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1.
Genet Med ; 2019 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-31578471

RESUMO

PURPOSE: Lamb-Shaffer syndrome (LAMSHF) is a neurodevelopmental disorder described in just over two dozen patients with heterozygous genetic alterations involving SOX5, a gene encoding a transcription factor regulating cell fate and differentiation in neurogenesis and other discrete developmental processes. The genetic alterations described so far are mainly microdeletions. The present study was aimed at increasing our understanding of LAMSHF, its clinical and genetic spectrum, and the pathophysiological mechanisms involved. METHODS: Clinical and genetic data were collected through GeneMatcher and clinical or genetic networks for 41 novel patients harboring various types of SOX5 alterations. Functional consequences of selected substitutions were investigated. RESULTS: Microdeletions and truncating variants occurred throughout SOX5. In contrast, most missense variants clustered in the pivotal SOX-specific high-mobility-group domain. The latter variants prevented SOX5 from binding DNA and promoting transactivation in vitro, whereas missense variants located outside the high-mobility-group domain did not. Clinical manifestations and severity varied among patients. No clear genotype-phenotype correlations were found, except that missense variants outside the high-mobility-group domain were generally better tolerated. CONCLUSIONS: This study extends the clinical and genetic spectrum associated with LAMSHF and consolidates evidence that SOX5 haploinsufficiency leads to variable degrees of intellectual disability, language delay, and other clinical features.

2.
Brain ; 141(11): 3160-3178, 2018 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-30351409

RESUMO

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels control neuronal excitability and their dysfunction has been linked to epileptogenesis but few individuals with neurological disorders related to variants altering HCN channels have been reported so far. In 2014, we described five individuals with epileptic encephalopathy due to de novo HCN1 variants. To delineate HCN1-related disorders and investigate genotype-phenotype correlations further, we assembled a cohort of 33 unpublished patients with novel pathogenic or likely pathogenic variants: 19 probands carrying 14 different de novo mutations and four families with dominantly inherited variants segregating with epilepsy in 14 individuals, but not penetrant in six additional individuals. Sporadic patients had epilepsy with median onset at age 7 months and in 36% the first seizure occurred during a febrile illness. Overall, considering familial and sporadic patients, the predominant phenotypes were mild, including genetic generalized epilepsies and genetic epilepsy with febrile seizures plus (GEFS+) spectrum. About 20% manifested neonatal/infantile onset otherwise unclassified epileptic encephalopathy. The study also included eight patients with variants of unknown significance: one adopted patient had two HCN1 variants, four probands had intellectual disability without seizures, and three individuals had missense variants inherited from an asymptomatic parent. Of the 18 novel pathogenic missense variants identified, 12 were associated with severe phenotypes and clustered within or close to transmembrane domains, while variants segregating with milder phenotypes were located outside transmembrane domains, in the intracellular N- and C-terminal parts of the channel. Five recurrent variants were associated with similar phenotypes. Using whole-cell patch-clamp, we showed that the impact of 12 selected variants ranged from complete loss-of-function to significant shifts in activation kinetics and/or voltage dependence. Functional analysis of three different substitutions altering Gly391 revealed that these variants had different consequences on channel biophysical properties. The Gly391Asp variant, associated with the most severe, neonatal phenotype, also had the most severe impact on channel function. Molecular dynamics simulation on channel structure showed that homotetramers were not conducting ions because the permeation path was blocked by cation(s) strongly complexed to the Asp residue, whereas heterotetramers showed an instantaneous current component possibly linked to deformation of the channel pore. In conclusion, our results considerably expand the clinical spectrum related to HCN1 variants to include common generalized epilepsy phenotypes and further illustrate how HCN1 has a pivotal function in brain development and control of neuronal excitability.

4.
J Clin Invest ; 128(12): 5368-5373, 2018 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-30226830

RESUMO

Hyperphosphatemic familial tumoral calcinosis (HFTC)/hyperostosis-hyperphosphatemia syndrome (HHS) is an autosomal recessive disorder of ectopic calcification due to deficiency of or resistance to intact fibroblast growth factor 23 (iFGF23). Inactivating mutations in FGF23, N-acetylgalactosaminyltransferase 3 (GALNT3), or KLOTHO (KL) have been reported as causing HFTC/HHS. We present what we believe is the first identified case of autoimmune hyperphosphatemic tumoral calcinosis in an 8-year-old boy. In addition to the classical clinical and biochemical features of hyperphosphatemic tumoral calcinosis, the patient exhibited markedly elevated intact and C-terminal FGF23 levels, suggestive of FGF23 resistance. However, no mutations in FGF23, KL, or FGF receptor 1 (FGFR1) were identified. He subsequently developed type 1 diabetes mellitus, which raised the possibility of an autoimmune cause for hyperphosphatemic tumoral calcinosis. Luciferase immunoprecipitation systems revealed markedly elevated FGF23 autoantibodies without detectable FGFR1 or Klotho autoantibodies. Using an in vitro FGF23 functional assay, we found that the FGF23 autoantibodies in the patient's plasma blocked downstream signaling via the MAPK/ERK signaling pathway in a dose-dependent manner. Thus, this report describes the first case, to our knowledge, of autoimmune hyperphosphatemic tumoral calcinosis with pathogenic autoantibodies targeting FGF23. Identification of this pathophysiology extends the etiologic spectrum of hyperphosphatemic tumoral calcinosis and suggests that immunomodulatory therapy may be an effective treatment.

