High risk of congenital hypothyroidism in multiple pregnancies.

CONTEXT: In Italy, the surveillance of congenital hypothyroidism (CH) is performed by the Italian National Registry of Infants with CH (INRICH). Up to now, about 3600 infants with CH are recorded in the INRICH, and a high number of twins are included. OBJECTIVE: Our objective was to estimate the risk of CH in multiple and single deliveries and to compare neonatal features of CH twins with twins from the general population. DESIGN: The Italian population of CH infants recorded in the INRICH from 1989-2000 was investigated. RESULTS: A more than 3-fold higher frequency of twins was found in the CH population than in the general population, and for the first time, it was possible to estimate the CH incidence in multiple (10.1 in 10,000) and single deliveries (3.2 in 10,000 live births). Significantly higher frequencies of in situ gland as well as lower TSH mean level at screening were found in twin than in singleton CH babies. The concordance rate for permanent CH was very low (4.3%) and due to only three concordant couples. However, a high recurrence risk for CH was estimated in siblings of affected babies recorded in the INRICH, including twins considered as siblings. CONCLUSIONS: The high CH incidence observed in twins is worthy of interest for the high number of induced pregnancies in Italy as well as in other Western countries. Moreover, the low concordance rate for CH among twins together with a high recurrence risk for the disease among siblings indicates that environmental risk factors may act as a trigger on a susceptible genetic background in the etiology of the disease.

Activating PTPN11 mutations play a minor role in pediatric and adult solid tumors.

The PTPN11 gene encodes SHP-2, a widely expressed cytoplasmic protein tyrosine phosphatase functioning as a signaling transducer. Germ-line PTPN11 mutations cause Noonan syndrome (NS), a developmental disorder characterized by an increased risk of malignancies. Recently, a novel class of activating mutations in PTPN11 has been documented as a somatic event in a heterogeneous group of leukemias. Because of the relatively higher prevalence of certain solid tumors in children with NS and the positive modulatory function of SHP-2 in RAS signaling, a wider role for activating PTPN11 mutations in cancer has been hypothesized. Here, we screened a number of solid tumors, including those documented in NS or in which deregulated RAS signaling occurs at significant frequency, for PTPN11 mutations. No disease-associated mutation was identified in rhabdomyosarcoma (n = 13), neuroblastoma (n = 32), melanoma (n = 50), thyroid (n = 85), and colon (n = 48) tumors; a novel missense change, promoting an increased basal phosphatase activity of SHP-2, was observed in one glioma specimen. Our data document that deregulated SHP-2 function does not represent a major molecular event in pediatric and adult tumors, further supporting our previous evidence indicating that the oncogenic role of PTPN11 mutations is cell-context specific.

Missense mutation in the transcription factor NKX2-5: a novel molecular event in the pathogenesis of thyroid dysgenesis.

CONTEXT: Congenital hypothyroidism (CH) is a common endocrine disorder with an incidence of 1:3000-4000 at birth. In 80-85% of cases, CH is caused by defects in thyroid organogenesis, resulting in absent, ectopically located, and/or severely reduced gland [thyroid dysgenesis (TD)]. Mutations in genes controlling thyroid development have demonstrated that in a few cases, TD is a Mendelian trait. However, accumulating evidence supports the view that the genetics of TD are complex, possibly with a polygenic/multifactorial basis. A higher prevalence of congenital heart disease has been documented in children with CH than in the general population. Such an association suggests a possible pathogenic role of genes involved in both heart and thyroid development. NKX2-5 encodes a homeodomain-containing transcription factor with a major role in heart development, and mutations affecting this gene have been reported in individuals with congenital heart disease. OBJECTIVE: In the present work we investigated the possible involvement of NKX2-5 mutations in TD. RESULTS: Our results indicate that Nkx2-5(-/-) embryos exhibit thyroid bud hypoplasia, providing evidence that NKX2-5 plays a role in thyroid organogenesis and that NKX2-5 mutations contribute to TD. NKX2-5 mutational screening in 241 patients with TD allowed the identification of three heterozygous missense changes (R25C, A119S, and R161P) in four patients with TD. Functional characterization of the three mutations demonstrated reduced DNA binding and/or transactivation properties, with a dominant-negative effect on wild-type NKX2-5. CONCLUSION: Our results suggest a previously unknown role of NKX2-5 in the pathogenesis of TD.

