Alison J. Black

Thiopurine Methyltransferase Genotype Predicts Therapy-Limiting Severe Toxicity from Azathioprine

Azathioprine has demonstrated disease-modifying activity in rheumatic disease and is an effective therapy for inflammatory bowel disease, multiple sclerosis, systemic vasculitis, chronic hepatitis, dermatologic disorders, and immunosuppression after solid-organ transplantation [1-6]. However, its use has been limited by a high incidence of hematologic toxicity and concern about long-term side effects [7]. Azathioprine is a relatively inexpensive therapeutic agent for common diseases, and methods for prospectively identifying patients at risk for acute toxicity may be useful for therapeutic decision making. Azathioprine is converted to 6-mercaptopurine in vivo, where it is metabolized to cytotoxic thioguanine nucleotides or is inactivated by xanthine oxidase or thiopurine methyltransferase. Thiopurine methyltransferase-catalyzed S-methylation shunts thiopurine to relatively inactive compounds and away from activation to thioguanine nucleotides [7]. Population studies have found activity of thiopurine methyltransferase in erythrocytes to be trimodal: Approximately 90% of persons have high activity, 10% have intermediate activity, and 0.3% have low or no activity [8]. Azathioprine-induced bone marrow suppression was more frequent in patients with a dermatologic condition and the intermediate phenotype, whereas patients with high thiopurine methyltransferase activity had a poor clinical response [6]. Patients with low thiopurine methyltransferase activity have severe or fatal hematopoietic toxicity in response to thiopurine-based therapies [7, 9]. However, azathioprine also induces activity of thiopurine methyltransferase in erythrocytes, making direct assessment of enzyme activity difficult [10]. The recent identification of three distinct thiopurine methyltransferase mutations (detected in 80% to 95% of white persons with low or intermediate thiopurine methyltransferase activity) has allowed the development of polymerase chain reaction (PCR)-based techniques for genotype analysis [11]. The most common variant allele in white persons contains point mutations at nucleotides 460 and 719 and has been named TPM*3A [11]. Alleles containing a mutation at nucleotide 238 are designated TPM*2 [11]. This discovery led to the hypothesis that molecular analysis of thiopurine methyltransferase may be a useful way to identify patients at risk for toxicity from thiopurine medication. Methods Patients Thiopurine methyltransferase genotype was assessed in consecutive patients who were seen at the rheumatology departments of the Aberdeen Royal Infirmary or the Glasgow Royal Infirmary in Glasgow, United Kingdom, over a 6-month period and were prescribed azathioprine. No patients were excluded on the basis of previous or current medication. Patients received azathioprine, 2 to 3 mg/kg of body weight per day, as second-line therapy; some patients were also prescribed oral corticosteroids. The patients had received stable doses of nonsteroidal anti-inflammatory drugs as first-line therapy. Blood counts and liver function test results were regularly monitored at least monthly. Reasons for discontinuation of azathioprine therapy were intolerance, progressively abnormal liver function test results, and reduction in total leukocyte count to less than 3.5 109/L or reduction in neutrophil count to less than 1.5 109/L. Clinicians caring for the patients were unaware of the thiopurine methyltransferase genotype. Analysis of Thiopurine Methyltransferase Genotype After patients gave written informed consent, genomic DNA was extracted from 5 mL of whole blood and was analyzed for the presence of mutations at nucleotides 238, 460, and 719 by using allele-specific PCR or PCR restriction fragment length polymorphism, as described elsewhere [11, 12]. In brief, mutation-specific PCR was performed by using pairs of oligonucleotides that specifically amplified a PCR product in the presence of guanine (wild-type allele) or cytosine (mutant allele) at nucleotide 238 [11, 12]. Diagnostic assays for the other alleles took advantage of alterations in the presence of a restriction enzyme cut-site when mutations occurred at nucleotides 460 or 719 [11, 12]. Molecular analysis was done without knowledge of clinical outcome, and both positive and negative (no DNA) controls were included in each assay. Statistical Analysis Differences in the duration of therapy between patients with wild-type alleles and those with mutant alleles was assessed by the log-rank test, accompanied by Kaplan-Meier curves. Results All 67 patients recruited for this study (55 women and 12 men; mean age SD, 51 13.4 years) were evaluable for toxicity and duration of therapy. Azathioprine was used for rheumatoid arthritis (n = 49), systemic lupus erythematosus (n = 7), or other rheumatic diseases (n = 11). Therapy was discontinued in 25 patients (37%) because of side effects (nausea, abnormal liver