5.
Genet Med ; 2018 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-30206421

RESUMO

PURPOSE: Variants in IQSEC2, escaping X inactivation, cause X-linked intellectual disability with frequent epilepsy in males and females. We aimed to investigate sex-specific differences. METHODS: We collected the data of 37 unpublished patients (18 males and 19 females) with IQSEC2 pathogenic variants and 5 individuals with variants of unknown significance and reviewed published variants. We compared variant types and phenotypes in males and females and performed an analysis of IQSEC2 isoforms. RESULTS: IQSEC2 pathogenic variants mainly led to premature truncation and were scattered throughout the longest brain-specific isoform, encoding the synaptic IQSEC2/BRAG1 protein. Variants occurred de novo in females but were either de novo (2/3) or inherited (1/3) in males, with missense variants being predominantly inherited. Developmental delay and intellectual disability were overall more severe in males than in females. Likewise, seizures were more frequently observed and intractable, and started earlier in males than in females. No correlation was observed between the age at seizure onset and severity of intellectual disability or resistance to antiepileptic treatments. CONCLUSION: This study provides a comprehensive overview of IQSEC2-related encephalopathy in males and females, and suggests that an accurate dosage of IQSEC2 at the synapse is crucial during normal brain development.

6.
Endocrinology ; 158(3): 470-476, 2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-28005411

RESUMO

Mutations in the dentin matrix protein 1 (DMP1) gene cause autosomal recessive hypophosphatemic rickets (ARHR). Hypophosphatemia in ARHR results from increased circulating levels of the phosphaturic hormone, fibroblast growth factor 23 (FGF23). Similarly, elevated FGF23, caused by mutations in the PHEX gene, is responsible for the hypophosphatemia in X-linked hypophosphatemic rickets (XLH). Previously, we demonstrated that a Phex mutation in mice creates a lower set point for extracellular phosphate, where an increment in phosphorus further stimulates Fgf23 production to maintain low serum phosphorus levels. To test the presence of the similar set point defect in ARHR, we generated 4- and 12-week-old Dmp1/Galnt3 double knockout mice and controls, including Dmp1 knockout mice (a murine model of ARHR), Galnt3 knockout mice (a murine model of familial tumoral calcinosis), and phenotypically normal double heterozygous mice. Galnt3 knockout mice had increased proteolytic cleavage of Fgf23, leading to low circulating intact Fgf23 levels with consequent hyperphosphatemia. In contrast, Dmp1 knockout mice had little Fgf23 cleavage and increased femoral Fgf23 expression, resulting in hypophosphatemia and low femoral bone mineral density (BMD). However, introduction of the Galnt3 null allele to Dmp1 knockout mice resulted in a significant increase in serum phosphorus and normalization of BMD. This increased serum phosphorus was accompanied by markedly elevated Fgf23 expression and circulating Fgf23 levels, an attempt to reduce serum phosphorus in the face of improving phosphorus levels. These data indicate that a Dmp1 mutation creates a lower set point for extracellular phosphate and maintains it through the regulation of Fgf23 cleavage and expression.


Assuntos
Líquido Extracelular/metabolismo , Proteínas da Matriz Extracelular/genética , Raquitismo Hipofosfatêmico Familiar/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Fosfatos/metabolismo , Animais , Densidade Óssea , Raquitismo Hipofosfatêmico Familiar/sangue , Feminino , Fêmur/crescimento & desenvolvimento , Masculino , Camundongos , Camundongos Knockout , Mutação
7.
J Bone Miner Res ; 31(10): 1845-1854, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27164190

