Maria Felicia Faienza

SOX2 Plays a Critical Role in the Pituitary, Forebrain, and Eye during Human Embryonic Development

CONTEXT Heterozygous, de novo mutations in the transcription factor SOX2 are associated with bilateral anophthalmia or severe microphthalmia and hypopituitarism. Variable additional abnormalities include defects of the corpus callosum and hippocampus. OBJECTIVE We have ascertained a further three patients with severe eye defects and pituitary abnormalities who were screened for mutations in SOX2. To provide further evidence of a direct role for SOX2 in hypothalamo-pituitary development, we have studied the expression of the gene in human embryonic tissues. RESULTS All three patients harbored heterozygous SOX2 mutations: a deletion encompassing the entire gene, an intragenic deletion (c.70_89del), and a novel nonsense mutation (p.Q61X) within the DNA binding domain that results in impaired transactivation. We also show that human SOX2 can inhibit beta-catenin-driven reporter gene expression in vitro, whereas mutant SOX2 proteins are unable to repress efficiently this activity. Furthermore, we show that SOX2 is expressed throughout the human brain, including the developing hypothalamus, as well as Rathkes pouch, the developing anterior pituitary, and the eye. CONCLUSIONS Patients with SOX2 mutations often manifest the unusual phenotype of hypogonadotropic hypogonadism, with sparing of other pituitary hormones despite anterior pituitary hypoplasia. SOX2 expression patterns in human embryonic development support a direct involvement of the protein during development of tissues affected in these individuals. Given the critical role of Wnt-signaling in the development of most of these tissues, our data suggest that a failure to repress the Wnt-beta-catenin pathway could be one of the underlying pathogenic mechanisms associated with loss-of-function mutations in SOX2.

Metabolic, inflammatory, endothelial and haemostatic markers in a group of Italian obese children and adolescents.

Childhood obesity and its related comorbidities are increasingly recognised in children, predisposing them to early cardiovascular disease and metabolic syndrome. The objective of the study was to investigate markers of metabolism, inflammation and haemostasis in a group of Italian obese children and adolescents. Fifty-nine obese and 40 non-obese subjects were recruited. Fasting glucose and insulin, total cholesterol, HDL and LDL cholesterol, triglycerides, high-sensitivity C-reactive protein (hsCRP), tumour necrosis factor alpha (TNF-α), and adiponectin were measured. Hypercoagulability was assessed by measuring the circulating levels of thrombin-antithrombin complex (TAT), D-dimer, fibrinogen, plasminogen activator inhibitor 1 (PAI-1) and von Willebrand Factor (vWF). A significant degree of insulin resistance was present in obese subjects compared with controls (p < 0.0001). The obese showed higher levels of total cholesterol, LDL cholesterol and triglycerides, and lower levels of HDL cholesterol than controls (p < 0.0001). Circulating levels of hsCRP and TNF-α were significantly higher in obese than in controls while serum adiponectin levels were significantly lower in obese than non-obese subjects (p < 0.001; p = 0.031; p < 0.0001, respectively). vWF, TAT, D-dimer, fibrinogen and PAI-1 levels were significant higher in obese subjects compared with control group (p = 0.02; p < 0.0001; p = 0.0037; p < 0.0001; p = 0.017, respectively). In conclusion, our results suggest that childhood obesity per se is associated with a proinflammatory and prothrombotic state.

SOS1 mutations in Noonan syndrome: molecular spectrum, structural insights on pathogenic effects, and genotype–phenotype correlations†

