Exome sequencing reveals three homozygous missense variants in SNRPA in two sisters with syndromic intellectual disability.

Splicing-related gene mutations might affect the expression of a single gene or multiple genes and cause clinically heterogeneous diseases. With the advent of next-generation sequencing, several splicing gene mutations have been exposed, yet most major spliceosome genes have no reports of germline mutations and therefore, their effects are largely unknown. We describe the previously unreported concurrence of intellectual disability, short stature, poor speech, and minor craniofacial and hand anomalies in 2 female siblings with 3 homozygous missense variants in SNRPA (a component of the U1 small nuclear ribonucleoprotein complex) characterized by homozygosity mapping and whole exome sequencing. Combined, c.97A>G, c.98T>C, and c.100T>A, in exon 2 of SNRPA lead to p.Ile33Ala and p.Phe34Ile exchanges, which were predicted in silico to be deleterious. Although both patients exhibited some clinical features seen in other spliceosomal disorders, their complete clinical phenotype appears to be rather uncommon, a finding that may further support the notion that mutations in components of the major spliceosome do not strictly lead to the same syndromes/phenotypes.

Adrenal hypofunction from histoplasmosis: a literature review from 1971 to 2012.

PURPOSE: While histoplasmosis has been reported from most continents, the disease is most often recognized in the midwestern United States. The recent diagnosis of adrenal hypofunction in two patients with progressive disseminated histoplasmosis (PDH) in our hospital led us to review the literature. METHODS: We reviewed PubMed using the search term "adrenal histoplasmosis" for the years 1971 to 2012. RESULTS: The results included 242 patients with adrenal histoplasmosis from either case reports or case series. Most of the reported patients were from countries not previously considered to be heavily endemic for histoplasmosis. In addition, 41.3 % of patients with adrenal involvement developed adrenal hypofunction. CONCLUSION: As modern technology elucidates more cases of adrenal histoplasmosis, the global boundaries of endemicity are being redefined.

Genetic contribution of CYP2C9, CYP2C19, and APOE variants in acenocoumarol response.

Oral anticoagulants of the coumarin type have an inconveniently narrow therapeutic window, making their use difficult. In Mexico, genetic variables that participate in the heterogeneity of the therapeutic response remain poorly investigated. With the focus on warfarin, extensive pharmacogenomic studies have been performed, including those on the CYP450 family and APOE. The objective of this study was to determine the contribution of CYP2C9, CYP2C19, and APOE polymorphisms to the variations in response to the doses of acenocoumarol, which is the main anticoagulant prescribed to the Mexican population. The polymerase chain reaction-restriction fragment length polymorphism method was applied to identify 2 and 3 of CYP2C9, 2 of CYP2C19, and APOE variants. The genetic distribution of every polymorphism tested showed high variability when compared with other populations worldwide. Our results showed statistical differences only in the CYP2C19 gene between the 1 1 and 1 2 groups, with effective acenocoumarol doses of 2.56 ± 1.34 mg/day vs 1.35 ± 0.84 mg/day (P = 0.005), respectively. Multiple regression analysis, including patient age and both the CYP2C9 and CYP2C19 genes, showed that these variables explained more than 20% of the dose variations. This is the first report in Mexico searching for the relationship between CYP450 and APOE polymorphisms and the dose requirements of acenocoumarol. Our results suggest that, in the Mexican population, CYP2C19 is more involved in acenocoumarol metabolism than CYP2C9 and APOE. Besides considering the age factor, pharmacogenetic testing for CYP2C19 2 before initiating acenocoumarol treatment could lead to a safer anticoagulation therapy in Mexican patients.

Factor V Leiden and thrombosis in patients with systemic lupus erythematosus: a meta-analysis.

