Lesley J. Hinks

Determinants of the rate of nicotine metabolism and effects on smoking behavior

Studies on cytochrome P450 (CYP) 2A6 suggest that genotype affects the rate of nicotine metabolism and, consequently, cigarette consumption. However, known alleles of CYP2A6 associated with fast or slow metabolism are relatively uncommon, and there remains considerable variation in metabolic activity among those with presumed wild‐type CYP2A6 alleles, suggesting that other genetic or environmental factors also influence the rate of nicotine metabolism.

The association of the PON1 Q192R polymorphism with coronary heart disease: findings from the British Women's Heart and Health cohort study and a meta-analysis

BackgroundThere have been inconsistent results from case-control studies assessing the association of the PON1 Q192R polymorphism with coronary heart disease (CHD). Most studies have included predominantly men and the association in women is unclear. Since lipid levels vary between the sexes the antioxidant effect of PON1 and any genes associated with it may also vary by sex. We have examined the association of the PON1 Q192R polymorphism with CHD in a large cohort of British women and combined the results from our cohort study with those from all other published studies.ResultsThe distribution of genotypes was the same among women with CHD and those without disease. The odds ratio (95% confidence interval) of having CHD comparing those with either the QR or RR genotype to those with QQ genotype (dominant model of association) was 1.03 (0.89, 1.21) and the per allele odds ratio was 0.98 (0.95, 1.01). In a meta-analysis of this and 38 other published studies (10,738 cases and 17,068 controls) the pooled odds ratio for the dominant effect was 1.14 (1.08, 1.20) and for the per allele effect was 1.10 (1.06, 1.13). There was evidence of small study bias in the meta-analyses and the dominant effect among those studies with 500 or more cases was 1.05 (0.96, 1.15). Ethnicity and reporting of whether the genotyping was done blind to the participants clinical status also contributed to heterogeneity between studies, but there was no difference in effect between studies with 50% or more women compared to those with fewer women and no difference between studies of healthy populations compared to those at high risk (with diabetes, renal disease of familial hypercholesterolaemia).ConclusionThere is no robust evidence that the PON1 Q192R polymorphism is associated with CHD risk in Caucasian women or men.

CYP2A6, MAOA, DBH, DRD4, and 5HT2A genotypes, smoking behaviour and cotinine levels in 1518 UK adolescents

Objectives Smoking is a major cause of death and often initiates in adolescence. Mutations in CYP2A6 slow metabolism of nicotine to cotinine. Haploinsufficiency in adults is associated with lower cigarette consumption, lower cotinine level and higher quit rates. Other genes are also implicated in smoking behaviour. We explored smoking behaviour and cotinine levels in relation to genotypes in adolescents. Methods 1518 subjects from the Ten Towns Heart Health Study were genotyped for CYP2A6 alleles *1A, *1B, *2, *4, *5, *9 and *12 to classify predicted nicotine metabolism rate. DBH(rs77905), MAOA(rs1801291+VNTR), DRD4(VNTR) and 5HT2A(rs6313) were also studied. Smoking status was established by questionnaire and salivary cotinine measurement at 13–15 and 18 years. Results No significant associations were identified for DBH, MAOA, DRD4 and 5HT2A markers, with smoking status or cotinine level at either age. At age 18, haploinsufficiency (HI) for CYP2A6 was associated with a higher odds of being a current smoker compared with the *1B carriers (WT1B) (OR=2.23 (1.16, 4.27) for current versus ex); *1A homozygotes (WT1A) were also at slightly higher risk (OR=1.44 (1.01, 2.06)). Partial haploinsufficiency (PHI) was not associated with being a current smoker. There were no significant associations at age 13–15. PHI and HI were associated with higher cotinine levels amongst smokers at both 13–15 and at 18 years compared with WT1B and WT1A groups. Conclusions CYP2A6 haploinsufficiency increases likelihood of continuing smoking in teenagers. We hypothesize an explanatory ‘occupancy’ model to explain why haploinsufficiency results in faster progression to nicotine dependence, but lower subsequent consumption.

Reduced plasma and white blood cell selenium levels in haemodialysis patients

Selenium concentrations have been measured in plasma and in blood leucocytes from 29 haemodialysis patients and from 25 healthy men. The selenium contents of the plasma and white blood cells of the dialysis patients were significantly reduced (p less than 0.001). Selenium deficiency in humans results in a congestive cardiomyopathy and is associated with increased risks of accelerated atherosclerosis and cancer. Each of these is found with abnormal frequency in haemodialysis patients.

Comparison of three methods for single nucleotide polymorphism typing for DNA bank studies: sequence-specific oligonucleotide probe hybridisation, TaqMan liquid phase hybridisation, and microplate array diagonal gel electrophoresis (MADGE).

In the near future the number of SNPs identified and mapped will increase and the need for high throughput SNP typing will be paramount for comprehensive examination by association of the role of genomic regions in disease traits. A range of higher throughput methods for typing SNPs is now in routine use in many laboratories worldwide. In this report, we analyse the relative advantages and disadvantages of three such methods, TaqMan, PCR‐SSOP, and ARMS‐MADGE, currently in use in our laboratories. Throughputs achievable are similar, but there are major differences in cost and time for set‐up, equipment, consumables, and staff time, which may determine the choice for individual laboratories. Hum Mutat 14:340–347, 1999.