RESUMO

Familial tumoral calcinosis (FTC)/hyperostosis-hyperphosphatemia syndrome (HHS) is a rare disorder caused by mutations in the genes encoding fibroblast growth factor-23 (FGF23), N-acetylgalactosaminyltransferase 3 (GALNT3), or KLOTHO. The result is functional deficiency of, or resistance to, intact FGF23 (iFGF23), causing hyperphosphatemia, increased renal tubular reabsorption of phosphorus (TRP), elevated or inappropriately normal 1,25-dihydroxyvitamin D3 (1,25D), ectopic calcifications, and/or diaphyseal hyperostosis. Eight subjects with FTC/HHS were studied and treated. Clinical manifestations varied, even within families, ranging from asymptomatic to large, disabling calcifications. All subjects had hyperphosphatemia, increased TRP, and elevated or inappropriately normal 1,25D. C-terminal FGF23 was markedly elevated whereas iFGF23 was comparatively low, consistent with increased FGF23 cleavage. Radiographs ranged from diaphyseal hyperostosis to massive calcification. Two subjects with severe calcifications also had overwhelming systemic inflammation and elevated C-reactive protein (CRP). GALNT3 mutations were identified in seven subjects; no causative mutation was found in the eighth. Biopsies from four subjects showed ectopic calcification and chronic inflammation, with areas of heterotopic ossification observed in one subject. Treatment with low phosphate diet, phosphate binders, and phosphaturia-inducing therapies was prescribed with variable response. One subject experienced complete resolution of a calcific mass after 13 months of medical treatment. In the two subjects with systemic inflammation, interleukin-1 (IL-1) antagonists significantly decreased CRP levels with resolution of calcinosis cutis and perilesional inflammation in one subject and improvement of overall well-being in both subjects. This cohort expands the phenotype and genotype of FTC/HHS and demonstrates the range of clinical manifestations despite similar biochemical profiles and genetic mutations. Overwhelming systemic inflammation has not been described previously in FTC/HHS; the response to IL-1 antagonists suggests that anti-inflammatory drugs may be useful adjuvants. In addition, this is the first description of heterotopic ossification reported in FTC/HHS, possibly mediated by the adjacent inflammation. © 2016 American Society for Bone and Mineral Research.


Assuntos
Calcinose , Fatores de Crescimento de Fibroblastos/genética , Glucuronidase/genética , Hiperostose Cortical Congênita , Hiperostose , Hiperfosfatemia , N-Acetilgalactosaminiltransferases/genética , Adolescente , Adulto , Calcinose/sangue , Calcinose/genética , Calcinose/patologia , Calcinose/terapia , Criança , Estudos de Coortes , Feminino , Humanos , Hiperostose/sangue , Hiperostose/genética , Hiperostose/patologia , Hiperostose/terapia , Hiperostose Cortical Congênita/sangue , Hiperostose Cortical Congênita/genética , Hiperostose Cortical Congênita/patologia , Hiperostose Cortical Congênita/terapia , Hiperfosfatemia/sangue , Hiperfosfatemia/genética , Hiperfosfatemia/patologia , Hiperfosfatemia/terapia , Masculino
8.
Oncotarget ; 6(23): 19647-60, 2015 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-25944690

RESUMO

Multiply myeloma (MM) grows in and destroys bone, where osteocytes secrete FGF23, a hormone which affects phosphate homeostasis and aging. We report that multiple myeloma (MM) cells express receptors for and respond to FGF23. FGF23 increased mRNA for EGR1 and its target heparanase, a pro-osteolytic factor in MM. FGF23 signals through a complex of klotho and a classical FGF receptor (FGFR); both were expressed by MM cell lines and patient samples. Bone marrow plasma cells from 42 MM patients stained positively for klotho, while plasma cells from 8 patients with monoclonal gammopathy of undetermined significance (MGUS) and 6 controls were negative. Intact, active FGF23 was increased 2.9X in sera of MM patients compared to controls. FGF23 was not expressed by human MM cells, but co-culture with mouse bone increased its mRNA. The FGFR inhibitor NVP-BGJ398 blocked the heparanase response to FGF23. NVP-BGJ398 did not inhibit 8226 growth in vitro but significantly suppressed growth in bone and induction of the osteoclast regulator RANK ligand, while decreasing heparanase mRNA. The bone microenvironment provides resistance to some anti-tumor drugs but increased the activity of NVP-BGJ398 against 8226 cells. The FGF23/klotho/heparanase signaling axis may offer targets for treatment of MM in bone.


Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Glucuronidase/metabolismo , Mieloma Múltiplo/enzimologia , Crânio/metabolismo , Adulto , Idoso , Animais , Estudos de Casos e Controles , Técnicas de Cocultura , Proteína 1 de Resposta de Crescimento Precoce/genética , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Feminino , Fatores de Crescimento de Fibroblastos/sangue , Regulação Neoplásica da Expressão Gênica , Glucuronidase/genética , Humanos , Células MCF-7 , Masculino , Camundongos , Pessoa de Meia-Idade , Mieloma Múltiplo/sangue , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Compostos de Fenilureia/farmacologia , Pirimidinas/farmacologia , Ligante RANK/metabolismo , RNA Mensageiro/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Crânio/efeitos dos fármacos , Fatores de Tempo , Microambiente Tumoral , Regulação para Cima
9.
Bone ; 67: 139-44, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25007710