Noonan syndrome (NS) is among the most common nonchromosomal disorders affecting development and growth. NS is caused by aberrant RAS‐MAPK signaling and is genetically heterogeneous, which explains, in part, the marked clinical variability documented for this Mendelian trait. Recently, we and others identified SOS1 as a major gene underlying NS. Here, we explored further the spectrum of SOS1 mutations and their associated phenotypic features. Mutation scanning of the entire SOS1 coding sequence allowed the identification of 33 different variants deemed to be of pathological significance, including 16 novel missense changes and in‐frame indels. Various mutation clusters destabilizing or altering orientation of regions of the protein predicted to contribute structurally to the maintenance of autoinhibition were identified. Two previously unappreciated clusters predicted to enhance SOS1s recruitment to the plasma membrane, thus promoting a spatial reorientation of domains contributing to inhibition, were also recognized. Genotype–phenotype analysis confirmed our previous observations, establishing a high frequency of ectodermal anomalies and a low prevalence of cognitive impairment and reduced growth. Finally, mutation analysis performed on cohorts of individuals with nonsyndromic pulmonic stenosis, atrial septal defects, and ventricular septal defects excluded a major contribution of germline SOS1 lesions to the isolated occurrence of these cardiac anomalies. Hum Mutat 32:760–772, 2011.

Analysis of cyclin-dependent kinase inhibitor genes (CDKN2A, CDKN2B, and CDKN2C) in childhood rhabdomyosarcoma.

p16INK4A, p15INK4B, and p18 proteins are highly specific inhibitors of cyclin‐dependent serine/threonine kinase (CDK) activities required for G1‐S transition in the eukaryotic cell division cycle. Mutations, mainly homozygous deletions, of the CDKN2A (p16INK4A/MTSI) gene have been recently found in tumor cell lines and in many primary tumors. We looked for homozygous deletions of CDKN2A, CDKN2B (p15INK4B), and CDKN2C (p18) in 12 primary rhabdomyosarcoma (RMS) specimens and in five cell lines established from this cancer type. By means of polymerase chain reaction (PCR) and PCR‐single strand conformation polymorphism (PCR‐SSCP), we analyzed the presence of biallelic gene deletion or point mutation causing gene function loss. All the examined tumor cell lines (100%) and three of 12 (25%) primary tumors showed homozygous deletion of CDKN2A. Furthermore, no aberrant bands in primary tumors were detected via SSCP, suggesting the absence of mutations in the coding region. In all cases the deleted area at 9p21 also involved the CDKN2B gene. Conversely, no homozygous deletion or point mutations were detected when CDKN2C was analyzed. Our results strongly indicate that the p16INK4A (and/or p15INK4B) protein plays a key role in the development and/or progression of childhood rhabdomyosarcoma and suggest that this CDK‐inhibitor protein might control proliferation and/or differentiation of human muscle cells. Moreover, alteration of CDKN2C does not appear to be involved in the genesis of rhabdomyosarcoma. Genes Chromosom Cancer 15:217–222 (1996).

Postmenopausal Osteoporosis: The Role of Immune System Cells

In the last years, new evidences of the relationship between immune system and bone have been accumulated both in animal models and in humans affected by bone disease, such as rheumatoid arthritis, bone metastasis, periodontitis, and osteoporosis. Osteoporosis is characterized by low bone mass and microarchitectural deterioration of bone tissue with a subsequent increase in bone fragility and susceptibility to fractures. The combined effects of estrogen deprivation and raising of FSH production occurring in menopause cause a marked stimulation of bone resorption and a rapid bone loss which is central for the onset of postmenopausal osteoporosis. This review focuses on the role of immune system in postmenopausal osteoporosis and on therapeutic strategies targeting osteoimmunology pathways.

Transient neonatal diabetes mellitus is associated with a recurrent (R201H) KCNJ11 (KIR6.2) mutation.

