Multisite Reproducibility of Results Obtained by the Broth Microdilution Method for Susceptibility Testing of Mycobacterium abscessus, Mycobacterium chelonae, and Mycobacterium fortuitum

A multicenter study was conducted to assess the interlaboratory reproducibility of broth microdilution testing of the more common rapidly growing pathogenic mycobacteria. Ten isolates (four Mycobacterium fortuitum group, three Mycobacterium abscessus, and three Mycobacterium chelonae isolates) were tested against amikacin, cefoxitin, ciprofloxacin, clarithromycin, doxycycline, imipenem, sulfamethoxazole, and tobramycin (M. chelonae only) in four laboratories. At each site, isolates were tested three times on each of three separate days (nine testing events per isolate) with a common lot of microdilution trays. Agreement among MICs (i.e., mode +/- 1 twofold dilution) varied considerably for the different drug-isolate combinations and overall was best for cefoxitin (91.7 and 97.2% for one isolate each and 100% for all others), followed by doxycycline, amikacin, and ciprofloxacin. Agreement based on the interpretive category, using currently suggested breakpoints, also varied and overall was best for doxycycline (97.2% for one isolate and 100% for the rest), followed by ciprofloxacin and clarithromycin. Reproducibility among MICs and agreement by interpretive category was most variable for imipenem. Based on results reported from the individual sites, it appears that inexperience contributed significantly to the wide range of MICs of several drugs, especially clarithromycin, ciprofloxacin, and sulfamethoxazole. New interpretive guidelines are presented for the testing of M. fortuitum against clarithromycin; M. abscessus and M. chelonae against the aminoglycosides; and all three species against cefoxitin, doxycycline, and imipenem.     

cDNA cloning and chromosome mapping of the human Fe65 gene: interaction of the conserved cytoplasmic domains of the human beta-amyloid precursor protein and its homologues with the mouse Fe65 protein

Using the yeast two hybrid system, a mouse embryo cDNA library was screened for proteins that interact with the C-terminus of the human beta-amyloid precursor protein (beta PP). A fusion protein was identified that interacts specifically with the cytoplasmic domain of beta PP and does not interact with the beta-amyloid region. The protein encoded by this partial mouse cDNA is identical to the C-terminus of the rat Fe65 protein. This mouse protein also interacts with the homologous C-terminal domains of the mouse amyloid precursor-like proteins, APLP1 and APLP2. These conserved cytoplasmic regions contain a common amino acid motif, Asn-Pro-Thr-Tyr, which has previously been shown to influence both the secretion and internalization of beta PP. Fe65 has been implicated in regulatory and cell signaling mechanisms because it contains two different motifs involved in protein binding, a WW domain (a variant of Src homology 3 domains) and a phosphotyrosine interaction domain (PID). Interestingly, the PID domain binds to the same motif present in the conserved cytoplasmic domains of the beta PP and beta PP-like proteins. RNA analyses reveal that Fe65 is predominantly expressed in brain and in the regions most affected by Alzheimer's disease (AD)-associated neuropathology. The human Fe65 mRNA was cloned from a fetal brain cDNA library. The message encodes a protein of 735 amino acids that is 95% identical to the rat Fe65 protein. The human Fe65 gene was mapped on human metaphase chromosomes to band 11p15 using fluorescence in situ hybridization.      

Congenital gastrointestinal defects in Down syndrome: a report from the Atlanta and National Down Syndrome Projects

We report Down syndrome (DS)-associated congenital gastrointestinal (GI) defects identified during a 15 year, population-based study of the etiology and phenotypic consequences of trisomy 21. Between 1989 and 2004, six sites collected DNA, clinical and epidemiological information on live-born infants with standard trisomy 21 and their parents. We used chi-squared test and logistic regression to explore relationships between congenital GI defects and infant sex, race, maternal age, origin of the extra chromosome 21, and presence of a congenital heart defect. Congenital GI defects were present in 6.7% of 1892 eligible infants in this large, ethnically diverse, population-based study of DS. Defects included esophageal atresia/tracheoesophageal fistula (0.4%), pyloric stenosis (0.3%), duodenal stenosis/atresia (3.9%), Hirschsprung disease (0.8%), and anal stenosis/atresia (1.0%). We found no statistically significant associations between these defects and the factors examined. Although not significant, esophageal atresia was observed more often in infants of younger mothers and Hispanics, Hirschsprung disease was more frequent in males and in infants of younger mothers and blacks, and anal stenosis/atresia was found more often among females and Asians.     

BRCA2 alteration is important in clear cell carcinoma of the ovary

BRCA2 has been shown to play a significant role in hereditary ovarian carcinoma. Several cases of clear cell carcinoma (CCC) of the ovary containing BRCA2 mutations have been identified. We hypothesize that sequence variants of the BRCA2 gene are common in CCC of the ovary. Multiple methods were utilized to detect BRCA2 genetic alterations in a cohort of 13 ovarian CCC. These included an LOH analysis for copy number, real-time and methylation-specific polymerase chain reaction (PCR) to probe for BRCA2 promoter methylation, in addition to protein truncation testing (PTT) gel screening for nonsense BRCA2 mutations, and finally direct gene sequencing to either confirm the nonsense mutations or to detect candidate missense mutations in the remaining tumor samples. Whenever a sequence variation was detected in a tumor sample, the corresponding region was sequenced from a blood sample to determine germline status. Seven BRCA2 sequence variations were identified in 6 of the 13 CCC (46%); three tumors contained an alteration in BRCA2 copy number. Only one subject carried a germline sequence variation that might alter BRCA2 function despite the fact that a family history of breast, ovarian or colon cancer was common in this population. The 5-year disease-specific survival probability for patients with a BRCA2 alteration is 87.5%, compared to only 40% for those patients without a BRCA2 alteration ( p = 0.39).
Alterations in BRCA2 gene sequence, copy number, or expression are extremely common in CCC and may contribute to a paradoxical better clinical outcome.