Clinical Abstracts
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June 2006 Editor: Cytogenetics and muscular dystrophy
A large proportion of eukaryotic genomes consists of repetitive DNA. The head-to-tail tandem repetitive sequences, categorized according to repeat-unit length, are highly polymorphic. Micro-, mini-, macro-, and mega-satellite repeats are distinguished, each with their own mutation characteristics. Several hypothetical rearrangement models have been proposed, mainly on the basis of micro- and mini-satellite instability. However, little is known about macro- and mega-satellite instability in human cells. Facioscapulohumeral muscular dystrophy, or FSHD, is an early-onset, autosomal dominant myopathy primarily characterized by a progressive and variable atrophy and weakness of the facial, shoulder, and upper arm muscles. The FSHD phenotype also presents with extra-muscular symptoms, such as retinovasculopathy, hearing loss, and, in severe cases, mental retardation. FSHD is associated with a macro-satellite repeat instability—the contraction of the subtelomeric D4Z4 repeat on 4q35. This polymorphic D4Z4 repeat is highly recombinogenic since somatic mosaicism for a rearrangement of D4Z4 is found in up to three percent of the general population. The authors of this study further examined the mechanism by which D4Z4 rearranges by using the 4qA/ 4qB polymorphism as a distal flanking marker in combination with a newly identified restriction fragment length polymorphism within D4Z4 proper, serving as a proximal flanking marker. Studying these proximal and distal markers provided insight into the mechanism and timing of D4Z4 rearrangements and may stand as a model for macro-satellite repeat mutation in general. Autosomal dominant facioscapulohumeral muscular dystrophy, or FSHD1A, is associated with contractions of the polymorphic D4Z4 repeat on chromosome 4qter. Almost half of new FSHD mutations occur postfertilization, resulting in somatic mosaicism for D4Z4. Detailed D4Z4 analysis of 11 mosaic individuals with FSHD revealed a mosaic mixture of a contracted FSHD-sized allele and the unchanged ancestral allele in eight cases, which is suggestive of a mitotic gene conversion without crossover. However, in three cases, the D4Z4 rearrangement resulted in two different-sized D4Z4 repeats, indicative of a gene conversion with crossover. In all cases, DNA markers proximal and distal to D4Z4 showed no allelic exchanges, suggesting that all rearrangements were intrachromosomal. The authors proposed that D4Z4 rearrangements occur via a synthesis-dependent strand-annealing model that allows for crossovers relatively frequently. Furthermore, the distribution of different cell populations in mosaic patients with FSHD suggests the mosaicism results from D4Z4 rearrangements occurring during the first few zygotic cell divisions after fertilization. Lemmers RJLF, Van Overveld PGM, Sandkuijl LA, et al. Mechanism and timing of mitotic rearrangements in the subtelomeric D4Z4 repeat involved in facioscapulohumeral muscular dystrophy. Am J Hum Genet. 2004;75:44–53. Reprints: Reprints: Dr. Silvère M. van der Maarel, Leiden University Medical Center, Dept. of Human and Clinical Genetics, Wassenaarseweg 72, 2333 AL Leiden, Netherlands; maarel@lumc.nl Cryopreservation involves three major phases—a prefreezing phase, in which the cells are exposed to a cold shock; a critical freezing phase, in which cell membranes are exposed to osmotic and thermal stresses; and a phase wherein reverse process osmotic and thermal stress occur. During these phase transitions, the cell membranes are highly vulnerable to variation in thermal and osmotic conditions. Oxygen-free radical generation during freezing contributes to cell damage. Current freezing protocols for hematopoietic cells generally involve using dimethyl sulfoxide (DMSO) as a cryoprotectant, slow cooling rate, and storage below –120°C. Because cyropreservation is indispensable in the banking of umbilical cord blood cells for use in unrelated human leukocyte antigen-matched procedures, the goal is to achieve optimal freezing protocols for improved recovery. The use of membrane stabilizer and bioantioxidant has been shown to afford cryoprotection. A combination of catalase and trehalose in conventional freezing medium containing 10 percent DMSO helped protect various functions of hematopoietic cells that are important in clinical settings, such as growth factor responsiveness, preservation of surface molecules, and long-term culture-forming ability. In this report, the authors provided evidence that the additives protect important biologic properties of frozen hematopoietic cells, namely, migration, adhesion, and homing. They studied homing-related properties like migration toward stromal-derived factor-1α (SDF-1α), adhesion on stroma and extracellular matrix, FLT3 responsiveness, and expression of FLT3R. Mononuclear and CD34+ cells isolated from cord blood were used as a source of hematopoietic cells. The KG1a cell line was used as a model system in adhesion assays. The cells were frozen in a programmable