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.      

Oocyte morphology predicts outcome of intracytoplasmic sperm injection

To examine the influence of cytoplasmic morphology on the success rate of intracytoplasmic sperm injection (ICSI), the morphology of 837 metaphase II oocytes was assessed after cumulus stripping. The main abnormalities detected were excessive granularity, cytoplasmic inclusions such as vacuoles, smooth endoplasmic reticulum clustering and refractile bodies. Microinjection was performed in 538 oocytes with normal cytoplasm, 142 out of 161 with excessive granularity and 112 out of 138 with cytoplasmic inclusions. Very poor oocytes were not injected. No difference was found in fertilization rate. The embryos achieved cleaved normally and a similar number of good quality embryos among the three groups was noted. The outcome of transfer of embryos derived solely from normal oocytes (group A: 72 patients, 183 embryos) was compared with those from oocytes with cytoplasmic abnormalities (group B: 34 patients, 85 embryos). In group A, 17 clinical pregnancies (24% per patient, implantation rate 10%) were established. In group B, only one clinical pregnancy (3% per patient, implantation rate 1%) was established, from the transfer of embryos derived from oocytes with homogeneous granularity of the cytoplasm. No pregnancy resulted following the transfer of embryos from eggs with cytoplasmic inclusions. The difference was statistically significant. The outcome of ICSI is dependent on the quality of the oocytes retrieved. Normal fertilization and early embryo development were achieved in oocytes with abnormal cytoplasm morphology, but the resulting embryos failed to demonstrate the same implantation potential as those derived from oocytes with normal cytoplasm.      

Isolation and characterization of human and rabbit sperm tail fibrous sheath

Using mechanical and chemical dissection methods, fibrous sheath was isolated both from normal ejaculated human spermatozoa and from rabbit cauda epididymal spermatozoa. The same techniques did not produce a pure preparation of fibrous sheath from ejaculated rabbit spermatozoa, suggesting that further cross-linking and stabilization of sperm structures occurs in response to components of the seminal plasma. The isolation procedures were monitored by phase contrast microscopy and the purity of the fibrous sheath was verified by electron microscopy. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of isolated human fibrous sheath revealed at least 14 protein bands of which the most intensely stained were of molecular weight 84, 72, 66.2, 57, 32 and 28.5 kDa. The rabbit fibrous sheath revealed at least 10 protein bands, of which the most intensely stained were 35.2, 32.7 and 28.5 kDa. The amino acid composition of the purified fibrous sheath from human and rabbit spermatozoa was similar, being high in aspartic acid and/or asparagine and glutamic acid and/or glutamine, serine, alanine, leucine, lysine and glycine, but low in histidine, tyrosine and isoleucine. This composition is similar to that reported for the rat and suggests that mammalian sperm tail fibrous sheaths are composed of similar types of proteins, although there are apparent differences in protein components between species.      

Respiratory control when measuring respiratory sinus arrhythmia during a talking task

The current study explored the effects of talking on respiratory sinus arrhythmia (RSA) during a semi-structured emotional interview (Adult Attachment Interview) using 76 female undergraduates. The effectiveness of 2 different methodological approaches (i.e. talking baseline or transfer function) was explored as respiratory control during talking tasks. RSA was collected during resting baseline, talking baseline, and interview conditions. Subjective reports of distress were higher in the interview than in the other 2 conditions. Mean RSA levels were significantly lower in the 2 talking tasks than in the resting baseline. After applying a transfer function for respiratory control, there were no significant differences between the 3 conditions. Moderator analyses yielded lower RSA values in the talking baseline and interview conditions for participants who reported greater distress during the interview. It was concluded that respiratory controls are likely necessary when using RSA in talking paradigms and that both approaches appeared to be adequate.     

Collaborative Initiative to Help End Chronic Homelessness: Introduction

The Collaborative Initiative to Help End Chronic Homelessness was a coordinated effort by the US Departments of Health and Human Services (HHS), Housing and Urban Development (HUD), and Veterans Affairs (VA), and the US Interagency Council on Homelessness to house and provide comprehensive supportive services to individuals with serious psychiatric, substance use, health, and related disabilities who were experiencing long-term chronic homelessness. Eleven communities received 3-year grants from HHS and VA (2003–2006) and up to 5-year grants from HUD (2003–2008) to implement the initiative. This article provides background on chronic homelessness, describes the federal collaboration to comprehensively address chronic homelessness, and introduces the seven articles in this special issue that describe the findings and lessons learned from the participating communities in addressing chronic homelessness. Collectively, these articles offer insight into the challenges and benefits of providing housing and services to individuals experiencing chronic homelessness.     

