Spleen phenotype in autosomal dominant polycystic kidney disease.

AIM: To evaluate splenic phenotype in autosomal dominant polycystic kidney disease (ADPKD) including presence of cysts and splenomegaly to determine if these are ADPKD related or represent unrelated incidental findings. MATERIALS AND METHODS: The axial/coronal T2-weighted images of ADPKD patients (n=215) and age/gender-matched controls (n=215) were evaluated for the presence of T2-bright splenic lesions by three blinded observers. Spleen volume (SV) was evaluated in the context of clinical and imaging features as well as results of gene testing for PKD1 and PKD2 mutations. RESULTS: T2-bright splenic lesions were found in 16 of 215 (7%) ADPKD patients compared to 11 of 215 (5%) control patients (p=0.32) and their prevalence was similar in patients with either PKD1 or PKD2 mutations. Median SV was significantly higher in ADPKD patients than controls (236 [182; 313 ml] versus 176 [129; 264 ml], p<0.0001). In multivariable analysis, height-adjusted SV (htSV) was not associated with the presence of liver cysts, haemorrhagic cysts, or infections; however, htSV was directly associated with height-adjusted total kidney volume (htTKV), a biomarker for ADPKD disease severity. CONCLUSIONS: The prevalence of T2-bright splenic lesions is similar in ADPKD patients and non-ADPKD controls, suggesting no relation to the diagnosis of ADPKD; however, splenic enlargement in ADPKD compared to controls could not be explained by liver cystic involvement, by infection/inflammatory conditions, or by haemorrhagic renal cysts. This combined with direct correlation of htSV with htTKV, a biomarker of ADPKD severity, suggests splenomegaly may be related to the pathogenesis of ADPKD.

Autosomal dominant polycystic kidney disease caused by somatic and germline mosaicism.

Autosomal dominant polycystic kidney disease (ADPKD) is a heterogeneous genetic disorder caused by loss of function mutations of PKD1 or PKD2 genes. Although PKD1 is highly polymorphic and the new mutation rate is relatively high, the role of mosaicism is incompletely defined. Herein, we describe the molecular analysis of ADPKD in a 19-year-old female proband and her father. The proband had a PKD1 truncation mutation c.10745dupC (p.Val3584ArgfsX43), which was absent in paternal peripheral blood lymphocytes (PBL). However, very low quantities of this mutation were detected in the father's sperm DNA, but not in DNA from his buccal cells or urine sediment. Next generation sequencing (NGS) analysis determined the level of this mutation in the father's PBL, buccal cells and sperm to be ∼3%, 4.5% and 10%, respectively, consistent with somatic and germline mosaicism. The PKD1 mutation in ∼10% of her father's sperm indicates that it probably occurred early in embryogenesis. In ADPKD cases where a de novo mutation is suspected because of negative PKD gene testing of PBL, additional evaluation with more sensitive methods (e.g. NGS) of the proband PBL and paternal sperm can enhance detection of mosaicism and facilitate genetic counseling.

Serum 25(OH)D concentrations in sunny Israel.