freezer and stored at –196°C. Various homing-related assays were carried out on the frozen cells. The authors reported that the two additives afford better preservation of adhesion- and migration-related properties of the frozen cells. The test cells frozen with additives resulted in improved migration toward SDF-1a and showed higher expression of its receptor CXCR4. Colony-forming unit assay of migrated test cells showed that these cells are early progenitors that have the capacity to give rise to all types of myeloid colonies. Test cells also show increased expression of FLT3R and improved responsiveness to FLT3 ligand, the homing-related cytokine. Adhesion to stroma and extracellular matrix was better in test cells than in control cells. The authors concluded that the data provide evidence that adding catalase and trehalose to the conventional freezing medium preserves migration- and adhesion-related properties of the hematopoietic graft. Sasnoor LM, Kale VP, Limaye LS. A combination of catalase and trehalose as additives to conventional freezing medium results in improved cryoprotection of human hematopoietic cells with reference to in vitro migration and adhesion properties. Transfusion. 2005;45:622–633. Reprints: Reprints: Lalita Shrikant Limaye, National Center for Cell Science, Ganeshkhind, Pune, 411007, India; Islimaye@nccs.res.in
Campylobacter isolation requires a simple medium that uses antibiotics as selective agents. These antibiotics vary from the cocktail of vancomycin, polymixin B, and trimethroprin found in Skirrow’s medium, to a single antibiotic (cefazolin) in CDA and cefoperazone in modified CDA. Less inhibitory agents have also been advocated, such as teicoplanin in CAT medium. However, Campylobacter jejuni and C. coli remain the two main pathogens responsible for Campylobacter enteritis, and early diagnosis by culture and isolation can enhance clinical investigation. The ability to recover campylobacters depends on the selective media and techniques used. Studies have found that filtration techniques are effective but time consuming and that molecular methods of detecting campylobacters using DNA probes are not ideal for routine clinical use. Because a preliminary study evaluating conventional methods was inconclusive, the author conducted a pilot study to establish an isolation-selective medium that would support the growth of most common enteric campylobacters. The study evaluated six antibiotics not previously used in campylobacter-selective medium with the hope that one antibiotic would prove active in microaerobic conditions, suppress gram-negative flora, and yet be noninhibitory to Campylobacter spp at the incubation temperature of 37°C. Aztreonam promised to fulfill these requirements. Vancomycin was also chosen because of its inhibitory effect on gram-positive contaminating flora and was assessed for its antagonism to aztreonam. Amphotericin was included in the selective combination for its activity against yeasts. This pilot study for aztreonam amphotericin vancomycin (AAV) medium used several challenge methods, and AAV was compared with modified CDA medium to verify its suitability as a selective medium for isolating Campylobacter spp. The AAV experimental campylobacter-selective medium showed growth microaerobically at 37°C of C. jejuni, C. coli, C. lari, C. hyointestinalis, C. fetus subsp. fetus, and C. jejuni subsp. Doylei after 24 to 48 hours of incubation. Six campylobacter NCTC strains demonstrated a minimum inhibitory concentration (MIC) of ±256 mg/L for vancomycin and aztreonam, whereas C. upsaliensis and two campylobacter-like strains now reclassified under genus helicobacter—H. cinaedi and H. fennelliae—had a MIC of 4 mg/L for vancomycin and aztreonam. In the pilot study of 150 samples, AAV medium (37°C) has a higher sensitivity for isolating campylobacters: 14 were isolated on AAV compared with 10 on modified CDA (43°C) over three days, and nine were isolated on AAV medium compared with five on modified CDA (43°C) after 24 hours of incubation. Contamination rates remained low. The medium was devised in a pilot study performed between 1990 and 1993; however, this is the first report of AAV medium used as a selective medium capable of growing six campylobacters of pathogenic importance at 37°C. The author concluded that further studies are indicated. Thomas GD. Pilot study for the development of a new campylobacter selective medium at 37°C using aztreonam. J Clin Pathol. 2005;58:413–416. Reprints: Reprints: G. D.Thomas at Ipi@Ipi37.com
