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1.
Nature ; 532(7598): 250-4, 2016 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-27042933

RESUMO

Cancer is a disease of ageing. Clinically, aged cancer patients tend to have a poorer prognosis than young. This may be due to accumulated cellular damage, decreases in adaptive immunity, and chronic inflammation. However, the effects of the aged microenvironment on tumour progression have been largely unexplored. Since dermal fibroblasts can have profound impacts on melanoma progression, we examined whether age-related changes in dermal fibroblasts could drive melanoma metastasis and response to targeted therapy. Here we find that aged fibroblasts secrete a Wnt antagonist, sFRP2, which activates a multi-step signalling cascade in melanoma cells that results in a decrease in ß-catenin and microphthalmia-associated transcription factor (MITF), and ultimately the loss of a key redox effector, APE1. Loss of APE1 attenuates the response of melanoma cells to DNA damage induced by reactive oxygen species, rendering the cells more resistant to targeted therapy (vemurafenib). Age-related increases in sFRP2 also augment both angiogenesis and metastasis of melanoma cells. These data provide an integrated view of how fibroblasts in the aged microenvironment contribute to tumour progression, offering new possibilities for the design of therapy for the elderly.


Assuntos
Envelhecimento/metabolismo , Resistencia a Medicamentos Antineoplásicos , Melanoma/tratamento farmacológico , Melanoma/patologia , Proteínas de Membrana/metabolismo , Metástase Neoplásica , Microambiente Tumoral , Adulto , Animais , Linhagem Celular Tumoral , Meios de Cultivo Condicionados/farmacologia , Dano ao DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Progressão da Doença , Fibroblastos/metabolismo , Humanos , Indóis/farmacologia , Indóis/uso terapêutico , Masculino , Melanoma/irrigação sanguínea , Melanoma/genética , Camundongos , Fator de Transcrição Associado à Microftalmia/metabolismo , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Neovascularização Patológica , Estresse Oxidativo , Fenótipo , Espécies Reativas de Oxigênio/metabolismo , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Vemurafenib , Via de Sinalização Wnt , Proteína Wnt1/antagonistas & inibidores , beta Catenina/metabolismo
2.
Am J Hum Genet ; 102(4): 696-705, 2018 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-29606302

RESUMO

AEBP1 encodes the aortic carboxypeptidase-like protein (ACLP) that associates with collagens in the extracellular matrix (ECM) and has several roles in development, tissue repair, and fibrosis. ACLP is expressed in bone, the vasculature, and dermal tissues and is involved in fibroblast proliferation and mesenchymal stem cell differentiation into collagen-producing cells. Aebp1-/- mice have abnormal, delayed wound repair correlating with defects in fibroblast proliferation. In this study, we describe four individuals from three unrelated families that presented with a unique constellation of clinical findings including joint laxity, redundant and hyperextensible skin, poor wound healing with abnormal scarring, osteoporosis, and other features reminiscent of Ehlers-Danlos syndrome (EDS). Analysis of skin biopsies revealed decreased dermal collagen with abnormal collagen fibrils that were ragged in appearance. Exome sequencing revealed compound heterozygous variants in AEBP1 (c.1470delC [p.Asn490_Met495delins(40)] and c.1743C>A [p.Cys581∗]) in the first individual, a homozygous variant (c.1320_1326del [p.Arg440Serfs∗3]) in the second individual, and a homozygous splice site variant (c.1630+1G>A) in two siblings from the third family. We show that ACLP enhances collagen polymerization and binds to several fibrillar collagens via its discoidin domain. These studies support the conclusion that bi-allelic pathogenic variants in AEBP1 are the cause of this autosomal-recessive EDS subtype.


Assuntos
Alelos , Carboxipeptidases/genética , Colágeno/metabolismo , Tecido Conjuntivo/patologia , Síndrome de Ehlers-Danlos/genética , Mutação/genética , Proteínas Repressoras/genética , Adulto , Sequência de Aminoácidos , Carboxipeptidases/química , Criança , Pré-Escolar , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Masculino , Domínios Proteicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/química , Pele/patologia , Pele/ultraestrutura , Adulto Jovem
3.
Am J Med Genet A ; 179(5): 797-802, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30793832

RESUMO

Vascular Ehlers-Danlos syndrome (vEDS) is a connective tissue disorder due to defective type III collagen production and is associated with arterial rupture, spontaneous intestinal perforation, and gravid uterine rupture. Spontaneous pneumothorax and/or hemothorax (P/HTX) also occurs in vEDS patients. The temporal relation of pulmonary manifestations to arterial and intestinal complications in vEDS has not been well described. This was investigated in a multi-institutional retrospective case series of vEDS patients with confirmatory testing for COL3A1 mutation between 2000 and 2012. Data abstracted included demographics, family histories, presentation, and management of associated complications. Ninety-six cases (39% males, mean age 38.6 ± 15.5 years, range 8-79) had confirmatory testing for vEDS. P/HTX was documented in 17 (17.7%) cases. Most P/HTX preceded the diagnosis of vEDS (81%). Diagnosis of vEDS was made after arterial or intestinal complications at a mean of 7 years (range 0-26) post the initial P/HTX. In conclusion, spontaneous P/HTX is an early manifestation of vEDS frequently preceding an arterial complication or intestinal perforation. Thus, a spontaneous P/HTX in a young patient should trigger a differential diagnosis that includes vEDS. This should lead to an investigation of other vEDS features and subsequent genetic testing if vEDS features are present.


