Eleonora Ferrarini

Genetic analysis of the PAX8 gene in children with congenital hypothyroidism and dysgenetic or eutopic thyroid glands: Identification of a novel sequence variant

Objective  To analyse the coding region of PAX8 in individuals with congenital (CH) or post neonatal hypothyroidism due to dysgenetic (TD) or eutopic thyroid glands.

Identification and Functional Analysis of Novel Dual Oxidase 2 (DUOX2) Mutations in Children with Congenital or Subclinical Hypothyroidism

CONTEXT Congenital hypothyroidism (CH) associated with goiter or a gland of normal size has been linked to dual oxidase 2 (DUOX2) mutations in the presence of iodide organification defect. OBJECTIVE Thirty unrelated children with CH or subclinical hypothyroidism (SH) identified during infancy with a eutopic thyroid gland, coming from our Screening Centre for CH or referred from other regions of Italy, were studied with the perchlorate discharge test to identify organification defects. Eleven children with iodide organification defect were considered for the genetic analysis of TPO, DUOX2, and dual oxidase maturation factor 2 (DUOXA2) genes. PATIENTS Eight children with CH and three with SH and eutopic thyroid gland were included in the study. After discontinuation of therapy, a partial or complete organification defect was shown after ¹²³I scintigraphy and perchlorate test. METHODS TPO, DUOX2, and DUOXA2 genes were analyzed, and functional activity of DUOX2 variants was studied in HeLa cells. RESULTS Sequencing of the DUOX2 gene revealed a deletion S965fsX994 in three children; two were euthyroid after 1 month of L-T₄ discontinuation but developed SH after 5 and 18 months, respectively, whereas the other child had SH. One child with SH showed H678R, R701Q, and P982A substitutions, and another child with SH showed only the P982A. One child with SH showed the Y1150C mutation, and another euthyroid child showed the A728T mutation. Functional studies confirmed that S965fsX994, Y1150C, and A728T mutations were responsible for the defect in H₂O₂ production, whereas H678R, R701Q, and P982A did not alter H₂O₂ production in vitro. CONCLUSIONS Genetic analysis of the DUOX2 gene was performed in 11 children with organification defect. Two new mutations (Y1150C and A728T) and the deletion S965FsX994 were responsible for the deficit in the organification process and the phenotypes. Three polymorphisms (H678R, P982A, and R701Q) were identified.

The sodium-iodide symporter expression in placental tissue at different gestational age: an immunohistochemical study

Background  Iodide (I−) is crucial for foetal thyroid function. Foetal iodide results from maternal circulating iodide and from deiodination of iodothyronines within the placenta. The Na+/I− symporter (NIS) localized in placental cells appears to be involved in iodide exchange. Low NIS expression has been reported in trophoblast cells from the first trimester and pregnancy at term.

Identification and Functional Studies of Two New Dual-Oxidase 2 (DUOX2) Mutations in a Child with Congenital Hypothyroidism and a Eutopic Normal-Size Thyroid Gland

CONTEXT Some cases of congenital hypothyroidism (CH) are associated with a gland of normal size. OBJECTIVE To explore the cause of organification defect in one child with CH and a eutopic thyroid gland, genetic analyses of TPO, DUOX2, and DUOXA2 genes were performed. PATIENT One child with CH, a eutopic thyroid gland, and a partial organification defect was shown after (123)I scintigraphy and perchlorate test. METHODS In the child with the organification defect, TPO, DUOX2, and DUOXA2 genes were analyzed. The functional activity of the DUOX2 mutants was studied after expression in eukaryotic cells. RESULTS No TPO or DUOXA2 gene mutations were identified. Direct sequencing of the DUOX2 gene revealed a compound heterozygous genotype for S911L and C1052Y substitutions. S911L and C1052Y caused a partial defect in H(2)O(2) production after transient expression in HeLa cells. CONCLUSIONS We performed a genetic analysis in one child with CH and a eutopic thyroid gland. Two new mutations in DUOX2 gene responsible for the partial deficit in the organification process were identified.

Clinical characteristics and genetic analysis in women with premature ovarian insufficiency.

