Christiaan W. Sies

High TPMT enzyme activity does not explain drug resistance due to preferential 6-methylmercaptopurine production in patients on thiopurine treatment

Up to 20% of patients on thiopurine therapy fail to achieve adequate drug response. Many of these patients preferentially produce the toxic 6‐methylmercaptopurine metabolites (6‐MMP) rather than the active 6‐thioguanine nucleotides (6‐TGN) resulting in a high 6‐MMP/6‐TGN ratio (>20) and increased risk of hepatotoxicity.

Trinucleotide repeat variants in the promoter of the thiopurine S-methyltransferase gene of patients exhibiting ultra-high enzyme activity.

Thiopurine S-methyl transferase (TPMT) is a cytosolic enzyme that catalyses the S-methylation of the thiopurine immunosuppressants. To date, 22 variants have been identified that are predictive of decreased TPMT activity. In contrast, no molecular explanation has been found for the 1–2% of Caucasians who exhibit ultra-high TPMT activity. Here, we report the characterization of polymorphisms within a trinucleotide (GCC) repeat element of the TPMT promoter in two patients with inflammatory bowel disease exhibiting the highest TPMT activity from two testing centres. The first patient was heterozygous for a variant allele carrying seven GCC repeats [(GCC)7], whereas the second patient was heterozygous for a variant allele containing five GCC repeats [(GCC)5]. Fifty patients with inflammatory bowel disease with normal TPMT activity were all homozygous for six GCC repeats [(GCC)6]. Of 200 healthy controls, five were found to be heterozygous for the (GCC)7 variant. Within in vitro reporter gene assays, the mean luciferase activities of the (GCC)6, (GCC)7, and (GCC)5 constructs were 8.0±0.26, 13.2±0.10 and 12.3±0.12, respectively. The significant increase in activity observed for (GCC)5 and (GCC)7 compared with (GCC)6 (P-value ≤0.001) strongly suggests that alteration in the number of trinucleotide repeats is responsible for the ultra-high TPMT activity observed in these patients.

Effect of atorvastatin on plasma levels of asymmetric dimethylarginine in patients with non-ischaemic heart failure

Elevated plasma levels of asymmetric dimethylarginine (ADMA), an endothelial nitric oxide synthase (eNOS) inhibitor, may contribute to endothelial dysfunction in chronic heart failure (CHF). Since statins upregulate eNOS and ameliorate endothelial dysfunction in non‐ischaemic CHF, we hypothesized that this may be in part through modification of ADMA.

Uncertainty of sweat chloride testing: does the right hand know what the left hand is doing?

Although analytical variation in sweat electrolyte testing can be easily estimated, there is limited data on total variation. This study aims to evaluate the total variation of the sweat test by measuring the difference between sweat electrolyte values in specimens obtained simultaneously from two sites. Chloride is recommended in published guidelines as the only discriminant for the diagnosis of cystic fibrosis, and sodium may be measured as a guide to the adequacy of collection and analysis. Both are reported here. Sweat was collected in patients by the Gibson Cooke method from two sites simultaneously. Coefficient of variation in this laboratory is 4.1 and 5% for chloride and sodium, respectively. 295 patients had sufficient sweat collected from both sites for analysis. The values for chloride and sodium were compared between the two sites. The total coefficient of variation (CVt) calculated for the whole group between the two sites was 20.2% for chloride and 16.9% for sodium, and the standard deviations 4.3 mmol/L and 4.8 mmol/L, respectively. In patients with intermediate chloride concentrations; in different age groups; and when those tests with a difference between sodium and chloride concentration of more than 15 were excluded, minimal differences in these figures were observed. Use of strictly defined cut-off points to discriminate between normal and intermediate electrolyte values, and between intermediate and raised electrolyte values, does not reflect the variation in sweat electrolyte content found within an individual patient. This has important implications for reporting.

Biological variation of thiopurine methyltransferase enzyme activity: when has a significant change taken place?

