Insights into genotype-phenotype correlations from CREBBP point mutation screening in a cohort of 46 Rubinstein-Taybi syndrome patients.

The genetic basis of Rubinstein-Taybi syndrome (RSTS), a rare, sporadic, clinically heterogeneous disorder characterized by cognitive impairment and a wide spectrum of multiple congenital anomalies, is primarily due to private mutations in CREBBP (approximately 55% of cases) or EP300 (approximately 8% of cases). Herein, we report the clinical and the genetic data taken from a cohort of 46 RSTS patients, all carriers of CREBBP point mutations. Molecular analysis revealed 45 different gene alterations including 31 inactivating (21 frameshift and 10 nonsense), 10 missense and 4 splicing mutations. Bioinformatic tools and transcript analyses were used to predict the functional effects of missense and splicing alterations. Of the 45 mutations, 42 are unreported and 3 were described previously. Recurrent mutations maybe a key tool in addressing genotype-phenotype correlations in patients sharing the same defects (at the genomic or transcript level) and specific clinical signs, demonstrated here in two cases. The clinical data of our cohort evidenced frequent signs such as arched eyebrows, epicanthus, synophrys and/or frontal hypertrichosis and broad phalanges that, previously overlooked in RSTS diagnosis, now could be considered. Some suggested correlations between organ-specific anomalies and affected CREB-binding protein domains broaden the RSTS clinical spectrum and perhaps will enhance patient follow-up and clinical care.

Clinical and molecular characterization of Rubinstein-Taybi syndrome patients carrying distinct novel mutations of the EP300 gene.

Rubinstein-Taybi syndrome (RSTS) is a rare congenital neurodevelopmental disorder characterized by postnatal growth deficiency, skeletal abnormalities, dysmorphic features and cognitive deficit. Mutations in two genes, CREBBP and EP300, encoding two homologous transcriptional co-activators, have been identified in ˜55% and ˜3-5% of affected individuals, respectively. To date, only eight EP300-mutated RSTS patients have been described and 12 additional mutations are reported in the database LOVD. In this study, EP300 analysis was performed on 33 CREBBP-negative RSTS patients leading to the identification of six unreported germline EP300 alterations comprising one deletion and five point mutations. All six patients showed a convincing, albeit mild, RSTS phenotype with minor skeletal anomalies, slight cognitive impairment and few major malformations. Beyond the expansion of the RSTS-EP300-mutated cohort, this study indicates that EP300-related RSTS cases occur more frequently than previously thought (˜8% vs 3-5%); furthermore, the characterization of novel EP300 mutations in RSTS patients will enhance the clinical practice and genotype-phenotype correlations.

Histone acetylation deficits in lymphoblastoid cell lines from patients with Rubinstein-Taybi syndrome.

BACKGROUND: Rubinstein-Taybi syndrome (RSTS) is a congenital neurodevelopmental disorder defined by postnatal growth deficiency, characteristic skeletal abnormalities and mental retardation and caused by mutations in the genes encoding for the transcriptional co-activators with intrinsic lysine acetyltransferase (KAT) activity CBP and p300. Previous studies have shown that neuronal histone acetylation is reduced in mouse models of RSTS. METHODS: The authors identified different mutations at the CREBBP locus and generated lymphoblastoid cell lines derived from nine patients with RSTS carrying distinct CREBBP mutations that illustrate different grades of the clinical severity in the spectrum of the syndrome. They next assessed whether histone acetylation levels were altered in these cell lines. RESULTS: The comparison of CREBBP-mutated RSTS cell lines with cell lines derived from patients with an unrelated mental retardation syndrome or healthy controls revealed significant deficits in histone acetylation, affecting primarily histone H2B and histone H2A. The most severe defects were observed in the lines carrying the whole deletion of the CREBBP gene and the truncating mutation, both leading to a haploinsufficiency state. Interestingly, this deficit was rescued by treatment with an inhibitor of histone deacetylases (HDACi). CONCLUSIONS: The authors' results extend to humans the seminal observations in RSTS mouse models and point to histone acetylation defects, mainly involving H2B and H2A, as relevant molecular markers of the disease.

Prediction of factor VIII inhibitor development in the SIPPET cohort by mutational analysis and factor VIII antigen measurement.

Essentials A residual factor VIII synthesis is likely to be protective towards inhibitor (INH) development. Mutation type-inhibitor risk association was explored in 231 patients with severe hemophilia A. A 2-fold increase in INH development for in silico null vs. non-null mutations was found. A 3.5-fold increase in INH risk for antigen negative vs. antigen positive mutations was found. SUMMARY: Background The type of F8 mutation is the main predictor of inhibitor development in patients with severe hemophilia A. Mutations expected to allow residual synthesis of factor VIII are likely to play a protective role against alloantibody development by inducing immune tolerance. According to the expected full or partial impairment of FVIII synthesis, F8 variants are commonly classified as null and non-null. Objectives To explore the mutation type-inhibitor risk association in a cohort of 231 patients with severe hemophilia A enrolled in the Survey of Inhibitors in Plasma-Product Exposed Toddlers (SIPPET) randomized trial. Methods The genetic defects in these patients, consisting of inversions of intron 22 (n = 110) and intron 1 (n = 6), large deletions (n = 16), and nonsense (n = 38), frameshift (n = 28), missense (n = 19) and splicing (n = 14) variants, of which 34 have been previously unreported, were reclassified according to two additional criteria: the functional effects of missense and splicing alterations as predicted by multiple in silico analyses, and the levels of FVIII antigen in patient plasma. Results A two-fold increase in inhibitor development for in silico null mutations as compared with in silico non-null mutations (hazard ratio [HR] 2.08, 95% confidence interval [CI] 0.84-5.17) and a 3.5-fold increase in inhibitor development for antigen-negative mutations as compared with antigen-positive mutations (HR 3.61, 95% CI 0.89-14.74] were found. Conclusions Our findings confirm an association between the synthesis of minute amounts of FVIII and inhibitor protection, and underline the importance of investigating the residual FVIII antigen levels associated with causative variants in order to understand their clinical relevance.