The aim of this study was to perform a meta-analysis of the association between the factor V Leiden polymorphism (FVL) and thrombosis among patients with systemic lupus erythematosus (SLE) and/or antiphospholipid antibody (aPL) positivity. Included studies recruited patients based on SLE or aPL-positive status, confirmed subjects' SLE diagnosis as defined by the American College of Rheumatology, and documented thrombotic events. Excluded studies were non-English or considered only arterial thrombosis. Individual patient data, available from 5 studies, together with unpublished data from 1210 European-American SLE patients from the UCSF Lupus Genetics Collection genotyped for FVL, were further analyzed. Seventeen studies (n=2090 subjects) were included in the initial meta-analysis. Unadjusted odds ratios (OR) were calculated to assess association of FVL with thrombosis. The OR for association of thrombosis with FVL was 2.88 (95% confidence interval (CI) 1.98-4.20). In the secondary analysis with our individual patient dataset (n=1447 European-derived individuals), SLE subjects with the FVL polymorphism still had more than two times the odds of thrombosis compared to subjects without this polymorphism, even when adjusting for covariates such as gender, age and aPL status. SLE and/or aPL-positive patients with the FVL variant have more than two times the odds of thrombosis compared to those without this polymorphism.

A BAC-based physical map of the major autosomes of Drosophila melanogaster.

We constructed a bacterial artificial chromosome (BAC)-based physical map of chromosomes 2 and 3 of Drosophila melanogaster, which constitute 81% of the genome. Sequence tagged site (STS) content, restriction fingerprinting, and polytene chromosome in situ hybridization approaches were integrated to produce a map spanning the euchromatin. Three of five remaining gaps are in repeat-rich regions near the centromeres. A tiling path of clones spanning this map and STS maps of chromosomes X and 4 was sequenced to low coverage; the maps and tiling path sequence were used to support and verify the whole-genome sequence assembly, and tiling path BACs were used as templates in sequence finishing.

Detailed genetic characterization of the interleukin-23 receptor in psoriasis.

Using a multi-tiered, case-control association design, scanning 25 215 gene-centric SNPs, we previously identified two psoriasis susceptibility genes: IL12B and IL23R. These results have recently been confirmed. To better characterize the IL23R psoriasis-association, we used a fine mapping strategy to identify 59 additional IL23R-linked SNPs, which were genotyped in our three independent, white North American sample sets (>2800 individuals in toto). A sliding window of haplotype association demonstrates colocalization of psoriasis susceptibility effects within the boundaries of IL23R across all sample sets, thereby decreasing the likelihood that neighboring genes, particularly IL12RB2, are driving the association at this region. Additional haplotype work identified two 5-SNP haplotypes with strong protective effects, consistent across our three sample sets (OR(common)=0.67; P(comb)=4.32E-07). Importantly, heterogeneity of effect was extremely low between sample sets for these haplotypes (P(Het)=0.961). Together, these protective haplotypes attain a frequency of 16% in controls, declining to 11% in cases. The characterization of association patterns within IL23R to specific predisposing/protective variants will play an important role in the elucidation of psoriasis etiology and other related phenotypes. Further, this work is essential to lay the foundation for the role of IL23R genetics in response to pharmaceutical therapy and dosage.

Variants in the 5q31 cytokine gene cluster are associated with psoriasis.

A multitiered genetic association study of 25 215 single-nucleotide polymorphisms (SNPs) in three case-control sample sets (1446 patients and 1432 controls) identified three IL13-linked SNPs (rs1800925, rs20541 and rs848) associated with psoriasis. Although the susceptibility effects at these SNPs were modest (joint allelic odds ratios (ORs): 0.76 to 0.78; P(comb): 1.3E-03 to 2.50E-04), the association patterns were consistent across the sample sets, with the minor alleles being protective. Haplotype analyses identified one common, susceptible haplotype CCG (joint allelic OR=1.27; P(comb)=1.88E-04) and a less common, protective haplotype TTT (joint allelic OR=0.74; P(comb)=7.05E-04). In combination with the other known genetic risk factors, HLA-C, IL12B and IL23R, the variants reported here generate an 11-fold psoriasis-risk differential. Residing in the 5q31 cytokine gene cluster, IL13 encodes an important T-cell-derived cytokine that regulates cell-mediated immunity. These results provide the foundation for additional studies required to fully dissect the associations within this cytokine-rich genomic region, as polymorphisms in closely linked candidate genes, such as IRF1, IL5 or IL4, may be driving these results through linkage disequilibrium.

