Bivariate genome-wide scan for metabolic phenotypes in non-diabetic Chinese individuals from the Stanford, Asia and Pacific Program of Hypertension and Insulin Resistance Family Study.

AIMS/HYPOTHESIS: Hypertension, obesity, impaired glucose tolerance and dyslipidaemia are metabolic abnormalities that often cluster together more often than expected by chance alone. Since these metabolic variables are highly heritable and are at least partially genetically determined, the clustering of defects in these traits implies that pleiotropic effects, where a common set of genes influences more than one trait simultaneously, are likely. METHODS: We conducted bivariate linkage analyses for highly correlated traits, aiming to dissect the genetic architecture affecting these traits, in 411 Chinese families participating in the Stanford Asia-Pacific Program of Hypertension and Insulin Resistance Study. RESULTS: We confirmed the pleiotropic effects of the locus at 37 cM on chromosome 20 on the following pairs: (1) fasting insulin and insulin AUC (empirical p = 0.0006); (2) fasting insulin and homeostasis model assessment of beta cell function (HOMA-beta) (empirical p = 0.0051); and (3) HOMA of insulin resistance (IR) and HOMA-beta (empirical p = 0.0044). In addition, the peak logarithm of the odds (LOD) scores of linkage between a chromosomal locus and a trait for the pair fasting insulin and HOMA-IR rose to 5.10 (equivalent LOD score in univariate analysis, LOD([1]) = 4.01, empirical p = 8.0 x 10(-5)) from 3.67 and 3.42 respectively for these two traits in univariate analysis. Additional significant linkage evidence, not shown in single-trait analysis, was identified at 45 cM on chromosome 16 for the pair 1 h insulin and the AUC for insulin, with a LOD score of 4.29 (or LOD([1]) = 3.27, empirical p = 2.0 x 10(-4)). This new locus is also likely to harbour the common genes regulating these two traits (p = 1.73 x 10(-6)). CONCLUSIONS/INTERPRETATION: These data help provide a better understanding of the genomic structure underlying the metabolic syndrome.

Combined effects of well-done red meat, smoking, and rapid N-acetyltransferase 2 and CYP1A2 phenotypes in increasing colorectal cancer risk.

Heterocyclic amines (HAAs) are suspected carcinogens that are formed in meat when it is cooked at high temperature for long durations. These compounds require metabolic activation by CYP1A2 and N-acetyltransferase (NAT) 2 or NAT1 before they can bind to DNA. It has been hypothesized that well-done meat increases the risk of colorectal cancer (CRC), especially in individuals with the rapid phenotype for CYP1A2 and NAT2. This association may be particularly strong in smokers because smoking is known to induce CYP1A2. We conducted a population-based case-control study on Oahu, Hawaii to specifically test this hypothesis. An in-person interview assessed the diet and preference for well-done red meat of 349 patients with CRC and 467 population controls. A urine collection after caffeine challenge and a blood collection were used to assess phenotype for CYP1A2 and NAT2 and genotype for NAT2 and NAT1, respectively. No statistically significant main effect association with CRC was found for red meat intake, preference for well-done red meat, the NAT2 rapid genotype, the CYP1A2 rapid phenotype or the NAT1*10 allele. However, in ever-smokers, preference for well-done red meat was associated with an 8.8-fold increased risk of CRC (95% confidence interval, 1.7-44.9) among subjects with the NAT2 and CYP1A2 rapid phenotypes, compared with smokers with low NAT2 and CYP1A2 activities who preferred their red meat rare or medium. No similar association was found in never-smokers, and there was no increased risk for well-done meat among smokers with a rapid phenotype for only one of these enzymes or for smokers with both rapid phenotypes who did not prefer their red meat well-done. These data provide additional support to the hypothesis that exposure to carcinogens (presumably HAAs) through consumption of well-done meat increases the risk of CRC, particularly in individuals who are genetically susceptible (as determined by a rapid phenotype for both NAT2 and CYP1A2) and suggest that smoking, by inducing CYP1A2, facilitates this effect.

Association of methylenetetrahydrofolate reductase polymorphism C677T and dietary folate with the risk of cervical dysplasia.