To the Editor: Neonatal diabetes mellitus (NDM) is a rare, monogenic form of diabetes currently defined as insulinrequiring hyperglycaemia within the first 3 months of life [1]. Neonatal diabetes can be either permanent (PNDM), requiring life-long insulin treatment, or transient (TNDM), the latter usually subsiding within 12 months of onset. In some patients with TNDM a relapse of diabetes can occur during adolescence. Recently, activating mutations of KCNJ11 (previously known as KIR6.2), encoded by the KCNJ11 gene, have been found to result in the permanent form of this condition [2]. In addition, KCNJ11 mutations with a milder effect can also give rise to remitting, relapsing, or transient neonatal diabetes [3]. In this study, the genetic basis of a case of neonatal diabetes with an atypical clinical course was investigated. The proband (referred to as nd-BA/2) is now 20 years old, and is the only child born to healthy, unrelated parents. She was delivered at term with a weight of 2,300 g (10th centile). Her random plasma glucose was found to be elevated during the 2nd day of life, with values ranging from 10.0 to 16.6 mmol/l without ketonuria. During the first 5 weeks of life the child was in good general health and showed a regular increase of body weight despite high plasma glucose levels. Fasting C-peptide was detectable (149 pmol/l, reference values: 178–680) and tests for anti-beta-cell autoantibodies were negative. Insulin therapy was not begun until the age of 37 days, when due to severe hyperglycaemia (32 mmol/l), ketonuria and a failure to thrive, a daily dose of 1.1 U kg day was administered. After stabilisation, insulin treatment was progressively reduced and stopped at the age of 29 months because of good metabolic control and episodes of hypoglycaemia. Two OGTTs performed 5 and 17 months after insulin withdrawal showed that the patient had progressed from IGT to normal glucose tolerance (Table 1). HbA1c, determined once a year during the following 4 years, was always below 7% (4.1–6.3%). An OGTT performed at the age of 7 years and 7 months showed a recurrence of diabetes (Table 1), and 6 months later insulin therapy was re-established (0.8 U kg day) because of persistently high HbA1c values (11.5%). At around the same age anti-gliadin autoantibodies were detected in the absence of clinical symptoms. Coeliac disease was confirmed by biopsy and the child was put on a glutenfree diet. In November 2004, informed consent for genetic analysis was obtained from the proband. The intronless KCNJ11 gene was amplified in three overlapping fragments (B, C and D), with a primer pair previously described for B and C fragments [4] and modified for the D fragment (D forward: 5′ ccg ctg atc atc tac cat gtc 3′; D reverse: 5′ tac cac atg gtc cgt gtg tac 3′). We identified a heterozygous R201H mutation (c.602 G→A) that arose de novo in the patient. C. Colombo . F. Barbetti Laboratory of Molecular Endocrinology and Metabolism, Bambino Gesù Children’s Hospital, Scientific Institute (IRCCS), Rome, Italy

Oxidative Stress in Obesity and Metabolic Syndrome in Children and Adolescents

Background/Aims: The aim of this study was to investigate the alterations in the oxidant/antioxidant status in obese children with and without metabolic syndrome (MetS). Methods: We recruited 25 Caucasian obese children with MetS, 30 Caucasian children with simple obesity and a control group of 30 Caucasian children. We performed diacron-reactive oxygen metabolites (d-ROMs) test and biological antioxidant potential (BAP) test in order to evaluate the oxidant-antioxidant status in recruited patients. Results: d-ROM level was significantly higher in obese children with and without MetS (p = 0.005). The total antioxidant capacity (BAP level) was reduced in MetS and noMetS children compared to controls (p = 0.009). The subjects without MetS had higher d-ROMs test and lower BAP/d-ROMs ratio than subjects with MetS (although not significant). The ratio BAP/d-ROMs was higher in controls than noMetS and MetS children (p < 0.0001). d-ROM level was higher in prepubertal subjects with MetS than pubertal ones (p = 0.03). A direct correlation was found between d-ROM levels and BMI SDS (p = 0.0005), while an inverse correlation was found between BAP and BMI SDS (p = 0.004) and BAP/d-ROMs and BMI SDS (p = 0.0001). Conclusions: This result confirms that fat accumulation plays a key role in the pathogenesis of systemic oxidative stress already during pediatric age.