Assuntos
Síndrome de Ehlers-Danlos/complicações , Síndrome de Ehlers-Danlos/genética , Hemotórax/etiologia , Fenótipo , Pneumotórax/etiologia , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Colágeno Tipo III/genética , Estudos Transversais , Síndrome de Ehlers-Danlos/diagnóstico , Feminino , Estudos de Associação Genética , Testes Genéticos , Hemotórax/diagnóstico , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Pneumotórax/diagnóstico , Prevalência , Adulto Jovem
5.
FASEB J ; 28(8): 3313-24, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24732132

RESUMO

Fibromuscular dysplasia (FMD) is a rare, nonatherosclerotic arterial disease for which the molecular basis is unknown. We comprehensively studied 47 subjects with FMD, including physical examination, spine magnetic resonance imaging, bone densitometry, and brain magnetic resonance angiography. Inflammatory biomarkers in plasma and transforming growth factor ß (TGF-ß) cytokines in patient-derived dermal fibroblasts were measured by ELISA. Arterial pathology other than medial fibrodysplasia with multifocal stenosis included cerebral aneurysm, found in 12.8% of subjects. Extra-arterial pathology included low bone density (P<0.001); early onset degenerative spine disease (95.7%); increased incidence of Chiari I malformation (6.4%) and dural ectasia (42.6%); and physical examination findings of a mild connective tissue dysplasia (95.7%). Screening for mutations causing known genetically mediated arteriopathies was unrevealing. We found elevated plasma TGF-ß1 (P=0.009), TGF-ß2 (P=0.004) and additional inflammatory markers, and increased TGF-ß1 (P=0.0009) and TGF-ß2 (P=0.0001) secretion in dermal fibroblast cell lines from subjects with FMD compared to age- and gender-matched controls. Detailed phenotyping of patients with FMD allowed us to demonstrate that FMD is a systemic disease with alterations in common with the spectrum of genetic syndromes that involve altered TGF-ß signaling and offers TGF-ß as a marker of FMD.


Assuntos
Fibroblastos/metabolismo , Displasia Fibromuscular/metabolismo , Fator de Crescimento Transformador beta1/biossíntese , Adulto , Idoso , Malformação de Arnold-Chiari/complicações , Biomarcadores/sangue , Densidade Óssea , Doenças Ósseas Metabólicas/etiologia , Estudos de Casos e Controles , Ciclo Celular , Linhagem Celular , Tecido Conjuntivo/patologia , Derme/patologia , Dilatação Patológica , Dura-Máter/patologia , Feminino , Displasia Fibromuscular/complicações , Displasia Fibromuscular/patologia , Humanos , Inflamação/sangue , Inflamação/etiologia , Mediadores da Inflamação/sangue , Instabilidade Articular/etiologia , Masculino , Pessoa de Meia-Idade , Fenótipo , Artéria Renal/patologia , Método Simples-Cego , Coluna Vertebral/patologia , Fator de Crescimento Transformador beta1/sangue , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta2/sangue , Fator de Crescimento Transformador beta2/metabolismo , Adulto Jovem
6.
J Vasc Surg ; 60(1): 160-9, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24650746

RESUMO

OBJECTIVE: The management of arterial pathology in individuals with vascular Ehlers-Danlos syndrome (vEDS) remains a challenge. Here we describe the correlation between COL3A1 gene mutation type and the clinical phenotype in individuals with vEDS. METHODS: Individuals with confirmed molecular diagnoses of vEDS were enrolled in a multi-institutional natural history study. Data collected included demographics, clinical and family histories, arterial pathology (aneurysm, dissection, and rupture), operative details, and autopsy reports. Individuals were classified into two cohorts by the type of COL3A1 mutations and their effect on the amount of normal collagen produced: those with mutations that lead to minimal (MIN) production (10%-15%) of normal type III collagen and those with haploinsufficiency (HI) mutations that lead to production of 50% of the normal type III collagen. RESULTS: A cohort of 68 individuals (72%) from 56 families had arterial pathology (44% male) with 13% HI. The HI group was older at the time of their first vascular event (mean, 42 [range, 26-58] years vs 33 [range, 8-62] years; P = .016) and had a higher incidence of aortic pathology than the MIN group (56% vs 21%; P = .025). Visceral arterial pathology was seen in 43 arteries in 23 individuals in the MIN group vs only one artery in five individuals in the HI group. Emergency surgical procedures were more likely to be undertaken when vEDS diagnosis was not known (81% vs 41%; P = .005), and 81% of these procedures were open surgical repair compared with 19% endovascular repairs (P = .019). Open and endovascular repairs were equally used in the elective setting. Postoperative complications were highest when the diagnosis of vEDS was not known (62% vs 14%; P < .001) and when procedures were undertaken in an emergency setting (5% vs 55% P < .001). Mortality due to arterial complications was 0% in the HI cohort and 21% in the MIN cohort (P = .132). CONCLUSIONS: Arterial pathology in vEDS individuals is related to the underlying COL3A1 mutation type. The arterial pathology in individuals with HI mutations occurs at later ages with a higher incidence of aortic disease compared with other COL3A1 mutation types. Molecular diagnosis is recommended because diagnosis confirmation, appropriate surveillance, and prophylactic interventions in an elective setting improve surgical outcomes.


Assuntos
Aneurisma Roto/genética , Aneurisma Aórtico/genética , Dissecção Aórtica/genética , Colágeno Tipo III/genética , Síndrome de Ehlers-Danlos/genética , Adolescente , Adulto , Dissecção Aórtica/cirurgia , Aneurisma Roto/cirurgia , Angioplastia/efeitos adversos , Aneurisma Aórtico/cirurgia , Artéria Celíaca/cirurgia , Criança , Colágeno Tipo III/biossíntese , Síndrome de Ehlers-Danlos/classificação , Síndrome de Ehlers-Danlos/diagnóstico , Síndrome de Ehlers-Danlos/mortalidade , Emergências , Feminino , Testes Genéticos , Genótipo , Artéria Hepática/cirurgia , Humanos , Aneurisma Ilíaco/genética , Aneurisma Ilíaco/cirurgia , Masculino , Artéria Mesentérica Superior/cirurgia , Pessoa de Meia-Idade , Mutação , Fenótipo , Artéria Renal/cirurgia , Artéria Esplênica/cirurgia , Resultado do Tratamento , Vísceras/irrigação sanguínea , Adulto Jovem
7.
FASEB J ; 26(2): 668-77, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22038052