Diversity and functional consequences of germline and somatic PTPN11 mutations in human disease.

Germline mutations in PTPN11, the gene encoding the protein tyrosine phosphatase SHP-2, cause Noonan syndrome (NS) and the clinically related LEOPARD syndrome (LS), whereas somatic mutations in the same gene contribute to leukemogenesis. On the basis of our previously gathered genetic and biochemical data, we proposed a model that splits NS- and leukemia-associated PTPN11 mutations into two major classes of activating lesions with differential perturbing effects on development and hematopoiesis. To test this model, we investigated further the diversity of germline and somatic PTPN11 mutations, delineated the association of those mutations with disease, characterized biochemically a panel of mutant SHP-2 proteins recurring in NS, LS, and leukemia, and performed molecular dynamics simulations to determine the structural effects of selected mutations. Our results document a strict correlation between the identity of the lesion and disease and demonstrate that NS-causative mutations have less potency for promoting SHP-2 gain of function than do leukemia-associated ones. Furthermore, we show that the recurrent LS-causing Y279C and T468M amino acid substitutions engender loss of SHP-2 catalytic activity, identifying a previously unrecognized behavior for this class of missense PTPN11 mutations.

Risk factors for congenital hypothyroidism: results of a population case-control study (1997-2003).

OBJECTIVE: To identify risk factors for permanent and transient congenital hypothyroidism (CH). DESIGN: A population-based case-control study was carried out by using the network created in Italy for the National Register of Infants with CH. METHODS: Four controls were enrolled for each new CH infant; 173 cases and 690 controls were enrolled in 4 years. In order to distinguish among risk factors for permanent and transient CH, diagnosis was re-evaluated 3 years after enrollment when there was a suspicion of transient CH being present. Familial, maternal, neonatal and environmental influences were investigated. RESULTS: An increased risk for permanent CH was detected in twins by a multivariate analysis (odds ratio (OR) = 12.2, 95% confidence interval (CI): 2.4-62.3). A statistically significant association with additional birth defects, female gender and gestational age >40 weeks was also confirmed. Although not significant, an increased risk of CH was observed among infants with a family history of thyroid diseases among parents (OR = 1.9, 95% CI: 0.7-5.2). Maternal diabetes was also found to be slightly associated with permanent CH (OR = 15.7, 95% CI: 0.9-523) in infants who were large for gestational age. With regard to transient CH, intrauterine growth retardation and preterm delivery were independent risk factors for this form of CH. CONCLUSION: This study showed that many risk factors contribute to the aetiology of CH. In particular, our results suggested a multifactorial origin of CH in which genetic and environmental factors play a role in the development of the disease.

Development of age-dependent glomerular lesions in galectin-3/AGE-receptor-3 knockout mice.

Aging is characterized by renal functional and structural abnormalities resembling those observed in diabetes. These changes have been related to the progressive accumulation of advanced glycation end-products (AGEs) and cumulative oxidative stress occurring in both conditions. We previously reported that galectin-3 ablation is associated with increased susceptibility to diabetes- and AGE-induced glomerulopathy, thus indicating a protective role of galectin-3 as an AGE receptor. To investigate the role of the AGE/AGE receptor pathway in the pathogenesis of age-related renal disease, we evaluated the development of glomerular lesions in aging galectin-3 knockout (KO) vs. wild-type (WT) mice and their relation to the increased AGE levels and oxidative stress characterizing the aging process. KO mice showed significantly more pronounced age-dependent increases in proteinuria, albuminuria, glomerular sclerosis, and glomerular and mesangial areas, starting at 18 mo, as well as renal extracellular matrix mRNA and protein expression, starting at 12 mo vs. age-matched WT mice. Circulating and renal AGEs, plasma isoprostane 8-epi-PGF2alpha levels, glomerular content of the glycoxidation and lipoxidation products N(epsilon)-carboxymethyllysine and 4-hydroxy-2-nonenal, and renal nuclear factor-kappaB activity also increased more markedly with age in KO than WT mice. AGE levels correlated significantly with renal functional and structural parameters. These data indicate that aging galectin-3 KO mice develop more pronounced changes in renal function and structure than coeval WT mice, in parallel with a more marked degree of AGE accumulation, oxidative stress, and associated low-grade inflammation, thus supporting the concept that the AGE/AGE receptor pathway is implicated in age-related renal disease.