SUMMARY: This study assesses vitamin D status in Israel. Serum 25(OH)D levels <25 and <50 nmol/L are common in Israel with noted differences between Arabs and Jews, Arab females were particularly at high risk. These findings may require public health intervention at the population level. INTRODUCTION: Small studies from Israel have suggested a high prevalence of hypovitaminosis D. The objective of this study was to evaluate the extent of hypovitaminosis D among demographic subgroups in Israel. METHODS: The data of this study are from the Clalit Health Services (CHS) which is a non-for-profit health maintenance organization (HMO) covering more than half of the Israeli population. We included all CHS members for whom a 25(OH)D test result in 2009 was available and who were not taking vitamin D supplements in 2008-2009 before that 25(OH)D result. Complete data were available for 198,834 members. RESULTS: The mean level of 25(OH)D was 51.9 ± 24.5 nmol/L and was higher in summer compared to winter (P < 0.0001). Level <25, <37.5, and <50 nmol/L were detected in 14.4%, 30.7%, and 49.9% of tests; 16.4% had levels >75 nmol/L. Females had higher prevalence of 25(OH)D levels < 50 nmol/L which were found in 51.8% of females versus 45.0% in males (P < 0.0001); 76.7% of the Arabs had levels <50 nmol/L versus 46.5% in Jews (P < 0.0001). Arabs females were particularly at high risk for 25(OH)D <50 nmol/L; 84.8% of them had levels <50 nmol/L versus 48.1% of Jewish females (P < 0.0001). The relation of 25(OH)D levels with age had a sinusoidal shape among Jews, a U-shape in Arab females, and inverse linear pattern in Arab males. CONCLUSIONS: 25(OH)D levels <25 and <50 nmol/L are common in Israel. Public health measures are needed for values lesser than about 30 nmol/L and further monitoring of concentrations between about 30 and 50 nmol/L to determine if there are adverse health effects.

Genetic polymorphisms in fatty acid metabolism genes and colorectal cancer.

Colorectal cancer (CRC) is a leading cause of cancer death worldwide. Epidemiological risk factors for CRC included dietary fat intake; consequently, the role of genes in the fatty acid biosynthesis and metabolism pathways is of particular interest. Moreover, hyperlipidaemia has been associated with different type of cancer and serum lipid levels could be affected by genetic factors, including polymorphisms in the lipid metabolism pathway. The aim of this study is to assess the association between single-nucleotide polymorphisms (SNPs) in fatty acid metabolism genes, serum lipid levels, body mass index (BMI) and dietary fat intake and CRC risk; 30 SNPs from 8 candidate genes included in fatty acid biosynthesis and metabolism pathways were genotyped in 1780 CRC cases and 1864 matched controls from the Molecular Epidemiology of Colorectal Cancer study. Information on clinicopathological characteristics, lifestyle and dietary habits were also obtained. Logistic regression and association analysis were conducted. Several LIPC (lipase, hepatic) polymorphisms were found to be associated with CRC risk, although no particular haplotype was related to CRC. The SNP rs12299484 showed an association with CRC risk after Bonferroni correction. We replicate the association between the T allele of the LIPC SNP rs1800588 and higher serum high-density lipoprotein levels. Weak associations between selected polymorphism in the LIPC and PPARG genes and BMI were observed. A path analysis based on structural equation modelling showed a direct effect of LIPC gene polymorphisms on colorectal carcinogenesis as well as an indirect effect mediated through serum lipid levels. Genetic polymorphisms in the hepatic lipase gene have a potential role in colorectal carcinogenesis, perhaps though the regulation of serum lipid levels.

Aberrant PKD2 splicing due to a presumed novel missense mutation in autosomal-dominant polycystic kidney disease.

Autosomal-dominant polycystic kidney disease (ADPKD) is a heterogeneous genetic disorder characterized by abnormal proliferation of renal tubular epithelium, leading to massive kidney enlargement and progressive chronic kidney disease. ADPKD is caused by mutations in PKD1 and PKD2 genes. Herein, we describe and characterize a novel missense mutation in the PKD2 gene (c.1320G>T) in a 41-year-old White man with kidney cysts and a family history of ADPKD. This mutation abolishes a conserved acceptor splice site of intron 5, resulting in a premature termination following the addition of three aberrant amino acids (PKD2 p.L441C fsX4). We demonstrate that the aberrantly spliced transcript is found in substantial amounts in the patient's peripheral blood leukocytes (PBL), and show that this alternative splicing of exon 6 occurs, to a lesser magnitude, in other patients with ADPKD and in normal control individuals. The biological and clinical significance of this splice variant in ADPKD is currently unknown.

Smoking attenuates the negative association between carotenoids consumption and colorectal cancer risk.