OBJECTIVE Premature ovarian insufficiency (POI) is defined as a primary ovarian defect characterized by absent menarche (primary amenorrhea) or premature depletion of ovarian follicles before the age of 40 (secondary amenorrhea) with hypergonadotropism and hypoestrogenism. METHODS We studied the clinical, biological, and genetic data related to 50 POI patients with a mean age of menopause of 29 years (94% with secondary amenorrhea, 6% with primary amenorrhea and 15% with a family history of POI). Seventeen patients were affected by endocrine autoimmune diseases, antral follicles were observed in 31 patients by ultrasonography. RESULTS Karyotype analysis did not show any abnormality of the X chromosome. No mutation in FSH receptor and GDF-9 genes was reported, while in one patient a variant of BMP-15 gene (A180T) was found. Four patients had fragile X mental retardation 1 gene (FMR1) premutation and one an intermediate sized CGG repeats of the same gene. Two patients with FMR1 premutation were sister and developed secondary amenorrhea at the age of 34 and 37 years. The other two patients presented with oligoamenorrhea at the age of 39 and 34 years. The patient harboured the intermediate sized CGG repeats developed secondary amenorrhea at the age of 33 years. CONCLUSIONS The genetic analysis performed on a cohort of patients with POI revealed that 8% had FMR1 premutation and only one patient a previously known variant of BMP-15 gene. No alteration of the karyotype and FSH receptor and GDF-9 genes was evidenced.

Gonadotrophin receptor blocking antibodies measured by the use of cell lines stably expressing human gonadotrophin receptors are not detectable in women with 46,XX premature ovarian failure

background  Premature ovarian failure (POF) is defined by cessation of ovarian function after puberty and before the age of 40. The syndrome is characterized by amenorrhoea, oestrogen deficiency and elevated levels of gonadotrophins. Autoimmunity has been proposed as a mechanism for some cases of destruction or malfunction of ovarian follicles. POF is often associated with type I and type II polyglandular autoimmune syndromes. It has also been postulated that receptors such as the LH and FSH receptors might become targets for blocking antibodies and such antibodies could be a cause of ovarian failure.

Congenital hypothyroidism caused by a novel homozygous mutation in the thyroglobulin gene

Congenital hypothyroidism (CH) due to thyroglobulin (TG) deficit is an autosomal recessive disease (OMIM #274700) characterized by hypothyroidism, goiter, low serum TG, and a negative perchlorate discharge test. The aim of this study was to perform the genetic analysis of the TG gene in two sisters born from consanguineus parents and affected by CH and low serum TG levels. The index patient and her sister were identified at neonatal screening for CH and treated with L-thyroxine (L-T4). After discontinuation of L-T4 therapy, hypothyroidism was confirmed, serum TG was undetectable, and no organification defect after 123I scintigraphy and perchlorate test was shown; thyroid ultrasound showed a eutopic gland of normal size. DNA was extracted from peripheral white blood cells of the two sisters and the father. All 48 exons of TG gene were amplified by polymerase chain reaction and subjected to direct sequencing. A novel homozygous point mutation in exon 10 of TG gene was identified in the patient and her sister. The mutation determined a stop codon at position 768 (R768X) resulting in an early truncated protein or in the complete absence of the protein. The father (euthyroid) was heterozygous carrier of the mutation. Conclusion: Genetic analysis of TG gene was performed in two sisters affected by CH. A novel point mutation of the TG gene determining a stop codon at position 768 of the protein was identified. The early truncated nonfunctioning protein or the absence of the protein due to the premature degradation of abnormal mRNA may be responsible of the observed phenotype.

Genetic analysis of the follicle stimulating hormone receptor gene in women with polycystic ovary syndrome

This study was designed to assess the relationship between mutations in the FSH receptor (FSHr) gene and polycystic ovary syndrome (PCOS) in Italian women. The study population included 50 patients with PCOS and 50 age- and body mass index (BMI)-matched controls. A complete anthropometrical, hormonal and pelvic ultrasonographic evaluation was performed in all subjects. Genomic DNA was extracted from peripheral lymphocytes and then each exon of the FSHr gene was amplified by PCR. The mutation identified was cloned and the functional properties were studied after transient expression in COS-7 cells. Direct sequencing of exons 1–10 of the FSHr gene revealed the presence of a heterozygous AAT/ATT mutation affecting the isoleucine residue at position 411, which was replaced by an asparagine, in the second transmembrane segment (I411N). This mutation was only found in one woman with PCOS and not in her parents. This mutation was not present in 50 age and BMI controls and in another 150 women not affected by PCOS. The functional study after transient expression in COS-7 cells revealed that this I411N had similar functional characteristics with respect to the wild type FSHr (wtFSHr). Genetic analyses of polymorphisms in the human FSHr gene were also performed. All 50 women with PCOS harbored the A307T polymorphic variant, 56% harbored N680S, 30% S680S and 14% N680N polymorphisms. In conclusion, the present study demonstrates that mutations of the FSHr gene are rare in Italian women. The only mutation that we found does not appear to have any pathophysiological significance in PCOS.

