A truncating TPO mutation (Y55X) in patients with hypothyroidism and total iodide organification defect.

UNLABELLED: Absract Purpose: Mutations in the TPO gene have been reported to cause congenital hypothyroidism (CH), and our aim in this study was to determine the genetic basis of congenital hypothyroidism in two affected children coming from a consanguineous family. METHODS: Since CH is usually inherited in autosomal recessive manner in consanguineous/multi case-families, we adopted a two-stage strategy of genetic linkage studies and targeted sequencing of the candidate genes. First we investigated the potential genetic linkage of the family to any known CH locus using microsatellite markers and then screened for mutations in linked-gene by Sanger sequencing. RESULTS: The family showed potential linkage to the TPO gene and we detected a non-sense mutation (Y55X) in both cases that had total iodode organification defect (TIOD). The mutation segregated with disease status in the family. Y55X is the only truncating mutation in the exon 2 of the TPO gene reported in the literature and results in the earliest stop codon known in the gene to date. CONCLUSIONS: This study confirms the pathogenicity of Y55X mutation and demonstrates that a nonsense mutation in the amino-terminal coding region of the TPO gene could totally abolish the function of the TPO enzyme leading to TIOD. Thus it helps to establish a strong genotype/phenotype correlation associated with this mutation. It also highlights the importance of molecular genetic studies in the definitive diagnosis and accurate classification of CH.

Thyroid dyshormonogenesis is mainly caused by TPO mutations in consanguineous community.

OBJECTIVE: In this study, we aimed to investigate the genetic background of thyroid dyshormonogenesis (TDH). CONTEXT: Thyroid dyshormonogenesis comprises 10-15% of all cases of congenital hypothyroidism (CH), which is the most common neonatal endocrine disorder, and might result from disruptions at any stage of thyroid hormone biosynthesis. Currently seven genes (NIS, TPO, PDS, TG, IYD, DUOX2 and DUOXA2) have been implicated in the aetiology of the disease. DESIGN: As TDH is mostly inherited in an autosomal recessive manner, we planned to conduct the study in consanguineous/multi-case families. PATIENTS: One hundred and four patients with congenital TDH all coming from consanguineous and/or multi-case families. MEASUREMENTS: Initially, we performed potential linkage analysis of cases to all seven causative-TDH loci as well as direct sequencing of the TPO gene in cases we could not exclude linkage to this locus. In addition, in silico analyses of novel missense mutations were carried out. RESULTS: TPO had the highest potential for linkage and we identified 21 TPO mutations in 28 TDH cases showing potential linkage to this locus. Four of 10 distinct TPO mutations detected in this study were novel (A5T, Y55X, E596X, D633N). CONCLUSIONS: This study underlines the importance of molecular genetic studies in diagnosis, classification and prognosis of CH and proposes a comprehensive mutation screening by new sequencing technology in all newly diagnosed primary CH cases.

Health impact and toxicological effects of nanomaterials in the lung.

The manufacture, use and disposal of nanomaterials will result in increased human exposures to engineered nanoparticles (ENPs), potentially via the lung. ENPs differ physically and chemically from natural- or combustion-derived nanoparticles (NP) in important respects. While there are parallels with ultrafine aerosol particles in the atmosphere and colloids in water, there remain some unique issues and impacts of engineered materials on lung health that require consideration and urgent study. The study of toxicity of nanomaterials in biological systems--nanotoxicology--emerged from the observed effects of inhaled particulate matter (PM) and NP. Some engineered nanomaterials deserve special toxicological examination because of their unique properties in biological systems; novel toxicological approaches may be required for their assessment. Translocation in biological systems--a key feature of ENPs--is dependent on ENP size and surface interactions with macromolecules at the portal of entry, upstream of cellular interaction. Of particular significance is the agglomeration processes associated with macromolecule adsorption at ENP surfaces, which determine clearance rates and cellular response. ENP toxicity is therefore dominated by three linked physico-chemical factors: size-shape, surface and 'corona' (formed by adhering macromolecules from the suscipient host). Measuring and predicting ENP translocation and effects following lung entry have proven to be particularly challenging, but understanding ENP behaviour in vivo is fundamental for safe design for effective and targeted drug delivery. Human exposures via medical and dental applications appear important in terms of dose and toxicity, and may need to be assessed for risk on a case-by-case basis.