RESUMO

The vascular type of the Ehlers-Danlos syndrome (vEDS) is caused by dominant-negative mutations in the procollagen type III (COL3A1) gene. Patients with this autosomal dominant disorder have a shortened life expectancy due to complications from ruptured vessels or hollow organs. We tested the effectiveness of allele-specific RNA interference (RNAi) to reduce the mutated phenotype in fibroblasts. Small-interfering RNAs (siRNAs) discriminating between wild-type and mutant COL3A1 allele were identified by a luciferase reporter gene assay and in primary fibroblasts from a normal donor and a patient with vEDS. The best discriminative siRNA with the mutation at position 10 resulted in >90% silencing of the mutant allele without affecting the wild-type allele. Transmission and immunogold electron microscopy of extracted extracellular matrices from untreated fibroblasts of the patient with vEDS revealed structurally abnormal fibrils. After siRNA treatment, collagen fibrils became similar to fibrils from fibroblasts of normal and COL3A1 haploinsufficient donors. In addition, it was shown that expression of mutated COL3A1 activates the unfolded protein response and that reduction of the amount of mutated protein by siRNA reduces cellular stress. Taken together, the results provide evidence that allele-specific siRNAs are able to reduce negative effects of mutated COL3A1 proteins. Thus, the application of allele-specific RNAi may be a promising direction for future personalized therapies to reduce the severity of vEDS.


Assuntos
Síndrome de Ehlers-Danlos/genética , Síndrome de Ehlers-Danlos/terapia , Técnicas de Silenciamento de Genes , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/uso terapêutico , Alelos , Substituição de Aminoácidos , Sequência de Bases , Células Cultivadas , Colágeno Tipo III/genética , Síndrome de Ehlers-Danlos/patologia , Matriz Extracelular/ultraestrutura , Fibroblastos/metabolismo , Fibroblastos/patologia , Genes Reporter , Terapia Genética/métodos , Haploinsuficiência , Humanos , Luciferases/genética , Microscopia Imunoeletrônica , Proteínas Mutantes/genética , Mutação , Mutação de Sentido Incorreto , Fenótipo , Medicina de Precisão , Interferência de RNA , Resposta a Proteínas não Dobradas/genética
8.
Hum Genet ; 131(12): 1889-94, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22886582

RESUMO

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD) is an autosomal recessive disorder of cortisol biosynthesis caused by CYP21A2 mutations. An increase in gene copy number variation (CNV) exists at the CYP21A2 locus. CNV of C4, a neighboring gene that encodes complement component 4, is associated with autoimmune disease susceptibility. In this study, we performed comprehensive genetic analysis of the RP-C4-CYP21-TNX (RCCX) region in 127 unrelated 21-OHD patients (100 classic, 27 nonclassic). C4 copy number was determined by Southern blot. C4 CNV and serum C4 levels were evaluated in relation to CYP21A2 mutations and relevant phenotypes. We found that the most common CYP21A2 mutation associated with the nonclassic form of CAH, V281L, was associated with high C4 copy number (p = 7.13 × 10(-16)). Large CYP21A2 deletion, a common mutation associated with the classic form of CAH, was associated with low C4 copy number (p = 1.61 × 10(-14)). Monomodular RCCX with a short C4 gene, a risk factor for autoimmune disease, was significantly less frequent in CAH patients compared to population estimates (2.8 vs. 10.6 %; p = 1.08 × 10(-4)). In conclusion, CAH patients have increased C4 CNV, with mutation-specific associations that may be protective for autoimmune disease. The study of CYP21A2 in relation to neighboring genes provides insight into the genetics of CNV hotspots, an important determinant of human health.


Assuntos
Hiperplasia Suprarrenal Congênita/genética , Complemento C4/genética , Variações do Número de Cópias de DNA , Esteroide 21-Hidroxilase/genética , Adolescente , Hiperplasia Suprarrenal Congênita/enzimologia , Hiperplasia Suprarrenal Congênita/imunologia , Adulto , Autoimunidade/genética , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Dosagem de Genes , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinases/genética , Tenascina/genética
9.
Clin Chem ; 58(2): 421-30, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22156666

RESUMO

BACKGROUND: Chimeric CYP21A1P/CYP21A2 genes, caused by homologous recombination between CYP21A2 (cytochrome P450, family 21, subfamily A, polypeptide 2) and its highly homologous pseudogene CYP21A1P (cytochrome P450, family 21, subfamily A, polypeptide 1 pseudogene), are common in patients with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD). A comprehensive junction site analysis of chimeric CYP21A1P/CYP21A2 genes is needed for optimizing genetic analysis strategy and determining clinical relevance. METHODS: We conducted a comprehensive genetic analysis of chimeric CYP21A1P/CYP21A2 genes in a cohort of 202 unrelated 21-OHD patients. Targeted CYP21A2 mutation analysis was performed, and genotyping of chimeric CYP21A1P/CYP21A2 genes was cross-confirmed with Southern blot, RFLP, and multiplex ligation-dependent probe amplification analyses. Junction sites of chimera genes were determined by sequencing the long-PCR products amplified with primers CYP779f and Tena32F. An updated bioinformatics survey of Chi-like sequences was also performed. RESULTS: Of 100 probands with a chimeric allele, 96 had a chimera associated with the severe classic salt-wasting form of CAH, and the remaining 4 carried an uncommon attenuated chimera with junction sites upstream of In2G (c.293-13A/C>G), which is associated with a milder phenotype. In addition to 6 of 7 reported chimeras, we identified a novel classic chimera (CH-8) and a novel attenuated chimera (CH-9). Attenuated chimeras explained prior genotype-phenotype discrepancies in 3 of the patients. Sequencing the CYP779f/Tena32F amplicons accurately differentiated between classic and attenuated chimeras. The bioinformatics survey revealed enrichment of Chi-like sequences within or in the vicinity of intron 2. CONCLUSIONS: Junction site analysis can explain some genotype-phenotype discrepancies. Sequencing the well-established CYP779f/Tena32F amplicons is an unequivocal strategy for detecting attenuated chimeric CYP21A1P/CYP21A2 genes, which are clinically relevant.