Immune responses to AAV in clinical trials

Recent findings in a clinical trial in which an adeno-associated virus (AAV) vector expressing coagulation factor IX (F.IX) was introduced into the liver of hemophilia B subjects highlighted a new issue previously not identified in animal studies. Upon AAV gene transfer to liver, two subjects enrolled in this trial developed transient elevation of liver enzymes, likely as a consequence of immune rejection of transduced hepatocytes mediated by AAV capsid-specific CD8(+) T cells. Studies in healthy donors showed that humans carry a population of antigen-specific memory CD8(+) T cells probably arising from wild-type AAV infections. The hypothesis formulated here is that these cells expanded upon re-exposure to capsid, i.e. upon AAV-2 hepatic gene transfer, and cleared AAV epitope-bearing transduced hepatocytes. Other hypotheses have been formulated which include specific receptor-binding properties of AAV-2 capsid, presence of capsid-expressing DNA in AAV vector preparations, and expression of alternative reading frames from the transgene. Absence of a valid animal model has prevented an in-depth mechanistic study of the phenomenon. Several possible solutions to the problem are discussed, including the administration of a short-term anti-T cell immunosuppression regimen concomitant with gene transfer. While more studies will be necessary to further define mechanisms and risks associated with capsid-specific immune responses in humans, monitoring of these responses in clinical trials will be essential to achieving the goal of long-term therapeutic gene transfer in humans.     

Bromodeoxyuridine incorporation and Ki 67 expression in oral hairy leukoplakia

OBJECTIVES: Oral hairy leukoplakia (HL) is an acan-thotic, hyperparakeratotic lesion characterised by the presence of a replicative Epstein-Barr virus (EBV) infection in the superficial and adjoining layers of the epithelium. EBV or its gene products are capable of modifying epithelial differentiation. The aim of this study was to establish whether the presence of EBV was associated with an alteration in cell turnover by assessing bromodeoxyuridine (BrdU) incorporation and Ki 67 expression in lesional tissue and control mucosa.
METHODS: Biopsies of HL together with age, site and sex matched controls ( n = 7 and 8 respectively) were incubated in 200 μM BrdU in vitro , fixed in methacarn and processed to paraffin wax. Following acid hydrolysis, incorporated BrdU and Ki 67 were identified in serial 5 fim sections using a three-stage immunoperoxidase technique and cell density expressed as the number of positive cells per mm basement membrane length.
RESULTS: Overall, there was no difference in the number of BrdU positive cells per mm basement membrane length between control and HL tissue. However, within HL alone, the presence'of focal EBV replication was associated with a significant reduction in the number of basal cells incorporating BrdU compared to adjacent EBV free areas (P < 0.05). There was no significant difference between Ki 67 positive cells in control and HL tissue and no evidence of a reduction of Ki 67 positive cells in areas associated with EBV replication.
CONCLUSIONS: These results suggest that there is no evidence of a generalised alteration of the proliferative capacity of basal cells in HL, although the focal reduction in BrdU incorporation may reflect subtle changes on cell turnover by EBV infection.     

新型藏药对快速进入高原者心电图改变的作用

目的 :研究藏药对部队进入高原增强习服的功效 ;方法 :1 71名进藏新兵 ,随机分为 6组 ,实验组用药分别为洛布桑胶囊、诺迪康胶囊、红景天胶囊、藏王宝胶囊、地奥心血康胶囊 ,空白对照组为医用淀粉胶囊 ,观察实验药物对进藏前后心电图的影响 ;结果 :进藏前实验组与对照组比较无差别 ,P >0 .0 5,进藏后对照组与各实验组心率、Q -T及QT TQ有显著差异 ;进藏后心率增快 ,Q -T、QT TQ比值增大 ,对照组与实验组存有显著差别 ,P <0 .0 5或P <0 .0 1 ;结论 :5种药物对心电图参数均有干预作用 ,以诺迪康、红景天及洛布桑作用较为显著 ,提示有增强高原习服作用