Epidemiological studies have been inconsistent regarding a role for folate in the etiology of cervical dysplasia. Methylenetetrahydrofolate reductase (MTHFR) catalyzes the synthesis of 5-methyltetrahydrofolate, which is involved in the methylation of homocysteine to methionine. A common variant of this enzyme, resulting from a 677C-->T (Ala-->Val) substitution in the gene, has been shown to have reduced activity and is associated with mild hyperhomocysteinemia. A multiethnic case-control study was used to examine the association of dietary folate and MTHFR genotype with the odds ratios (ORs) for cervical dysplasia among women identified from several clinics on Oahu, Hawaii, between 1992 and 1996. We collected blood samples for DNA extraction, cervical smears for cytological diagnosis, exfoliated cervical cells for human papillomavirus (HPV) DNA testing, and personal interviews from 150 women with squamous intraepithelial lesions (SILs) and from 179 women with cytologically normal (Pap) smears. We found a positive, monotonic trend (P = 0.02) in the ORs for cervical SILs associated with the number of variant MTHFR T alleles, after multivariate adjustment. Women with the heterozygous CT genotype had twice the risk of cervical SILs [OR, 2.0; 95% confidence interval (CI), 1.1-3.7], and women with the homozygous TT genotype had almost three times the risk of SILs (OR, 2.9; 95% CI, 1.0-8.8) compared to women with the homozygous MTHFR CC genotype. The dietary intakes of folate, vitamin B(6), and vitamin B(12) were inversely related to the ORs for cervical SILs, after adjustment for HPV DNA and other confounders. The OR among women in the highest quartile compared with women in the lowest quartile of folate intake was 0.3 (95% CI, 0.1-0.7; P for trend = 0.002). Women with the variant T allele and folate intakes below the median were at significantly elevated risk of cervical SILs (OR, 5.0; 95% CI, 2.0-12.2) compared to women with CC alleles and folate intakes above the median. HPV infection was a strong risk factor for cervical dysplasia, particularly among women with the variant T allele (OR, 46.6; 95% CI, 15.9-136.2). All associations of MTHFR genotype with the ORs for cervical SILs were independent of other risk factors under study. These findings suggest that the MTHFR T allele and reduced dietary folate may increase the risk for cervical SILs.

Case-control study of ovarian cancer and polymorphisms in genes involved in catecholestrogen formation and metabolism.

Steroid hormones, such as estrogens, appear to be associated with ovarian carcinogenesis, but the precise biological mechanisms are unclear. Polymorphisms in genes that regulate the concentration of estrogens and their metabolites may contribute directly to the individual variation in ovarian cancer risk through a mechanism involving oxidative stress or indirectly by influencing ovarian cancer susceptibility associated with ovulation and reproduction. We conducted a population-based, case-control study of primary ovarian cancer between 1993 and 1999 in Hawaii to test several genetic and related hypotheses. A personal interview and blood specimen were obtained in the subjects' homes. In a sample of 129 epithelial ovarian cancer cases and 144 controls, we compared the frequencies of several polymorphisms in genes that regulate steroid hormone metabolism and catecholestrogen formation. Multivariate unconditional logistic regression was used to model the association of each genetic polymorphism separately after adjusting for age, ethnicity, and other covariates. The high-activity Val432 allele of the CYP1B1 gene, which may be linked to oxidative stress through elevated 4-hydroxylated catecholestrogen formation, was associated with an increased risk of ovarian cancer. The Val/Leu genotype for CYP1B1 was associated with an odds ratio of 1.8 (95% confidence interval, 1.0-3.3) and the Val/Val genotype with an odds ratio of 3.8 (95% confidence interval, 1.2-11.4) compared with the Leu/Leu genotype (P = 0.005). Tobacco smokers with at least one CYP1A1 (MspI) m2 allele, one CYP1B1 Val allele, one COMT Met allele, or two CYP1A2 A alleles were at significantly increased risk of ovarian cancer compared to never-smokers with CYP1A1 (MspI) ml/ml, CYP1B1 Leu/Leu, COMT Val/Val, or CYP1A2 A/A genotypes, respectively. We found a positive statistical interaction (P = 0.03) between tobacco smoking and the CYP1A1 (MspI) polymorphism on the risk of ovarian cancer. None of the other gene-environment (pregnancy, oral contraceptive pill use) or gene-gene interactions were statistically significant. Although not significant, there was a suggestion that the effect of the CYP1B1 Val allele was reduced substantially in the presence of the high-activity COMT Met allele. These findings suggest that the CYP1B1-Val allele and perhaps other genetic polymorphisms in combination with environmental or hormonal exposures are susceptibility factors for ovarian cancer.