Assuntos
Hiperplasia Suprarrenal Congênita/genética , Proteínas Mutantes Quiméricas/genética , Esteroide 21-Hidroxilase/genética , Sequência de Bases , Estudos de Coortes , Biologia Computacional , Genótipo , Humanos , Dados de Sequência Molecular , Mutação , Pseudogenes
10.
J Pharmacol Exp Ther ; 337(3): 621-7, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21363928

RESUMO

The vascular form of Ehlers-Danlos syndrome (vEDS), a rare disease with grave complications resulting from rupture of major arteries, is caused by mutations of collagen type III [α1 chain of collagen type III (COL3A1)]. The only, recently proven, preventive strategy consists of the reduction of arterial wall stress by ß-adrenergic blockers. The heterozygous (HT) Col3a1 knockout mouse has reduced expression of collagen III and recapitulates features of a mild presentation of the disease. The objective of this study was to determine whether changing the balance between synthesis and degradation of collagen by chronic treatment with doxycycline, a nonspecific matrix metalloproteinase (MMP) inhibitor, could prevent the development of vascular pathology in HT mice. After 3 months of treatment with doxycycline or placebo, 9-month-old HT or wild-type (WT) mice were subjected to surgical stressing of the aorta. A 3-fold increase in stress-induced aortic lesions found in untreated HT mice 1 week after intervention (cumulative score 4.5 ± 0.87 versus 1.3 ± 0.34 in WT, p < 0.001) was fully prevented in the doxycycline-treated group (1.1 ± 0.56, p < 0.001). Untreated HT mice showed increased MMP-9 activity in the carotid artery and decreased collagen content in the aorta; however, in doxycycline-treated animals there was normalization to the levels observed in WT mice. Doxycycline treatment inhibits the activity of tissue MMP and attenuates the decrease in the collagen content in aortas of mice haploinsufficient for collagen III, as well as prevents the development of stress-induced vessel pathology. The results suggest that doxycycline merits clinical testing as a treatment for vEDS.


Assuntos
Doenças da Aorta/prevenção & controle , Colágeno Tipo III/metabolismo , Doxiciclina/farmacologia , Síndrome de Ehlers-Danlos/tratamento farmacológico , Síndrome de Ehlers-Danlos/patologia , Inibidores de Metaloproteinases de Matriz , Inibidores de Proteases/farmacologia , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Aorta/patologia , Aorta/fisiopatologia , Doenças da Aorta/patologia , Artérias Carótidas/efeitos dos fármacos , Artérias Carótidas/enzimologia , Colo/efeitos dos fármacos , Colo/fisiologia , Síndrome de Ehlers-Danlos/fisiopatologia , Proteínas da Matriz Extracelular/metabolismo , Feminino , Metaloproteinases da Matriz/sangue , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Knockout , Pele/efeitos dos fármacos , Pele/enzimologia
11.
Circulation ; 120(6): 526-32, 2009 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-19635970

RESUMO

BACKGROUND: Marfan syndrome (MFS) is caused by mutations in the fibrillin-1 gene and dysregulation of transforming growth factor-beta (TGF-beta). Recent evidence suggests that losartan, an angiotensin II type 1 blocker that blunts TGF-beta activation, may be an effective treatment for MFS. We hypothesized that dysregulation of TGF-beta might be mirrored in circulating TGF-beta concentrations. METHODS AND RESULTS: Serum obtained from MFS mutant mice (Fbn1(C1039G/+)) treated with losartan was analyzed for circulating TGF-beta1 concentrations and compared with those from placebo-treated and wild-type mice. Aortic root size was measured by echocardiography. Data were validated in patients with MFS and healthy individuals. In mice, circulating total TGF-beta1 concentrations increased with age and were elevated in older untreated Fbn1(C1039G/+) mice compared with wild-type mice (P=0.01; n=16; mean+/-SEM, 115+/-8 ng/mL versus n=17; mean+/-SEM, 92+/-4 ng/mL). Losartan-treated Fbn1(C1039G/+) mice had lower total TGF-beta1 concentrations compared with age-matched Fbn1(C1039G/+) mice treated with placebo (P=0.01; n=18; 90+/-5 ng/mL), and circulating total TGF-beta1 levels were indistinguishable from those of age-matched wild-type mice (P=0.8). Correlation was observed between circulating TGF-beta1 levels and aortic root diameters in Fbn1(C1039G/+) and wild-type mice (P=0.002). In humans, circulating total TGF-beta1 concentrations were elevated in patients with MFS compared with control individuals (P<0.0001; n=53; 15+/-1.7 ng/mL versus n=74; 2.5+/-0.4 ng/mL). MFS patients treated with losartan (n=55) or beta-blocker (n=80) showed significantly lower total TGF-beta1 concentrations compared with untreated MFS patients (P< or =0.05). CONCLUSIONS: Circulating TGF-beta1 concentrations are elevated in MFS and decrease after administration of losartan, beta-blocker therapy, or both and therefore might serve as a prognostic and therapeutic marker in MFS.


Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Biomarcadores/sangue , Losartan/farmacologia , Síndrome de Marfan/sangue , Síndrome de Marfan/tratamento farmacológico , Fator de Crescimento Transformador beta1/sangue , Adulto , Animais , Aorta/diagnóstico por imagem , Ecocardiografia , Feminino , Heterozigoto , Humanos , Masculino , Síndrome de Marfan/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Pessoa de Meia-Idade , Prognóstico
12.
J Vasc Surg ; 51(1): 131-8; discussion 138-9, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19879095

RESUMO

OBJECTIVES: There has been debate regarding the safety of performing elective procedures in patients with vascular manifestations associated with Ehlers-Danlos syndrome (EDS). The purpose of this study was to review the surgical management and clinical outcomes of EDS patients undergoing vascular procedures at a tertiary medical center with multimodality expertise in connective tissue disorders. METHODS: All patients with EDS undergoing endovascular and open vascular procedures at a single-institution academic medical center from 1994 to 2009 were retrospectively reviewed. Clinical data were evaluated including patient demographics, length of stay (LOS), and mortality outcomes during hospital course and long-term follow-up. RESULTS: A total of 40 patients with EDS were identified, including individuals diagnosed with classic (n = 15), hypermobility (n = 16), and vascular (n = 9) types of EDS. These patients collectively underwent 45 endovascular and 18 open procedures for vascular disease during the time period, including embolization (n = 37), angioplasty (n = 8), arterial bypass (n = 5), and aortic aneurysm repair (n = 13). All cases were performed electively, except for one (2%) urgent endovascular and one (5%) emergent open procedure. Endovascular procedures were associated with a median LOS (interquartile range [IQR]) of 2 (1 to 3) days with no procedure-related mortality or in-hospital deaths among all EDS types, whereas open vascular procedures had median LOS (IQR) of 6 (5 to 8) days with one (6%) in-hospital death occurring in a vascular EDS patient. Survival free of any complication at 5 years was 85% and 54% following endovascular and open procedures, respectively. CONCLUSIONS: The elective surgical management of vascular disorders in EDS patients using open and endovascular procedures has been associated with good outcomes. Our results suggest that vascular interventions in these EDS patients can be safely performed and should not be withheld until rupture or acute symptoms arise.


Assuntos
Síndrome de Ehlers-Danlos/complicações , Embolização Terapêutica , Doenças Vasculares/terapia , Procedimentos Cirúrgicos Vasculares , Adolescente , Adulto , Angioplastia , Síndrome de Ehlers-Danlos/diagnóstico por imagem , Síndrome de Ehlers-Danlos/economia , Síndrome de Ehlers-Danlos/mortalidade , Síndrome de Ehlers-Danlos/terapia , Embolização Terapêutica/efeitos adversos , Embolização Terapêutica/economia , Embolização Terapêutica/mortalidade , Feminino , Mortalidade Hospitalar , Humanos , Estimativa de Kaplan-Meier , Longevidade , Masculino , Procedimentos Cirúrgicos Minimamente Invasivos , Estudos Retrospectivos , Medição de Risco , Fatores de Tempo , Tomografia Computadorizada por Raios X , Resultado do Tratamento , Doenças Vasculares/diagnóstico por imagem , Doenças Vasculares/economia , Doenças Vasculares/etiologia , Doenças Vasculares/mortalidade , Procedimentos Cirúrgicos Vasculares/efeitos adversos , Procedimentos Cirúrgicos Vasculares/economia , Procedimentos Cirúrgicos Vasculares/mortalidade , Adulto Jovem
13.
Am J Med Genet A ; 149A(12): 2803-8, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19921645

RESUMO

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is an autosomal recessive disorder and is the most common cause of ambiguous genitalia in the newborn. The genes encoding 21-hydroxylase, CYP21A2, and tenascin-X (TNX), TNXB, are located within the HLA complex, in a region of high gene density termed the RCCX module. The module has multiple pseudogenes as well as tandem repeat sequences that promote misalignment during meiosis leading to complex gene rearrangements, deletions and gene conversion events. CYP21A2 mutations cause CAH, and TNX deficiency has been identified as a cause of hypermobility type Ehlers-Danlos syndrome (EDS). Here we report on a three-generation family with a heterozygous deletion encompassing CYP21A2 and TNXB that initially came to medical attention due to the diagnosis of CAH in the proposita. Southern blotting and PCR-based analysis of the RCCX module revealed a CYP21A2 deletion extending into TNXB in one allele and a CYP21A2 point mutation in the other allele. Family history is notable for joint hypermobility. Additional radiological and clinical investigations showed a quadricuspid aortic valve, single kidney, bicornuate uterus and a bifid uvula in the proposita, and mitral valve prolapse in her mother. These findings further delineate the phenotype of the CAH-TNX contiguous gene deletion syndrome and point to an intersection of connective tissue dysplasias with a common gene-mediated endocrine disorder.


Assuntos
Valva Aórtica/anormalidades , Deleção de Genes , Doenças das Valvas Cardíacas/genética , Esteroide 21-Hidroxilase/genética , Tenascina/genética , Adulto , Southern Blotting , Criança , Feminino , Genoma Humano/genética , Haplótipos/genética , Doenças das Valvas Cardíacas/diagnóstico por imagem , Humanos , Recém-Nascido , Imageamento por Ressonância Magnética , Masculino , Linhagem , Fenótipo , Reação em Cadeia da Polimerase , Ultrassonografia
14.
J Neurosurg Spine ; 7(6): 601-9, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18074684