RESUMO

Mutations in the GALNT3 gene result in familial tumoral calcinosis, characterized by persistent hyperphosphatemia and ectopic calcific masses in soft tissues. Since calcific masses often recur after surgical removal, a more permanent solution to the problem is required. Nicotinamide is reported to lower serum phosphate by decreasing sodium-dependent phosphate co-transporters in the gut and kidney. However, its effectiveness in tumoral calcinosis remains unknown. In this study, we investigated nicotinamide as a potential therapy for tumoral calcinosis, using a murine model of the disease-Galnt3 knockout mice. Initially, five different doses of nicotinamide were given to normal heterozygous mice intraperitoneally or orally. Treatment had no effect on serum phosphate levels, but serum levels of a phosphaturic hormone, fibroblast growth factor 23 (Fgf23), decreased in a dose-dependent manner. Subsequently, high-dose nicotinamide (40mM) was tested in Galnt3 knockout mice fed a high phosphate diet. The radiographic data pre- and post-treatment showed that nicotinamide did not reverse the calcification. However, the treatment retarded calcification growth after 4weeks, while in the untreated animals, calcifications increased in size. The therapy did not affect serum phosphate levels, but intact Fgf23 decreased in the treated mice. The treated mice also had increased calcium in the heart. In summary, nicotinamide did not alter serum phosphate levels, likely due to compensatory decrease in Fgf23 to counteract the phosphate lowering effect of nicotinamide. Although increased calcium accumulation in the heart is a concern, the therapy appears to slow down the progression of ectopic calcifications.


Assuntos
Calcinose/sangue , Calcinose/tratamento farmacológico , Cálcio/metabolismo , Fatores de Crescimento de Fibroblastos/sangue , Niacinamida/uso terapêutico , Animais , Calcinose/genética , Calcinose/metabolismo , Masculino , Camundongos , N-Acetilgalactosaminiltransferases/genética
10.
Endocrinology ; 155(10): 3891-8, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25051439

RESUMO

Fibroblast growth factor 23 (FGF23) is a hormone that inhibits renal phosphate reabsorption and 1,25-dihydroxyvitamin D biosynthesis. The FGF23 subtilisin-like proprotein convertase recognition sequence ((176)RHTR(179)↓) is protected by O-glycosylation through ppGalNAc-T3 (GALNT3) activity. Thus, inactivating GALNT3 mutations render FGF23 susceptible to proteolysis, thereby reducing circulating intact hormone levels and leading to hyperphosphatemic familial tumoral calcinosis. To further delineate the role of glycosylation in the Fgf23 function, we generated an inducible FGF23 transgenic mouse expressing human mutant FGF23 (R176Q and R179Q) found in patients with autosomal dominant hypophosphatemic rickets (ADHR) and bred this animal to Galnt3 knockout mice, a model of familial tumoral calcinosis. Due to the low intact Fgf23 level, Galnt3 knockout mice with wild-type Fgf23 alleles were hyperphosphatemic. In contrast, carriers of the mutant FGF23 transgene, regardless of Galnt3 mutation status, had significantly higher serum intact FGF23, resulting in severe hypophosphatemia. Importantly, serum phosphorus and FGF23 were comparable between transgenic mice with or without normal Galnt3 alleles. To determine whether the presence of the ADHR mutation could improve biochemical and skeletal abnormalities in Galnt3-null mice, these mice were also mated to Fgf23 knock-in mice, carrying heterozygous or homozygous R176Q ADHR Fgf23 mutations. The knock-in mice with functional Galnt3 had normal Fgf23 but were slightly hypophosphatemic. The stabilized Fgf23 ADHR allele reversed the Galnt3-null phenotype and normalized total Fgf23, serum phosphorus, and bone Fgf23 mRNA. However, the skeletal phenotype was unaffected. In summary, these data demonstrate that O-glycosylation by ppGaINAc-T3 is only necessary for proper secretion of intact Fgf23 and, once secreted, does not affect Fgf23 function. Furthermore, the more stable Fgf23 ADHR mutant protein could normalize serum phosphorus in Galnt3 knockout mice.


Assuntos
Raquitismo Hipofosfatêmico Familiar/genética , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , N-Acetilgalactosaminiltransferases/genética , N-Acetilgalactosaminiltransferases/metabolismo , Raquitismo Hipofosfatêmico/genética , Raquitismo/genética , Substituição de Aminoácidos , Animais , Modelos Animais de Doenças , Raquitismo Hipofosfatêmico Familiar/metabolismo , Raquitismo Hipofosfatêmico Familiar/terapia , Feminino , Terapia Genética , Glicosilação , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Raquitismo/metabolismo , Raquitismo/terapia , Transgenes
11.
Bone ; 64: 166-72, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24747200

RESUMO

Osteoporosis is a common complex disorder with reduced bone mineral density (BMD) and increased susceptibility to fracture. Peak BMD is one of the primary determinants of osteoporotic fracture risk, and is under substantial genetic control. Extracellular matrix, a major component of the bone, influences BMD by regulating mineral deposition and maintaining cellular activity. It contains several SIBLING family proteins, null mutations of which cause mineralization defects in humans. In this study, we tested 59 single-nucleotide polymorphisms (SNPs) located in the 5 SIBLING family genes (DSPP, DMP1, IBSP, MEPE and SPP1) for association with normal variation in peak BMD in healthy men and women. We measured femoral neck (FN) and lumbar spine (LS) areal BMD by dual energy x-ray absorptiometry (DXA) in 1692 premenopausal European-American women, 512 premenopausal African-American women and 715 European-American men. SNPs were tested for association with FN and LS-BMD in the 3 subsamples. In the European-American women, we observed association (p≤0.005) with LS-BMD for SNPs in DSPP, IBSP and MEPE, and for FN-BMD with SNPs in DMP1 and IBSP. Allele-specific regulation of gene expression (ASE) is an important mechanism in which an allele giving rise to modest influence in transcript abundance might result in a predisposition to disease. To identify whether there was ASE of SIBLING family genes at these SNPs, we examined 52 human bone samples obtained from the femoral neck during surgical hip replacement (27 female, 25 male; 44 European-American and 8 African-American). We observed unidirectional ASE for the IBSP gene, with lower expression of the G allele compared to the A allele for SNP rs17013181. Our data suggest that SNPs within the SIBLING genes may contribute to normal variation of peak BMD. Further studies are necessary to identify the functional variants and to determine the mechanisms underlying the differences in ASE and how these differences relate to the pathophysiology of osteoporosis.


