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.      

Antibody avidity: use for the diagnosis of HIV early infection

Determination of IgG avidity is useful to distinguish primary infection from reactivation or reinfection in viral, parasitic or bacterial infections. For diagnosis of HIV type 1 primary infection, the detection of IgM antibodies is often useless since they are also found in chronic infection. The usual serology (Elisa, western-blot, p24 antigen) may present no interest if done too late (more than 2 or 3 months after infection). Therefore, we have developed a test to determine the avidity of anti-HIV1 antibodies, using 1 M guanidine as denaturing agent. We have adapted the measurement of avidity to the Axsym automatic system for a routine use. Indeed, since requests for avidity determinations are sporadic, the use of microplates is not convenient. Using this assay, we found a low avidity (less than 50%) in immunocompetent and recent infected patients (less than 6 months), compared to old infected patients (more than 12 months) who had high avidity (80 to 100%). However, early treated patients (in the 6 months after contamination) had also low avidities but with a slower development of antibody maturation (8 to 27 months versus 2 to 8 months in non treated patients). To conclude, the determination of the anti-HIV1 avidity, according to the proper procedures explained here (notion of treatment and/or serious immunodepression), may help the physician to date the infection in each new infected patient who might benefit from an early treatment.     

Intermediate filament expression by normal and diseased human corneal epithelium

Cicatricial conjunctivitis may be a sequel to systemic disorders (eg, Stevens-Johnson syndrome, cicatricial pemphigoid) or local disorders such as chemical burns. The cicatrisation is often associated with corneal epithelial changes that cause visual loss. These have been attributed to encroachment of the conjunctival epithelium over the cornea. However, the epithelial anomalies are poorly understood. We investigated the corneal epithelial changes in cicatricial conjunctivitis with an immunohistochemical study of intermediate filaments in normal and pathological specimens. Our results show that the normal corneal epithelium is immunoreactive for cytokeratin 3 (CK 3) but not cytokeratin 19 (CK 19), whereas normal conjunctival epithelium is CK 3 negative and CK 19 positive. Conjunctiva artificially transposed over the cornea (after therapeutic conjunctival flap reconstruction) retained the normal pattern of conjunctival cytokeratin expression (CK 3 negative, CK 19 positive). Conversely, the entire corneal epithelium exhibited the normal cytokeratin pattern (CK 3 positive, CK 19 negative) in 82% of Stevens-Johnson, 80% of cicatricial pemphigoid, and 69% of chemical burns specimens. The findings suggest that conjunctival encroachment is not responsible for the changes at the corneal surface in cicatricial conjunctivitis and that the abnormal corneal epithelium is derived from native corneal cells in these diseases.     

乳头腺管口挑拨治疗急性乳腺炎32例

0　引言　急性乳腺炎是产后妇女的常见病 .我们采用针头挑拨治疗急性乳腺炎 32例 ,疗效显著 .1　临床资料　患者 32例 ,年龄 2 3～ 34 (平均 2 5 .3)岁 ,均为产后哺乳产妇 .其中患乳胀痛为共有症状 .伴发热 10例 ;右侧乳腺炎 14例 ,左侧乳腺炎 16例 ,双侧乳腺炎 2例 ;外上象限胀痛 6例 ,外下象限胀痛 8例 ,内上象限胀痛 7例 ,内下象限胀痛 6例 ,二象限同时胀痛 5例 .乳头均无破损 .患者取卧位 ,显露患乳 ,乳头以碘伏严密消毒后 ,术者持五号针头以指腹常规触诊患乳 ,触摸到肿胀部位后 ,沿乳腺管走行方向找到对应的乳头腺管口 ,以针尖轻轻挑拨…     

Glucuronidation of drugs by hepatic microsomes derived from healthy and cirrhotic human livers

Pharmacokinetic studies demonstrated that the decrease in drug biotransformation in hepatic failure depends on the metabolic pathways involved. To test whether glucuronidation reactions supported by UDP-glucuronosyltransferases are differentially affected in such conditions, we investigated the in vitro glucuronidation of four selected drugs and xenobiotics (zidovudine, oxazepam, lamotrigine, and umbelliferone) by using microsomes from human healthy and unhealthy (cirrhosis, hepatitis) livers as enzyme sources. Theses substances are glucuronidated by several UDP-glucuronosyltransferase isoforms. Lidocaine N-deethylation activity measured concomitantly was used as a positive control, because the inhibition of this reaction in patients with hepatic diseases is well documented. The metabolic clearances of zidovudine and lidocaine were decreased significantly in liver cirrhosis (0.17 versus 0.37 microliter/min/mg protein and 0.40 versus 2.73 microliter/min/mg protein, respectively) as a consequence of a decrease of their corresponding Vmax of metabolism. By contrast, the metabolic clearances of oxazepam, umbelliferone, and lamotrigine glucuronidation remained unchanged. Previous studies reported that the in vivo oral clearances of zidovudine and lidocaine were decreased by 70% and 60%, respectively, in cirrhotic livers, whereas those of lamotrigine and oxazepam were not affected. Consequently, it is likely that the in vitro metabolic data, which support the in vivo results, therefore could contribute to reasonably predict the level of impairment of hepatic clearance in patients with liver cirrhosis.     

Randomized comparison of transurethral electroresection and holmium: YAG laser vaporization for symptomatic benign prostatic hyperplasia

OBJECTIVE: To evaluate the safety and efficacy of holmium:YAG laser vaporization v transurethral electroresection (TURP) for benign prostatic hyperplasia. PATIENTS AND METHODS: Thirty-six patients were randomized. Two laser procedures (60 to 80 W) were performed for one TURP. Symptom Score, peak flow rate, potency, and ejaculation status were measured at baseline and at 1, 3, 6, and 12 months. RESULTS: The mean operative time was 75 minutes for laser and 56 minutes for TURP (P = 0.0407). With a mean laser energy delivered of 103.6 kJ, hemostasis was satisfactory during vaporization. The mean catheterization time was 1.7 and 2.1 days in the laser and TURP group, respectively. For the laser and TURP groups, the mean AUA Score improved from 20 preoperatively to 7 and from 24.1 to 5, respectively, at 12 months. The mean peak flow increased from 8.4 to 19.5 mL/sec and from 7.6 to 16.8 ml/sec, respectively, at 12 months. These results are not statistically different. No significant initial dysuria occurred. No significant difference between the groups appeared in potency or ejaculatory status during the follow-up. One patient in the laser group (Day 5) and two in the TURP group (2nd and 6th month) had to undergo a second procedure to relieve obstruction. CONCLUSION: Although taking slightly longer to accomplish, holmium:YAG laser vaporization of BPH provides early results very similar to those of TURP with a shorter catheterization time and no initial dysuria or pain.