Relationships of TP53 codon 72 and HRAS1 polymorphisms with lung cancer risk in an ethnically diverse population.

Tobacco smoking is a strong cause of lung cancer. However, because only a small proportion of smokers develop the disease, other factors, including genetic susceptibility, may be important in determining lung cancer risk. Polymorphisms in the TP53 tumor suppressor gene and HRAS1 proto-oncogene have been associated in some studies with this cancer; we sought to replicate these associations in an ethnically diverse population in Hawaii. We conducted a population-based case-control study among 334 incident lung cancer cases and 446 controls of Caucasian, Japanese, or Native Hawaiian origin. In-person interviews collected detailed information on lifestyle risk factors. DNA was extracted from peripheral blood leukocytes, and genotyping was performed using a PCR-based assay for the TP53 codon 72 polymorphism and Southern blot analysis and PCR for allelic polymorphisms in the HRAS1 minisatellite. Logistic regression analyses were used to compute odds ratios (ORs) and 95% confidence intervals (CIs) adjusting for smoking and other risk factors. The presence of two rare HRAS1 alleles was associated with a 2.2-fold (95% CI, 1.0-5.0) increased lung cancer risk for all ethnic groups combined. The association was present in Native Hawaiians (OR, 5.2; 95% CI, 1.1-24.4) and was suggested for Japanese (OR, 2.8; 95% CI, 0.6-12.5); no association was observed in Caucasians (OR, 0.8; 95% CI, 0.2-3.6). This association was also observed for each lung cancer cell type. The presence of only one rare allele did not increase risk for any ethnic group or cell type. No significant association was found between the TP53 codon 72 polymorphism and lung cancer [OR, 1.4 (95% CI, 0.8-2.4) for the Pro/Pro genotype compared with the Arg/Arg genotype]. This study suggests that the presence of two rare HRAS1 alleles confers an increased lung cancer risk in Native Hawaiians and Japanese but possibly not in Caucasians. The amino acid replacement of arginine by proline at codon 72 of TP53 appears not to be important in determining lung cancer risk in this population.

Correlation between growth control, neoplastic potential and endogenous connexin43 expression in HeLa cell lines: implications for tumor progression.

A HeLa cell line, obtained from the ATCC, was cloned and found to exhibit a spectrum of in vitro and in vivo growth characteristics as well as variable expression of endogenous connexin43 (Cx43), a widely expressed gap junction protein implicated in growth control. The majority of clones expressed functional Cx43, which contrasted with previous studies reporting that HeLa cells are completely negative for Cx43 mRNA/protein expression. This endogenous Cx43 expression correlated with increased growth control: Cx43-positive clones exhibited a decreased saturation density and a diminished growth capacity when in co-culture with growth-controlled normal cells in constrast to Cx43-negative clones. Endogenous Cx43 expression was negatively correlated with neoplastic potential as evidenced by attenuated anchorage-independent growth and decreased tumorigenicity in immunodeficient mice. Treatment of Cx43-negative cells with 5-aza-2'-deoxycytidine resulted in expression of Cx43, suggesting gene silencing via DNA methylation. These results support the concept of growth control via junctionally transmitted signals and suggest an epigenetic mechanism for tumor cells to circumvent this control during carcinogenesis. Moreover, the heterogeneous nature of this cell line and the ease of connexin43 gene induction suggest caution in the interpretation of results involving gene transfection using noninducible gene expression systems.

Determination of the breakpoint of the common alpha-thalassaemia deletion in Filipinos in Hawaii.

The region of the crossover causing the Filipino type of alpha thalassaemia has been determined by examining similarity between the regions which had been indicated as involved in the crossover points by restriction mapping, using the published alpha-globin region DNA sequence. The crossover point was found in 21 base pairs between two Alu sequences using PCR primers flanking these Alu sequences. A simple PCR multiplex assay has been devised to detect heterozygotes and homozygotes.

Prader-Willi syndrome is caused by disruption of the SNRPN gene.