Somatic PTPN11 mutations in childhood acute myeloid leukaemia.

Somatic mutations in PTPN11, the gene encoding the transducer SHP-2, have emerged as a novel class of lesions that upregulate RAS signalling and contribute to leukaemogenesis. In a recent study of 69 children and adolescents with de novo acute myeloid leukaemia (AML), we documented a non-random distribution of PTPN11 mutations among French-American-British (FAB) subtypes. Lesions were restricted to FAB-M5 cases, where they were relatively common (four of 12 cases). Here, we report on the results of a molecular screening performed on 181 additional unselected patients, enrolled in participating institutions of the Associazione Italiana Ematologia Oncologia Pediatrica-AML Study Group, to provide a more accurate picture of the prevalence, spectrum and distribution of PTPN11 mutations in childhood AML and to investigate their clinical relevance. We concluded that PTPN11 defects do not represent a frequent event in this heterogeneous group of malignancies (4.4%), although they recur in a considerable percentage of patients with FAB-M5 (18%). PTPN11 lesions rarely occur in other subtypes. Within the FAB-M5 group no clear association of PTPN11 mutations with any clinical variable was evident. Nearly two third of the patients with this subtype were found to harbour an activating mutation in PTPN11, NRAS, KRAS2 or FLT3.

Development of diabetic nephropathy in the Milan normotensive strain, but not in the Milan hypertensive strain: possible permissive role of hemodynamics.

BACKGROUND: Rats of the Milan normotensive strain develop spontaneous glomerulosclerosis, whereas those of the Milan hypertensive strain are resistant to renal disease, possibly due to intrarenal artery hypertrophy protecting from systemic hypertension. To assess the role of hemodynamic versus metabolic factors in diabetic nephropathy, we investigated whether streptozotocin-induced diabetes accelerates glomerulosclerosis in Milan normotensive and/or removes (the hemodynamic) protection in Milan hypertensive rats by reducing preglomerular vascular resistance. METHODS: Diabetic and nondiabetic Milan normotensive, hypertensive, and progenitor Wistar rats were followed for 6 months for the assessment of renal function and structure. RESULTS: Proteinuria increased in nondiabetic and diabetic normotensive and, to a lesser extent, in diabetic Wistar, but not hypertensive rats. Serum creatinine increased and creatinine clearance decreased in nondiabetic and diabetic normotensive rats at 6 months. At 1.5 months, diabetic normotensive, but not hypertensive rats showed increased glomerular filtration rate and filtration fraction, suggesting glomerular hypertension. Diabetic nephropathy was detected in diabetic normotensive and Wistar, but not hypertensive rats. Glomerular extracellular matrix and TGF-beta mRNA levels increased with diabetes (and age) in normotensive, but not hypertensive rats. Arterioles and interlobular arteries showed increased media thickness in hypertensive versus normotensive rats, with diabetes reducing it only in the normotensive. CONCLUSION: These data show that Milan hypertensive rats are not susceptible to diabetic nephropathy, at variance with glomerulosclerosis-prone Milan normotensive rats, thus indicating the importance of genetic background. Our study suggests that the nature of this (genetic) protection might be hemodynamic, with intrarenal artery hypertrophy preventing diabetes-induced loss of autoregulation.