OBJECTIVES: Consumption of vegetables and fruits, physical activity, obesity and caloric intake are all strongly related to the risk of colorectal cancer (CRC). The association between dietary intake of carotenoids from vegetables/fruits and risk of CRC in the context of cigarette smoking was studied in a nutritionally diverse population. METHODS: The study included 1,817 age sex residence-matched case-control pairs from a population-based study in Northern Israel. Data were acquired by food-frequency questionnaire. Individual intake of carotenoid isomers was calculated using an Israeli food content database. Odds ratios (ORs) were calculated using conditional logistic regression models adjusted for known risk factors. RESULTS: Strong inverse associations were found with consumption of 9-cis-beta-carotene (OR = 0.35, 0.26-0.47), all-trans-beta-carotene (OR = 0.58, 0.44-0.76), cis-beta-cryptoxanthin (OR = 0.67, 0.50-0.90), all-trans-zeaxanthin (OR = 0.64, 0.48-0.86), and lutein (OR = 0.74, 0.57-0.96). Lycopene (OR = 2.22, 1.71-2.89) and all-trans-beta-cryptoxanthin (OR = 2.01, 1.48-2.73) were associated with increased risk of CRC. Inverse associations of most carotenoids with CRC, demonstrated in non-smokers, were much attenuated or reversed in past or current smokers with a highly significant interaction term. CONCLUSIONS: Consumption of most dietary carotenoids was found to be strongly associated with reduced risk of CRC. However, smoking significantly attenuated or reversed this observed protective effect on CRC occurrence. Smokers should be advised that smoking also hampers the potential health promoting effects of high fruit and vegetable consumption.

Long-term (10-year) outcome in patients with unstable angina pectoris treated by coronary balloon angioplasty.

OBJECTIVES: We sought to examine completed 10-year survival and event-free survival in patients with stable and unstable angina pectoris treated by coronary balloon angioplasty. BACKGROUND: Patients with unstable angina are at increased risk for recurrent acute coronary events. METHODS: The study included 208 consecutive patients (133 with stable and 75 with unstable angina pectoris) undergoing angioplasty from 1984 to 1986. The balloon crossed the lesion in 185 patients (121 with stable and 64 with unstable angina pectoris). Angioplasty was performed in patients with unstable angina pectoris 12+/-15 days (median 8) after symptom onset. Patients with unstable angina pectoris were classified retrospectively into Braunwald class I (n=3), class II (n=20), class III (n=28), class B (n=52) and class C (n=12). Follow-up data were obtained from hospital charts, telephone interview and official death certificates where applicable. The study had >80% power to detect a clinically significant 20% difference in survival and a 20% difference in event-free survival between the stable and unstable patient groups. RESULTS: Despite similar baseline characteristics, early (40-day) mortality was slightly higher in patients with unstable angina (4.7% [3 of 64 patients] vs. 0.8% [1 of 121 patients], p=NS). Long-term outcome was not different, because survival curves were parallel thereafter (10-year survival was 83% for those with stable and 77% for those with unstable angina, p=NS). Survival free of myocardial infarction or coronary artery bypass graft surgery at 10 years was 53% in patients with stable and 47% in patients with unstable angina (p=NS), and survival free of infarction, bypass surgery or repeat angioplasty was 32% for both groups at 10 years. In patients with Braunwald class III unstable angina, 10-year survival was 80%, as compared with 85% in other patients with unstable angina, due to the early hazard (p=NS). Survival and event-free survival were similar in patients who had had a recent myocardial infarction (Braunwald class C) and in patients with acute electrocardiographic changes. Repeat hospital admissions were not more frequent in patients with unstable angina (3.1+/-3.5 vs. 3.0+/-2.6, p=NS). CONCLUSIONS: Ten-year survival and event-free survival were similar in patients with stable and unstable angina pectoris treated by coronary balloon angioplasty, with no evidence of an increased rate of recurrent cardiovascular events in the unstable group.

Differences in the characteristics of families with BRCA1 and BRCA2 mutations in Israel.