Mild and severe congenital primary hypothyroidism in two patients by thyrotropin receptor (TSHR) gene mutation.

Congenital hypothyroidism (CH) is the most commonly encountered endocrinological birth defect, with an incidence of approximately 1 in 3000-4000 live births. It could be sporadic or familial as well as goitrous or non-goitrous. Inactivating mutations of TSHR , which is one of the genes responsible for non-goitrogenic congenital hypothyroidism, are mostly inherited autosomal recessively and result in a wide clinical spectrum owing to the extent of receptor function loss. Here, we report detailed clinical features of two CH cases with TSHR mutations. The first case was diagnosed before the initiation of the national screening program and had a severe clinical phenotype associated with a homozygous inactivating TSHR mutation (P556R), whereas the second case was diagnosed after the introduction of the national screening program and showed a mild clinical presentation and carried another homozygous missense mutation (P162A) in the TSHR gene. We compared the clinical features of our cases with those of previously reported patients with TSHR mutations to enhance the genotype/phenotype correlations between these mutations and corresponding clinical phenotypes.

TSHR is the main causative locus in autosomal recessively inherited thyroid dysgenesis.

Congenital hypothyroidism (CH) is the most common neonatal endocrine disorder and results in mental retardation if untreated. Eighty-five percent of CH cases are due to disruptions in thyroid organogenesis and are mostly sporadic, but about 2% of thyroid dysgenesis is familial, indicating the involvement of genetic factors in the aetiology of the disease. In this study, we aimed to investigate the Mendelian (single-gene) causes of non-syndromic and non-goitrous congenital hypothyroidism (CHNG) in consanguineous or multi-case families. Here we report the results of the second part (n=105) of our large cohort (n=244), representing the largest such cohort in the literature, and interpret the overall results of the whole cohort. Additionally, 50 sporadic cases with thyroid dysgenesis and 400 unaffected control subjects were included in the study. In familial cases, first, we performed potential linkage analysis of four known genes causing CHNG (TSHR, PAX8, TSHB, and NKX2-5) using microsatellite markers and then examined the presence of mutations in these genes by direct sequencing. In addition, in silico analyses of the predicted structural effects of TSHR mutations were performed and related to the mutation specific disease phenotype. We detected eight new TSHR mutations and a PAX8 mutation but no mutations in TSHB and NKX2-5. None of the biallelic TSHR mutations detected in familial cases were present in the cohort of 50 sporadic cases. Genotype/phenotype relationships were established between TSHR mutations and resulting clinical presentations. Here we conclude that TSHR mutations are the main detectable cause of autosomal recessively inherited thyroid dysgenesis. We also outline a new genetic testing strategy for the investigation of suspected autosomal recessive non-goitrous CH.

Novel TSHR mutations in consanguineous families with congenital nongoitrous hypothyroidism.

OBJECTIVE: Nonsyndromic autosomal recessively inherited nongoitrous congenital hypothyroidism (CHNG) can be caused by mutations in TSHR, PAX8, TSHB and NKX2-5. We aimed to investigate mutational frequencies of these genes and genotype/phenotype correlations in consanguineous families with CHNG. DESIGN: Because consanguinity in individuals with a presumptive genetic condition is often an indicator of an autosomal recessive inheritance and allows firmer correlations to be established between genotype and phenotype, we planned to execute our study in consanguineous families. PATIENTS: Hundred and thirty-nine children with CHNG phenotype born to consanguineous families. MEASUREMENTS: First, we investigated cases for evidence of linkage to the four known CHNG genes by microsatellite marker analysis. Mutation analysis by direct sequencing was then performed in those cases in whom linkage to the relevant candidate gene could not be excluded. In addition, in silico analysis of the predicted structural effects of TSHR mutations was performed and related to the mutation-specific disease phenotype. RESULTS: Homozygous germline TSHR mutations were detected in six families (5%), but no mutations were detected in PAX8, TSHB and NKX2-5. Four of TSHR mutations had not previously been described. Genotype-phenotype correlations were established and found to be related to the predicted structural effects of the mutations. CONCLUSIONS: Known causative genes account for the development of CHNG only in a minority of cases, and our cohort should provide a powerful resource to identify novel causative genes and to delineate the extent of locus heterogeneity in autosomal recessively inherited CHNG.