Assuntos
Alelos , Densidade Óssea/genética , Regulação da Expressão Gênica , Absorciometria de Fóton , Adulto , Idoso , Idoso de 80 Anos ou mais , Grupo com Ancestrais do Continente Europeu , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único
12.
J Bone Miner Res ; 29(9): 2017-23, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24692172

RESUMO

Familial tumoral calcinosis is characterized by ectopic calcifications due to persistent hyperphosphatemia. The most common genetic cause of the disease is mutations in GALNT3, encoding a glycosyltransferase involved in a posttranslational modification of fibroblast growth factor 23 (FGF23). The Galnt3 knockout mouse we developed was hyperphosphatemic due to low intact Fgf23 levels, but did not develop any apparent calcifications on a standard rodent diet. We therefore tested the hypothesis that a further challenge with a high phosphate diet could induce ectopic calcifications in Galnt3 knockout mice. Mice were fed either normal (0.6%) or high (1.65%) phosphate diet for 20 weeks beginning from weaning at 3 weeks. The high phosphate diet did not affect serum phosphorus concentration. However, regardless of the dietary phosphate contents, serum phosphorus levels were consistently elevated in Galnt3 knockout mice. The mice on the high phosphate diet had slightly low serum calcium, but significantly high alkaline phosphatase, parathyroid hormone (PTH), and calcium in the kidney. Although none of Galnt3 knockout mice on the normal phosphate diet developed calcifications, calcifications appeared in approximately one-half of the mice on the high phosphate diet by 12 weeks. Calcified masses were most often found around the neck and on the back and as large as 9.9 mm in length. These data indicate that dietary phosphate load has major impact on the development of ectopic calcifications in tumoral calcinosis.


Assuntos
Calcinose/etiologia , Dieta , Fosfatos/efeitos adversos , Animais , Calcinose/sangue , Calcinose/diagnóstico por imagem , Calcinose/urina , Modelos Animais de Doenças , Rim/metabolismo , Rim/patologia , Camundongos , Camundongos Knockout , Miocárdio/metabolismo , Miocárdio/patologia , N-Acetilgalactosaminiltransferases/deficiência , N-Acetilgalactosaminiltransferases/metabolismo , Radiografia
13.
J Am Soc Nephrol ; 25(10): 2366-75, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24700880

RESUMO

Compound heterozygous and homozygous (comp/hom) mutations in solute carrier family 34, member 3 (SLC34A3), the gene encoding the sodium (Na(+))-dependent phosphate cotransporter 2c (NPT2c), cause hereditary hypophosphatemic rickets with hypercalciuria (HHRH), a disorder characterized by renal phosphate wasting resulting in hypophosphatemia, correspondingly elevated 1,25(OH)2 vitamin D levels, hypercalciuria, and rickets/osteomalacia. Similar, albeit less severe, biochemical changes are observed in heterozygous (het) carriers and indistinguishable from those changes encountered in idiopathic hypercalciuria (IH). Here, we report a review of clinical and laboratory records of 133 individuals from 27 kindreds, including 5 previously unreported HHRH kindreds and two cases with IH, in which known and novel SLC34A3 mutations (c.1357delTTC [p.F453del]; c.G1369A [p.G457S]; c.367delC) were identified. Individuals with mutations affecting both SLC34A3 alleles had a significantly increased risk of kidney stone formation or medullary nephrocalcinosis, namely 46% compared with 6% observed in healthy family members carrying only the wild-type SLC34A3 allele (P=0.005) or 5.64% in the general population (P<0.001). Renal calcifications were also more frequent in het carriers (16%; P=0.003 compared with the general population) and were more likely to occur in comp/hom and het individuals with decreased serum phosphate (odds ratio [OR], 0.75, 95% confidence interval [95% CI], 0.59 to 0.96; P=0.02), decreased tubular reabsorption of phosphate (OR, 0.41; 95% CI, 0.23 to 0.72; P=0.002), and increased serum 1,25(OH)2 vitamin D (OR, 1.22; 95% CI, 1.05 to 1.41; P=0.008). Additional studies are needed to determine whether these biochemical parameters are independent of genotype and can guide therapy to prevent nephrocalcinosis, nephrolithiasis, and potentially, CKD.


