious mutations in the TG gene have been identified and characterized. The purpose of the present study was to identify and characterize new mutations in the TG gene. We report eight patients from seven unrelated families with goiter, hypothyroidism and low levels of serum TG. All patients underwent clinical, biochemical and image evaluation. Sequencing of DNA, genotyping, as well as bioinformatics analysis were performed. Molecular analyses revealed three novel inactivating TG mutations: c.5560G>T [p.E1835*], c.7084G>C [p.A2343P] and c.7093T>C [p.W2346R], and four previously reported mutations: c.378C>A [p.Y107*], c.886C>T [p.R277*], c.1351C>T [p.R432*] and c.7007G>A [p.R2317Q]. Two patients carried homozygous mutations (p.R277*/p.R277*, p.W2346R/p.W2346R), four were compound heterozygous mutations (p.Y107*/p.R277* (two unrelated patients), p.R432*/p.A2343P, p.Y107*/p.R2317Q) and two siblings from another family had a single p.E1835* mutated allele. Additionally, we include the analysis of 48 patients from 31 unrelated families with TG mutations identified in our present and previous studies. Our observation shows that mutations in both TG alleles were found in 27 families (9 as homozygote and 18 as heterozygote compound), whereas in the remaining four families only one mutated allele was detected. The majority of the detected mutations occur in exons 4, 7, 38 and 40. 28 different mutations were identified, 33 of the 96 TG alleles encoded the change p.R277*. In conclusion, our results confirm the genetic heterogeneity of TG defects and the pathophysiological importance of the predicted TG misfolding and therefore thyroid hormone formation as a consequence of truncated TG proteins and/or missense mutations located within its ACHE-like domain.

Thyroglobulin reference values in a pediatric infant population.

Few reports have addressed normal serum thyroglobulin (Tg) values in newborns and infants. In the present study, serum Tg was measured in 228 normal children (110 females and 118 males) aged from 3 to 180 days of life, all presenting normal age-related serum thyrotropin (thyroid-stimulating hormone [TSH]) values and negative anti-Tg and antithyroperoxidase antibodies. Serum Tg was measured by Radioimmunoassay (RIA) (two methods) and Immunometricassay (IMA) (three methods). Mean Tg values measured by the five methods exhibited among-method biases, although a significant positive correlation was observed. Serum Tg levels measured by the five methods showed a correlation with age, but not with TSH or gender. During the first days of life, relatively high mean Tg levels were observed, which progressively decreased until they reached a plateau. Therefore, with the aim of establishing reference values, the population was separated into two age groups: from 3 to 15 days of life (group A) and from 16 to 180 days of life (group B). Mean Tg concentration in group A was statistically higher than in group B. Tg centile distributions were calculated with the aim of establishing the normal levels of serum Tg for each method. We conclude that for a correct interpretation of serum Tg levels, the age and the methods used should be considered.

Compound heterozygous DUOX2 gene mutations (c.2335-1G>C/c.3264_3267delCAGC) associated with congenital hypothyroidism. Characterization of complex cryptic splice sites by minigene analysis.

Iodide Organification defects (IOD) represent 10% of cases of congenital hypothyroidism (CH) being the main genes affected that of TPO (thyroid peroxidase) and DUOX2 (dual oxidasa 2). From a patient with clinical and biochemical criteria suggestive with CH associated with IOD, TPO and DUOX2 genes were analyzed by means of PCR-Single Strand Conformation Polymorphism analysis and sequencing. A novel heterozygous compound to the mutations c.2335-1G>C (paternal mutation, intron 17) and c.3264_3267delCAGC (maternal mutation, exon 24) was identified in the DUOX2 gene. Ex-vivo splicing assays and subsequent RT-PCR and sequencing analyses were performed on mRNA isolated from the HeLa cells transfected with wild-type and mutant pSPL3 expression vectors. The wild-type and c.2335-1G>C mutant alleles result in the complete inclusion or exclusion of exon 18, or in the activation of an exonic cryptic 5' ss with the consequent deletion of 169 bp at the end of this exon. However, we observed only a band of the expected size in normal thyroid tissue by RT-PCR. Additionally, the c.2335-1G>C mutation activates an unusual cryptic donor splice site in intron 17, located at position -14 of the authentic intron 17/exon 18 junction site, with an insertion of the last 14 nucleotides of the intron 17 in mutant transcripts with complete and partial inclusion of exon 18. The theoretical consequences of splice site mutation, predicted with the bioinformatics NNSplice, Fsplice, SPL, SPLM and MaxEntScan programs were investigated and evaluated in relation with the experimental evidence. These analyses confirm that c.2335-1G>C mutant allele would result in the abolition of the authentic splice acceptor site. The results suggest the coexistence in our patient of four putative truncated proteins of 786, 805, 806 and 1105 amino acids, with conservation of peroxidase-like domain and loss of gp91(phox)/NOX2-like domain. In conclusion a novel heterozygous compound was identified being responsible of IOD. Cryptic splicing sites have been characterized in DUOX2 gene for the first time. The use of molecular biology techniques is a valuable tool for understanding the molecular pathophysiology of this type of thyroid defects.

Five new cases of 46,XX aromatase deficiency: clinical follow-up from birth to puberty, a novel mutation, and a founder effect.

CONTEXT: Aromatase is the key enzyme for estrogen biosynthesis and is encoded by the CYP19A1 gene. Since 1991, several molecular CYP19A1 gene alterations associated with aromatase deficiency have been described in both sexes. OBJECTIVE: The objective of the study was to detect CYP19A1 mutations in five aromatase-deficient 46,XX patients, to describe the clinical follow-up from birth to puberty and to perform haplotype analysis associated with the high-frequency c.628G>A splice mutation in Argentinean patients. DESIGN: The design of the study was the sequencing of the coding and flanking intronic regions of the CYP19A1 gene in all patients and parents. Haplotype analysis of patients carrying the c.628G>A mutation was also performed. PATIENTS: Clinical and biochemical findings in five new cases and one previously reported female aromatase-deficient patient (46,XX) are described. All patients presented with ambiguous genitalia at birth. Congenital adrenal hyperplasia due to 21-hydroxylase deficiency as well as other steroidogenic defects were ruled out. RESULTS: Phenotypic variability among the affected patients was found during follow-up. Direct sequencing of the CYP19A1 gene from genomic DNA revealed one novel mutation (c.574C>T) in two patients. In silico analysis predicted the c.574C>T mutation to be probably damaging. Four of six nonrelated patients presented with the c.628G>A splice mutation. Haplotype analysis showed that the c.628G>A splice mutation is associated with the same haplotype in our population. CONCLUSIONS: Increased knowledge on phenotypical variability found in female aromatase-deficient patients is useful to improve the detection rate in this disorder. In our population, a genetic founder defect has probably contributed to an increase in the incidence of the c.628G>A splice mutation.