function test results, and low leukocyte count) and in 18 patients (27%) because of lack or loss of efficacy. Six patients (9%) were heterozygous for TPM*3A; TPM*2 was not detected in this study sample. No patients were homozygous for low-activity thiopurine methyltransferase alleles. Of the 6 patients heterozygous for TPM*3A, 5 discontinued azathioprine therapy within 1 month of starting this therapy because of reduced total leukocyte counts (range, 0.9 to 2.7 109/L) (Figure 1). The sixth patient had a well-documented history of noncompliance with drug therapy and on subsequent questioning was found not to be taking azathioprine. Figure 1. Influence of thiopurine methyltransferase genotype on the duration of azathioprine therapy (61 patients had the wild-type TPM allele and 5 patients had the mutant TPM allele). Duration of therapy was significantly longer for patients with wild-type thiopurine methyltransferase alleles (median, 39 weeks [range, 6 to 180 weeks]) than for those with mutant alleles (median, 2 weeks [range, 2 to 4 weeks]) (P = 0.018). Within the first 2 months of therapy, liver function test results were abnormal in 6 patients with wild-type thiopurine methyltransferase alleles and 1 patient with mutant alleles. All other side effects were mild; the most frequent side effect was gastrointestinal upset. No hematologic abnormalities occurred in patients with wild-type alleles. Discussion Our study provides the first analysis of thiopurine methyltransferase genotype in patients with rheumatic disease and identifies the incidence of heterozygous genotype in this population. The clinical significance of this finding is shown in Figure 1: All patients with mutant thiopurine methyltransferase alleles were forced to discontinue azathioprine therapy within 1 month of initiating therapy because of serious hematologic side effects. The rate of abnormal liver function test results caused by azathioprine therapy was not associated with mutant alleles. These data strongly suggest that prospective analysis of thiopurine methyltransferase genotype may allow pretherapy identification of patients at risk for serious toxicity from azathioprine. Azathioprine therapy is active in patients with various inflammatory disorders and may still be useful in patients with variant thiopurine methyltransferase genotypes. However, substantial reductions in azathioprine dosage would be required [7]. Polymerase chain reaction-based assays are routinely used in many laboratory medicine departments; this availability facilitates the use of molecular therapeutic tests, such as thiopurine methyltransferase genotyping. Use of this approach may be appropriate from a health economic standpoint; at our center, for example, the cost of blood monitoring and supportive care exceeds that of PCR analysis by several-fold. This suggests that analysis of all azathioprine recipients to identify the 10% of the population at risk for thiopurine methyltransferase-mediated toxicity would be cost-effective. However, each individual center would need to verify this. Our study shows the ability of molecular analysis of thiopurine methyltransferase to identify patients at risk for hematologic toxicity from azathioprine. The use of such a test to avoid harmful, and potentially fatal, side effects is especially important because azathioprine is primarily used in ambulatory patients who do not have a terminal illness. Genotype analysis of thiopurine methyltransferase does not consider other variables regulating in vivo azathioprine activity, such as activation to thiopurine nucleotides, inactivation by xanthine oxidase, or dephosphorylation by 5-nucleotidase [13]. Assessment of the activity of thiopurine methyltransferase in erythrocytes may also prove useful. This testing will identify patients with high thiopurine methyltransferase activity who are resistant to therapy because thiopurine is shunted away from activation to thioguanine nucleotides [6, 14]. Alternatively, thioguanine nucleotides could be directly measured to determine the quantity of active metabolites formed, thereby predicting the dose necessary for clinical effectiveness [7]. However, only a few research centers have assays for enzyme activity or thioguanine nucleotides; this limits the widespread application of these assays as a screening test for azathioprine. Analysis of thiopurine methyltransferase genotype is a quick and relatively inexpensive way to identify patients at risk for acute toxicity from azathioprine and should also be applicable to related thiopurine drugs, such as 6-mercaptopurine. It may be a helpful tool for the clinical management of the many patients treated with azathioprine for inflammatory disorders. Drs. McLeod, Collie-Duguid, and Reid, Mr. Powrie, Mr. Matowe, and Mr. Pritchard: Department of Medicine & Therapeutics, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, United Kingdom. Dr. Capell: Centre for Rheumatic Disease, Glasgow Royal Infirmary, Glasgow G31 2ER, United Kingdom.