A Prader-Willi syndrome patient is described who has a de novo balanced translocation, (4;15)(q27;q11.2)pat, with breakpoints lying between SNRPN exons 2 and 3. Parental-origin studies indicate that there is no uniparental disomy and no apparent deletion. This patient expresses ZNF127, SNRPN exons 1 and 2, IPW, and D15S227E (PAR1) but does not express either SNRPN exons 3 and 4 or D15S226E (PAR5), as assayed by reverse transcription-PCR, of peripheral blood cells. Methylation studies showed normal biparental patterns of inheritance of loci DN34/ZNF127, D15S63, and SNRPN exon 1. Results for this patient and that reported by Sun et al. support the contention that an intact genomic region and/or transcription of SNRPN exons 2 and 3 play a pivotal role in the manifestations of the major clinical phenotype in Prader-Willi syndrome.

Microchimerism and tolerance following intrauterine transplantation and transfusion for alpha-thalassemia-1.

A fetus homozygous for alpha-thalassemia-1 was given haploidentical paternal CD34 cells at 13, 19 and 24 weeks' gestation and supported through pregnancy by blood transfusion. The fetal hematocrit ranged between 27 and 47% and between one half and three quarters of this hemoglobin was of recipient (Bart's) type. Intrauterine growth proceeded normally and no significant fetal hydrops was detected. Tests for donor HLA antigens, and alpha-globin DNA, were negative on fetal blood samples drawn before birth. A positive signal for alpha-globin DNA was obtained from cord blood and from marrow obtained at 3 months of age, suggesting that some donor stem cells had persisted in the recipient. The infant's blood mononuclear cells showed little proliferative and no cytotoxic response to the donor while responses to a third party were present. Additional paternal CD34 cells given at 3 months age did not reduce transfusion dependency in the subsequent 6 months. Our results show that repeated transfusions can support an alpha-thalassemia-1 fetus through pregnancy, in this instance without significant birth defects or apparent hypoxic tissue injury. The donor stem cells did not have a survival advantage compared with endogenous stem cells, but appeared to survive in the recipient as judged by the persistence of an alpha-globin DNA signal. In vitro studies of alloreactivity suggest tolerization of the host to the donor's MHC disparity. Future efforts will focus on exploiting this tolerance to improve the level of donor chimerism.

Non-mosaic trisomy 16 in a third-trimester fetus.

BACKGROUND: Trisomy 16 in the most common trisomy first-trimester spontaneous abortions, suggesting a high rate of non-disjunction of this chromosome. Deoxyribonucleic acid studies in aborted conceptuses with trisomy 16 have demonstrated a maternal origin in all cases. There have been cases of confined placental mosaicism, fetal mosaicism, and partial trisomy involving chromosome 16 reported in term fetuses. However, to our knowledge, there have been no previous reports of a near-term fetus with full trisomy 16 since the advent of modern chromosomal banding techniques. CASE: A 25-year-old Filipino woman underwent obstetric sonographic evaluation at 32 weeks' gestation; results were remarkable for oligohydramnios, severe growth restriction, and multiple dysmorphic features. Percutaneous umbilical blood sampling was performed for rapid karyotyping, viral serology, and blood profiles. The fetal karyotype was 47, XY+16; the remainder of the laboratory analysis was unremarkable. The patient went into spontaneous labor at 35 weeks' gestation and delivered a stillborn female fetus (birth weight 783 g). Chromosomes from skin, brain, and chorionic villi were examined and all demonstrated trisomy 16 (47, XX,+16). Deoxyribonucleic acid primers for known polymorphic regions of chromosome 16 were used and determined the origin of the extra chromosome to be non-disjunction during paternal meiosis. CONCLUSION: Previously, full trisomy 16 has been thought to be incompatible with fetal survival past the early second trimester. This case also contrasts with previously reported experience with trisomy 16 in that parental origin studies determined that the extra chromosome 16 originated from the father, suggesting that paternal derivation of the additional chromosome may play a role in the ultimate phenotypic expression.

Effects of granulocyte colony-stimulating factor therapy on in vitro hemopoiesis in myelodysplastic syndromes.