Platelet aggregation induced by polystyrene and platinum nanoparticles is dependent on surface area.

Nanoparticles are key components underlying recent technological advances in various industrial and medical fields, and thus understanding their mode of interaction with biological systems is essential. However, while several nanoparticle systems have been shown to interact with blood platelets, many questions remain concerning the mechanisms of platelet activation and the role that the physicochemical properties of nanoparticles play in inducing platelet aggregation. Here, using negatively charged polystyrene nanoparticles with sizes of 25, 50, 119, 151, 201 nm and negatively charged platinum nanoparticles with sizes of 7 and 73 nm, we show that it is not the size of the nanoparticles but rather the nanoparticle surface area that is critical in mediating the effects on platelet activation. The nanoparticles stimulate platelet aggregation through passive (agglutination) and activation of integrin αIIbß3 through a pathway regulated by Src and Syk tyrosine kinase.

'Bio-nano interactions: new tools, insights and impacts': summary of the Royal Society discussion meeting.

Bio-nano interactions can be defined as the study of interactions between nanoscale entities and biological systems such as, but not limited to, peptides, proteins, lipids, DNA and other biomolecules, cells and cellular receptors and organisms including humans. Studying bio-nano interactions is particularly useful for understanding engineered materials that have at least one dimension in the nanoscale. Such materials may consist of discrete particles or nanostructured surfaces. Much of biology functions at the nanoscale; therefore, our ability to manipulate materials such that they are taken up at the nanoscale, and engage biological machinery in a designed and purposeful manner, opens new vistas for more efficient diagnostics, therapeutics (treatments) and tissue regeneration, so-called nanomedicine. Additionally, this ability of nanomaterials to interact with and be taken up by cells allows nanomaterials to be used as probes and tools to advance our understanding of cellular functioning. Yet, as a new technology, assessment of the safety of nanomaterials, and the applicability of existing regulatory frameworks for nanomaterials must be investigated in parallel with development of novel applications. The Royal Society meeting 'Bio-nano interactions: new tools, insights and impacts' provided an important platform for open dialogue on the current state of knowledge on these issues, bringing together scientists, industry, regulatory and legal experts to concretize existing discourse in science law and policy. This paper summarizes these discussions and the insights that emerged.

Nanoparticle growth and surface chemistry changes in cell-conditioned culture medium.

When biomolecules attach to engineered nanoparticle (ENP) surfaces, they confer the particles with a new biological identity. Physical format may also radically alter, changing ENP stability and agglomeration state within seconds. In order to measure which biomolecules are associated with early ENP growth, we studied ENPs in conditioned medium from A549 cell culture, using dynamic light scattering (DLS) and linear trap quadrupole electron transfer dissociation mass spectrometry. Two types of 100 nm polystyrene particles (one uncoated and one with an amine functionalized surface) were used to measure the influence of surface type. In identically prepared conditioned medium, agglomeration was visible in all samples after 1 h, but was variable, indicating inter-sample variability in secretion rates and extracellular medium conditions. In samples conditioned for 1 h or more, ENP agglomeration rates varied significantly. Agglomerate size measured by DLS was well correlated with surface sequestered peptide number for uncoated but not for amine coated polystyrene ENPs. Amine-coated ENPs grew much faster and into larger agglomerates associated with fewer sequestered peptides, but including significant sequestered lactose dehydrogenase. We conclude that interference with extracellular peptide balance and oxidoreductase activity via sequestration is worthy of further study, as increased oxidative stress via this new mechanism may be important for cell toxicity.

Nanoparticles modulate surfactant protein A and D mediated protection against influenza A infection in vitro.

