Isolation of a peptide inhibitor of human rhinovirus

Cell culture-based transdominant genetic techniques provide new methods for discovering peptide/RNA modulators of cellular pathways. We applied this technology to isolate a peptide inhibitor of human rhinovirus. A green fluorescent protein (GFP)-scaffolded library of cDNA fragments was expressed in HeLa cells from a retroviral vector and screened for inhibitors of rhinovirus-mediated cell killing. A DNA clone, I421, increased cell survival in an HRV14 challenge assay from less than 0.5% to greater than 60%. It encodes a 53-amino-acid C-terminal extension of the GFP scaffold. Particular subclones of Hela cells expressing I421 (exemplified by I421dp3) show a delay in virus production and a 50-fold decrease in viral RNA levels at 6-8 h postinfection. HRV2, HRV14, and HRV16 show a dramatic decrease in plaque-forming ability on I421dp3 while Coxsackievirus B3 showed a small reduction. Levels of ICAM-1, the receptor for the main rhinovirus serotype, are not altered in I421dp3.     

NF1 gene analysis based on DHPLC

The high mutation rate at the NF1 locus results in a wide range of molecular abnormalities. The majority of these mutations are private and rare, generating elevated allelic diversity with a restricted number of recurrent mutations. In this study, we have assessed the efficacy of denaturing high-performance liquid chromatography (DHPLC), for detecting mutation in the NF1 gene. DHPLC is a fast and highly sensitive technique based on the detection of heteroduplexes in PCR products by ion pair reverse-phase HPLC under partially denaturing conditions. We established theoretical conditions for DHPLC analysis of all coding exons and splice junctions of the NF1 gene using the WAVEmaker software version 4.1.40 and screened for mutations a panel of 40 unrelated NF1 patients (25 sporadic and 15 familial), genetically uncharacterized. Disruptive mutations were identified in 29 individuals with an overall mutation detection rate of 72.5%. The mutations included eight deletions (exons 4b, 7, 10a, 14, 26, and 31), one insertion (exon 8), nine nonsense mutation (exons 10a, 13, 23.1, 27a, 29, 31, and 36), six missense mutations (exons 15, 16, 17, 24, and 31), four splice errors (exons 11, 14, 36, and 40) and a complex rearrangement within exon 16. Eighteen (62%) of the identified disruptive mutations are novel. Seven unclassified and three previously reported polymorphisms were also detected. None of the missense mutations identified in this study were found after screening of 150 controls. Our results suggest that DHPLC provides an accurate method for the rapid identification of NF1 mutations.     

Risk factors,clinical course and outcomes of pregnancy-related group A streptococcal infections: retrospective 13-year cohort study

Objectives

To investigate the incidence, risk factors, clinical course and outcomes of pregnancy-related group A streptococcus (GAS) infection.

Implementation of HaemScreen, a workplace-based genetic screening program for hemochromatosis

There is debate as to whether community genetic screening for the mutation(s) causing hereditary hemochromatosis (HH) should be implemented, due to issues including disease penetrance, health economic outcomes, and concerns about community acceptance. Hemochromatosis is a common preventable iron overload disease, due in over 90% of cases to C282Y homozygosity in the HFE gene. We are, therefore, piloting C282Y screening to assess understanding of genetic information and screening acceptability in the workplace setting. In this program, HaemScreen, education was by oral or video presentation in a group setting. C282Y status was assessed by polymerase chain reaction (PCR) and melt-curve analysis on DNA obtained by cheek-brush sampling. Of eligible participants, 5.8% (1.5-15.8%) attended information and screening sessions, of whom 97.7% (5571 individuals) chose to be tested. Twenty-two C282Y (1 : 253) homozygotes were identified and offered clinical follow-up. There were 638 heterozygotes (1 : 8.7). The determinants for participation have been analyzed in terms of the principles outlined in the Health Belief Model. Widespread screening for HH is readily accepted in a workplace setting, and a one-to-many education program is effective. The level of participation varies greatly and the advertizing and session logistics should be adapted to the specific features of each workplace.     

Novel mutations, including the second most common in Japan, in the β-hexosaminidase α subunit gene, and a simple screening of Japanese patients with Tay-Sachs disease

Two novel mutations of the β-hexosaminidase α subunit gene were identified in Japanese patients with the infantile form of Tay-Sachs disease. One mutation was a one-base deletion at nt613C, which generated a stop codon at two codons downstream, in three unrelated patients. The other mutation was a one-base substitution of G-to-A at IVS 5, +1, which caused a splicing abnormality, in one patient. A missense mutation of R170W, which has already been reported in other ethnic groups, was also newly identified in one patient. In 1993, the most common mutation (IVS 5, −1G → T) in Japanese patients with Tay-Sachs disease was reported as the major mutation in Japan accounting for 80% of 56 mutant alleles from 28 unrelated patients. The deletion of nt613C was the second most common mutation, accounting for 5% of the mutant alleles. The previously reported mutation IVS 5, −1G → T and the nt613C deletion found in this study together accounted for 85% of the mutations causing Tay-Sachs disease among Japanese. Since these two mutations were located in or close to exon 6 and since they abolish Fok I (IVS 5, −1G → T) and Sfa NI (nt613C deletion) restriction sites, respectively, they were screened rapidly by single polymerase chain reaction followed by digestion with these enzymes. Received: November 10, 1998 / Accepted November 14, 1998     