Three specific mutations in the BRCA1 (185delAG, 5382insC) and BRCA2 (6174delT) genes have been reported to be of high prevalence in the Jewish Ashkenazi population. We studied the differences in phenotype of families carrying these mutations. All consecutive families found by the CHS Familial Cancer Service to carry one of the three 'Jewish' mutations of the BRCA1/BRCA2 genes were evaluated for phenotypic characteristics. Chi-squared and Student's t-test statistics were employed to study differences in a variety of clinical and demographic parameters. A total of 111 families with 1499 family members were included. Among them 454 cases of cancer (297 in breast/ovary) were reported. Ovarian cancer, but not breast cancer, was detected at a significantly younger age among carriers of 185delT compared with other mutation carriers. In families with 185delAG, 5382insC and 6174delT mutations, breast cancer was found in 20.2, 39.4 and 24.1% of all identified women (born between 1900 and 1975), correspondingly. The corresponding figures for ovarian cancer were 13.9, 6.8 and 4.9%. Families carrying the 5382insC mutation had the highest probability of expressing bilateral breast cancer (38.9% of families, 15.4% of women with cancer, 6.1% of all women in family) and metachronous breast and ovary tumours (22.2, 9.8 and 4.5% correspondingly). Other tumours were reported in 7.9, 9.1 and 12.0% of women and 9.5, 12.9 and 15.8% of men in families with 185delAG, 5382insC, 6174delT, correspondingly. Marked phenotypic differences were found between families carrying different BRCA mutations warranting mutation-specific counselling to families seeking risk-reduction advice. 5382insC emerged as a most aggressive mutation.

Generation of regulatory oxysterols: 26-hydroxylation of cholesterol by ovarian mitochondria.

De novo synthesis of cholesterol and low-density lipoprotein (LDL) receptor levels are suppressed in the presence of cholesterol. Recent evidence suggests that a cholesterol metabolite (possibly a hydroxysterol), not cholesterol per se, is the effector that inhibits transcription of genes encoding enzymes involved in sterol synthesis and LDL receptors. We found that 26-hydroxycholesterol inhibits human ovarian cell sterol synthesis, and that luteinized human granulosa cells contain 26-hydroxylase messenger RNA (mRNA). We proceeded to characterize the enzyme generating 26-hydroxycholesterol in the rat ovary. Mitochondria derived from ovaries of PMSG-human CG (hCG) primed immature rats (day 3 post-hCG) metabolized [3H] cholesterol into [3H]26-hydroxycholesterol in the presence of nicotinamide adenine dinucleotide phosphate and aminoglutethimide (100 micrograms/ml), added to inhibit metabolism of sterols by the cholesterol side-chain cleavage system. The identity of the product was confirmed by chromatography in several systems; recrystallization to constant specific activity and mass spectrometry. Negligible 26-hydroxylase activity was detected in other ovarian subcellular fractions. 26-Hydroxycholesterol formation progressed at a linear rate for up to 40 min and was linearly related to mitochondrial protein added to the incubation mixture. 26-Hydroxylase was markedly stimulated (5-fold) by calcium (0.2 mM). Maximal rates of 26-hydroxycholesterol formation observed were 1 pmol/min.mg protein. This activity is substantially lower than cholesterol side-chain cleavage measured in the absence of aminoglutethimide. Ketoconazole (1-100 microM) inhibited 26-hydroxylase in a dose-dependent manner. Pregnenolone (1-1000 microM) and progesterone (1-100 microM) inhibited 26-hydroxylase in a dose-dependent manner, with appreciable inhibitory effects in the 1-10 microM range. We suggest that 26-hydroxycholesterol is an intracrine regulator that controls cellular sterol metabolism. Formation of 26-hydroxcholesterol in ovarian cells may be regulated by steroidogenic activity in such a way as to ensure availability of steroid hormone precursors. When steroidogenesis is active, 26-hydroxylase is inhibited by products of the side-chain cleavage system, allowing increased de novo sterol synthesis and LDL uptake. With reduced steroidogenic activity and less demand for cholesterol, 26-hydroxylase is not blocked, permitting formation of 26-hydroxycholesterol with attendant reduction in sterol synthesis and LDL receptor gene expression.

