QF-PCR-based prenatal detection of aneuploidy in a southeast Asian population.

OBJECTIVES: We have investigated the efficacy of using quantitative fluorescent polymerase chain reaction (QF-PCR) for the prenatal recognition of aneuploidy in chromosomes 13, 18, 21, X and Y. A total of 1115 samples, from mainly southeast Asian patients, were analysed and compared in a blind trial to the results previously obtained cytogenetically. METHODS: A multiplex PCR involving 15 short tandem repeat (STR) sequences was used. The probability of two or more of these markers being informative was calculated, and this required the multiplex PCR to be modified. RESULTS: The QF-PCR and previous cytogenetic results concurred, except for two products of conception (POC). One of these may be a case of complete uniparental disomy that was not recognized cytogenetically. The other was tetraploid, and as such appeared normal using QF-PCR. A mosaic trisomy 18 was correctly identified. The population sample was of a mainly Chinese, ethnic origin, and the allele frequency, size and heterozygosity appeared more restricted than the population groups analysed hitherto. CONCLUSION: The QF-PCR methodology is an efficient cost-effective method of screening for major chromosome aneuploidy, and, for certain referral categories, could be used alone. It also appears to be applicable to patients of different ethnic origins.

Coordinate changes in Myosin heavy chain isoform gene expression are selectively associated with alterations in dilated cardiomyopathy phenotype.

BACKGROUND: The most common cause of chronic heart failure in the US is secondary or primary dilated cardiomyopathy (DCM). The DCM phenotype exhibits changes in the expression of genes that regulate contractile function and pathologic hypertrophy. However, it is unclear if any of these alterations in gene expression are disease producing or modifying. MATERIALS AND METHODS: One approach to providing evidence for cause-effect of a disease-influencing gene is to quantitatively compare changes in phenotype to changes in gene expression by employing serial measurements in a longitudinal experimental design. We investigated the quantitative relationships between changes in gene expression and phenotype n 47 patients with idiopathic DCM. In endomyocardial biopsies at baseline and 6 months later, we measured mRNA expression of genes regulating contractile function (beta-adrenergic receptors, sarcoplasmic reticulum Ca(2) + ATPase, and alpha- and beta-myosin heavy chain isoforms) or associated with pathologic hypertrophy (beta-myosin heavy chain and atrial natriuretic peptide), plus beta-adrenergic receptor protein expression. Left ventricular phenotype was assessed by radionuclide ejection fraction. RESULTS: Improvement in DCM phenotype was directly related to a coordinate increase in alpha- and a decrease in beta-myosin heavy chain mRNA expression. In contrast, modification of phenotype was unrelated to changes in the expression of beta(1)- or beta(2)-adrenergic receptor mRNA or protein, or to the mRNA expression of sarcoplasmic reticulum Ca(2) + ATPase and atrial natriuretic peptide. CONCLUSION: We conclude that in human DCM, phenotypic modification is selectively associated with myosin heavy chain isoform changes. These data support the hypothesis that myosin heavy chain isoform changes contribute to disease progression in human DCM.

Altered autonomic support of arterial blood pressure with age in healthy men.