Aberrant retinal projections to midbrain targets mediate spared visual orienting function in hamsters with neonatal lesions of superior colliculus

Summary Rodents, cats, and most nonmammalian vertebrates with bilateral tectal deafferentation or ablation in adulthood are extremely deficient at orienting to visual stimuli; yet animals with neonatal lesions of superficial layers of the superior colliculus (SC) show partial sparing of this response, particularly for targets in the central visual field. In this study, we sought to determine whether these spared orienting abilities are mediated by aberrant retinal projections to the remaining intermediate layers of the SC, or whether visual cortex (VC) mechanisms or alternative behavioral strategies are responsible. Neonatal golden hamsters received either bilateral heat lesions of the SC (rlSC), or a heat lesion of the right SC and enucleation of the right eye (rSCrE). This latter procedure causes axons from the left eye to recross the tectal midline and terminate in the wrong (left) SC (Schneider 1973). As adults, both groups of hamsters were extremely deficient in visually guided approach to stationary targets, although rlSC-lesioned hamsters showed some sparing for central field targets and rSCrE-lesioned hamsters often made wrong-direction turns for targets in the left peripheral field. We then subjected both groups of neonatally lesioned hamsters to bilateral aspiration lesions of the VC. Retesting showed no change in visual orienting behavior as a result of the cortical lesions. Labeling of the optic tract with horseradish peroxidase (HRP) revealed abundant aberrant retinal projections to remaining intermediate layers of the SC and thalamic nucleus lateralis posterior (LP), as well as supernormal innervation of pretectal nuclei, the dorsal terminal nucleus of the accessory optic tract, and the ventral nucleus of the lateral geniculate body (LGv). We conclude that the spared visual orienting capabilities of hamsters with rlSC and rSCrE lesions are mediated by the aberrant midbrain projections, and that cortical mechanisms are not involved in spared visual orienting functions following these neonatal lesions.     

Allele-specific PCR and Next-generation sequencing based genetic screening for Congenital Adrenal Hyperplasia in India

Genetic screening of Congenital Adrenal Hyperplasia (CAH) is known to be challenging due to the complexities in CYP21A2 genotyping and has not been the first-tier diagnostic tool in routine clinical practice. Also, with the advent of massive parallel sequencing technology, there is a need for investigating its utility in screening extended panel of genes implicated in CAH. In this study, we have established and utilized an Allele-Specific Polymerase Chain Reaction (ASPCR) based approach for screening eight common mutations in CYP21A2 gene followed by targeted Next Generation Sequencing (NGS) of CYP21A2, CYP11B1, CYP17A1, POR, and CYP19A1 genes in 72 clinically diagnosed CAH subjects from India. Through these investigations, 88.7% of the subjects with 21 hydroxylase deficiency were positive for eight CYP21A2 mutations with ASPCR. The targeted NGS assay was sensitive to pick up all the mutations identified by ASPCR. Utilizing NGS in subjects negative for ASPCR, five study subjects were homozygous positive for other CYP21A2 variants: one with a novel c.1274G>T, three with c.1451G>C and one with c.143A>G variant. One subject was compound heterozygous for c.955C>T and c.1042G>A variants identified using ASPCR and NGS. One subject suspected for a Simple Virilizing (SV) 21 hydroxylase deficiency was positive for a CYP19A1:c.1142A>T variant. CYP11B1 variants (c.1201-1G>A, c.1200+1del, c.412C>T, c.1024C>T, c.1012dup, c.623G>A) were identified in all six subjects suspected for 11 beta-hydroxylase deficiency. The overall mutation positivity was 97.2%. Our results suggest that ASPCR followed by targeted NGS is a cost-effective and comprehensive strategy for screening common CYP21A2 mutations and the CAH panel of genes in a clinical setting.     

Characterization of a cosmid library from flow-sorted chromosomes 11

A cosmid library specific for human chromosome 11 has been constructed from flow-sorted chromosomes. The flow-purified chromosomes were prepared from the hamster/human hybrid line J1 which contains chromosome 11 as the only human chromosome. Individual clones were sampled in 187 microtitre plates, resulting in a total of 17 952 colonies. Hybridization analysis revealed that 83.7% of these clones were of human and 10.4% of hamster origin. The average insert size was estimated at 33.6 kb, and only 2.4% of insert fragments appear to be rearranged. This should result in 494 487 kb of cloned human DNA representing 3.5 chromosome 11 equivalents. We have prepared high-density nylon membranes of the arrayed library containing 1 536 single colonies per filter. We have demonstrated the usefulness of the library in the molecular genetic analysis of human chromosome 11 by testing for the presence of possibly polymorphic simple repeat motifs, by identifying cosmids that contain inserts from the telomeric ends of chromosome 11 and by assessing the potential of the library for rapid chromosome walking.     

Evaluating plasma holds in the presence of multiple infections

To protect plasma supplies, donors are screened for infectiousdiseases. As an added measure of protection, donations are identifiedand stored for a period of time to allow future discard in theevent that an identified donor subsequently tests positive forsome screened disease. Previous models for evaluating such plasmaholds have only addressed the case of a single infectious disease.This paper extends the analysis to the case of multiple infections.Given knowledge of the marginal incidence rates for those infectionschecked, upper and lower bounds for important quantities suchas the probability of interdicting an infectious but undetecteddonation, the expected number of infections interdicted perdonation, and the net economic benefits of the holding policyare developed. Several examples are developed, illustratinghow the models can be used to evaluate the consequences of aplasma hold.