High throughput parallel analysis of hundreds of patient samples for more than 100 mutations in multiple disease genes

As more mutations are identified in genes of known sequence, there is a crucial need in the areas of medical genetics and genome analysis for rapid, accurate and cost-effective methods of mutation detection. We have developed a multiplex allele-specific diagnostic assay (MASDA) for analysis of large numbers of samples (> 500) simultaneously for a large number of known mutations (> 100) in a single assay. MASDA utilizes oligonucleotide hybridization to interrogate DNA sequences. Multiplex DNA samples are immobilized on a solid support and a single hybridization is performed with a pool of allele-specific oligonucleotide (ASO) probes. Any probes complementary to specific mutations present in a given sample are in effect affinity purified from the pool by the target DNA. Sequence-specific band patterns (fingerprints), generated by chemical or enzymatic sequencing of the bound ASO(s), easily identify the specific mutation(s). Using this design, in a single diagnostic assay, we tested samples for 66 cystic fibrosis (CF) mutations, 14 beta-thalassemia mutations, two sickle cell anemia (SCA) mutations, three Tay-Sachs mutations, eight Gaucher mutations, four mutations in Canavan disease, four mutations in Fanconi anemia, and five mutations in BRCA1. Each mutation was correctly identified. Finally, in a blinded study of 106 of these mutations in > 500 patients, all mutations were properly identified. There were no false positives or false negatives. The MASDA assay is capable of detecting point mutations as well as small insertion or deletion mutations. This technology is amenable to automation and is suitable for immediate utilization for high-throughput genetic diagnostics in clinical and research laboratories.      

Stimulation of β-adrenoceptors inhibits store-operated channel currents via a cAMP-dependent protein kinase mechanism in rabbit portal vein myocytes

Previously we have described the properties of store-operated channel currents (SOCs) in freshly dispersed rabbit portal vein smooth muscle cells. In addition to Ca²⁺ store depletion these SOCs could also be activated by α-adrenoceptor stimulation and diacylglycerol (DAG) via a protein kinase C (PKC)-dependent mechanism. In the present study we have investigated the effect of β-adrenoceptor stimulation on SOCs in rabbit portal vein myocytes. With whole-cell recording the selective β-adrenoceptor agonist isoprenaline reduced the current evoked by cyclopiazonic acid (CPA, sarcoplasmic/endoplasmic reticulum ATPase inhibitor) by over 85%. With cell-attached patch recording, bath application of isoprenaline produced a pronounced inhibition of SOC activity evoked by either CPA or the acetoxymethyl ester form of BAPTA (BAPTA-AM). SOC activity evoked by CPA, the DAG analogue, 1-oleoyl-acetyl- sn -glycerol (OAG) or the phorbol ester, phorbol-12,13-dibutyrate (PDBu) was also markedly inhibited by the adenylate cyclase activator, forskolin, and the cell-permeable non-hydrolysable analogue of cyclic adenosine monophosphate (cAMP), 8-Br-cAMP. With inside-out patches, bath application of PDBu evoked channel currents with similar properties to SOCs which were inhibited by over 90% by a catalytic subunit of protein kinase A (PKA) and by 8-Br-cAMP. Moreover bath application of PKA inhibitors, H-89, KT5720 and an inhibitory peptide to quiescent cell-attached or inside-out patches, activated channel currents with similar properties to SOCs. These data suggest that in rabbit portal vein myocytes, stimulation of β-adrenoceptors inhibits SOC activity via a cAMP-dependent protein kinase signal transduction cascade. In addition it is concluded that constitutive PKA activity has a profound inhibitory effect on SOC activity in this vascular preparation.     

Distribution of mutations in the PEX gene in families with X-linked hypophosphataemic rickets (HYP)

Mutations in the PEX gene at Xp22.1 (phosphate-regulating gene with homologies to endopeptidases, on the X-chromosome), are responsible for X-linked hypophosphataemic rickets (HYP). Homology of PEX to the M13 family of Zn2+ metallopeptidases which include neprilysin (NEP) as prototype, has raised important questions regarding PEX function at the molecular level. The aim of this study was to analyse 99 HYP families for PEX gene mutations, and to correlate predicted changes in the protein structure with Zn2+ metallopeptidase gene function. Primers flanking 22 characterised exons were used to amplify DNA by PCR, and SSCP was then used to screen for mutations. Deletions, insertions, nonsense mutations, stop codons and splice mutations occurred in 83% of families screened for in all 22 exons, and 51% of a separate set of families screened in 17 PEX gene exons. Missense mutations in four regions of the gene were informative regarding function, with one mutation in the Zn2+-binding site predicted to alter substrate enzyme interaction and catalysis. Computer analysis of the remaining mutations predicted changes in secondary structure, N-glycosylation, protein phosphorylation and catalytic site molecular structure. The wide range of mutations that align with regions required for protease activity in NEP suggests that PEX also functions as a protease, and may act by processing factor(s) involved in bone mineral metabolism.      