Assuntos
Cálculos Renais/genética , Nefrocalcinose/genética , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIc/genética , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Mutação de Sentido Incorreto
14.
Bone ; 59: 66-75, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24185277

RESUMO

Autosomal dominant osteopetrosis type II (ADO2) is a heritable osteosclerotic disorder dependent on osteoclast impairment. In most patients it results from heterozygous missense mutations in the chloride channel 7 (CLCN7) gene, encoding for a 2Cl(-)/1H(+) antiporter. By a knock-in strategy inserting a missense mutation in the Clcn7 gene, our two research groups independently generated mouse models of ADO2 on different genetic backgrounds carrying the homolog of the most frequent heterozygous mutation (p.G213R) in the Clcn7 gene found in humans. Our results demonstrate that the heterozygous model holds true presenting with higher bone mass, increased numbers of poorly resorbing osteoclasts and a lethal phenotype in the homozygous state. Considerable variability is observed in the heterozygous mice according with the mouse background, suggesting that modifier genes could influence the penetrance of the disease gene.


Assuntos
Genes Dominantes , Osteopetrose/genética , Animais , Sequência de Bases , Biomarcadores/sangue , Densidade Óssea/genética , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Células Cultivadas , Canais de Cloreto/genética , Modelos Animais de Doenças , Feminino , Técnicas de Introdução de Genes , Heterozigoto , Homozigoto , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Neurônios/metabolismo , Osteoclastos/metabolismo , Osteoclastos/patologia , Fenótipo , Microtomografia por Raio-X
15.
Bone ; 59: 53-6, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24176905

RESUMO

Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is a rare metabolic disorder, characterized by hypophosphatemia, variable degrees of rickets/osteomalacia, and hypercalciuria secondary to increased serum 1,25-dihydroxyvitamin D [1,25(OH)2D] levels. HHRH is caused by mutations in the SLC34A3 gene, which encodes sodium-phosphate co-transporter type IIc. A 6-1/2-year-old female presented with a history of nephrolithiasis. Her metabolic evaluation revealed increased 24-hour urine calcium excretion with high serum calcium, low intact parathyroid hormone (PTH), and elevated 1,25(OH)2D. In addition, the patient had low to low-normal serum phosphorus with high urine phosphorus. The patient had normal stature; without rachitic or boney deformities or a history of fractures. Genetic analysis of SLC34A3 revealed the patient to be a compound heterozygote for a novel single base pair deletion in exon 12 (c.1304delG) and 30-base pair deletion in intron 6 (g.1440-1469del). The single-base pair mutation causes a frameshift, which results in premature stop codon. The intronic deletion is likely caused by misalignment of the 4-basepair homologous repeats and results in the truncation of an already small intron to 63bp, which would impair proper RNA splicing of the intron. This is the fourth unique intronic deletion identified in patients with HHRH, suggesting the frequent occurrence of sequence misalignments in SLC34A3 and the importance of screening introns in patients with HHRH.


Assuntos
Raquitismo Hipofosfatêmico Familiar/complicações , Raquitismo Hipofosfatêmico Familiar/genética , Hipercalciúria/complicações , Hipercalciúria/genética , Íntrons/genética , Deleção de Sequência/genética , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIc/genética , Sequência de Bases , Criança , Análise Mutacional de DNA , Demografia , Feminino , Predisposição Genética para Doença , Humanos , Dados de Sequência Molecular
16.
Calcif Tissue Int ; 93(2): 155-62, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23700148

RESUMO

X-linked hypophosphatemia (XLH) is caused by mutations in the PHEX gene, which increase circulating levels of the phosphaturic hormone, fibroblast growth factor 23 (FGF23). Because XLH is a dominant disease, one mutant allele is sufficient for manifestation of the disease. However, the dosage effect of a PHEX mutation in XLH is not completely understood. To examine the effect of Phex genotypes, we compared serum biochemistries and skeletal measures between all five possible genotypes of a new murine model of XLH (Phex (K496X) or Phex (Jrt) ). Compared to sex-matched littermate controls, all Phex mutant mice had hypophosphatemia, mild hypocalcemia, and increased parathyroid hormone and alkaline phosphatase levels. Furthermore, mutant mice had markedly elevated serum Fgf23 levels due to increased Fgf23 expression and reduced cleavage of Fgf23. Although females with a homozygous Phex mutation were slightly more hypocalcemic and hypophosphatemic than heterozygous females, the two groups had comparable intact Fgf23 levels. Similarly, there was no difference in intact Fgf23 or phosphorus concentrations between hemizygous males and heterozygous females. Compared to heterozygous females, homozygous counterparts were significantly smaller and had shorter femurs with reduced bone mineral density, suggesting the existence of dosage effect in the skeletal phenotype of XLH. However, overall phenotypic trends in regards to mineral ion homeostasis were mostly unaffected by the presence of one or two mutant Phex allele(s). The lack of a gene dosage effect on circulating Fgf23 (and thus phosphorus) levels suggests that a Phex mutation may create the lower set point for extracellular phosphate concentrations.