Vitamin D3 supplementation has no effect on conventional cardiovascular risk factors: a parallel-group, double-blind, placebo-controlled RCT.

CONTEXT Observational studies show an association between low vitamin D status assessed by circulating 25-hydroxyvitamin D and cardiovascular events and mortality. Data from randomized controlled trials are limited. OBJECTIVE The aim of this study was to test whether daily doses of vitamin D(3) at 400 or 1000 IU/d for 1 yr affected conventional markers of cardiovascular disease (CVD) risk. DESIGN We conducted a parallel-group, double-blind, placebo-controlled randomized controlled trial. Randomization was computer generated. Participants and study investigators were blinded to intervention groupings throughout the trial. SETTING The study was conducted at the Clinical Research Facility, University of Aberdeen, United Kingdom. PARTICIPANTS A total of 305 healthy postmenopausal women aged 60-70 yr were recruited for the study. INTERVENTION Each woman received a daily capsule of 400 or 1000 IU vitamin D(3) or placebo randomly allocated. MAIN OUTCOME MEASURES Primary outcomes were serum lipid profile [total, high-density lipoprotein, and low-density lipoprotein cholesterol; triglycerides; and apolipoproteins A-1 and B100], insulin resistance (homeostatic model assessment), inflammatory biomarkers (high-sensitivity C-reactive protein, IL-6, soluble intracellular adhesion molecule-1), and blood pressure. RESULTS A total of 265 (87%) participants completed all study visits. Small differences between groups for serum apolipoprotein B100 change [repeated measures ANOVA, P=0.04; mean (sd), -1.0 (10.0) mg/dl (400 IU); -1.0 (10.0) mg/dl (1000 IU); and +0.02 (10.0) mg/dl (placebo)] were not considered clinically significant. Other systemic markers for CVD risk remained unchanged. There was significant seasonal variation in systolic and diastolic blood pressure independent of vitamin D dose (P<0.001, linear mixed model). Mean (sd) reduction in systolic blood pressure from winter to summer was -6.6 (10.8) mm Hg. CONCLUSIONS Improving vitamin D status through dietary supplementation is unlikely to reduce CVD risk factors. Confounding of seasonality should be recognized and addressed in future studies of vitamin D.

Vitamin D status in postmenopausal women living at higher latitudes in the UK in relation to bone health, overweight, sunlight exposure and dietary vitamin D

For 5 months a year the UK has insufficient sunlight for cutaneous synthesis of vitamin D and winter requirements are met from stores made the previous summer. Although there are few natural dietary sources, dietary intake may help maintain vitamin D status. We investigated the relationship between 25-hydroxyvitamin D (25(OH)D), bone health, overweight, sunlight exposure and dietary vitamin D in 3113 women (age 54.8 [SD 2.3] years) living at latitude 57 degrees N between 1998-2000. Serum 25(OH)D was measured by high performance liquid chromatography (HPLC), dietary intakes (food frequency questionnaire, n=2598), sunlight exposure (questionnaire, n=2402) and bone markers were assessed. Bone mineral density (BMD) was measured by dual x-ray absorptiometry in all women at the sampling visit and 6 years before. Seasonal variation in 25(OH)D was not substantial with a peak in the autumn (23.7 [9.9] ng/ml) and a nadir in spring (19.7 [7.6] ng/ml). Daily intake of vitamin D was 4.2 [2.5] mug from food only and 5.8 [4.0] mug including vitamin D from cod liver oil and multivitamins. The latter was associated with 25(OH)D at each season whereas vitamin D simply from food was associated with 25(OH)D in winter and spring only. Sunlight exposure was associated with 25(OH)D in summer and autumn. 25(OH)D was negatively associated with increased bone resorption and bone loss (P<0.05) remaining significant after adjustment for confounders (age, weight, height, menopausal status/HRT use, physical activity and socio-economic status). Using an insufficiency cut-off of <28 ng/ml 25(OH)D, showed lower concentrations of bone resorption markers in the upper category (fDPD/Cr 5.1 [1.7] nmol/mmol compared to 5.3 [2.1] nmol/mmol, P=0.03) and no difference in BMD or bone loss. 25(OH)D was lower (P<0.01) and parathyroid hormone higher (P<0.01) in the top quintile of body mass index. In conclusion, low vitamin D status is associated with greater bone turnover, bone loss and obesity. Diet appears to attenuate the seasonal variation of vitamin D status in early postmenopausal women at northerly latitude where quality of sunlight for production of vitamin D is diminished.