Controlled clinical trial of peritoneal lavage for the treatment of severe acute pancreatitis

We performed a multicenter, randomized, controlled clinical trial of therapeutic peritoneal lavage (2 liters per hour for three days) in 91 patients with severe acute pancreatitis. Patients were entered into the study if severe pancreatitis was indicated by multiple laboratory criteria or diagnostic peritoneal lavage. All patients received full supportive treatment. The median time between the onset of symptoms and randomization was 38 hours. Forty-six patients were assigned to the control group and 45 to the lavage group. There were 13 deaths (28 per cent) and 16 patients with major complications (35 per cent) in the control group, as compared with 12 deaths (27 per cent) and 17 patients with major complications (38 per cent) in the lavage group. Lavage did not appear to modify the length of survival, the incidence of pancreatic collections (pseudocysts or abscesses), or the plasma amylase concentration. Considering the statistical power of the design, we conclude that the outcome of severe pancreatitis was not greatly, if at all, influenced by the regimen of peritoneal lavage used in this study.     

Limiting factors for cytopathological diagnosis of high-grade squamous intraepithelial lesions: a cytohistological correlation between findings in cervical smears and loop electrical excision procedure.

The present study sought possible factors leading to the cytological diagnosis of atypical squamous cells of uncertain significance (ASCUS) in cases of high-grade squamous intraepithelial lesions (HSIL). Based on retrospective histopathological analysis of loop electrical excision procedure (LEEP) products that diagnosed HSIL, two study groups were randomly selected. The first was consisted of cases with two consecutive Papanicolaou (Pap) smears with the diagnosis of ASCUS. The second (control) group was represented by cases diagnosed as HSIL by cytology. From the Pap smears diagnosed as ASCUS, the sampling limitations was different from control group (P < 0.05). The median size of the largest lesion in each case with ASCUS was 2.66 mm (+/- 1.71 mm). In the control group, the median size of the largest lesion was 5.15 mm (+/-2.58 mm) (P < 0.05). The size of the lesion and sample limitations led patients with cervical intraepithelial neoplasms to be diagnosed as ASCUS for two consecutive times, after a 6-mo period.     

Wrist arthrodesis following ulnar bar excision in Fibrodysplasia ossificans progressiva

Fibrodysplasia ossificans progressiva is a rare disorder characterized by the progressive development of heterotopic bone in the connective tissues of skeletal muscle, ligaments and tendons. Surgical trauma is one of the most potent stimuli for ossification and surgical treatment is generally considered to be contraindicated in this condition. We report a good functional result in a patient with severe hand disability secondary to an ulna-carpal bar in fibrodysplasia ossificans progressiva.     

Increased expression of α2-6 linked sialic acids by nasal mucins in allergic rhinitis

Mucins have a polypeptide backbone, oligosaccharide side-chains and peripheral structures that include sialic acids. Several pathogens have specific receptors for sialic acids, including human strains of influenza A virus which preferentially recognise and bind α2-6 linked rather than α2-3 linked sialic acids.¹ 1 NElson J., COuceiro S.S., PAulson J.C. & BAum L. (1993) Influenza virus strains selectively recognise sialyloligosaccharides on human respiratory epithelium; the role of the host cell in selection of haemagglutinin receptor specificity. The aim of this study was to identify possible disease-related changes in the expression of sialic acids in nasal mucins. Nasal mucosal samples were placed in organ culture. Metabolically-labelled mucins were purified by gel filtration, blotted on to nitrocellulose membranes and probed with the sialic acid-binding lectins Sambucus nigra and Maackia amurensis. Ninety-five mucosal samples were collected (49 turbinates, 31 nasal polyps, 15 samples from FESS). Lectin binding, expressed as optical density, showed significantly increased binding of S. nigra to cellular (P = 0.02; Kruskal–Wallis) and secreted (P = 0.045) mucin from allergic mucosa compared to non-allergic mucosa. No significant differences were found in the binding patterns of M. amurensis. This study has demonstrated increased expression of α2-6 linked sialic acids in the mucins synthesised and secreted by allergic compared to non-allergic nasal mucosa. This may cause a change in the way mucins and pathogens interact in allergic rhinitis, leading to altered susceptibility to upper respiratory tract infection.     