Numerous epidemiological and toxicological studies have indicated that respiratory infections are exacerbated following enhanced exposure to airborne particulates. Surfactant protein A (SP-A) and SP-D form an important part of the innate immune response in the lung and can interact with nanoparticles to modulate the cellular uptake of these particles. We hypothesize that this interaction will also affect the ability of these proteins to combat infections. TT1, A549 and differentiated THP-1 cells, representing the predominant cell types found in the alveolus namely alveolar type I (ATI) epithelial cells, ATII cells and macrophages, were used to examine the effect of two model nanoparticles, 100 nm amine modified (A-PS) and unmodified polystyrene (U-PS), on the ability of SP-A and SP-D to neutralize influenza A infections in vitro. Pre-incubation of low concentrations of U-PS with SP-A resulted in a reduction of SP-A anti-influenza activity in A549 cells, whereas at higher concentrations there was an increase in SP-A antiviral activity. This differential pattern of U-PS concentration on surfactant protein mediated protection against IAV was also shown with SP-D in TT1 cells. On the other hand, low concentrations of A-PS particles resulted in a reduction of SP-A activity in TT1 cells and a reduction in SP-D activity in A549 cells. These results indicate that nanoparticles can modulate the ability of SP-A and SP-D to combat viral challenges. Furthermore, the nanoparticle concentration, surface chemistry and cell type under investigation are important factors in determining the extent of these modulations.

Surfactant protein A (SP-A) inhibits agglomeration and macrophage uptake of toxic amine modified nanoparticles.

The lung provides the main route for nanomaterial exposure. Surfactant protein A (SP-A) is an important respiratory innate immune molecule with the ability to bind or opsonise pathogens to enhance phagocytic removal from the airways. We hypothesised that SP-A, like surfactant protein D, may interact with inhaled nanoparticulates, and that this interaction will be affected by nanoparticle (NP) surface characteristics. In this study, we characterise the interaction of SP-A with unmodified (U-PS) and amine-modified (A-PS) polystyrene particles of varying size and zeta potential using dynamic light scatter analysis. SP-A associated with both 100 nm U-PS and A-PS in a calcium-independent manner. SP-A induced significant calcium-dependent agglomeration of 100 nm U-PS NPs but resulted in calcium-independent inhibition of A-PS self agglomeration. SP-A enhanced uptake of 100 nm U-PS into macrophage-like RAW264.7 cells in a dose-dependent manner but in contrast inhibited A-PS uptake. Reduced association of A-PS particles in RAW264.7 cells following pre-incubation of SP-A was also observed with coherent anti-Stokes Raman spectroscopy. Consistent with these findings, alveolar macrophages (AMs) from SP-A(-/-) mice were more efficient at uptake of 100 nm A-PS compared with wild type C57Bl/6 macrophages. No difference in uptake was observed with 500 nm U-PS or A-PS particles. Pre-incubation with SP-A resulted in a significant decrease in uptake of 100 nm A-PS in macrophages isolated from both groups of mice. In contrast, increased uptake by AMs of U-PS was observed after pre-incubation with SP-A. Thus we have demonstrated that SP-A promotes uptake of non-toxic U-PS particles but inhibits the clearance of potentially toxic A-PS particles by blocking uptake into macrophages.

Comparisons of the dust/smoke particulate that settled inside the surrounding buildings and outside on the streets of southern New York City after the collapse of the World Trade Center, September 11, 2001.

The collapse of the World Trade Center (WTC) on September 11, 2001, generated large amounts of dust and smoke that settled in the surrounding indoor and outdoor environments in southern Manhattan. Sixteen dust samples were collected from undisturbed locations inside two uncleaned buildings that were adjacent to Ground Zero. These samples were analyzed for morphology, metals, and organic compounds, and the results were compared with the previously reported outdoor WTC dust/smoke results. We also analyzed seven additional dust samples provided by residents in the local neighborhoods. The morphologic analyses showed that the indoor WTC dust/smoke samples were similar to the outdoor WTC dust/smoke samples in composition and characteristics but with more than 50% mass in the <53-microm size fraction. This was in contrast to the outdoor samples that contained >50% of mass above >53 microm. Elemental analyses also showed the similarities, but at lower concentrations. Organic compounds present in the outdoor samples were also detected in the indoor samples. Conversely, the resident-provided convenience dust samples were different from either the WTC indoor or outdoor samples in composition and pH, indicating that they were not WTC-affected locations. In summary, the indoor dust/smoke was similar in concentration to the outdoor dust/smoke but had a greater percentage of mass <53 microm in diameter.

One Base Deletion (c.2422delT) in the TPO Gene Causes Severe Congenital Hypothyroidism.