Assuntos
Raquitismo Hipofosfatêmico Familiar/genética , Dosagem de Genes , Mutação , Endopeptidase Neutra Reguladora de Fosfato PHEX/genética , Endopeptidase Neutra Reguladora de Fosfato PHEX/metabolismo , Alelos , Animais , Densidade Óssea , Feminino , Fêmur/anatomia & histologia , Fatores de Crescimento de Fibroblastos/sangue , Genótipo , Heterozigoto , Masculino , Camundongos , Fenótipo , Fosfatos/química
17.
J Cell Biochem ; 113(7): 2432-41, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22573557

RESUMO

X-linked hypophosphatemic rickets (XLH) is a dominantly inherited disease characterized by renal phosphate wasting, aberrant vitamin D metabolism, and defective bone mineralization. It is known that XLH in humans and in certain mouse models is caused by inactivating mutations in PHEX/Phex (phosphate-regulating gene with homologies to endopeptidases on the X chromosome). By a genome-wide N-ethyl-N-nitrosourea (ENU)-induced mutagenesis screen in mice, we identified a dominant mouse mutation that exhibits the classic clinical manifestations of XLH, including growth retardation, skeletal abnormalities (rickets/osteomalacia), hypophosphatemia, and increased serum alkaline phosphatase (ALP) levels. Mapping and sequencing revealed that these mice carry a point mutation in exon 14 of the Phex gene that introduces a stop codon at amino acid 496 of the coding sequence (Phex(Jrt) also published as Phex(K496X) [Ichikawa et al., 2012]). Fgf23 mRNA expression as well as that of osteocalcin, bone sialoprotein, and matrix extracellular phosphoglycoprotein was upregulated in male mutant long bone, but that of sclerostin was unaffected. Although Phex mRNA is expressed in bone from mutant hemizygous male mice (Phex(Jrt)/Y mice), no Phex protein was detected in immunoblots of femoral bone protein. Stromal cultures from mutant bone marrow were indistinguishable from those of wild-type mice with respect to differentiation and mineralization. The ability of Phex(Jrt)/Y osteoblasts to mineralize and the altered expression levels of matrix proteins compared with the well-studied Hyp mice makes it a unique model with which to further explore the clinical manifestations of XLH and its link to FGF23 as well as to evaluate potential new therapeutic strategies.


Assuntos
Osso e Ossos/patologia , Modelos Animais de Doenças , Raquitismo Hipofosfatêmico Familiar , Doenças Genéticas Ligadas ao Cromossomo X , Endopeptidase Neutra Reguladora de Fosfato PHEX/genética , Mutação Puntual , Animais , Sequência de Bases , Células da Medula Óssea , Osso e Ossos/metabolismo , Calcificação Fisiológica/genética , Calcificação Fisiológica/fisiologia , Células Cultivadas , Mapeamento Cromossômico , Etilnitrosoureia , Proteínas da Matriz Extracelular/biossíntese , Raquitismo Hipofosfatêmico Familiar/genética , Raquitismo Hipofosfatêmico Familiar/metabolismo , Raquitismo Hipofosfatêmico Familiar/patologia , Feminino , Glicoproteínas/biossíntese , Sialoproteína de Ligação à Integrina/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutagênicos/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteocalcina/biossíntese , Fosfoproteínas/biossíntese , RNA Mensageiro/biossíntese , Análise de Sequência de DNA , Células Estromais
18.
Bioinformatics ; 28(14): 1879-86, 2012 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-22611130

RESUMO

MOTIVATION: One of the fundamental questions in genetics study is to identify functional DNA variants that are responsible to a disease or phenotype of interest. Results from large-scale genetics studies, such as genome-wide association studies (GWAS), and the availability of high-throughput sequencing technologies provide opportunities in identifying causal variants. Despite the technical advances, informatics methodologies need to be developed to prioritize thousands of variants for potential causative effects. RESULTS: We present regSNPs, an informatics strategy that integrates several established bioinformatics tools, for prioritizing regulatory SNPs, i.e. the SNPs in the promoter regions that potentially affect phenotype through changing transcription of downstream genes. Comparing to existing tools, regSNPs has two distinct features. It considers degenerative features of binding motifs by calculating the differences on the binding affinity caused by the candidate variants and integrates potential phenotypic effects of various transcription factors. When tested by using the disease-causing variants documented in the Human Gene Mutation Database, regSNPs showed mixed performance on various diseases. regSNPs predicted three SNPs that can potentially affect bone density in a region detected in an earlier linkage study. Potential effects of one of the variants were validated using luciferase reporter assay.


