RESUMEN
The TSC1 and TSC2 gene products interact to form the tuberous sclerosis complex (TSC), an important negative regulator of the mechanistic target of rapamycin complex 1 (TORC1). Inactivating mutations in TSC1 or TSC2 cause TSC, and the identification of a pathogenic TSC1 or TSC2 variant helps establish a diagnosis of TSC. However, it is not always clear whether TSC1 and TSC2 variants are inactivating. To determine whether TSC1 and TSC2 variants of uncertain clinical significance affect TSC complex function and cause TSC, in vitro assays of TORC1 activity can be employed. Here we combine genetic, functional, and structural approaches to try and classify a series of 15 TSC2 VUS. We investigated the effects of the variants on the formation of the TSC complex, on TORC1 activity and on TSC2 pre-mRNA splicing. In 13 cases (87%), the functional data supported the hypothesis that the identified TSC2 variant caused TSC. Our results illustrate the benefits and limitations of functional testing for TSC.
Asunto(s)
Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Mutación , Fenotipo , Proteína 2 del Complejo de la Esclerosis Tuberosa/química , Proteína 2 del Complejo de la Esclerosis Tuberosa/genética , Sustitución de Aminoácidos , Técnicas de Silenciamiento del Gen , Estudios de Asociación Genética/métodos , Genotipo , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Empalme del ARN , Relación Estructura-Actividad , Proteína 2 del Complejo de la Esclerosis Tuberosa/metabolismoRESUMEN
INTRODUCTION: Diagnostic evaluation of patients with a bleeding tendency remains challenging, as no disorder is identified in approximately 50% of patients. An impaired interplay of several haemostatic factors might explain bleeding phenotype in these patients. OBJECTIVE: To investigate whether global haemostasis assays are able to identify haemostatic abnormalities in patients with a bleeding tendency unexplained by current diagnostic laboratory tests. MATERIALS AND METHODS: Patients of ≥12 years with a bleeding tendency were included from a tertiary outpatient clinic. Bleeding phenotype was assessed with the ISTH-BAT. Patients were classified as having bleeding of unknown cause (BUC) or a mild bleeding disorder (MBD) based on abnormalities assessed by routine haemostatic tests. Global haemostasis tests (rotational thromboelastometry (ROTEM), thrombin generation test (TG) and plasma clot lysis time (CLT)) were measured in all patients. The results were compared with 76 controls. RESULTS: One hundred and eighty-one patients were included, and 60% (109/181) was classified as having BUC. BUC patients demonstrated a significantly prolonged lag time in TG (median 7.7 minutes, IQR 6.7-8.7) and a significantly prolonged CLT (median 60.5 minutes, IQR 54.7-66.1) compared to controls. No differences in ROTEM variables were found. Patients with MBD showed an impaired thrombin generation with a significantly decreased ETP (median 1024 nmol/L*min, IQR 776-1355) and peak height (median 95 nmol/L, IQR 76-138), compared to BUC patients and controls. CONCLUSION: No major differences were found in ROTEM and TG variables in BUC patients compared to controls. BUC patients did have a significantly prolonged clot lysis time. The underlying mechanism for this finding is unknown.