OBJECTIVE: Congenital hypothyroidism (CH) is the most common neonatal endocrine disorder and mutations in the TPO gene have been reported to cause CH. Our aim in this study was to determine the genetic basis of CH in two affected individuals coming from a consanguineous family. METHODS: Since CH is usually inherited in autosomal recessive manner in consanguineous/multi-case families, we adopted a two-stage strategy of genetic linkage studies and targeted sequencing of the candidate genes. First, we investigated the potential genetic linkage of the family to any known CH locus using microsatellite markers and then screened for mutations in linked-gene by Sanger sequencing. RESULTS: The family showed potential linkage to the TPO gene and we detected a deletion (c.2422delT) in both cases. The mutation segregated with disease status in the family. CONCLUSION: This study demonstrates that a single base deletion in the carboxyl-terminal coding region of the TPO gene could cause CH and helps to establish a genotype/phenotype correlation associated with the mutation. The study also highlights the importance of molecular genetic studies in the definitive diagnosis and accurate classification of CH.

Novel truncating thyroglobulin gene mutations associated with congenital hypothyroidism.

Mutations in the thyroglobulin (TG) gene have been reported to cause congenital hypothyroidism (CH) and we have been investigating the genetic architecture of CH in a large cohort of consanguineous/multi-case families. Our aim in this study was to determine the genetic basis of CH in four affected individuals coming from two separate consanguineous families. Since CH is usually inherited in autosomal recessive manner in consanguineous/multi-case families, we adopted a two-stage strategy of genetic linkage studies and targeted sequencing of the TG gene. First we investigated the potential genetic linkage of families to any known CH locus using microsatellite markers and then determined the pathogenic mutations in linked-genes by Sanger sequencing. Both families showed potential linkage to TG locus and we detected two previously unreported nonsense TG mutations (p.Q630X and p.W637X) that segregated with the disease status in both families. This study highlights the importance of molecular genetic studies in the definitive diagnosis and classification of CH, and also adds up to the limited number of nonsense TG mutations in the literature. It also suggests a new clinical testing strategy using next-generation sequencing in all primary CH cases.

A common thyroid peroxidase gene mutation (G319R) in Turkish patients with congenital hypothyroidism could be due to a founder effect.

The most common congenital endocrine disorder is congenital hypothyroidism (CH), which can lead to mental retardation if untreated. Majority of the patients have been found to have defects in thyroid development and migration disorders (dysgenesis), and the remaining ones have thyroid hormone synthesis defects (dyshormonogenesis). One of the most common mechanisms to cause dyshormonogenesis is a defect in the thyroid peroxidase (TPO) enzyme. In familial cases, mutations in the TPO gene are fairly prevalent. To date, more than 80 mutations have been identified, which result in variably decreasing TPO bioactivities. Clinical manifestations of TPO defects are typically permanent CH and with or without goiter. In this report, we presented two children with CH who were born to consanguineous parents and were homozygous carriers of a missense (G319R) TPO mutation, the mutation segregated with the disease status in the families confirming its pathogenicity. G319R mutation seemed to be a common cause of CH in Turkish population, which could originate from a common founder ancestor. Moreover, our results also confirmed the phenotypic variability associated with different TPO mutations.

An essential splice site mutation (c.317+1G>A) in the TSHR gene leads to severe thyroid dysgenesis.

Congenital hypothyroidism (CH) is the most common neonatal endocrine disorder and 2% of cases have familial origin. Our aim in this study was to determine the genetic alterations in two siblings with CH coming from a consanguineous family. Because CH is often inherited in autosomal recessive manner in consanguineous/multicase-families, we first performed genetic linkage studies to all known causative CH loci followed by conventional sequencing of the linked gene. The family showed potential linkage to the TSHR locus, and we detected an essential splice site mutation (c.317+1G>A) in both siblings. RT-PCR analysis confirmed the functionality of the mutation. The mutation was homozygous in the cases whereas heterozygous in carrier parents and an unaffected sibling. Here we conclude that thyroid agenesis in both siblings in this study originates from c.317+1G>A splice site mutation in the TSHR gene, and this study underlines the importance of detailed molecular genetic studies in the definitive diagnosis and classification of CH.