Schilbach-Rott syndrome in a third family: further delineation of an autosomal dominant trait.

We describe a father-son Mexican pair with typical features of Schilbach-Rott syndrome (SRS): ocular hypotelorism, cleft palate, hypospadias (only in the child), and microcephaly. This observation documents for the first time a male to male transmission and therefore confirms that the SRS is inherited as an autosomal dominant trait with variable expressivity.

Enzymatic inactivation of human antithrombin III. Limited proteolysis of the inhibitor by snake venom proteinases in the presence of heparin.

Incubation of human plasma antithrombin III with Crotalid, Viperid and Colubrid snake venoms resulted in the enzymatic inactivation of the inhibitor, as evidenced by a gradual loss of inhibitory activity against trypsin and thrombin. This indicates that proteinases which selectively inactivate antithrombin III are widespread among the families of poisonous snakes. The inactivation was due to metalloproteinases present in the venoms, since the reaction could be terminated by the addition of EDTA. Elapid venoms were tested and shown to be devoid of activity on antithrombin III. Preincubation of the anthrombin III with heparin accelerated the reaction, and less venom was required to achieve total inactivation. Several venoms had very little effect on antithrombin III in the absence of heparin, but inactivated the inhibitor completely within 2 h when heparin was present. Optimal rates of inactivation were observed with antithrombin III: heparin ratios of approx. 3 : 1. When heparin was present in excess, the inactivation was retarded. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the antithrombin III/venom proteinase reaction mixtures indicated that intact antithrombin III (63 000 daltons) was converted to an inactive form (57 500 daltons) by limited proteolysis. No complex formation between antithrombin III and venom proteinases was detectable. The inactivating cleavage occurred within a disulfide loop of the antithrombin III molecule, since the lower molecular weight species was detected only under reducing conditions.

Enzymatic inactivation of human plasma C1-inhibitor and alpha 1-antichymotrypsin by Pseudomonas aeruginosa proteinase and elastase.

Two major human plasma proteinase inhibitors, C1-inhibitor and alpha 1-antichymotrypsin, were enzymatically inactivated by Pseudomonas aeruginosa elastase and proteinase. Incubation of C1-inhibitor with the Pseudomonas enzymes at inhibitor/enzyme molar ratios of 1000:1 (elastase) or 22:1 (proteinase) resulted in cleavage of the 104 kDa intact inhibitor to an 89 kDa intermediate which retained full inhibitory activity against plasmin and plasma kallikrein. The intermediate was then cleaved to an 83 kDa inactive product. The initial non-inactivating cleavage of C1-inhibitor occurred in a region of the molecule readily accessible to limited proteolysis by both enzymes. The inactivating cleavage, however, occurred more readily with the elastase. alpha 1-Antichymotrypsin was inactivated by P. aeruginosa proteinase and elastase by limited proteolysis at inhibitor/enzyme molar ratios of 14 000:1. The 64 kDa intact inhibitor was cleaved to form an inactive 60 kDa product, and a low molecular mass peptide fragment was observed. No stable enzyme-inhibitor complexes were detected, and no random proteolysis of the inactivated inhibitors was noted, even after prolonged incubation. Catalytic inactivation of C1-inhibitor and alpha 1-antichymotrypsin by P. aeruginosa proteinase and elastase may contribute to the tissue damage and hemorrhagic lesions which occur during pseudomonal infections.