Large-Scale Population-Based Study Shows No Evidence of Association Between Common Polymorphism of the VDR Gene and BMD in British Women

The VDR is a candidate gene for osteoporosis. Here we studied five common polymorphisms of VDR in relation to calcium intake and vitamin D status in a population‐based cohort of 3100 British women, but found no significant association with bone mass, bone loss, or fracture.

Bone mineral density in osteoarthritis.

The inverse relation between osteoporosis and osteoarthritis has long been considered in the literature. This review looks at current evidence to support this relation, concentrating on studies published since 1998. The review also summarizes previous large studies investigating this relation. Recent studies indicate higher bone mineral density as measured by dual energy x-ray absorptiometry in subjects with osteoarthritis at a distant site, but suggest less association with hand osteoarthritis. Genetic work has sought to explain this association and this too is discussed. There is some indication that a higher bone density may not protect against fracture in these subjects, due to the increased risk of falls.

Hip bone loss is attenuated with 1000 IU but not 400 IU daily vitamin D3: a 1-year double-blind RCT in postmenopausal women.

Few year‐long vitamin D supplementation trials exist that match seasonal changes. The aim of this study was to determine whether daily oral vitamin D3 at 400 IU or 1000 IU compared with placebo affects annual bone mineral density (BMD) change in postmenopausal women in a 1‐year double‐blind placebo controlled trial in Scotland. White women aged 60 to 70 years (n = 305) were randomized to one of two doses of vitamin D or placebo. All participants started simultaneously in January/February 2009, attending visits at bimonthly intervals with 265 (87%) women attending the final visit and an additional visit 1 month after treatment cessation. BMD (Lunar iDXA) and 1,25‐dihydroxyvitamin D[1,25(OH)2D], N‐terminal propeptide of type 1 collagen [P1NP], C‐terminal telopeptide of type I collagen [CTX], and fibroblast growth factor‐23 [FGF23] were measured by immunoassay at the start and end of treatment. Circulating PTH, serum Ca, and total 25‐hydroxyvitamin D [25(OH)D] (latter by tandem mass spectrometry) were measured at each visit. Mean BMD loss at the hip was significantly less for the 1000 IU vitamin D group (0.05% ± 1.46%) compared with the 400 IU vitamin D or placebo groups (0.57% ± 1.33% and 0.60% ± 1.67%, respectively) (p < 0.05). Mean (± SD) baseline 25(OH)D was 33.8 ± 14.6 nmol/L; comparative 25(OH)D change for the placebo, 400 IU, and 1000 IU vitamin D groups was −4.1 ± 11.5 nmol/L, +31.6 ± 19.8 nmol/L, and +42.6 ± 18.9 nmol/L, respectively. Treatment did not change markers of bone metabolism, except for a small reduction in PTH and an increase in serum calcium (latter with 1000 IU dose only). The discordance between the incremental increase in 25(OH)D between the 400 IU and 1000 IU vitamin D and effect on BMD suggests that 25(OH)D may not accurately reflect clinical outcome, nor how much vitamin D is being stored.