A nonsense thyrotropin receptor gene mutation (R609X) is associated with congenital hypothyroidism and heart defects.

Congenital hypothyroidism (CH), one of the most important preventable causes of mental retardation, is a clinical condition characterized by thyroid hormone deficiency in newborns. CH is most often caused by defects in thyroid development leading to thyroid dysgenesis. The thyroid-stimulating hormone receptor (TSHR) is the main known gene causing thyroid dysgenesis in consanguineous families with CH. In this study, we aim to determine the genetic alteration in a case with congenital hypothyroidism and heart defects coming from a consanguineous family. We utilized genetic linkage analysis and direct sequencing to achieve our aim. Our results revealed that the family showed linkage to the TSHR locus, and we detected a homozygous nonsense mutation (R609X) in the case. Apart from other cases with the same mutation, our case had accompanying cardiac malformations. Although cardiac malformations are not uncommon in sporadic congenital hypothyroidism, here, they are reported for the first time with R609X mutation in a familial case.

A truncating DUOX2 mutation (R434X) causes severe congenital hypothyroidism.

Mutations in DUOX2 have been reported to cause congenital hypothyroidism (CH), and our aim in this study was to determine the genetic basis of CH in two affected individuals coming from a consanguineous family. Because CH is usually inherited in autosomal recessive manner in consanguineous/multicase families, we adopted a two-stage strategy of genetic linkage studies and targeted sequencing of the candidate genes. First, we investigated the potential genetic linkage of the family to any known CH locus using microsatellite markers and then screened for mutations in linked genes by Sanger sequencing. The family showed potential linkage to DUOX2 locus and we detected a nonsense mutation (R434X) in both cases and the mutation segregated with disease status in the family. This study highlights the importance of molecular genetic studies in the definitive diagnosis and classification of CH, and it also suggests a new clinical testing strategy using next-generation sequencing in all primary CH cases.

A deletion including exon 2 of the TSHR gene is associated with thyroid dysgenesis and severe congenital hypothyroidism.

Congenital hypothyroidism (CH) is the most common neonatal endocrine disorder and 2% of cases have a familial origin. Our aim in this study was to determine the genetic alterations in two siblings with CH coming from a consanguineous family. As CH is often inherited in an autosomal recessive manner in consanguineous/multi case-families, we first performed genetic linkage studies to all known causative CH loci followed by conventional sequencing of the linked gene. The family showed potential linkage to the TSHR locus and our attempts to amplify and sequence exon 2 of the TSHR gene continuously failed. Subsequent RT-PCR analysis using mRNA and corresponding cDNA showed a large deletion including the exon 2 of the gene. The deletion was homozygous in affected cases whilst heterozygous in carrier parents. Here we conclude that CH in both siblings of this study originates from a large deletion including the exon 2 of the TSHR gene. This study demonstrates that full sequence analysis in a candidate CH gene might not always be enough to detect genetic alterations, and additional analyses such as RT-PCR and MLPA might be necessary to describe putative genetic causes of the disease in some cases. It also underlines the importance of detailed molecular genetic studies in the definitive diagnosis and classification of CH.

Surfactant protein D (SP-D) alters cellular uptake of particles and nanoparticles.

Surfactant protein D (SP-D) is primarily expressed in the lungs and modulates pro- and anti-inflammatory processes to toxic challenge, maintaining lung homeostasis. We investigated the interaction between NPs and SP-D and subsequent uptake by cells involved in lung immunity. Dynamic light scattering (DLS) and scanning electron microscopy (SEM) measured NP aggregation, particle size and charge in native human SP-D (NhSP-D) and recombinant fragment SP-D (rfhSP-D). SP-D aggregated NPs, especially following the addition of calcium. Immunohistochemical analysis of A549 epithelial cells investigated the co-localization of NPs and rfhSP-D. rfhSP-D enhanced the co-localisation of NPs to epithelial A549 cells in vitro. NP uptake by alveolar macrophages (AMs) and lung dendritic cells (LDCs) from C57BL/6 and SP-D knock-out mice were compared. AMs and LDCs showed decreased uptake of NPs in SP-D deficient mice compared to wild-type mice. These data confirmed an interaction between SP-D and NPs, and subsequent enhanced NP uptake.