Assuntos
Biologia Computacional/métodos , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Área Sob a Curva , Sítios de Ligação , Bases de Dados Genéticas , Ligação Genética , Genoma Humano , Estudo de Associação Genômica Ampla , Células HEK293 , Humanos , Fenótipo , Curva ROC
19.
J Bone Miner Res ; 27(2): 453-60, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22006791

RESUMO

Mutations in the PHEX gene cause X-linked hypophosphatemia (XLH). Hypophosphatemia in XLH results from increased circulating levels of a phosphaturic hormone, fibroblast growth factor 23 (FGF23), which inhibits renal phosphate reabsorption and 1,25-dihydroxyvitamin D (calcitriol) synthesis. The current standard therapy for XLH--high-dose phosphate and calcitriol--further increases FGF23 concentrations, suggesting that patients with XLH may have an altered response to extracellular phosphate. To test for the presence of abnormal phosphate responsiveness, we compared serum biochemistries and femoral Fgf23 mRNA expression between wild-type mice, murine models of XLH (Phex(K496X)) and hyperphosphatemic tumoral calcinosis (Galnt3(-/-)), and Galnt3/Phex double-mutant mice. Phex mutant mice had not only increased Fgf23 expression but also reduced proteolytic cleavage of intact Fgf23 protein, resulting in markedly elevated intact Fgf23 levels and consequent hypophosphatemia. In contrast, despite markedly increased Fgf23 expression, Galnt3 knockout mice had significantly high proteolytic cleavage of Fgf23 protein, leading to low intact Fgf23 concentrations and hyperphosphatemia. Galnt3/Phex double-mutant mice had an intermediate biochemical phenotype between wild-type and Phex mutant mice, including slightly elevated intact Fgf23 concentrations with milder hypophosphatemia. Despite the hypophosphatemia, double-mutant mice attempted to reduce serum phosphate back to the level of Phex mutant mice by upregulating Fgf23 expression as much as 24-fold higher than Phex mutant mice. These data suggest that Phex mutations alter the responsiveness of bone cells to extracellular phosphate concentrations and may create a lower set point for "normal" phosphate levels.


Assuntos
Raquitismo Hipofosfatêmico Familiar/enzimologia , Raquitismo Hipofosfatêmico Familiar/genética , Doenças Genéticas Ligadas ao Cromossomo X , Mutação/genética , Osteócitos/efeitos dos fármacos , Osteócitos/metabolismo , Endopeptidase Neutra Reguladora de Fosfato PHEX/genética , Fosfatos/farmacologia , Animais , Modelos Animais de Doenças , Raquitismo Hipofosfatêmico Familiar/sangue , Feminino , Fêmur/efeitos dos fármacos , Fêmur/metabolismo , Fêmur/patologia , Fatores de Crescimento de Fibroblastos/sangue , Fatores de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , N-Acetilgalactosaminiltransferases/deficiência , N-Acetilgalactosaminiltransferases/metabolismo , Osteócitos/patologia , Endopeptidase Neutra Reguladora de Fosfato PHEX/metabolismo , Fenótipo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
20.
Endocrinology ; 152(12): 4504-13, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22009723

RESUMO

Mutations in the GALNT3 gene cause tumoral calcinosis characterized by ectopic calcifications due to persistent hyperphosphatemia. We recently developed Galnt3 knockout mice in a mixed background, which had hyperphosphatemia with increased bone mineral density (BMD) and infertility in males. To test the effect of dietary phosphate intake on their phenotype, Galnt3 knockout mice were generated in the C57BL/6J strain and fed various phosphate diets: 0.1% (low), 0.3% (low normal), 0.6% (normal), and 1.65% (high). Sera were analyzed for calcium, phosphorus, alkaline phosphatase, creatinine, blood urine nitrogen, 1,25-dihydroxyvitamin D, osteocalcin, tartrate-resistant acid phosphatase 5b, and fibroblast growth factor 23 (Fgf23). Femurs were evaluated by dual-energy x-ray absorptiometry, dynamic histomorphometry, and/or microcomputed tomography. Galnt3 knockout mice in C57BL/6J had the same biochemical phenotype observed in our previous study: hyperphosphatemia, inappropriately normal 1,25-dihydroxyvitamin D level, decreased alkaline phosphatase activity, and low intact Fgf23 concentration but high Fgf23 fragments. Skeletal analyses of their femurs revealed significantly high BMD with increased cortical bone area and trabecular bone volume. On all four phosphate diets, Galnt3 knockout mice had consistently higher phosphorus levels and lower alkaline phosphatase and intact Fgf23 concentrations than littermate controls. The low-phosphate diet normalized serum phosphorus, alkaline phosphatase, and areal BMD but failed to correct male infertility in Galnt3 knockout mice. The high-phosphate diet did not increase serum phosphorus concentration in either mutant or control mice due to a compensatory increase in circulating intact Fgf23 levels. In conclusion, dietary phosphate restriction normalizes biochemical and skeletal phenotypes of Galnt3 knockout mice and, thus, can be an effective therapy for tumoral calcinosis.


Assuntos
Calcinose/dietoterapia , N-Acetilgalactosaminiltransferases/fisiologia , Neoplasias/patologia , Fosfatos/farmacologia , Animais , Densidade Óssea/efeitos dos fármacos , Osso e Ossos , Calcinose/prevenção & controle , Dieta , Modelos Animais de Doenças , Infertilidade Masculina/tratamento farmacológico , Masculino , Camundongos , Camundongos Knockout , N-Acetilgalactosaminiltransferases/deficiência , Fosfatos/administração & dosagem
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