Retrospective clinical study on the notable efficacy and related factors of infliximab therapy in a rheumatoid arthritis management group in Japan: one-year clinical outcomes (RECONFIRM-2)

Biologics targeting TNF have brought about a paradigm shift in the treatment of rheumatoid arthritis (RA) and infliximab, anti-TNF-α chimeric monoclonal antibody, was marketed in 2003 in Japan. We previously reported on the RECONFIRM study, a retrospective clinical study on the efficacy of infliximab therapy in a RA management group in Japan, where we evaluated the clinical response after 22 weeks of the therapy in 258 patients. The study reported here was aimed at reconfirming the clinical efficacy of the infliximab therapy and demographic factors related to the efficacy over a 54-week study period in 410 RA patients in the same study group. Infliximab was infused according to the domestically approved method, and the clinical response was evaluated following 54 weeks of infliximab therapy using the European League Against Rheumatism (EULAR) response criteria. Disease activity was assessed by DAS28-CRP (Disease Activity Score including a 28-joint count/C-reactive protein). Infliximab was discontinued in 24.4% of the 410 patients at 54 weeks and 9.3% and 8.1% discontinued the therapy due to adverse events and inefficiency, respectively. Average DAS28-CRP decreased from 5.5 at week 0 to 3.1 at week 54 after the therapy. Patients in remission and those showing low-, moderate-, and high-disease activity changed from 0.0, 1.0, 9.0 and 90.0%, respectively, at the start of the study to 27.6, 11.7, 34.4 and 26.3%, respectively, at week 54. Younger age, RF-negativity and low scores of DAS28-CRP showed significant correlations with remission at week 54. EULAR response criteria—good, moderate, and no response to infliximab—were 37.0, 41.7 and 21.2%, respectively. In conclusion, we reconfirmed the clinical efficacy of infliximab and demographic factors related to the efficacy over a 54-week study period in 410 Japanese patients with RA using DAS28-CRP and EULAR response criteria.
Introduction
Rheumatoid arthritis (RA) is a chronic, systemic inflammatory disease that causes significant morbidity and mortality. RA patients should be started with DMARDs as early as possible, and among multiple DMARDs methotrexate (MTX) is considered the anchor drug and should be used first of all. However, even the use of MTX often fails to control disease activity and to prevent structural damage, and so more effective treatment strategies are needed. TNF-α plays a pivotal role in the pathological processes of RA through the accumulation of inflammatory cells and the self-perpetuation of inflammation, leading to cartilage and bone destruction. The combinational use of biologics targeting TNF-α and MTX has revolutionized the treatment of RA, producing significant improvements in clinical, radiographic, and functional outcomes that were not previously observed [1–5].
Infliximab, anti-TNF-α chimeric monoclonal antibody, has been marketed since July 2003 in Japan and currently provides high efficacy and potential adverse events. Although global evidence of the efficacy and safety of infliximab has accumulated, including the ATTRACT study, ASPIRE study and many others [6–10], there is no well-established firm evidence of the efficacy of this agent in Japan. Only the RECONFIRM study, a retrospective clinical study on the notable efficacy and related factors of infliximab therapy in a RA management group in Japan, has reported clinical evidence on its efficacy and safety; this study was performed in several major rheumatology centers in Japan [11]. However, the RECONFIRM study evaluated the clinical response following only 22 weeks of infliximab therapy in 258 patients.
In this RECONFIRM-2 study, we assessed the clinical efficacy and safety of infliximab and MTX over a 54-week study period when used in 410 RA patients in the same group, in order to reconfirm not only its clinical efficacy but also demographic factors related to the efficacy.
Patients and methods
Data and information on RA patients that fulfilled the diagnostic criteria of the American College of Rheumatology (ACR) were collected from three major rheumatology centers in Japan, including the First Department of Internal Medicine, School of Medicine, University of Occupational and Environmental Health Japan, Kitakyushu; the Division of Rheumatology and Clinical Immunology, Department of Internal Medicine, Saitama Medical Center, Saitama Medical University, Saitama; and the Institute of Rheumatology, Tokyo Women’s Medical University. All patients that received infliximab treatment in each institution by December 2005 were registered with this retrospective study. Demographic data, including disease duration and concomitant therapy, were collected from medical charts. The following parameters were evaluated before and at 54 weeks after the initial infliximab infusion: tender joint count (TJC) 28, swollen joint count (SJC) 28, patient’s assessment of pain on a visual analog scale (patient’s pain VAS), patient’s global assessment of disease activity (patient’s global VAS), physician’s global assessment of disease activity (physician’s global VAS), and C-reactive protein (CRP).