Large-scale population-based study shows no association between common polymorphisms of the TGFB1 gene and BMD in women

The TGFB1 gene is a strong functional candidate for regulating genetic susceptibility to osteoporosis. We studied five common polymorphisms of TGFB1 in relation to osteoporosis‐related phenotypes in a population‐based cohort of 2975 British women, but found no significant association with bone mass, bone loss, bone markers, or fracture.

Effect of Pregnancy on Bone Mineral Density and Biochemical Markers of Bone Turnover in a Patient With Juvenile Idiopathic Osteoporosis

Juvenile idiopathic osteoporosis (JIO) is rare, presenting with vertebral fractures in the immediate prepubertal years; however, recovery is normally observed. We report the case of a 19‐year‐old pregnant woman previously diagnosed with JIO. She experienced three vertebral fractures in the third trimester of pregnancy. She delivered by caesarean section at 38 weeks gestation. Spinal bone mineral density decreased by 25%, hip bone mineral density by 10%, and forearm bone mineral density by 3% during pregnancy. Bone resorption markers, free pyridinoline and deoxypyridinoline (fPYD and fDPD), were elevated at baseline and markedly increased during pregnancy (fPYD/fDPD at 0, 10, 15, 20, and 28 weeks and immediately postpartum: 36.2/11.5, 52.9/15.8, 54.3/13.3, 51.1/13.3, 90/21.8, and 95.6/22.7 nmol/mmol creatinine, respectively) The bone formation marker, bone‐specific alkaline phosphatase (BSAP), was within the reference range at baseline and increased in the third trimester. (BSAP at 0, 10, 15, 20, and 28 weeks and immediately postpartum: 20.5, 18.3, 17.7, 19.8, 26.9, and 30.0 U/liter, respectively). Parathyroid hormone (PTH) was measured by two methods to assess the possible effect of PTH fragments. PTH(1–84) (Roche) showed little change during the pregnancy, whereas the Nichols assay [(1–84) and(7–84) PTH fragment], revealed increases paralleling the changes in bone resorption. This young womans bone turnover showed an exaggerated response to pregnancy, with bone resorption predominating over formation. PTH fragments may have partially mediated this effect.

Seasonal variation in 25(OH)D at Aberdeen (57°N) and bone health indicators--could holidays in the sun and cod liver oil supplements alleviate deficiency?

Vitamin D has been linked with many health outcomes. The aim of this longitudinal study, was to assess predictors of seasonal variation of 25-hydroxy-vitamin D (25(OH)D) (including use of supplements and holidays in sunny destinations) at a northerly latitude in the UK (57°N) in relation to bone health indicators. 365 healthy postmenopausal women (mean age 62.0 y (SD 1.4)) had 25(OH)D measurements by immunoassay, serum C-telopeptide (CTX), estimates of sunlight exposure (badges of polysulphone film), information regarding holidays in sunny destinations, and diet (from food diaries, including use of supplements such as cod liver oil (CLO)) at fixed 3-monthly intervals over 15 months (subject retention 88%) with an additional 25(OH)D assessment in spring 2008. Bone mineral density (BMD) at the lumbar spine (LS) and dual hip was measured in autumn 2006 and spring 2007 (Lunar I-DXA). Deficiency prevalence (25(OH)D<25 nmol/L) was reduced in women who went on holiday to sunny destinations 3 months prior to their visit, compared to women who did not go on holidays [5.4% vs. 24.6% in Spring (p<0.001) and 3.8% vs. 25.6% in Winter (p = 0.001), respectively]. Similarly deficiency was lower amongst those who took CLO supplements compared to women that did not consume these supplements [2.0% vs. 23.7% in Spring (p = 0.001) and 4.5% vs. 24.8% in winter (p = 0.005), respectively]. There was no seasonal variation in CTX; 25(OH)D predicted a small proportion (1.8% variation) of LS BMD in spring 2007 [unstandardized β (SE): 0.039 (0.016), p = 0.017]. Seasonal variation of 25(OH)D had little effect on BMD and no effect on CTX. It appears that small increments in vitamin D (e.g. those that can be achieved by cod liver oil supplements of 5 µg/day) are sufficient to ensure that 25(OH)D is above 25 nmol/L for most people throughout the year. Similarly, holidays in sunny destinations show benefit.