Population colorectal cancer screening with fecal occult blood test.

Screening for early detection of colorectal cancer using fecal occult blood testing has been shown to be effective in reducing mortality from this disease. The largest Health Maintenance Organization in Israel initiated the use of Hemoccult Sensa in 1992 to evaluate the field performance of this test. All primary care physicians were invited to order home-based tests for their asymptomatic patients 50-74 years of age. This report summarizes the results of 45,166 tests performed, 22,193 in the prevalence round. Seventy-eight cancers, 60 patients with adenomas, and 163 patients with polyps were detected, yielding a cancer detection rate of 2.61/1,000 screened in the prevalence round. Of these, 21.6% were in the right colon. Of screen-detected cancers, 44.5% and 58.9% were detected in Dukes' A and in situ stages in the prevalence and incidence rounds, correspondingly. The overall estimated sensitivity of the test (median follow-up, 35 months) was 85.3% for the prevalence round with a specificity of 95.5%. The sensitivity for left-side tumors (87.9%) was higher than for right-side or rectal tumors (78.6%). The positive predictive value for cancer increased with increasing number of positive fields. Four or more positive fields had a positive predictive value for cancer of 16-26% and a positive predictive value of 46-71% for all tumors combined. Population screening with a sensitive fecal occult blood test performs well outside a trial setting, detecting a high proportion of expected tumors with favorable stage distribution. Given its proven power to significantly reduce mortality, use of this test is strongly advised to both medical organizations and the healthy population at average risk.

Arthrogryposis due to infantile neuronal degeneration associated with deletion of the SMNT gene.

To determine whether SMNT deletion may be associated with arthrogryposis, we tested DNA extracted from paraffin blocks for deletion of SMNT (exons 7 and 8). Analysis of the DNA showed an SMNT deletion in two of four infants with neurogenic arthrogryposis. In addition to loss of anterior horn cells, patients with SMNT deletion had degeneration of central sensory neurons in Clarke's column and the thalamus. Although one of the patients with no deletion also had cortical pathology, clinical and pathologic characteristics of the two patients without deletion were otherwise similar to the two patients with deletion. Arthrogryposis and degeneration of sensory neurons may be associated with deletion of SMNT.

Heteroduplex formation in SMN gene dosage analysis.

Most spinal muscular atrophy patients lack both copies of SMN1 exon 7 and most carriers have only one copy of SMN1 exon 7. We investigated the effect of SMN1/SMN2 heteroduplex formation on SMN gene dosage analysis, which is an assay to determine copy number of SMN1 exon 7 that utilizes multiplex quantitative polymerase chain reaction (PCR) with DraI digestion to differentiate SMN1 from SMN2. Heteroduplex formation in PCR is a well-described phenomenon. In addition to demonstrating the presence of heteroduplexes by sequence analysis of purified SMN1 bands, we compared the SMN1 signals in various genotype groups (total n = 260) to those in a group lacking SMN2 (n = 13), and we estimated the relative amounts of SMN1/SMN2 heteroduplexes. The SMN1 signal increased as SMN2 copy number increased despite a constant SMN1 copy number, although not all pairwise comparisons showed a statistically significant difference in the SMN1 signal. In conclusion, SMN1/SMN2 heteroduplexes form in SMN gene dosage analysis, falsely increasing the SMN1 signal. External controls for SMN gene dosage analysis should be chosen carefully with regard to SMN2 copy number. The effect of heteroduplex formation should be considered when performing quantitative multiplex PCR.

Similar late revascularization rates 10 to 12 years after angioplasty or bypass surgery for multivessel coronary artery disease: a report from the Lady Davis Carmel Medical Center (LDCMC) Registry.

