Feasibility study of repeated fluorescent in-situ hybridization in the same human blastomeres for preimplantation genetic diagnosis

In order to increase the number of chromosomes examined in each blastomere, we have developed a repeated fluorescent in-situ hybridization (FISH) procedure by which six or more chromosomes can be analysed per blastomere of a human embryo. Three consecutive FISH procedures with directly-labelled fluorescent Vysis DNA probes were carried out for examination of chromosomes X, Y, 11, 13, 18 and 21 in the same blastomeres (n = 126) and lymphocytes (n = 164). Based on the initial number of nuclei, the percentages of nuclear loss and presence of signals were 3 and 92% respectively in blastomeres; 6 and 91% respectively in lymphocytes after the first FISH; 7 and 87% respectively in blastomeres and 10 and 86% respectively in lymphocytes, after the second FISH. These percentages were 13 and 78% respectively in blastomeres and 14 and 81% respectively in lymphocytes after the third FISH. The FISH procedure was repeated successfully in a couple for preimplantation genetic diagnosis of chromosomal aneuploidies in biopsied blastomeres of their embryos in our clinic. In conclusion, it is feasible to carry out repeated FISH procedures in the same blastomeres. Six or more chromosomes of a single blastomere may be examined using this procedure.      

Hirschsprung disease in MEN 2A: increased spectrum of RET exon 10 genotypes and strong genotype-phenotype correlation

The RET proto-oncogene encodes a transmembrane receptor with tyrosine kinase activity. Germline mutations in RET are responsible for a number of inherited diseases. These include the dominantly inherited cancer syndromes multiple endocrine neoplasia types 2A and 2B (MEN 2A and MEN 2B) and familial medullary thyroid carcinoma (FMTC), as well as some cases of familial Hirschsprung disease (HSCR1). RET mutations in HSCR1 have been shown to cause a loss of RET function, while the cancer syndromes result in RET oncogenic activation. Occasionally MEN 2A or FMTC occurs in association with HSCR1, albeit with low penetrance. An initial report linked HSCR1 in MEN 2A solely to the C618R and C620R RET mutations. In this study we have analyzed 44 families with MEN 2A. HSCR1 co-segregated with MEN 2A in seven (16%) of the 44 families. The predisposing RET mutation in all seven families had been previously reported in MEN 2A or FMTC and occurred in exon 10 at codons 609, 618 or 620, resulting in C609Y, C618S, C620R or C620W substitution. MEN 2A families with RET exon 10 Cys mutations had a substantially greater risk of developing HSCR1 than those with the more common RET exon 11 Cys634 or exon 14 c804 mutations (P = 0.0005). These findings suggest that expression of HSCR1 in MEN 2A may be peculiar to RET exon 10 Cys mutations . However, HSCR1 in MEN 2A is not exclusive to C618R or C620R RET mutations and can occur with other exon 10 Cys amino acid substitutions. The strong correlation between disease phenotype and position of the MEN 2A RET mutation suggests that oncogenic activation of RET alone is insufficient to account for co-expression of the diseases.      

TUBERCULOSIS IN BCG VACCINATED AND UNVACCINATED CHILDREN

One hundred, non-consecutive, non-randomized, cases of tuberculosis divided in 2 groups i.e. Group A including 50 BCG vaccinated children and Group B including 50 unvaccinated children were studied to determine the pattern of tuberculosis and the role of protein energy malnutrition in the pathogenesis of tuberculosis. Thirty four per cent of Group A and 52 per cent of Group B had severe protein energy malnutrition. Sixty eight per cent in Group A and 76 per cent in Group B had intrathoracic forms of tuberculosis. Twelve (24%) patients in Group A and 11 (22%) in Group B suffered from serious forms of tuberculosis including tubercular meningitis, miliary tuberculosis and disseminated tuberculosis. The difference was not statistically different (p>0.05). However, in severe form of tuberculosis, the morbidity in vaccinated group was less. Sixty six per cent of vaccinated children with disseminated forms of tuberculosis had features of severe protein energy malnutrition. BCG is not effective in preventing tubercular infection in children of preschool age. It is effective to a certain extent in localizing the infection to a particular organ. Severe protein energy malnutrition is a contributing factor in the genesis of tuberculosis in preschool children vaccinated with BCG at or immediately after birth.KEY WORDS: BCG vaccine, Protein energy malnutrition, Tuberculosis     

Socioeconomic status, hostility, and risk factor clustering in the normative aging study: Any help from the concept of allostatic load?