We evaluated the effects of 2 months of G-CSF treatment on in vitro hematopoiesis in 17 patients with myelodysplastic syndromes (MDS). Although in vitro marrow myeloid progenitor cell (CFU-GM) growth stimulated by G-CSF generally remained subnormal, in the majority of neutrophil responders significantly augmented incremental change (termed AIC) of CFU-GM numbers occurred after treatment, as did neutrophilic differentiation. The neutrophil non-responders had less prominent in vitro myeloid responses and lower basal neutrophil levels (p < 0.05). Following G-CSF treatment, the initially subnormal erythroid burst-forming unit (BFU-E) values underwent AIC in five of 11 patients along with increased reticulocyte responses in vivo, whereas four of the five patients who lacked AIC of BFU-E did not. Three patients with persisting cytogenetic abnormalities and increased neutrophilic differentiation in vitro also responded in vivo, suggesting that G-CSF induced in vivo cellular differentiation from the abnormal clone. Two of the three patients who developed blastic responses in vivo had increased CFU-GM growth pre- and post-therapy. These results indicate in vivo-in vitro correlations for myeloid and erythroid responses of MDS marrow cells which related to treatment with G-CSF.

Deletion involving D15S113 in a mother and son without Angelman syndrome: refinement of the Angelman syndrome critical deletion region.

Deletions of 15q11-q13 typically result in Angelman syndrome when inherited from the mother and Prader-Willi syndrome when inherited from the father. The critical deletion region for Angelman syndrome has recently been restricted by a report of an Angelman syndrome patient with a deletion spanning less than 200 kb around the D15S113 locus. We report here on a mother and son with a deletion of chromosome 15 that includes the D15S113 locus. The son has mild to moderate mental retardation and minor anomalies, while the mother has a borderline intellectual deficit and slightly downslanting palpebral fissures. Neither patient has the seizures, excessive laughter and hand clapping, ataxia or the facial anomalies which are characteristic of Angelman syndrome. The proximal boundary of the deletion in our patients lies between the D15S10 and the D15S113 loci. Our patients do not have Angelman syndrome, despite the deletion of the D15S113 marker. This suggests that the Angelman syndrome critical deletion region is now defined as the overlap between the deletion found in the previously reported Angelman syndrome patient and the region that is intact in our patients.

Characterization of spontaneous factor-independent cell lines derived from the human leukemic cell line TF-1: a dominant event.

Uncontrolled proliferation of acute myeloid leukemia (AML) cells is an important step during leukemogenesis. However, little is known about the mechanisms leading to growth autonomy. Studies using immortalized murine hematopoietic cell lines have suggested that autocrine production of growth factors, or the constitutive activation of molecules in growth factor signalling pathways, are involved. We have established six spontaneous factor-independent cell lines from the human growth factor-dependent TF-1 cell line. The factor-independent cells showed no detectable growth factor activity. Immunoblotting analyses of tyrosine phosphorylation, Raf-1 and extracellular signal-regulated kinase 2 (ERK-2) showed a similar pattern in all the cell lines including TF-1 cells. Furthermore, somatic-cell hybrids between TF-1 and the factor-independent cells grew in absence of growth factor. Taken together this data demonstrates that the factor independence in this system is dominant and suggests that the molecular event is located either downstream of the Raf-1 and MAP kinases pathway or on an alternative pathway. Finally, the karyotype analysis of one factor-independent cell line TF-1i1 and TF-1H- (G418 resistant, HAT sensitive TF-1 cells) and their hybrids demonstrated an unstable derivative chromosome [der(19) t(19;?) (q13.1;?)] which seemed to correlate with the factor-independence capacity. This model may help in our understanding of autonomous proliferation by human myeloid leukemias.

Small nuclear ribonucleoprotein polypeptide N (SNRPN), an expressed gene in the Prader-Willi syndrome critical region.

Prader-Willi syndrome (PWS) is associated with paternally derived chromosomal deletions in region 15q11-13 or with maternal disomy for chromosome 15. Therefore, loss of the expressed paternal alleles of maternally imprinted genes must be responsible for the PWS phenotype. We have mapped the gene encoding the small nuclear RNA associated polypeptide SmN (SNRPN) to human chromosome 15q12 and a processed pseudogene SNRPNP1 to chromosome region 6pter-p21. Furthermore, SNRPN was mapped to the minimal deletion interval that is critical for PWS. The fact that the mouse Snrpn gene is maternally imprinted in brain suggests that loss of the paternally derived SNRPN allele may be involved in the PWS phenotype.