We compared completed long-term outcome and late repeat revascularization rates in 272 consecutive patients with multivessel coronary disease who underwent revascularization (95 angioplasty cohort, 177 surgical cohort) between 1984 and 1986. Long-term survival was similar at 12 years in the angioplasty (70%) and surgical (74%) cohorts (p = NS), and repeat revascularization, although more frequent in the angioplasty patients during the first 5 years of follow-up, was performed equally in the 2 patient cohorts after 10 to 12 years of follow-up.

Late-onset heart failure as a mechanism for adverse long-term outcome in diabetic patients undergoing revascularization (a 13-year report from the Lady Davis Carmel Medical Center registry).

The adverse long-term prognosis following myocardial revascularization in diabetic patients has been ascribed to accelerated coronary disease, a higher incidence of late coronary restenosis after revascularization, and myocardial dysfunction. To examine the development of heart failure and its prognostic implications in diabetic patients, we analyzed the long-term (13-year) follow-up data of 363 patients-193 percutaneous transluminal coronary angioplasties and 170 coronary artery bypass operations-revascularized in a single cardiovascular center from 1984 to 1986. Baseline characteristics (age, previous infarction, baseline ventricular function) were similar in the 80 diabetic and 283 nondiabetic patients; multivessel disease and hypertension were marginally more common in diabetics (p = NS). Cumulative incidence of hospitalization for heart failure was high in the diabetic cohort (25% vs 11%, p = 0.001), with a rapidly increasing incidence after 5 years. Survival after first hospitalization for heart failure was markedly reduced in diabetics (11 of 20 [55%] vs 25 of 31 [81%] at 3 years; p = 0.04), as was survival free of further hospitalization for heart failure (5 of 20 [25%] vs 20 of 30 [63%]; p <0.005). Long-term 13-year survival (43% vs 78%, p <0.0001) and survival free of heart failure (33% vs 71%, p <0.0001) were decreased in diabetics, especially those with reduced ventricular function at baseline (17% vs 42%, p = 0.07). Multivariate analysis showed diabetes to be the strongest independent predictor of decreased survival (odds ratio 3. 6, 95% confidence interval 2.0 to 6.2; p <0.0001) and survival free of heart failure (odds ratio 4.0, 95% confidence interval 2.2 to 7. 1; p <0.0001) in patients undergoing revascularization. In summary, late-onset heart failure was frequent in diabetic patients after percutaneous transluminal coronary angioplasty or coronary artery bypass grafting, and once present heralded an unrelenting progressive downhill clinical course.

Relation of serum total cholesterol and high-density lipoprotein cholesterol percentage to the incidence of definite coronary events: twenty-year follow-up of the Donolo-Tel Aviv Prospective Coronary Artery Disease Study.

This 20-year follow-up report presents the incidence of definite coronary events and its relation to serum total cholesterol (TC) and alpha-lipoprotein cholesterol percentage (percentage of serum TC bound with alpha-lipoprotein), now usually called high-density lipoprotein cholesterol percentage (HDL-C%). The cohort consists of 1,454 men and 1,481 women, presumably healthy, aged 35 to 64 years at the time of entrance examination in 1964. During the follow-up period 123 men and 44 women died from coronary disease. Ninety-six men and 42 women had nonfatal myocardial infarctions. The incidence of definite coronary events increased from 6% in men with TC levels of less than 200 mg/dl to 25% in men with levels of more than 264 mg/dl. The corresponding figures in women were 3% and 10%. The incidence of definite coronary events was inversely correlated to HDL-C%. The incidence of definite coronary events in 225 men with HDL-C% of less than 14% was 28%, and in 669 men with HDL-C% of 21% or more, 7%. The corresponding figures in 234 women with HDL-C% of less than 17% was 14%, and in 853 women with HDL-C% of 23% or more, 2%. Two HDL-C% subsets were assessed: "low" and "high" subsets refer to HDL-C% of less than 21% and 21% or more, respectively, in men and less than 23% and 23% or more in women.(ABSTRACT TRUNCATED AT 250 WORDS)

Calmodulin inhibitors and 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate do not prevent the inhibitory effect of prostaglandin F2 alpha on cyclic AMP production in isolated rat corpora lutea.