Analysis of the effects of snake venom proteinases on the activity of human plasma C1 esterase inhibitor, alpha 1-antichymotrypsin and alpha 2-antiplasmin.

Incubation of C1 esterase inhibitor with Crotalid, Viperid and Colubrid snake venoms resulted in enzymatic inactivation of the inhibitor. Intact inhibitor (104 kDa) was converted into an active intermediate species of 89 kDa and then a further cleavage resulted in formation of an 86-kDa inactive inhibitor. In contrast, C1 esterase inhibitor did not lose activity during incubation with Elapid venoms; however, the intact inhibitor was gradually converted to an active species of 89 kDa during the incubation. Human alpha 1-antichymotrypsin was inactivated by all venoms tested, including those from the Elapid family. The 67-kDa intact inhibitor was converted by the venom proteinases to an inactive 63-kDa form. The results suggest that this acute-phase plasma protein is readily susceptible to inactivation by venom proteinases. Human alpha 2-antiplasmin (68 kDa) was cleaved to form a 61-kDa active intermediate, which then underwent a second cleavage to produce an inactive 53-kDa product. Elapid venoms had no effect on alpha 2-antiplasmin activity and did not cleave this inhibitor. All inhibitors were inactivated with catalytic amounts of venom proteinases. No stable proteinase-proteinase inhibitor complexes were detected, and no random proteolysis of the inhibitors occurred.

Semiautomated clone verification by real-time PCR using molecular beacons.

Conventional, high-throughput PCR analysis of common elements utilizing numerous primer sets and template DNA requires multiple rounds of PCR to ensure optimal conditions. Laborious gel electrophoresis and staining is then necessary to visualize amplification products. We propose novel multicolor molecular beacons, to establish a high-throughput, PCR-based sequence tagged site (STS) detection system that swiftly and accurately confirms marker content in template containing common repeat elements. A simple, one-tube, real-time PCR assay system was developed to specifically detect regions containing CA and GATA repeats. Ninety-six samples can be confirmed for marker content in a closed-tube format in 3 h, eliminating product confirmation on agarose gels and avoiding crossover contamination. Multiple STSs can be detected simultaneously in the same reaction tube by utilizing molecular beacons labeled with multicolor fluorophores. Template DNA from 260 RPCI-11 bacterial artificial chromosome (BAC) clones was examined for the presence of CA and/or GATA repeats using molecular beacon PCR and compared with conventional PCR results of the same clones. Of the 205 clones containing CA and GATA repeats, we were able to identify 129 clones (CA, n = 99; GATA, n = 30) by using molecular beacons and only 121 clones (CA, n = 92; GATA, n = 29) by conventional PCR amplification. As anticipated, 55 clones that contained sequences other than CA or GATA failed molecular beacon detection. Molecular beacon PCR, employing beacons specific for tandem repeat elements, provides a fast, accurate, and sensitive multiplex detection assay that will expedite verification of marker content in a multitude of template containing these repeats.

Heterogeneity of the mechanical properties of demineralized bone.

Knowledge of the mechanical properties of the collagenous component of bone is required for composite modeling of bone tissue and for understanding the age- and disease-related reductions in the ductility and strength of bone. The overall goal of this study was to investigate the heterogeneity of the mechanical properties of demineralized bone which remains unexplained and may be due to differences in the collagen structure or organization or in experimental protocols. Uniaxial tension tests were conducted to measure the elastic and failure properties of demineralized human femoral (n = 10) and tibial (n = 13) and bovine humeral (n = 8) and tibial (n = 8) cortical bone. Elastic modulus differed between groups (p = 0.02), varying from 275 +/- 94 MPa (mean +/- SD) to 450 + 50 MPa. Similarly, ultimate stress varied across groups from 15 + 4.2 to 26 + 4.7 MPa (p = 0.03). No significant differences in strain-to-failure were observed between any groups in this study (pooled mean of 8.4 +/- 1.6%; p = 0.42). However, Bowman et al. (1996) reported an average ultimate strain of 12.3 +/- 0.5% for demineralized bovine humeral bone, nearly 40% higher than our value. Taken together, it follows that all the monotonic mechanical properties of demineralized bone can display substantial heterogeneity. Future studies directed at explaining such differences may therefore provide insight into aging and disease of bone tissue.