OBJECTIVE: To examine the relationships between socioeconomic status (SES), psychosocial vulnerability (hostility), and allostatic load. Allostatic load refers to the cumulative physiological cost of adaptation to stress. METHOD: We examined the relationships between SES (as measured by educational attainment), hostility, and allostatic load in the Normative Aging Study, a longitudinal study of community-dwelling men aged 21 to 80 years and free of known chronic medical conditions at entry in the 1960s. In 1986, the revised Minnesota Multiphasic Personality Inventory was administered by mail, from which a hostility measure was derived by summing the scores from three Cook-Medley subscales: Hostile Affect, Hostile Attribution, Aggressive Responding. An index of allostatic load was constructed from data collected during physical exams conducted between 1987 and 1990 (i.e. measures reflecting "wear and tear" on the cardiovascular, endocrine, and metabolic systems). Cross-sectional relationships between education, hostility, and allostatic load were examined in 818 men. RESULTS: Separate linear regression analyses indicated that lower levels of educational attainment and greater hostility were both associated with higher allostatic load scores (p < .05 and p < .01, respectively). Less education was also associated with higher hostility (p < .001). When allostatic load was regressed simultaneously on education and hostility, the effect of education was attenuated, while hostility (p < .05) maintained an independent effect. CONCLUSIONS: Our findings suggest that lower levels of education and greater hostility are associated with greater "wear and tear" on the body. The effects of education on allostatic load may be mediated by hostility.     

Clinical evaluation of scanning laser polarimetry: I Intraoperator reproducibility and design of a blood vessel removal algorithm

AIMS—To evaluate the reproducibility of the retardation values (change in polarisation) obtained with the scanning laser polarimeter in a series of normal subjects and glaucoma patients. To improve the analysis of the raw data by devising and evaluating a blood vessel removal algorithm.
METHODS—Scanning laser polarimetry was performed on 10 normal subjects and 10 glaucoma patients. A series of six images was obtained from each eye. The normal subjects were re-imaged 3 months after their initial assessment. The retardation values obtained from each eye were analysed using the authors' own methods, including the use of an algorithm to remove blood vessels from the polar profiles. The reproducibility of these measurements and the performance of the blood vessel removal algorithm were assessed.
RESULTS—The "individual point" coefficient of variation was approximately 12.5% for normal subjects and 17.0% for glaucoma patients. The "integral" coefficient of variation for these groups was approximately 5.5% and 9.5% respectively. The reproducibility of the measurements did not improve with an increased number of measurements. There was no difference in the reproducibility of the measurements in normal subjects over time. The blood vessel removal algorithm improved the reproducibility of the measurements when the shape of the profile was assessed.
CONCLUSION—The intraoperator reproducibility of retardation values obtained with the scanning laser polarimeter is satisfactory for its use as a clinical tool. The use of a blood vessel removal algorithm improves the reproducibility of the measurements and also assists the clinician in the interpretation of the polar profiles. Furthermore, it allows the construction of normal database polar profiles, thereby enabling the identification, location and quantification of retinal nerve fibre layer damage in an "at risk" individual's polar profile.

Keywords: scanning laser polarimetry; glaucoma; reproducibility; algorithm     

Topographical distribution of blood supply to the anal canal

BACKGROUND: It has been suggested that anal fissure is an ischaemic ulcer caused by a combination of poor blood supply to the posterior midline of the anal canal and spasm of the internal anal sphincter. This study investigated the topographical distribution of blood supply to quadrants of the anal canal above and below the dentate line. METHODS: Cadaveric anal canals were removed and 1-cm blocks were cut above and below the dentate line. Blocks were sectioned at 10 microm and every 25th section was mounted. Using the technique of systematic random sampling, fields in the subanodermal space and the internal anal sphincter in posterior, lateral and anterior quadrants of the anal canal were chosen. The numbers of small arterioles in each field were counted. Mean counts were compared for both subanodermal space and internal anal sphincter between quadrants and levels above and below the dentate line using Page's L test for trends. RESULTS: Anal canals from eight cadavers were examined. There was a significant trend to an increasing number of arterioles from posterior to anterior in the subanodermal space at all levels and at two of three levels in the internal anal sphincter. CONCLUSION: The arteriolar density is less in the posterior quadrant throughout the anal canal. It may be that this poor blood supply predisposes to the development of anal fissures at their most common site in the posterior midline.