RESUMO

OBJECT: Chiari malformation Type I (CM-I) is generally regarded as a disorder of the paraxial mesoderm. The authors report an association between CM-I and hereditary disorders of connective tissue (HDCT) that can present with lower brainstem symptoms attributable to occipitoatlantoaxial hypermobility and cranial settling. METHODS: The prevalence of HDCT was determined in a prospectively accrued cohort of 2813 patients with CM-I. All patients underwent a detailed medical and neuroradiological workup that included an assessment of articular mobility. Osseous structures composing the craniocervical junction were investigated morphometrically using reconstructed 3D computed tomography and plain x-ray images in 114 patients with HDCT/CM-I, and the results were compared with those obtained in patients with CM-I (55 cases) and healthy control individuals (55 cases). RESULTS: The diagnostic criteria for Ehlers-Danlos syndrome and related HDCT were met in 357 (12.7%) of the 2813 cases. Hereditability was generally compatible with a pattern of autosomal dominant transmission with variable expressivity. The diagnostic features of HDCT/CM-I were distinguished from those of CM-I by clinical stigmata of connective tissue disease, a greater female preponderance (8:1 compared with 3:1, p < 0.001), and a greater incidence of lower brainstem symptoms (0.41 compared with 0.11, p < 0.001), retroodontoid pannus formation (0.71 compared with 0.11, p < 0.001), and hypoplasia of the oropharynx (0.44 compared with 0.02, p < 0.001). Measurements of the basion-dens interval, basion-atlas interval, atlas-dens interval, dens-atlas interval, clivus-atlas angle, clivus-axis angle, and atlas-axis angle were the same in the supine and upright positions in healthy control individuals and patients with CM-I. In patients with HDCT/CM-I, there was a reduction of the basion-dens interval (3.6 mm, p < 0.001), an enlargement of the basion-atlas interval (3.0 mm, p < 0.001), and a reduction of the clivus-axis angle (10.8 degrees, p < 0.001), clivus-atlas angle (5.8 degrees, p < 0.001), and atlas-axis angle (5.3 degrees, p < 0.001) on assumption of the upright position. These changes were reducible by cervical traction or returning to the supine position. CONCLUSIONS: The identification of HDCT in 357 patients with CM-I establishes an association between two presumably unrelated mesodermal disorders. Morphometric evidence in this cohort-cranial settling, posterior gliding of the occipital condyles, and reduction of the clivus-axis angle, clivus-atlas angle, and atlas-axis angle in the upright position-suggests that hypermobility of the occipitoatlantal and atlantoaxial joints contributes to retroodontoid pannus formation and symptoms referable to basilar impression.


Assuntos
Malformação de Arnold-Chiari/complicações , Articulação Atlantoaxial , Articulação Atlantoccipital , Doenças do Tecido Conjuntivo/complicações , Doenças do Tecido Conjuntivo/genética , Instabilidade Articular/etiologia , Crânio/diagnóstico por imagem , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Malformação de Arnold-Chiari/diagnóstico por imagem , Malformação de Arnold-Chiari/terapia , Articulação Atlantoaxial/diagnóstico por imagem , Articulação Atlantoccipital/diagnóstico por imagem , Criança , Pré-Escolar , Estudos de Coortes , Doenças do Tecido Conjuntivo/terapia , Síndrome de Ehlers-Danlos/complicações , Síndrome de Ehlers-Danlos/genética , Feminino , Genes Dominantes , Humanos , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Instabilidade Articular/diagnóstico por imagem , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Síndrome , Tomografia Computadorizada por Raios X , Tração
15.
J Clin Endocrinol Metab ; 100(8): E1143-52, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26075496

RESUMO

CONTEXT: The contiguous gene deletion syndrome (CAH-X) was described in a subset (7%) of congenital adrenal hyperplasia (CAH) patients with a TNXA/TNXB chimera, resulting in deletions of CYP21A2, encoding 21-hydroxylase necessary for cortisol biosynthesis, and TNXB, encoding the extracellular matrix glycoprotein tenascin-X (TNX). This TNXA/TNXB chimera is characterized by a 120-bp deletion in exon 35 and results in TNXB haploinsufficiency, disrupted TGF-ß signaling, and an Ehlers Danlos syndrome phenotype. OBJECTIVE: The objective of the study was to determine the genetic status of TNXB and resulting protein defects in CAH patients with a CAH-X phenotype but not the previously described TNXA/TNXB chimera. Design, Settings, Participants, and Intervention: A total of 246 unrelated CAH patients were screened for TNXB defects. Genetic defects were investigated by Southern blotting, multiplex ligation-dependent probe amplification, Sanger, and next-generation sequencing. Dermal fibroblasts and tissue were used for immunoblotting, immunohistochemical, and coimmunoprecipitation experiments. MAIN OUTCOME MEASURES: The genetic and protein status of tenascin-X in phenotypic CAH-X patients was measured. RESULTS: Seven families harbor a novel TNXB missense variant c.12174C>G (p.C4058W) and a clinical phenotype consistent with hypermobility-type Ehlers Danlos syndrome. Fourteen CAH probands carry previously described TNXA/TNXB chimeras, and seven unrelated patients carry the novel TNXB variant, resulting in a CAH-X prevalence of 8.5%. This highly conserved pseudogene-derived variant in the TNX fibrinogen-like domain is predicted to be deleterious and disulfide bonded, results in reduced dermal elastin and fibrillin-1 staining and altered TGF-ß1 binding, and represents a novel TNXA/TNXB chimera. Tenascin-X protein expression was normal in dermal fibroblasts, suggesting a dominant-negative effect. CONCLUSIONS: CAH-X syndrome is commonly found in CAH due to 21-hydroxylase deficiency and may result from various etiological mechanisms.


Assuntos
Hiperplasia Suprarrenal Congênita/complicações , Síndrome de Ehlers-Danlos/classificação , Síndrome de Ehlers-Danlos/complicações , Adolescente , Hiperplasia Suprarrenal Congênita/epidemiologia , Hiperplasia Suprarrenal Congênita/genética , Adulto , Idoso , Criança , Pré-Escolar , Estudos de Coortes , Síndrome de Ehlers-Danlos/epidemiologia , Síndrome de Ehlers-Danlos/genética , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Fenótipo , Adulto Jovem
16.
Eur J Med Genet ; 57(2-3): 95-102, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24380766