Diagnosis of pulmonary embolism: Ventilation perfusion scintigraphy versus helical computed tomography pulmonary angiography

The present study compared the accuracy of ventilation perfusion scintigraphy (VQS) and CT pulmonary angiography (CTPA) for the diagnosis of pulmonary embolism. This was a prospective observational study of 112 patients with suspected pulmonary embolism (PE) who could be studied with both investigations within 24 h. Results were compared to final diagnosis at completion of 6-month follow up, using receiver operating characteristic (ROC) analysis. Pulmonary embolism was diagnosed in 27 referred patients (24%). The sensitivity and specificity of VQS and CTPA were similar to that reported from the literature. A normal VQ scan had the highest negative predictive value (100%), while a high-probability VQ scan had the highest positive predictive value (92%). There was no overall difference (area under the ROC curve (AUC)) between VQS (AUC (95% CI) = 0.82 (0.75,0.89)) and CTPA (AUC = 0.88 (0.81,0.94)) for the diagnosis of PE. Among patients with abnormal chest X-rays, CTPA (AUC 0.90 (0.83,0.97)) appeared somewhat better than VQS (AUC 0.78 (0.68,0.88)) but this difference did not reach statistical significance. In this instance, CTPA is at least as accurate as VQS and may provide an opportunity to make alternative diagnoses.     

Assessment of Hageman factor activation in human plasma: quantification of activated Hageman factor-C1 inactivator complexes by an enzyme- linked differential antibody immunosorbent assay

An enzyme-linked immunosorbent assay has been developed for the quantitation of activated Hageman factor-C1 inactivator (HF-C1 INH) complexes. Addition of increasing quantities of either of the major forms of activated Hageman factor (HFa or HFf) to normal plasma or to Hageman factor-deficient plasma leads to a dose-dependent increase in activated HF-C1 INH complexes. As little as 0.5 micrograms/mL of activated HF added to plasma can be detected, corresponding to activation of approximately 2% of plasma HF. The sensitivity of the assay is increased at least tenfold when complexes are formed in HF- deficient plasma, indicating competition between unactivated HF and activated HF-C1 INH complexes for binding to the antibody. Specificity is demonstrated in that addition of activated HF to hereditary angioedema plasma yields less than 1% of the activated HF-C1 INH complex formation obtained with normal plasma. Kaolin activation of HF- deficient plasma yields no detectable complex formation. Kaolin activation of prekallikrein-deficient plasma demonstrates a time- dependent increase in formation of activated HF-C1 INH complex consistent with the ability of HF in this plasma to autoactivate as the time of incubation with the surface is increased. Kaolin treatment of high-molecular weight (HMW) kininogen-deficient plasma yields an even more profound abnormality in the rate of formation of activated HF-C1 INH complexes reflecting the complex role of HMW kininogen in the initiation of contact activation. Although addition of corn inhibitor to plasma prevents activated HF-C1 INH complex formation, it does not inhibit activated HF sufficiently fast to prevent prekallikrein activation.     

Sera from patients with heparin-induced thrombocytopenia generate platelet-derived microparticles with procoagulant activity: an explanation for the thrombotic complications of heparin-induced thrombocytopenia

Heparin-induced thrombocytopenia is characterized by moderate thrombocytopenia and thrombotic complications, whereas quinine/quinidine-induced thrombocytopenia usually presents with severe thrombocytopenia and bleeding. Using flow cytometry and assays of procoagulant activity, we investigated whether sera from patients with these immune drug reactions could stimulate normal platelets to generate platelet-derived microparticles with procoagulant activity. Sera or purified IgG from patients with heparin-induced thrombocytopenia stimulated the formation of platelet-derived microparticles in a heparin-dependent fashion. Further studies showed that heparin-induced thrombocytopenia sera also produced a marked increase in procoagulant activity. In contrast, sera from patients with quinine- or quinidine-induced thrombocytopenia did not generate platelet-derived microparticles nor generate increased procoagulant activity. However, quinine/quinidine-induced thrombocytopenia sera produced a significant increase in the binding of IgG to platelets in a drug-dependent fashion, whereas sera from patients with heparin-induced thrombocytopenia demonstrated no drug-dependent binding of IgG to platelets. We also observed increased levels of circulating microparticles in patients with acute heparin-induced thrombocytopenia compared with control patients. Our observations indicate that the generation of procoagulant platelet-derived microparticles in vivo is a plausible explanation for the thrombotic complications observed in some patients with heparin-induced thrombocytopenia.