Background Thiopurine methyltransferase (TPMT) enzyme activity is measured before initiating thiopurine therapy to reduce the risk of severe drug-associated myelotoxicity in patients with low enzyme activity. TPMT activity may vary over time in relation to drug treatment and patient clinical condition. What constitutes a significant change in TPMT activity can be derived from biological variation and analytical imprecision. Methods A large national laboratory database was used to identify patients with three or more TPMT activity measurements. Variance of TPMT activity was analysed by determining the total coefficient of variation (CVTOT) of repeated measurements and by correlation with parameters including gender and follow-up time. Between-run analytical imprecision (CVa) was determined by replicate analysis (n = 314). Results Of 7383 patients with TPMT measurements, 136 were identified as having three or more measurements over time (range 3–14). Median CVTOT for individual patient results was 14.5% (range 2.5–36.7%). Analytical imprecision (CVa) was 10.3%. A reference change value (or critical difference) with 95% probability was calculated as 42%. Therefore, a change in measured TPMT activity above 42% should lead to considering sources of variation other than biological variation and analytical imprecision. Conclusions TPMT enzyme activity needs to change by at least 42% to determine that a true change has taken place beyond biological variation and analytical imprecision. A single measurement of TPMT activity is sufficient for most clinical purposes.

Dual porphyria with mutations in both the UROD and HMBS genes

The porphyrias are a group of inborn or acquired disorders of haem synthesis that can result in neurovisceral or dermatological symptoms. Diagnosis is usually made using a combination of clinical presentation and biochemical parameters. This case report describes a 25-year-old woman clinically presenting with a rash and then found to have elevated porphobilinogen concentrations in her urine. The initial presumptive diagnosis of variegate porphyria was not supported by analysis of her plasma, urine and faeces, which suggested a combination of acute intermittent porphyria and porphyria cutanea tarda. Sequencing of the hydroxymethylbilane synthase and uroporphyrinogen decarboxylase genes confirmed the relatively rare diagnosis of dual porphyria, and revealed a novel uroporphyrinogen decarboxylase mutation.

Preanalytical stringency: what factors may confound interpretation of thiopurine S-methyl transferase enzyme activity?

Background Measurement of red blood cell thiopurine S-methyl transferase (TPMT) enzyme activity before commencing thiopurine therapy is recommended to avoid severe bone marrow suppression in TPMT-deficient patients. Patients samples go through preanalytical transportation and storage steps before measurement. We studied patients TPMT activity sample data to assess the effect of preanalytical variables including transportation time. Methods A total of 8524 TPMT enzyme activity analyses were conducted from 2002 to 2010 in a single laboratory, with samples sent from seven centres throughout New Zealand. TPMT activity was correlated with time of arrival at the reference laboratory, patient gender and age and centre of sample collection. Results The 6348 (74%) selected TPMT measurements that fulfilled selection criteria ranged from 0 to 25.8 IU/mL. Median delay to sample analysis was 42 h. Median TPMT activity was significantly lower for all centres compared with the reference centre (P < 0.001). Delay in sample arrival was significantly and independently correlated with TPMT enzyme activity (ANCOVA; P < 0.001), which showed a 0.011 (95% CI, 0.008–0.014) IU/mL decrease per extra hour of delay. After correcting for these data, one centre still had a significantly lower TPMT enzyme activity compared with the reference centre. Conclusions There was a significant negative correlation between TPMT enzyme activity and delay from sample collection to analysis. Transportation time is therefore an important preanalytical variable influencing TPMT activity. Samples from one centre had a lower TPMT activity after correcting for transportation delay, suggesting that other factors related to sample processing may also be relevant.

Urinary VMA, dopamine and the likelihood of neuroblastoma: a preferred way of reporting laboratory results?