Osteoporosis and obesity: Role of Wnt pathway in human and murine models

Studies concerning the pathophysiological connection between obesity and osteoporosis are currently an intriguing area of research. Although the onset of these two diseases can occur in a different way, recent studies have shown that obesity and osteoporosis share common genetic and environmental factors. Despite being a risk factor for health, obesity has traditionally been considered positive to bone because of beneficial effect of mechanical loading, exerted by high body mass, on bone formation. However, contrasting studies have not achieved a clear consensus, suggesting instead that excessive fat mass derived from obesity condition may not protect against osteoporosis or, even worse, could be rather detrimental to bone. On the other hand, it is hitherto better established that, since adipocytes and osteoblasts are derived from a common mesenchymal stem cell precursor, molecules that lead to osteoblastogenesis inhibit adipogenesis and vice versa. Here we will discuss the role of the key molecules regulating adipocytes and osteoblasts differentiation, which are peroxisome proliferators activated receptor-γ and Wnts, respectively. In particular, we will focus on the role of both canonical and non-canonical Wnt signalling, involved in mesenchymal cell fate regulation. Moreover, at present there are no experimental data that relate any influence of the Wnt inhibitor Sclerostin to adipogenesis, although it is well known its role on bone metabolism. In addition, the most common pathological condition in which there is a simultaneous increase of adiposity and decrease of bone mass is menopause. Given that postmenopausal women have high Sclerostin level inversely associated with circulating estradiol level and since the sex hormone replacement therapy has proved to be effective in attenuating bone loss and reversing menopause-related obesity, we hypothesize that Sclerostin contribution in adipogenesis could be an active focus of research in the coming years.

Clinical, endocrine, and molecular findings in 17β -hydroxysteroid dehydrogenase type 3 deficiency

The 17β-hydroxysteroid dehydrogenases (17βHSD) gene family comprises different enzymes involved in the biosynthesis of active steroid hormones. The 17βHSD type 3 (17βHSD3) isoenzyme catalyzes the reductive conversion of the inactive C19-steroid, Δ4-androstenedione (Δ4- A), into the biologically active androgen, testosterone (T), in the Leydig cells of the testis. It is encoded by the 17β-hydroxysteroid dehydrogenase type 3 (HSD17B3) gene, which maps to chromosome 9q22. Mutations in the HSD17B3 gene are associated with a rare form of 46,XY disorder of sex development referred to as 17βHSD3 deficiency (or as 17-ketosteroid reductase deficiency), due to impaired testicular conversion of Δ4-A into T. 46,XY patients with 17βHSD3 deficiency are usually classified as female at birth, raised as such, but develop secondary male features at puberty. Diagnosis, and consequently early treatment, is difficult because clinical signs from birth until puberty may be mild or absent. Biochemical diagnosis of 17βHSD3 deficiency requires measurement of serum T/Δ4-A ratio after hCG stimulation test in pre-pubertal subjects, while baseline values seem to be informative in early infancy and adolescence. However, low basal T/Δ4-A ratio is not specific for 17βHSD3 deficiency, being sometimes also found in patients with other defects in T synthesis or with Leydig cells hypoplasia. Mutational analysis of the 17HSDB3 gene is useful in confirming the clinical diagnosis of 17βHSD3 deficiency. This review describes clinical findings, diagnosis, and molecular basis of this rare disease.

Neonatal hyperbilirubinemia and Gilbert's syndrome.

Abstract The role of Gilberts syndrome (GS) in neonatal hyperbilirubinemia, characterized by bilirubin levels higher than 223 μMol/L during the first seven days of life, has been investigated, evaluating the frequency of GS genotype (A(TA)7TAA polymorphism in the promoter of the gene encoding UGT1). The frequency of GS was significantly higher in the hyperbilirubinemic group, even though neither the peak of bilirubin, nor the day on which the highest value was found, differed according to genotype. The normalization of bilirubin levels was slower in neonates with GS. These results confirm the idea that GS is one of the factors contributing to neonatal hyperbilirubinemia, but that other factors play a role in determining neonatal jaundice. The slower decrease of bilirubin levels in A(TA)7TAA homozygous neonates confirms that GS is an important factor in determining a prolonged neonatal jaundice.