In the rat corpus luteum, prostaglandin F2 alpha (PGF2 alpha) rapidly inhibits LH-induced cyclic AMP (cAMP) production when given in vivo or to isolated corpora lutea, but not to broken-cell preparations. The suggestion that increased cytosolic calcium concentration mediates PGF2 alpha action was investigated in corpora lutea of pseudopregnancy induced in immature rats by administration of pregnant mare serum gonadotrophin (15 i.u.). Isolated 10-day-old corpora lutea were incubated for 90 min with LH (5 micrograms/ml), PGF2 alpha (10 mumol/l) and other additions, and cAMP concentration in the tissue was estimated. The putative inhibitor of intracellular calcium release or action, 8-(n,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8; 30 or 150 mumol/l), did not abolish the effect of PGF2 alpha. Similarly ineffective was the combination of TMB-8 (150 mumol/l) and calcium-depleted medium (free ionized calcium concentration, 30 nmol/l). Calmodulin inhibitors of three different chemical structures were then tested. The phenothiazine trifluoperazine, at 300 as well as 30 mumol/l, did not interfere with the inhibitory effect of PGF2 alpha on cAMP, while suppressing (at 300 mumol/l) progesterone secretion in LH-treated tissue. Furthermore, inhibition by PGF2 alpha was not impaired by pimozide, a diphenylbutylpiperidine (25 and 50 mumol/l) nor by N-(6-aminohexyl)-5-chloro-1-naphthalene sulphonamide (W-7; 15 and 45 mumol/l). In the presence of LH alone, W-7 (45 mumol/l) inhibited and TMB-8 (30 mumol/l augmented cAMP accumulation, indicating that the luteal tissue was effectively exposed to these compounds. Thus, drugs known to inhibit calcium- and calmodulin-dependent processes in a variety of tissues did not abolish the inhibitory action of PGF2 alpha on luteal cAMP production.

Duplications and de novo deletions of the SMNt gene demonstrated by fluorescence-based carrier testing for spinal muscular atrophy.

Approximately 95% of individuals with spinal muscular atrophy (SMA) lack both copies of the SMNt gene at 5q13. The presence of a nearly identical centromeric homolog of the SMNt gene, SMNc, necessitates a quantitative polymerase chain reaction approach to direct carrier testing. Adapting a radioactivity-based method described previously, multiplex polymerase chain reaction was performed using fluorescently labeled primers followed by analysis on an ABI 373a DNA sequencer. The SMNt copy number was calculated from ratios of peak areas using both internal and genomic standards. Samples from 60 presumed carriers (50 parents of affected individuals and 10 relatives implicated by linkage analysis) and 40 normal control individuals were tested. Normalized results (to the mean of five or more control samples harboring two copies of the SMNt gene) were consistently within the ranges of 0.4 to 0.6 for carriers (one copy) and 0.8 to 1.2 for normal controls (two copies), without overlap. Combining linkage analyses with direct carrier test results demonstrated de novo deletions associated with crossovers, unaffected individuals carrying two SMNt gene copies on one chromosome and zero SMNt gene copies on the other chromosome, and unaffected individuals with three copies of the SMNt gene. This report demonstrates that fluorescence-based carrier testing for SMA is accurate, reproducible, and useful for genetic risk assessment, and that carrier testing may need to be combined with linkage analysis in certain circumstances.