BACKGROUND: Primary aging is associated with changes in the autonomic nervous system (ANS), but the functional significance of these changes for systemic circulatory control of arterial blood pressure (BP) is unknown. We tested the hypothesis that ANS support of BP is altered in healthy older humans. METHODS AND RESULTS: A total of 23 young (aged 24+/-1 years; systolic/diastolic BP, 126+/-2/66+/-1 mm Hg) and 16 older (aged 65+/-1 years; systolic/diastolic BP, 125+/-3/62+/-2 mm Hg) healthy men were studied before and during ganglionic blockade (intravenous trimethaphan). The reduction in mean BP (radial artery catheter) with trimethaphan was almost twice as great in the older men (-33+/-2 versus -19+/-2 mm Hg; -40% versus -22% of baseline; P<0.01) due to a lack of increase in heart rate (3+/-2 versus 25+/-2 bpm; P<0.001) and cardiac output (-0.42+/-0.19 versus 1.01+/-0.26 L/min; P<0.001); the decreases in systemic vascular resistance were not different. The absence of tachycardia in the older men was associated with reduced baseline heart rate variability (HRV, P<0.05); the change in heart rate with trimethaphan correlated with the standard deviation of the R-R intervals (HRV(SD R-R interval); r=0.57, P<0.001). Among individual subjects (pooled groups), the reductions in mean BP with trimethaphan were most strongly related to measures of sympathetic activity (r=0.58 to 0.67, P<0.005), change in mean BP with intravenous phenylephrine (r=0.57, P<0.001), and HRV(SD R-R interval) (r=-0.40, P<0.01). CONCLUSIONS: ANS support of BP is altered with age in healthy men due to less cardiac vagal inhibition of heart rate and cardiac output. Basal sympathetic activity and alpha-adrenergic vascular sensitivity are also key physiological correlates of ANS support of BP in healthy men.

Twenty-four hour, non-invasive, neonatal chromosome analysis--application in a case of mixed gonadal dysgenesis.

INTRODUCTION: With the advent of interphase molecular fluorescent in-situ hybridisation (FISH), buccal mucosa can be used to provide an highly accurate assessment of those chromosomes most commonly causing abnormality in live-born children. CLINICAL PICTURE: A newborn child presented with ambiguous genitalia. The phallus-looking enlarged "clitoris" had a urethral opening at the ventral surface near the tip and the "labial" folds were completely fused. No definite gonads were palpable. Differential diagnostic possibilities included sex chromosome or a single gene abnormality such as congenital adrenal hyperplasia. Thus, one initial objective was to investigate the sex chromosomes. Buccal mucosa was used in conjunction with fluorescent molecular probes for the X and Y. This methodology enabled a firm diagnosis of a 45,X/46,XY mosiac to be made within 24 hours. Decisions could then be made concerning gender assignment. TREATMENT AND OUTCOME: Intervention by means of reconstructive surgery of the external genitalia would be made available at a later date. CONCLUSIONS: The use of buccal mucosa is non-invasive, easy to obtain and, when combined with molecular techniques, is reliable and accurate. The clinical implication of this methodology is that it will be especially useful in gender assignment or when rapid decisions on live-saving surgery have to be made in cases of possible aneuploidy.

Identification of risk factors for increased cost, charges, and length of stay for cardiac patients.

BACKGROUND: In this study we explored different risk model options to provide clinicians with predictions for resource utilization. The hypotheses were that predictors of mortality are not predictive of resource consumption, and that there is a correlation between cost estimates derived using a cost-to-charge ratio or a product-line costing approach. METHODS: From March 1992 to June 1995, 2,481 University of Colorado Hospital patients admitted for ischemic heart disease were classified by diagnosis-related group code as having undergone or experienced coronary bypass procedures (CBP), percutaneous cardiovascular procedures (PCVP), acute myocardial infarction (AMI), and other cardiac-related discharges (Other). For each diagnosis-related group, Cox proportional hazards models were developed to determine predictors of cost, charges, and length of stay. RESULTS: The diagnosis groups differed in the clinical factors that predicted resource use. As the two costing methods were highly correlated, either approach may be used to assess relative resource consumption provided costs are reconciled to audited financial statements. CONCLUSIONS: To develop valid prediction models for costs of care, the clinical risk factors that are traditionally used to predict risk-adjusted mortality may need to be expanded.

Rapid prenatal diagnosis of chromosome abnormalities.

The aim of the present work was to examine the efficacy of using FISH for the rapid prenatal diagnosis of common chromosome aneuploidies. A total of 100 analyses over a six month period were included in the study. Diagnosis was possible in all cases. A mosaic for trisomy 21 proved, by comparison with an extensive analysis of long term cultures, to be an apparent false positive. Otherwise the technique was reliable, accurate and relatively straightforward to perform. Results could be available within 24 hrs. In most cases an additional long term full analysis was also done, so as to exclude rarer aneuploidies and structural rearrangements. This methodology is seen as a useful addition to the prenatal diagnostic repertoire.