Background: Neuroblastoma patients may be classified as normal or abnormal depending on reference interval and decision points for urine catecholamine metabolites. We therefore evaluated the utility of positive likelihood ratios (LR+) based on data from patients in whom the diagnosis was suspected. Methods: Urine samples from 249 patients (122 male, 127 female) suspected of neuroblastoma were assayed for VMA by spectrophotometry and dopamine by HPLC. Ratios of VMA to creatinine (VMA/Cr) and dopamine to creatinine (DA/Cr) were calculated and age-related median scores derived relative to patients without neuroblastoma. Receiver operator characteristic (ROC) curve analysis was undertaken for the ability of median scores to identify neuroblastoma. Results: Of the 249 patients, there were 20 confirmed cases of neuroblastoma, with ages ranging from 0 (congenital tumour) to 8.4 years. From ROC curves, VMA/Cr was found to have an area under the curve 0.96 (95% confidence interval [CI] 0.92-0.98) compared with 0.72 (95% CI 0.66-0.77) for DA/Cr, P=0.001. At the optimal decision point for VMA/Cr, LR+ was 7.2, identifying cases with a sensitivity of 95% and a specificity of 86%, and comparing favourably with published intervals. Conclusions: VMA/Cr is more accurate than DA/Cr for the diagnosis of neuroblastoma. Reporting LR+ may also be more informative than using reference intervals and decision points.

A Right Royal Porphyria Fallacy

To the Editor: We were intrigued by the cover image for the November 2011 issue of Clinical Chemistry , which showed a cover picture claiming that Mary Queen of Scots had a diagnosis of acute porphyria. The diagnosis of porphyria is usually assigned when a presenting patient is currently symptomatic; the range of indicative symptoms is diverse but relatively well defined. The 3 most common acute porphyrias are hereditary coproporphyria, variegate porphyria, and acute intermittent porphyria, all inherited as autosomal dominant disorders. Many individuals who inherit the enzyme abnormalities remain asymptomatic through out life. Abdominal pain is the most common symptom of an …

Re-emergence of thiamine deficiency disease in the Pacific islands (2014–15): A case-control study

Background From late 2014 multiple atolls in Kiribati reported an unusual and sometimes fatal illness. We conducted an investigation to identify the etiology of the outbreak on the most severely affected atoll, Kuria, and identified thiamine deficiency disease as the cause. Thiamine deficiency disease has not been reported in the Pacific islands for >5 decades. We present the epidemiological, clinical, and laboratory findings of the investigation. Methodology/Principal findings We initially conducted detailed interviews and examinations on previously identified cases to characterize the unknown illness and develop a case definition. Active and passive surveillance was then conducted to identify additional cases. A questionnaire to identify potential risk factors and blood samples to assay biochemical indices were collected from cases and asymptomatic controls. Thiamine hydrochloride treatment was implemented and the response to treatment was systematically monitored using a five-point visual analogue scale and by assessing resolution of previously abnormal neurological examination findings. Risk factors and biochemical results were assessed by univariate and multivariate analyses. 69 cases were identified on Kuria (7% attack rate) including 34 confirmed and 35 unconfirmed. Most were adults (median age 28 years [range 0–62]) and 83% were male. Seven adult males and two infants died (13% case fatality rate). Resolution of objective clinical signs (78%) or symptoms (94%) were identified within one week of starting treatment. Risk factors included having a friend with thiamine deficiency disease and drinking kava; drinking yeast alcohol reduced the risk of disease. Higher chromium (p<0·001) but not thiamine deficiency (p = 0·66) or other biochemical indices were associated with disease by univariate analyses. Chromium (p<0·001) and thiamine deficiency (p = 0·02) were associated with disease by multivariate analysis. Conclusions/Significance An outbreak of thiamine deficiency disease (beriberi) in Kiribati signals the re-emergence of a classic nutritional disease in the Pacific islands after five decades. Although treatment is safe and effective, the underlying reason for the re-emergence remains unknown. Chromium was highly and positively correlated with disease in this study raising questions about the potential role of factors other than thiamine in the biochemistry and pathophysiology of clinical disease.