Treatment of v-Ki-ras-transformed SVC1 cells with low retinoic acid induces malignancy reversion associated with ras p21 down-regulation.

The effect of nontoxic, low concentrations (10(-8) M) of retinoic acid (RA) for a relatively long time (28 days) on a Kirsten ras-virus transformed cell line (Ki-SVC1), derived from the rat seminal vesicle epithelium, was investigated. In these experimental conditions, the cell treatment with RA induced a decrease of the proliferation rate, apoptosis and a marked reduction of both anchorage-independent growth and tumorigenicity. These biological responses were either preceded or associated with important changes in adenylate cyclase/protein kinase C signaling pathways, the activation of important apoptosis-linked genes and a marked decrease of the v-Ki-ras p21 protein. The significance of these findings is discussed.

Molecular genetics of quantitative fibrinogen disorders.

Fibrinogen is a complex glycoprotein involved in the final step of the coagulation cascade as the precursor of fibrin monomers that participate in the formation of the haemostatic plug. Three genes (FGA, FGB, and FGG) clustered on chromosome 4q31.3-4q32.1 encode the three polypeptide chains (Aalpha, Bbeta, and gamma), which in a pairwise fashion form the hexameric circulating molecule. Among congenital fibrinogen deficiencies, quantitative defects (also called type I deficiencies; i.e. congenital afibrino-genemia [CAF] and hypofibrinogenemia) are characterized by the concomitant absence or reduction of coagulant activity and immunoreactive protein, while qualitative defects (type II deficiencies; i.e. dysfibrinogenemia and hypodysfibrino-genemia) show low clotting protein in contrast with normal or moderately reduced antigen. Patients affected by CAF (Mendelian Inheritance in Man, [MIM] #202400) or severe hypofibrinogenemia (MIM+134820, *134830, and *134850) may experience bleeding manifestations varying from mild to catastrophic. Although many cases of fibrinogen deficiencies have been described from a clinical point of view, only in a minority of cases the causal mutation was identified. The genetic defects so far described, most unique for any analyzed family, are invariantly located in the fibrinogen cluster; for only few of them the pathogenic role either at the protein or at the mRNA level has been investigated. This review, besides providing a concise description of the main structural and functional properties of fibrinogen and giving an overview of the clinical manifestations, the laboratory diagnosis and therapeutic approches, will be focused on the present knowledge on the genetic basis of quantitative fibrinogen deficiencies. Our systematic analysis of the available clinical and genetic data on these disorders evidences their high allelic heterogeneity, the existence of different pathogenic mechanisms, and the absence of strong genotype/phenotype correlations.

Anti-HCV antibody presence in hemodialyzed patients: assessment after one year regarding some preventive measures chosen.

The authors report on the presence of anti-HCV antibodies in a uremic chronic (UC) patient population after 6 months and 1 year with regard to certain selective preventive measures. The nonincreased number of HCV-positive subjects among the UC population could lead to the conclusion that the risk of environmental spreading of HCV is less than the risk of HBV diffusion.

Congenital afibrinogenemia: mutations leading to premature termination codons in fibrinogen A alpha-chain gene are not associated with the decay of the mutant mRNAs.

Congenital afibrinogenemia is a rare coagulation disorder with autosomal recessive inheritance, characterized by the complete absence or extremely reduced levels of fibrinogen in patients' plasma and platelets. Eight afibrinogenemic probands, with very low plasma levels of immunoreactive fibrinogen were studied. Sequencing of the fibrinogen gene cluster of each proband disclosed 4 novel point mutations (1914C>G, 1193G>T, 1215delT, and 3075C>T) and 1 already reported (3192C>T). All mutations, localized within the first 4 exons of the A alpha-chain gene, were null mutations predicted to produce severely truncated A alpha-chains because of the presence of premature termination codons. Since premature termination codons are frequently known to affect the metabolism of the corresponding messenger RNAs (mRNAs), the degree of stability of each mutant mRNA was investigated. Cotransfection experiments with plasmids expressing the wild type and each of the mutant A alpha-chains, followed by RNA extraction and semiquantitative reverse-transcriptase-polymerase chain reaction analysis, demonstrated that all the identified null mutations escaped nonsense-mediated mRNA decay. Moreover, ex vivo analysis at the protein level demonstrated that the presence of each mutation was sufficient to abolish fibrinogen secretion.

SV-IV, a major protein secreted from rat seminal vesicle epithelium, promotes lymphocyte cytotoxic activity against the lymphoblastoid Raji cell line in human peripheral blood mononuclear cells.

The treatment of human peripheral blood mononuclear cells (PBMC) with micromolar concentrations of SV-IV, a major protein secreted from the rat seminal vesicle epithelium, promotes in this cell population a marked cytotoxic activity against the Raji lymphoblastoid cell line. This activity is apparently due to cell-to-cell contact interactions. The expression of HLA DR on Raji cells has a modulatory effect on the SV-IV-induced cytotoxic activity. The experimental evidence strongly suggests that the cytotoxic effector cells are functionally activated NK cells.