RESUMO

Patients with congenital adrenal hyperplasia (CAH) with tenascin-X deficiency (CAH-X syndrome) have both endocrine imbalances and characteristic Ehlers Danlos syndrome phenotypes. Unlike other subtypes, tenascin-X-related Ehlers Danlos syndrome is caused by an extracellular matrix protein deficiency rather than a defect in fibrillar collagen or a collagen-modifying enzyme, and the understanding of the disease mechanisms is limited. We hypothesized that transforming growth factor-ß pathway dysregulation may, in part, be responsible for connective tissue phenotypes observed in CAH-X, due to this pathway's known role in connective tissue disorders. Fibroblasts and direct tissue from human skin biopsies from CAH-X probands and age- and sex-matched controls were screened for transforming growth factor-ß biomarkers known to be dysregulated in other hereditary disorders of connective tissue. In CAH-X fibroblast lines and dermal tissue, pSmad1/5/8 was significantly upregulated compared to controls, suggesting involvement of the bone morphogenetic protein pathway. Additionally, CAH-X samples compared to controls exhibited significant increases in fibroblast-secreted TGF-ß3, a cytokine important in secondary palatal development, and in plasma TGF-ß2, a cytokine involved in cardiac function and development, as well as palatogenesis. Finally, MMP-13, a matrix metalloproteinase important in secondary palate formation and tissue remodeling, had significantly increased mRNA and protein expression in CAH-X fibroblasts and direct tissue. Collectively, these results demonstrate that patients with CAH-X syndrome exhibit increased expression of several transforming growth factor-ß biomarkers and provide a novel link between this signaling pathway and the connective tissue dysplasia phenotypes associated with tenascin-X deficiency.


Assuntos
Hiperplasia Suprarrenal Congênita/genética , Hiperplasia Suprarrenal Congênita/metabolismo , Síndrome de Ehlers-Danlos/genética , Síndrome de Ehlers-Danlos/metabolismo , Transdução de Sinais , Tenascina/deficiência , Fator de Crescimento Transformador beta/metabolismo , Adolescente , Hiperplasia Suprarrenal Congênita/diagnóstico , Adulto , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Criança , Pré-Escolar , Síndrome de Ehlers-Danlos/diagnóstico , Feminino , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 13 da Matriz/metabolismo , Fator de Crescimento Transformador beta2/sangue , Fator de Crescimento Transformador beta2/metabolismo , Fator de Crescimento Transformador beta3/biossíntese , Adulto Jovem
17.
Circ Cardiovasc Genet ; 7(1): 80-8, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24399159

RESUMO

BACKGROUND: Vascular Ehlers-Danlos syndrome (VEDS) causes reduced life expectancy because of arterial dissections/rupture and hollow organ rupture. Although the causative gene, COL3A1, was identified >20 years ago, there has been limited progress in understanding the disease mechanisms or identifying treatments. METHODS AND RESULTS: We studied inflammatory and transforming growth factor-ß (TGF-ß) signaling biomarkers in plasma and from dermal fibroblasts from patients with VEDS. Analyses were done in terms of clinical disease severity, genotype-phenotype correlations, and body composition and fat deposition alterations. VEDS subjects had increased circulating TGF-ß1, TGF-ß2, monocyte chemotactic protein-1, C-reactive protein, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and leptin and decreased interleukin-8 versus controls. VEDS dermal fibroblasts secreted more TGF-ß2, whereas downstream canonical/noncanonical TGF-ß signaling was not different. Patients with COL3A1 exon skipping mutations had higher plasma intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, and VEDS probands had abnormally high plasma C-reactive protein versus affected patients identified through family members before any disease manifestations. Patients with VEDS had higher mean platelet volumes, suggesting increased platelet turnover because of ongoing vascular damage, as well as increased regional truncal adiposity. CONCLUSIONS: These findings suggest that VEDS is a systemic disease with a major inflammatory component. C-reactive protein is linked to disease state and may be a disease activity marker. No changes in downstream TGF-ß signaling and increased platelet turnover suggest that chronic vascular damage may partially explain increased plasma TGF-ß1. Finally, we found a novel role for dysregulated TGF-ß2, as well as adipocyte dysfunction, as demonstrated through reduced interleukin-8 and elevated leptin in VEDS.


Assuntos
Síndrome de Ehlers-Danlos/sangue , Inflamação/sangue , Fator de Crescimento Transformador beta/sangue , Adipocinas/sangue , Adolescente , Adulto , Biomarcadores/análise , Biomarcadores/sangue , Composição Corporal , Proteína C-Reativa/análise , Criança , Colágeno Tipo III/antagonistas & inibidores , Colágeno Tipo III/genética , Colágeno Tipo III/metabolismo , Síndrome de Ehlers-Danlos/etiologia , Síndrome de Ehlers-Danlos/genética , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Estudos de Associação Genética , Humanos , Inflamação/genética , Masculino , Pessoa de Meia-Idade , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/análise , Fator de Crescimento Transformador beta1/análise , Fator de Crescimento Transformador beta1/sangue , Fator de Crescimento Transformador beta2/análise , Fator de Crescimento Transformador beta2/sangue , Adulto Jovem
18.
J Mol Diagn ; 15(6): 745-53, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24071710

RESUMO

Congenital adrenal hyperplasia, due to 21-hydroxylase deficiency (21-OHD) is an autosomal recessive disorder of adrenal steroidogenesis caused by mutations in the CYP21A2 gene. Direct comparison of established and novel methodologies of CYP21A2 genetic analysis in a large cohort representing a wide range of genotypes has not been previously reported. We genotyped a cohort of 129 unrelated patients with 21-OHD, along with 145 available parents, using Southern blot (SB) analysis, multiplex ligation-dependent probe amplification (MLPA), PCR-based restriction fragment length polymorphism (RFLP) analysis, multiplex minisequencing and conversion-specific PCR, duplication-specific amplification, and DNA sequencing. CYP21A2 genotyping identified four duplicated CYP21A2 genes (1.53%) and 79 chimeric CYP21A1P/CYP21A2 genes (30.15%). Parental SB data were essential for determining the CYP21 haplotype in three cases, whereas PCR-based RFLP analysis was necessary for MLPA results to be accurately interpreted in the majority of cases. The comparison of different methods in detecting deletion and duplication showed that MLPA with PCR-based RFLP was comparable with SB analysis, with parental data of 100% sensitivity and specificity. DNA sequencing was required for the identification of 16 (6.1%) rare point mutations and determination of clinically significant chimera junction sites. MLPA with PCR-based RFLP analysis is an excellent substitute for SB analysis in detecting CYP21A2 deletion and duplication and a combination of MLPA, PCR-based RFLP, duplication-specific amplification, and DNA sequencing is a convenient and comprehensive strategy for mutation analysis of the CYP21A2 gene in patients with 21-OHD.