Interstitial deletion of chromosome 5 in a neonate due to maternal insertion, ins(8;5)(p23;q33q35).

We describe an infant girl with an interstitial deletion of chromosome bands 5q33 to 5q35 inherited from a maternal interchromosomal insertion ins(8;5)(p23;q33q35) which was demonstrated by fluorescent in situ hybridization with whole chromosome paints. Physical anomalies included hypertonicity, microcephaly, short neck, apparently low-set ears, micrognathia, camptodactyly, mild rocker bottom feet, and hammer toe. Cardiac anomalies included a large ventricular septal defect, patent ductus arteriosus, pulmonary hypertension and hypoplastic right ventricle. She died at age 3 months.

Right ventricular phenotypic characteristics in subjects with primary pulmonary hypertension or idiopathic dilated cardiomyopathy.

BACKGROUND: Studies of animal models and human subjects with cardiomyopathies suggest that cardiac myocyte and ventricular chamber remodeling show distinct phenotypic characteristics that may be dependent on specific signaling pathways. METHODS AND RESULTS: In this study, we characterize right ventricular (RV) chamber size, end-diastolic thickness, myocardial mass, and ejection fraction (EF) in human subjects with chronic heart failure from primary pulmonary hypertension (PPH; n = 10) and idiopathic dilated cardiomyopathy (IDC; n = 10). Subjects underwent gated cardiac magnetic resonance imaging (MRI), and the RVs were phenotypically classified based on the presence or absence of hypertrophy (increased mass), systolic dysfunction (reduced EF), and degree of wall thickness (concentric v eccentric pattern of hypertrophy). Within this schema, five abnormal phenotypes could be identified. In PPH subjects, in whom the RV is subjected to the uniform insult of chronic pressure overload, four different abnormal phenotypes were identified. CONCLUSIONS: These data indicate that distinct structural/functional ventricular chamber phenotypes may be classified by MRI, and that a uniform insult can result in multiple RV phenotypes.

Effects of carvedilol on right ventricular function in chronic heart failure.

This study investigated the effects of carvedilol on right ventricular (RV) volume and systolic function in chronic heart failure patients. Carvedilol treatment resulted in a significant improvement of RV ejection fraction and systolic performance, which paralleled the improvement of systolic function demonstrated in the left ventricle.

Changes in gene expression in the intact human heart. Downregulation of alpha-myosin heavy chain in hypertrophied, failing ventricular myocardium.

Using quantitative RT-PCR in RNA from right ventricular (RV) endomyocardial biopsies from intact nonfailing hearts, and subjects with moderate RV failure from primary pulmonary hypertension (PPH) or idiopathic dilated cardiomyopathy (IDC), we measured expression of genes involved in regulation of contractility or hypertrophy. Gene expression was also assessed in LV (left ventricular) and RV free wall and RV endomyocardium of hearts from end-stage IDC subjects undergoing heart transplantation or from nonfailing donors. In intact failing hearts, downregulation of beta1-receptor mRNA and protein, upregulation of atrial natriuretic peptide mRNA expression, and increased myocyte diameter indicated similar degrees of failure and hypertrophy in the IDC and PPH phenotypes. The only molecular phenotypic difference between PPH and IDC RVs was upregulation of beta2-receptor gene expression in PPH but not IDC. The major new findings were that (a) both nonfailing intact and explanted human ventricular myocardium expressed substantial amounts of alpha-myosin heavy chain mRNA (alpha-MHC, 23-34% of total), and (b) in heart failure alpha-MHC was downregulated (by 67-84%) and beta-MHC gene expression was upregulated. We conclude that at the mRNA level nonfailing human heart expresses substantial alpha-MHC. In myocardial failure this alteration in gene expression of MHC isoforms, if translated into protein expression, would decrease myosin ATPase enzyme velocity and slow speed of contraction.