Evidence showing that folic acid protects against the first occurrence and recurrence of neural tube defects led expert committees worldwide to issue folic acid guidelines recommending that women of child-bearing age, who are capable of becoming pregnant, take 400 μg/day folic acid. In general, it is recommended that this be done from before conception until the end of the first trimester. The issue of whether women should be supplemented with folic acid in later pregnancy is not addressed in official recommendations; therefore, practice is likely to be highly variable. Recently, it has been reported that, at as early as 15 weeks of gestation, women who subsequently go on to have severe preeclampsia have significantly increased plasma total homocysteine (tHcy) concentrations compared with control women of similar gestational age. Folate is the major determinant of tHcy concentration, with well-established evidence that supplementation with folic acid has a marked homocysteine-lowering effect. Several studies have reported that tHcy concentrations are lower during pregnancy than during the nonpregnant state. The authors of this study assessed the role of folate status in modulating tHcy concentration during pregnancy in a longitudinal study. Specifically, they sought to address whether folic acid supplementation in later pregnancy was of potential benefit to maternal health with respect to tHcy concentrations and folate status. In a longitudinal study, they determined tHcy and corresponding folate status in 101 pregnant women at 12, 20, and 35 weeks of gestation, in 35 nonpregnant controls sampled concurrently, and in a subgroup (n=21 pregnant women and 19 nonpregnant controls) at three days postpartum. They found that plasma tHcy was significantly lower throughout pregnancy than in nonpregnant controls, with values lowest in the second trimester before increasing toward nonpregnant values in the third trimester. Importantly, mean tHcy concentrations were lower in pregnant women taking folic acid supplements than in those who were not, an effect that reached significance in the third trimester (5.45 versus 7.40 μmol/L; P<0.05). During the third trimester, tHcy concentrations were significantly higher in pregnant women with a history of miscarriage than in women with no previous history (8.15 versus 6.38 μmol/L; P<0.01). The authors determined that this is the first longitudinal study to show that homocysteine concentrations increase in late pregnancy toward nonpregnant values; an increase that can be limited by enhancing folate status through continued folic acid supplementation. These results indicate a potential role for continued folic acid supplementation in reducing pregnancy complications associated with hyperhomocysteinemia. Holmes VA, Wallace JMW, Alexander HD, et al. Homocysteine is lower in the third trimester of pregnancy in women with enhanced folate status from continued folic acid supplementation. Clin Chem. 2005;51:629–634. Reprints: Julie M.W. Wallace, Northern Ireland Centre for Food and Health (NICHE), School of Biomedical Sciences, University of Ulster, Coleraine, Co Londonderry BT52 1SA, United Kingdom; j.wallace@ulster.ac.uk
Alterations of white blood cell count are well recognized features of sepsis, and raised white blood cell count and neutrophilia are associated with bacteremia in adults and children. A rapid, profound lymphopenia occurs in primates with experimental bacteremia. Recent clinical studies, performed on groups at low risk of human immunodeficiency virus, suggest that lymphopenia may define patients at high risk of adverse outcome. For example, lymphopenia is a risk factor for intensive care in unwell infants and for death in nursing home residents with pneumonia. In view of this, the authors investigated whether lymphopenia was useful for predicting bacteremia clinically, an activity that remains imprecise even after detailed study. It is difficult to evaluate the literature on the relation between lymphopenia and bacteremia. Two observational studies involving about 750 elderly patients have noted lymphopenia in some patients with bacteremia, but specificity varied between the studies, and the distributions of the counts were not reported. Generalization of the published data to other populations is further complicated by the gradual decline in lymphocyte counts that occurs as normal adults age. The authors attempted to clarify the relation between age, lymphocyte count, neutrophil count, and bacteremia by studying a large cohort of adults with medical emergencies in a region that had a low prevalence of HIV. Their two-year cohort study was carried out in a teaching hospital in Oxford, United Kingdom, and involved 21,331 adult emergency admissions to general medical or infectious disease wards. The authors found that neutrophilia and lymphopenia were associated with bacteremia. Lymphopenia was the better predictor in this cohort. Neutrophilia and lymphopenia were more predictive of bacteremia than total white blood cell count. The authors concluded that lymphocyte and neutrophil counts, rather than total white blood cell count, should be considered in adult medical admissions with suspected bacteremia. Wyllie DH, Bowler ICJW, Peto TEA. Relation between lymphopenia and bacteraemia in UK adults with medical emergencies. J Clin Pathol. 2004;57:950–955. Reprints: Dr. D.H. Wyllie, Nuffield Dept. of Clinical Laboratory Sciences, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom; david.wyllie@ndcls.ox.ac.uk Dr. Bissell is Professor and Director of Clinical Services and Vice Chair, Department of Pathology, Ohio State University Medical Center, Columbus. |