Evidence for early hematopoietic progenitor cell involvement in acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) represents a subtype of acute myeloid leukemia with characteristic morphologic, molecular, and immunophenotypic features. Previous immunophenotypic analyses have shown that leukemic cells in APL typically express the myeloid markers CD33 and CD13 but lack expression of the early hematopoietic progenitor cell antigens CD34 and HLA-DR. We analyzed selected immunophenotypic features of APL by flow cytometry and showed that 7 (41%) of 17 cases contained significant subsets of CD34+ leukemic cells: CD34+ myeloid cells predominated in 2 APL cases. By using a fluorescence-activated cell sorter-fluorescence in situ hybridization approach, we confirmed that the CD34+ cells harbored the t(15;17) translocation characteristic of APL. By using the same experimental approach, CD34+ populations were stratified into primitive CD34+ CD38- and committed CD34+ CD38+ progenitor cell subpopulations; cells in both subsets contained the t(15;17) translocation. The knowledge that APL may be partly or largely CD34+ is important for proper diagnosis. Furthermore, identification of the t(15;17) translocation in CD34+ CD38- blasts indicates that, in at least some cases, the leukemogenic mutation in APL occurs within primitive hematopoietic progenitor cells.

Sterol carrier protein 2: a role in steroid hormone synthesis?

The intracellular movement of cholesterol is an important regulated step in the process of steroidogenesis. However, the molecular mechanisms by which cholesterol is translocated to key organelles, including the mitochondria, remains poorly understood. Lipid transfer proteins may have an important function in this process. One candidate lipid transfer protein is sterol carrier protein 2 (SCP2). This 13.2 kDa protein enhances the movement of cholesterol between vesicles and isolated mitochondria. It also stimulates mitochondrial pregnenolone synthesis. When introduced into intact cells, anti-SCP2 antibodies reduce steroid secretion. Moreover, expression of SCP2 in COS cells engineered to produce progestins increases steroid formation. SCP2 is abundant in steroidogenic glands and the pattern of SCP2 gene expression is consistent with a role for the protein in hormone synthesis: SCP2 transcripts are more prominent in the most steroidogenic compartments of the ovary and tropic hormones that stimulate steroidogenesis increase SCP2 gene expression. Other evidence that suggests that SCP2 plays important roles in cellular function includes a remarkable conservation of primary structure across species. The mechanisms by which SCP2 promotes intracellular sterol movement have not been elucidated. The protein appears to bind sterols and is synthesized with a 20 amino acid N-terminal "pro-" sequence that may serve to target SCP2 to mitochondria. In addition, the C-terminus of SCP2 contains a peroxisome-targeting sequence. SCP2 is derived from a large gene that encodes transcripts that are translated into larger proteins of 30 and 58 kDa. The 58 kDa protein, which has some structural homologies with thiolases, seems to be specifically targeted to peroxisomes whereas SCP2 has a broader subcellular distribution. The significance of the peroxisome association of SCP2 and steroidogenesis has not been disclosed. However, diseases of peroxisome function, including adrenoleukodystrophy and Zellweger syndrome, have notable deficits in steroid and bile acid metabolism, thus linking peroxisomes and steroidogenesis. SCP2 is deficient in fibroblasts of patients with these diseases.

The human "peripheral-type" benzodiazepine receptor: regional mapping of the gene and characterization of the receptor expressed from cDNA.

A cDNA for the human "peripheral-type" benzodiazepine receptor (PBR) was isolated from a liver cDNA library. The 851-nucleotide probe hybridized with a approximately 1 kb mRNA in Northern blots of RNA extracted from various human tissues and cell lines. The human PBR probe was hybridized to DNA from a somatic cell hybrid mapping panel to determine that the gene maps to chromosome 22. With a regional mapping panel for chromosome 22, we localized the gene within band 22q13.31. The ligand-binding properties of the receptor expressed from the cDNA were examined in transient expression experiments and compared to the endogenous human PBR. The PBR ligand [3H]PK 11195 had high affinity for the expressed receptor in COS-1 cells, but the affinities of a pair of isoquinoline propanamide enantiomers differed remarkably in expressed and endogenous human PBR. These findings reveal that the host cell and/or post-translational modification may have an important influence on PBR function.