DTPA aerosol in ventilation/perfusion scintigraphy for diagnosing pulmonary embolism.

UNLABELLED: The use of lung scintigraphy in evaluating suspected pulmonary embolism (PE) is controversial. Several diagnostic methods have been described for lung scans, of which the most widely applied uses 99mTc-MAA for perfusion, 133Xe for ventilation and PIOPED diagnostic criteria. This study evaluates the accuracy of lung scintigraphy using an alternative ventilation agent, 99mTc-diethylenetriamine pentacetic acid (DTPA) aerosol, and specific criteria. METHODS: Diagnostic criteria for DTPA aerosol ventilation were prospectively applied to 5017 patients over a 9-yr period. Lung scan interpretations were analyzed for frequency of occurrence, and results were compared to those of angiography in 455 patients. RESULTS: Scans were interpreted as normal, low or high probability in 79% of patients and as either indeterminate or medium probability in 21% of patients. Three patients had normal scans and negative angiography. In patients with low-probability scans, 111 angiograms were performed: 103 (93%) were negative, and 8 (7%) were positive. In patients with indeterminate scans, 114 angiograms were performed: 85 (75%) were negative, and 29 (25%) were positive. In patients with medium-probability scans, 149 angiograms were performed: 86 (58%) were negative, and 63 (42%) were positive. In patients with high-probability scans, 78 angiograms were performed: 6 (8%) were negative, and 72 (92%) were positive. CONCLUSION: These results indicate that lung scintigraphy using DTPA aerosol and our criteria is accurate in diagnosing and stratifying risk of pulmonary embolic disease. Compared with 133Xe and PIOPED criteria, DTPA ventilation and our criteria reduced the false-negative rate in low-probability scans (7% versus 16%, p < 0.005) and decreased the fraction of intermediate-probability scans (21 % versus 39%, p < 0.01).

Centromeric inactivation in a dicentric human Y;21 translocation chromosome.

A de novo dicentric Y;21 (q11.23;p11) translocation chromosome with one of its two centromeres inactive has provided the opportunity to study the relationship between centromeric inactivation, the organization of alphoid satellite DNA and the distribution of CENP-C. The proband, a male with minor features of Down's syndrome, had a major cell line with 45 chromosomes including a single copy of the translocation chromosome, and a minor one with 46 chromosomes including two copies of the translocation chromosome and hence effectively trisomic for the long arm of chromosome 21. Centromeric activity as defined by the primary constriction was variable: in most cells with a single copy of the Y;21 chromosome, the Y centromere was inactive. In the cells with two copies, one copy had an active Y centromere (chromosome 21 centromere inactive) and the other had an inactive Y centromere (chromosome 21 centromere active). Three different partial deletions of the Y alphoid array were found in skin fibroblasts and one of these was also present in blood. Clones of single cell origin from fibroblast cultures were analysed both for their primary constriction and to characterise their alphoid array. The results indicate that (1) each clone showed a fixed pattern of centromeric activity; (2) the alphoid array size was stable within a clone; and (3) inactivation of the Y centromere was associated with both full-sized and deleted alphoid arrays. Selected clones were analysed with antibodies to CENP-C, and staining was undetectable at both intact and deleted arrays of the inactive Y centromeres. Thus centromeric inactivation appears to be largely an epigenetic event.

Effects of carvedilol on systolic and diastolic left ventricular performance in idiopathic dilated cardiomyopathy or ischemic cardiomyopathy.