Differences between male and female patients with regard to baseline demographics and clinical outcomes in the Asymptomatic Cardiac Ischemia Pilot (ACIP) Trial.

BACKGROUND: Coronary artery disease (CAD) is a common problem in men and women; however, men and women with similar clinical presentations of myocardial ischemia may receive different revascularization treatments. HYPOTHESIS: Using the data base of the Asymptomatic Cardiac Ischemia Pilot (ACIP) trial, this study was undertaken to compare by gender the baseline demographic data and the clinical outcome results in patients randomized to various treatments in the ACIP study. METHODS: This randomized trial compared three treatment regimens [pharmacologic management of angina, pharmacologic management of angina and ambulatory electrocardiographic (ECG) evidence of ischemia, and revascularization--that is, angioplasty and coronary artery bypass surgery], in patients with known CAD, positive stress ECG tests, and ECG evidence of ischemia during 48 h ambulatory monitoring. In all, 558 patients were randomized, 79 of whom were women (mean age: men 61.6 years, women 60.6 years) Ambulatory ECG evidence of ischemia, clinical events, that is, death, myocardial infarction, hospital admission for coronary events, and exercise performance were monitored. RESULTS: Although of the same age as men at baseline, women had a higher prevalence of hypertension and diabetes. Women had less severe CAD by angiography and higher left ventricular ejection fractions. Men had longer exercise tolerance times on the treadmill. However, men and women had similar numbers and duration of ambulatory ECG ischemic abnormalities. Regarding revascularization, men more commonly underwent coronary artery bypass surgery (p = 0.025) while women underwent percutaneous transluminal coronary angioplasty more frequently (p = 0.10). Clinical outcomes were comparable in men and women, although the numbers of events were relatively small. CONCLUSIONS: Men and women of comparable age manifest CAD with similar ischemic ECG abnormalities seen on both exercise tolerance and ambulatory ECG examinations. In ACIP, women tended to have more risk factors for CAD and less severity in anatomical disease, which may explain why women are less likely than men to have coronary bypass surgery.

Genetic variants associated with deep vein thrombosis: the F11 locus.

BACKGROUND: Recent studies have found associations between deep vein thrombosis (DVT) and single nucleotide polymorphisms (SNPs) in a 4q35.2 locus that contains genes encoding factor XI (F11), a cytochrome P450 family member (CYP4V2), and prekallikrein (KLKB1). OBJECTIVE: We investigated which of the common SNPs in this locus are independently associated with DVT. METHODS: The study populations were the Leiden Thrombophilia Study (LETS) (443 DVT cases and 453 controls) and the Multiple Environmental and Genetic Assessment of risk factors for venous thrombosis (MEGA study) (2712 DVT cases and 4634 controls). We assessed the association between DVT and 103 SNPs in a 200 kb region using logistic regression. RESULTS: We found that two SNPs (rs2289252 and rs2036914 in F11) were independently associated with DVT. After adjusting for age, sex, and the other SNP, the odds ratios (risk vs. non-risk homozygotes) of these two SNPs were 1.49 for rs2289252 (95% CI, 1.25-1.76) and 1.33 for rs2036914 (95% CI, 1.11-1.59). We found that rs2289252 was also associated with FXI levels, as has been previously reported for rs2036914; these two SNPs remained associated with DVT with somewhat attenuated risk estimates after adjustment for FXI levels. CONCLUSION: Two SNPs, rs2289252 and rs2036914 in F11, appear to independently contribute to the risk of DVT, a contribution that is explained at least in part by an association with FXI levels.