The association of the paraoxonase (PON1) Q192R polymorphism with depression in older women: findings from the British Women’s Heart and Health Study

Background: The association between the R allele of PON1 Q192R and symptoms reported by sheep dippers and Gulf War veterans has been used to suggest a biological basis for these symptoms. In the absence of such studies in non-occupational populations, these conclusions may not be valid. Objective: To examine the association of paraoxonase (PON1) Q192R with a report of ever being diagnosed with depression among a random sample of 3266 British women, aged 60–79 years. Results: The R allele of PON1 Q192R was associated with depression: per-allele odds ratio 1.22 (95% confidence interval: 1.05 to 1.41) in this population. Conclusions: These findings suggest that the association of PON1 Q192R with symptoms of depression in occupationally exposed groups may be driven by exposure to toxins that everyone in the general population is exposed to rather than exposure to toxins specifically used by sheep dippers or Gulf War veterans, or that other mechanisms underlie the association. This is because the study population in which we have found an association consisted of British women aged 60–79 years, few of whom were sheep dippers or Gulf War veterans. When using genotype–outcome associations to infer causality with respect to an environmental exposure modified by the genotype, it is important to examine these associations in general populations and in those specifically exposed to the putative agent. The possible role of PON1 Q192R in psychiatric morbidity requires further examination.

Zinc Absorption in Haemodialysis Patients

The gastrointestinal absorption of zinc by haemodialysis patients has been investigated. Subnormal plasma zinc responses were observed in nine haemodialysis patients during oral zinc tolerance tests. These patients also had reduced leucocyte zinc concentrations which did not respond when 50 mg (0·77 mmol) of zinc was given daily by mouth for a period of 4 months. White cell zinc concentrations measured in another group of 10 haemodialysis patients were reduced initially, but rose to normal levels over 4 months when zinc was added to the fluid used for dialysis. These data suggest that the intestinal absorption of zinc was defective in the dialysis patients participating in this investigation.

Reduced leucocyte zinc and albumin-bound zinc in blood of haemodialysis patients

The total, albumin-bound, and α2-macroglobulin-bound zinc concentrations in serum samples obtained from 20 haemodialysis patients have been measured. Blood leucocyte zinc contents were also determined. The total serum zinc concentrations in these samples were low, and in each case this was due to reduction in the zinc associated with albumin, although the concentrations of albumin itself were normal. There was no abnormality of the zinc bound to α2-macroglobulin. These data demonstrate a disturbance of the plasma transport of zinc in haemodialysis patients which might be due either to a reduced affinity of albumin for zinc in uraemic subjects, or the result of zinc deficiency. The zinc contents of white blood cells were also significantly reduced, supporting the possibility that these patients were indeed zinc deficient.

Aminoterminal procollagen III peptide elevation in alcoholics who are selenium and vitamin E deficient

Serum aminoterminal procollagen III peptide (PIIIP) was measured in 36 alcoholic subjects. There was a significant elevation of PIIIP in subjects with proven liver disease (median 17.5 ng/ml, n = 24) compared to those without liver disease (median 4.7 ng/ml, n = 12). Those subjects with raised serum transaminase values (AST) had elevated PIIIP values (median 13.7 ng/ml, n = 22) compared to those with normal transaminase values (median 3.7 ng/ml, n = 14). In those alcoholic subjects who were deficient in both selenium and vitamin E there was a significant elevation (p less than 0.01) of PIIIP values (median 26.4 ng/ml, n = 7) compared to subjects with normal levels (median 7 ng/ml, n = 11). Subjects deficient in selenium alone had PIIIP values in an intermediate range. Selenium and vitamin E, as important free radical scavengers, may protect the liver in alcoholic subjects from oxidative damage leading to hepatic fibrosis.

Microplate-array diagonal-gel electrophoresis (MADGE) systems for high-throughput electrophoresis

▼Microplate-array diagonal-gel electrophoresis (MADGE) was invented by Day in 1994 (Ref. 1, 2) to enable compatibility between microplate-based liquid phase reactions and polyacrylamide or agarose-gel electrophoretic analysis. In MADGE, a microplate-compatible array of wells is turned on a diagonal relative to the line of electrophoresis so that samples can run on a long track between the wells (Fig. 1). This means that the 96-well format can be coupled to high sample resolution. Microplates occupy an analytical ‘middle ground’ in throughput between test tubes and microarrays, and MADGE can now meet the associated electrophoretic requirements at this level. The natural scalability of MADGE enables high throughput in large laboratories while its simplicity facilitates distributability between smaller laboratories. The set-up and running costs of MADGE compare favourably with other techniques (Ref. 3) and most electrophoresis-based analysis should be feasible using MADGE. The invention of MADGE was driven by the higher-throughput requirements in human singlenucleotide polymorphism (SNP) genotyping, and similar requirements for de novo mutation scanning have further spawned the invention of melt-MADGE. This set of core protocols reflects the origins of MADGE but the scope of MADGE extends in principle to any higher throughput genomic analysis. In a short series of four articles for Technical Tips Online, we describe the developed components of MADGE. This first article reviews the use of MADGE for analysing simple patterns of polymorphism such as those generated in