Acquired PTPN11 mutations occur rarely in adult patients with myelodysplastic syndromes and chronic myelomonocytic leukemia.

Myelodysplastic syndromes (MDS) are comprised of a heterogeneous group of stem cell disorders characterized by ineffective hematopoiesis and susceptibility to transform to acute myeloid leukemia. The molecular pathways underlying disease initiation and evolution are still largely unknown. We recently demonstrated that acquired mutations in PTPN11 are a major event in JMML and occur with variable prevalence in children with other hematologic malignancies, including MDS. Here, we investigated contribution of PTPN11 mutations to adult MDS and CMML pathogenesis. Our results indicate that PTPN11 lesions might play a role in adult MDS/CMML pathogenesis but do not represent a major molecular event.

Galectin-3/AGE-receptor 3 knockout mice show accelerated AGE-induced glomerular injury: evidence for a protective role of galectin-3 as an AGE receptor.

We previously showed that mice lacking galectin-3/AGE-receptor 3 develop accelerated diabetic glomerulopathy. To further investigate the role of galectin-3/AGE-receptor function in the pathogenesis of diabetic renal disease, galectin-3 knockout (KO) and coeval wild-type (WT) mice were injected for 3 months with 30 microg/day of N(epsilon)-carboxymethyllysine (CML)-modified or unmodified mouse serum albumin (MSA). Despite receiving equal doses of CML, KO had higher circulating and renal AGE levels and showed more marked renal functional and structural changes than WT mice, with significantly higher proteinuria, albuminuria, glomerular, and mesangial area and glomerular sclerosis index. Renal 4-hydroxy-2-nonenal content and NFkappaB activation were also more pronounced in KO-CML vs. WT-CML. Kidney mRNA levels of fibronectin, laminin, collagen IV, and TGF-beta were up-regulated, whereas those of matrix metalloproteinase-2 and -14 were down-regulated, again more markedly in KO-CML than WT-CML mice. Basal and CML-induced RAGE and 80K-H mRNA levels were higher in KO vs. WT mice. MSA injection did not produce any significant effect in both genotypes. The association of galectin-3 ablation with enhanced susceptibility to AGE-induced renal disease, increased AGE levels and signaling, and altered AGE-receptor pattern indicates that galectin-3 is operating in vivo as an AGE receptor to afford protection toward AGE-dependent tissue injury.

Paternal germline origin and sex-ratio distortion in transmission of PTPN11 mutations in Noonan syndrome.

Germline mutations in PTPN11--the gene encoding the nonreceptor protein tyrosine phosphatase SHP-2--represent a major cause of Noonan syndrome (NS), a developmental disorder characterized by short stature and facial dysmorphism, as well as skeletal, hematologic, and congenital heart defects. Like many autosomal dominant disorders, a significant percentage of NS cases appear to arise from de novo mutations. Here, we investigated the parental origin of de novo PTPN11 lesions and explored the effect of paternal age in NS. By analyzing intronic portions that flank the exonic PTPN11 lesions in 49 sporadic NS cases, we traced the parental origin of mutations in 14 families. Our results showed that all mutations were inherited from the father, despite the fact that no substitution affected a CpG dinucleotide. We also report that advanced paternal age was observed among cohorts of sporadic NS cases with and without PTPN11 mutations and that a significant sex-ratio bias favoring transmission to males was present in subjects with sporadic NS caused by PTPN11 mutations, as well as in families inheriting the disorder.

Genetic evidence for lineage-related and differentiation stage-related contribution of somatic PTPN11 mutations to leukemogenesis in childhood acute leukemia.