Recent evidence has shown that improvement in left ventricular (LV) systolic function in patients with New York Heart Association class II to III heart failure occurs with beta-adrenergic blocking agents. However the specific effects on LV diastolic function have been subjected to only limited examination. This study investigated the effects of the combined beta blocker/vasodilator, carvedilol, on systolic and diastolic LV performance in dilated cardiomyopathy. Thirty-six patients with New York Heart Association II to III heart failure and LV ejection fraction < or = 0.35 were entered into either arm of this placebo-controlled, double-blind 4-month trial. Twenty-one subjects were entered into the carvedilol treatment arm and 15 patients were entered into the placebo arm in a 3:2 ratio. Carvedilol therapy resulted in a significant improvement in LV ejection fraction, from 0.22 +/- 0.02 to 0.30 +/- 0.02 when compared with the placebo group (0.19 +/- 0.02 to 0.21 +/- 0.02 at baseline and after 4 months of therapy, respectively; p = 0.0001). However, no significant change in radionuclide parameters of LV diastolic function, including peak filling rate or time to peak filling rate, was observed. LV end-diastolic volume index did not change with carvedilol therapy, whereas end-diastolic volume index increased in the placebo group, although the difference between groups at 4 months was significant (p = 0.02). In conjunction with these changes, end-systolic volume index was smaller at 4 months after carvedilol treatment compared with that of the placebo group (p = 0.04). Thus, these results demonstrate that in moderate chronic heart failure, systolic LV performance improves but diastolic LV function does not improve when compared with placebo after treatment with carvedilol.

Angiotensin II formation in the intact human heart. Predominance of the angiotensin-converting enzyme pathway.

It has been proposed that the contribution of myocardial tissue angiotensin converting enzyme (ACE) to angiotensin II (Ang II) formation in the human heart is low compared with non-ACE pathways. However, little is known about the actual in vivo contribution of these pathways to Ang II formation in the human heart. To examine angiotensin II formation in the intact human heart, we administered intracoronary 123I-labeled angiotensin I (Ang I) with and without intracoronary enalaprilat to orthotopic heart transplant recipients. The fractional conversion of Ang I to Ang II, calculated after separation of angiotensin peptides by HPLC, was 0.415 +/- 0.104 (n = 5, mean +/- SD). Enalaprilat reduced fractional conversion by 89%, to a value of 0.044 +/- 0.053 (n = 4, P = 0.002). In a separate study of explanted hearts, a newly developed in vitro Ang II-forming assay was used to examine cardiac tissue ACE activity independent of circulating components. ACE activity in solubilized left ventricular membrane preparations from failing hearts was 49.6 +/- 5.3 fmol 125I-Ang II formed per minute per milligram of protein (n = 8, +/- SE), and 35.9 +/- 4.8 fmol/min/mg from nonfailing human hearts (n = 7, P = 0.08). In the presence of 1 microM enalaprilat, ACE activity was reduced by 85%, to 7.3 +/- 1.4 fmol/min/mg in the failing group and to 4.6 +/- 1.3 fmol/min/mg in the nonfailing group (P < 0.001). We conclude that the predominant pathway for angiotensin II formation in the human heart is through ACE.

The spectrum of beta thalassaemia mutations in the UAE national population.

The beta thalassaemia alleles in 50 beta thalassaemia heterozygotes originating from many parts of the United Arab Emirates (UAE) have been characterised using the allele specific priming technique of the polymerase chain reaction (PCR). The IVSI-5 (G-->C) mutation was found to be present in 66%, while six other alleles occurred at the much lower frequencies of 2% to 8%. These were codon 8/9 (+G), IVSI-1, 3' end (-25 bp), codon 5 (-CT), IVSII-1 (G-->A), codon 30 (G-->C), and codon 15 (G-->A). The mutation types and percentages are compared with other Mediterranean Arab countries and neighbouring areas. It is proposed that IVSI-5 and other Asian Indian mutations were introduced into the UAE by population migration from the region previously known as Baluchistan. These findings should be useful for genetic counselling and the development of a first trimester prenatal diagnosis programme based on direct detection of mutations in the UAE.

Two sibs with unbalanced translocations in the Waardenburg gene region.

We report two sibs with unbalanced translocations between chromosomes 2 and 11, both products of a paternal balanced reciprocal translocation involving bands 2q37.3 and 11q23.3