Assuntos
Hiperplasia Suprarrenal Congênita/diagnóstico , Esteroide 21-Hidroxilase/genética , Hiperplasia Suprarrenal Congênita/genética , Análise Mutacional de DNA , Dosagem de Genes , Humanos , Técnicas de Diagnóstico Molecular , Estudos Observacionais como Assunto , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição
19.
J Clin Endocrinol Metab ; 98(2): E379-87, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23284009

RESUMO

CONTEXT: The gene for congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency, CYP21A2, is flanked by the gene encoding tenascin-X (TNXB), a connective tissue extracellular matrix protein that has been linked to both autosomal dominant and autosomal recessive Ehlers-Danlos syndrome (EDS). A contiguous deletion of CYP21A2 and TNXB has been described. OBJECTIVE: The objective of the study was to determine the frequency and clinical significance of TNXB haploinsufficiency in CAH patients. DESIGN, SETTING, AND PARTICIPANTS: A total of 192 consecutive unrelated CAH patients being seen as part of an observational study at the National Institutes of Health Clinical Center (Bethesda, MD) were prospectively studied during 2006-2010. Patients were evaluated for clinical evidence of EDS, including cardiac evaluation. DNA was analyzed by PCR, multiplex ligation-dependent probe amplification, Southern blot, and TNXB sequencing. Tenascin-X expression was evaluated by Western blot analysis of fibroblasts and immunostaining of the skin. CAH patients with TNXB haploinsufficiency were compared with age-matched CAH patients with normal TNXB (controls). Phenotyping of 7 parents with TNXB haploinsufficiency was performed. MAIN OUTCOME MEASURES: The frequency of TNXB haploinsufficiency among CAH patients and the frequency of EDS symptomatology among CAH patients with TNXB haploinsufficiency and controls. RESULTS: TNXB haploinsufficiency, here termed CAH-X syndrome, was present in 7% of CAH patients. Twelve of 91 patients carrying a CYP21A2 deletion (13%) carried a contiguous deletion that extended into TNXB. One patient carried a TNXB premature stop codon. Twelve of 13 patients with CAH-X had EDS clinical features. Patients with CAH-X were more likely than age-matched controls to have joint hypermobility (P < .001), chronic joint pain (P = .003), multiple joint dislocations (P = .004), a structural cardiac valve abnormality by echocardiography (P = .02), and reduced tenascin-X expression by Western blot and immunostaining. A subset of parents had clinical findings. CONCLUSIONS: Clinical evaluation for connective tissue dysplasia should be routinely performed in CAH patients, especially those harboring a CYP21A2 deletion.


Assuntos
Hiperplasia Suprarrenal Congênita/genética , Síndrome de Ehlers-Danlos/genética , Haploinsuficiência , Esteroide 21-Hidroxilase/genética , Tenascina/genética , Adolescente , Hiperplasia Suprarrenal Congênita/fisiopatologia , Adulto , Idoso , Criança , Pré-Escolar , Síndrome de Ehlers-Danlos/fisiopatologia , Feminino , Humanos , Lactente , Luxações Articulares/genética , Luxações Articulares/fisiopatologia , Instabilidade Articular/genética , Instabilidade Articular/fisiopatologia , Masculino , Pessoa de Meia-Idade , Mutação , Dor/genética , Dor/fisiopatologia
20.
J Clin Endocrinol Metab ; 96(1): E161-72, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20926536

RESUMO

BACKGROUND: Genetic analysis is commonly performed in patients with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency. STUDY OBJECTIVE: The objective of the study was to describe comprehensive CYP21A2 mutation analysis in a large cohort of CAH patients. METHODS: Targeted CYP21A2 mutation analysis was performed in 213 patients and 232 parents from 182 unrelated families. Complete exons of CYP21A2 were sequenced in patients in whom positive mutations were not identified by targeted mutation analysis. Copy number variation and deletions were determined using Southern blot analysis and PCR methods. Genotype was correlated with phenotype. RESULTS: In our heterogeneous U.S. cohort, targeted CYP21A2 mutation analysis did not identify mutations on one allele in 19 probands (10.4%). Sequencing identified six novel mutations (p.Gln262fs, IVS8+1G>A, IVS9-1G>A, p.R408H, p.Gly424fs, p.R426P) and nine previously reported rare mutations. The majority of patients (79%) were compound heterozygotes and 69% of nonclassic (NC) patients were compound heterozygous for a classic and a NC mutation. Duplicated CYP21A2 haplotypes, de novo mutations and uniparental disomy were present in 2.7% of probands and 1.9 and 0.9% of patients from informative families, respectively. Genotype accurately predicted phenotype in 90.5, 85.1, and 97.8% of patients with salt-wasting, simple virilizing, and NC mutations, respectively. CONCLUSIONS: Extensive genetic analysis beyond targeted CYP21A2 mutational detection is often required to accurately determine genotype in patients with CAH due to the high frequency of complex genetic variation.


Assuntos
Hiperplasia Suprarrenal Congênita/genética , Esteroide 21-Hidroxilase/genética , Adolescente , Adulto , Idoso , Alelos , Southern Blotting , Criança , Pré-Escolar , Análise Mutacional de DNA , Feminino , Estudos de Associação Genética , Testes Genéticos , Genótipo , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Mutação , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estados Unidos
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