SHP-2 is a protein tyrosine phosphatase functioning as signal transducer downstream to growth factor and cytokine receptors. SHP-2 is required during development, and germline mutations in PTPN11, the gene encoding SHP-2, cause Noonan syndrome. SHP-2 plays a crucial role in hematopoietic cell development. We recently demonstrated that somatic PTPN11 mutations are the most frequent lesion in juvenile myelomonocytic leukemia and are observed in a smaller percentage of children with other myeloid malignancies. Here, we report that PTPN11 lesions occur in childhood acute lymphoblastic leukemia (ALL). Mutations were observed in 23 of 317 B-cell precursor ALL cases, but not among 44 children with T-lineage ALL. In the former, lesions prevalently occurred in TEL-AML1(-) cases with CD19(+)/CD10(+)/cyIgM(-) immunophenotype. PTPN11, NRAS, and KRAS2 mutations were largely mutually exclusive and accounted for one third of common ALL cases. We also show that, among 69 children with acute myeloid leukemia, PTPN11 mutations occurred in 4 of 12 cases with acute monocytic leukemia (FAB-M5). Leukemia-associated PTPN11 mutations were missense and were predicted to result in SHP-2 gain-of-function. Our findings provide evidence for a wider role of PTPN11 lesions in leukemogenesis, but also suggest a lineage-related and differentiation stage-related contribution of these lesions to clonal expansion.

Oxidative stress in diabetes-induced endothelial dysfunction involvement of nitric oxide and protein kinase C.

Reactive oxygen species (ROS) formation plays a major role in diabetes-induced endothelial dysfunction, though the molecular mechanism(s) involved and the contribution of nitric oxide (NO) are still unclear. This study using bovine retinal endothelial cells was aimed at assessing (i) the role of oxygen-dependent vs. NO-dependent oxidative stress in the endothelial cell permeability alterations induced by the diabetic milieu and (ii) whether protein kinase C (PKC) activation ultimately mediates these changes. Superoxide, lipid peroxide, and PKC activity were higher under high glucose (HG) vs. normal glucose throughout the 30 d period. Nitrite/nitrate and endothelial NO synthase levels increased at 1 d and decreased thereafter. Changes in monolayer permeability to 125I-BSA induced by 1 or 30 d incubation in HG or exposure to advanced glycosylation endproduct were reduced by treatment with antioxidants or PKC inhibitors, whereas NO blockade prevented only the effect of 1 d HG. HG-induced changes were mimicked by a PKC activator, a superoxide generating system, an NO and superoxide donor, or peroxynitrite (attenuated by PKC inhibition), but not a NO donor. The short-term effect of HG depends on a combined oxidative and nitrosative stress with peroxynitrite formation, whereas the long-term effect is related to ROS generation; in both cases, PKC ultimately mediates permeability changes.

Role of galectin-3 in diabetic nephropathy.

The advanced glycosylation end products (AGE) participate in the pathogenesis of nephropathy and other diabetic complications through several mechanisms, including their binding to cell surface receptors. The AGE receptors include RAGE, the macrophage scavenger receptors, OST-48 (AGE-R1), 80K-H (AGE-R2), and galectin-3 (AGE-R3). Galectin-3 interacts with the beta-galactoside residues of cell surface and matrix glycoproteins via the carbohydrate recognition domain and with intracellular proteins via peptide-peptide associations mediated by its N-terminus domain. These structural properties enable galectin-3 to exert multiple functions, including the mRNA splicing activity, the control of cell cycle, the regulation of cell adhesion, the modulation of allergic reactions, and the binding of AGE. The lack of transmembrane anchor sequence or signal peptide suggests that it is associated with other AGE receptors, possibly AGE-R1 and AGE-R2, to form an AGE-receptor complex, rather than playing an independent role. In target tissues of diabetic vascular complications, such as the endothelium and mesangium, galectin-3 is weakly expressed under basal conditions and is markedly upregulated by the diabetic milieu (and to a lesser extent by aging). Galectin-3-deficient mice were found to develop accelerated diabetic glomerulopathy versus the wild-type animals, as evidenced by the more pronounced increase in proteinuria, mesangial expansion, and matrix gene expression. This was associated with a more marked renal/glomerular AGE accumulation, suggesting that it was attributable to the lack of galectin-3 AGE-receptor function. These data indicate that galectin-3 is upregulated under diabetic conditions and is operating in vivo to provide protection toward AGE-induced tissue injury, as opposed to RAGE.