Presence of a putative steroidal allosteric site on glycoprotein hormone receptors.

In a previous work we found that the insecticide 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), inhibits the accumulation of cAMP as induced by the bovine thyroid stimulating hormone (bTSH) in cells transfected with the TSH receptor. In this work, we demonstrate that the DDT molecular analogues, diethylstilbestrol and quercetine, are more potent inhibitors of the TSH receptor activity than DDT itself. The notion that all these compounds interfere with nuclear estrogen receptors, as either agonists (DDT and diethylstilbestrol) or antagonists (quercetin), prompted us to test the ability of the steroid hormone 17-beta-estradiol to inhibit the TSH receptor activity. We found that estrogen exposure causes a modest but significant inhibition of the bTSH induced cAMP accumulation both in transfected CHO-TSH receptor and Fischer Rat Thyroid Low Serum 5% (FRTL-5) cells. When applied to CHO cells transfected with the luteinizing hormone receptor, 17-beta-estradiol proved capable of inhibiting the hCG induced cAMP accumulation at a concentration as low as 10nM, though the effect was not greater than 35%. The effect of 17-beta-estradiol was not estrogen receptors mediated, as co-transfection of the estrogen receptor alpha and beta subunits with LH receptor caused cAMP to increase above the level attained by the sole hCG stimulation, and not to decrease it as expected. These data suggest the presence of a steroidal-like allosteric binding site on glycoprotein hormone receptors.

Prevalence of activating thyrotropin receptor and Gsα gene mutations in paediatric thyroid toxic adenomas: A multicentric Italian study

Toxic thyroid adenomas or functioning nodules are rare in children and adolescents, representing less than 3% of all causes of hyperthyroidism. Somatic gain-of-function mutations of the TSHr gene (accession number NM_00369) have been identified as a cause of toxic thyroid adenomas and functioning nodules of toxic multinodular goitre in the adult. In patients with toxic thyroid adenomas coming from an area of iodine deficiency, activating somatic mutations in the TSHr gene have been detected in up to 80% and in the Gsa gene up to 25%, suggesting that these genetic anomalies may play a role in the pathogenesis of functioning nodules in adults. In contrast, the incidence of gain-of-function somatic mutations of the TSHr or Gsa genes in rare toxic thyroid adenoma in the paediatric population is poorly understood. In this study, we searched for somatic mutations of TSHr and Gsa genes in a group of nine children affected by toxic thyroid adenomas or functioning nodules that had undergone surgery. Nine children from five Italian paediatric Endocrinology Units who underwent surgery for toxic thyroid adenoma or functioning nodules were included in this study. At diagnosis, six patients were clinically thyrotoxic and three had normal FT4 and FT3 and low TSH serum levels. Thyroid glands were studied by clinical examination, thyroid ultrasound, scintiscan imaging using iodine-123 (I) or technetium-99 m (Tc) and histology. Thyroid autoimmunity was excluded by the absence of circulating antithyroid antibodies. Genomic DNA was extracted from formalin-fixed paraffin-embedded thyroid sections, and direct sequencing of exons 9 and 10 of the TSHr gene and exons 8 and 9 of the Gsa gene was performed. The TSHr gene mutant harbouring the single-nucleotide missense substitution was generated by site-directed mutagenesis; COS-7 cells were transiently transfected using the DEAE-Dextran technique and used for cAMP production assay, I-bTSH binding and microchip flow cytometry analysis. Two heterozygous TSHr gene point mutations were identified in genomic DNA from nodular tissues with a prevalence of two of 9 (about 22%). No alteration was identified in normal tissues or blood cells. A residue change at position 568 of the TSHr (I568F) due to the heterozygous nucleotide substitution ATC/ TTC (Fig. 1a) was revealed in one nodule. In genomic DNA from another nodule, a residue change at position 453 of the TSHr (M453T) due to the heterozygous nucleotide substitution ATG/ACG (Fig. 1a) was identified. Sequence of exons 8 and 9 of the Gsa gene was normal in each patient. The I568F mutant was functionally characterized in this study, while the functional characteristics of M453T mutation were well known. The I568F mutant was characterized by increased basal constitutive activity in terms of cAMP accumulation when compared with the