Infliximab therapy
Infliximab was infused to patients at zero, two, and six weeks and thereafter every eight weeks at a dose of 3 mg/kg according to the drug labeling and the guidelines of the Infliximab Study Group in the Ministry of Health, Welfare and Labor in Japan [12]. Concomitant use of MTX was instituted in all cases, although the dose of MTX was determined by each attending physician.
Therapeutic response
Disease activity was assessed by Disease Activity Score, including a 28-joint count (DAS28)-CRP that was calculated according to the authorized formula (http://www.das-score.nl/). The value of DAS28-CRP is reported to be less than the original DAS28 using the erythrocyte sedimentation rate (ESR), and we used a threshold of 4.1 instead of the original 5.1 as the cut-off for high activity and 2.7 instead of 3.2 as the cut-off for low activity. Thus, we defined a value of DAS28-CRP >4.1 as high activity, 2.7–4.1 as moderate activity, <2.7 as low activity, with <2.3 being defined as remission [9]. The response to infliximab therapy at 22 weeks was evaluated by the European League Against Arthritis (EULAR) response criteria using 4.1 and 2.7 as the thresholds for the high and low disease activities, respectively [13]. Secondary insufficiency to infliximab was defined as patients with good or moderate EULAR response at week 22, and whose DAS28 increased to >0.6 or >1.2 from week 22 to 54.
Discontinued subjects
The continuation rate of infliximab therapy was calculated by all causes of discontinuation. Cumulative hazards and associated factors were analyzed in relation to three types of discontinuation: adverse events, inefficacy, and remission.
Statistical analysis
Baseline characteristics of patients are summarized in Table 1 using the mean, standard deviation, median, 25%, and 75% values for continuous variables. The continuation rate of infliximab therapy was calculated using the Kaplan–Meier estimator in pooled samples of three institutes. The discontinuation-cause specific hazards were estimated using the Nelson–Aalen estimator. Next, risk factors for the discontinuations were evaluated using the stratified proportional hazards model in order to adjust for differences among institutions. The clinical efficacies of infliximab were evaluated using a multivariate logistic regression to adjust for institutional effects. In this analysis, the last observed DAS28-CRP values were used for discontinued patients. All multivariate analyses were conducted using the variables gender, age, duration of disease, positive/negative rheumatoid factor (RF), concomitant MTX dose, concomitant prednisolone (PSL) dose, and DAS28-CRP at baseline. All reported P-values are two-sided and are not adjusted for multiple testing. The significance level was set at a P value of <0.05.
Table 1Baseline characteristics of patients in three institutions of rheumatology in JapanMeanSDMin25%Median75%MaxFemale (%)87.6––––––Age53.112.71946556280Duration9.48.8036.61354Stage3.01.012344Class2.20.512224RF positive (%)87.6––––––RF (titer)213331139962412980MTX dose7.82.0068820PSL dose3.83.7004522.5CRP3.32.801.182.74.713.7TJC2810.57.30591528SJC2810.66.106101428GH63.121.9049.36680100DAS28-CRP5.51.11.94.85.66.38.0MTX methotrexate, PSL prednisolone, RF rheumatoid factor, CRP C-reactive protein, GH general health, TJC tender joint count, SJC swollen joint count, DAS disease activity score
Results
Baseline demographic and clinical characteristics
The baseline demographic and clinical characteristics of 410 patients receiving infliximab therapy are summarized in Table 1. Patients had active disease at baseline as evidenced by high mean counts of tender and swollen joints, CRP levels, and the DAS28. Age, sex, and disease duration were similar among these three institutes, while the %user and dose of MTX or PSL were divergent and the disease activities as assessed by TJC, SJC, GH, and DAS28 was also different.
Continuation of infliximab therapy
Infliximab was discontinued in 100 cases (24.4%) among 410 patients during a 54-week period and the survival rate for infliximab use was comparable among three institutes by week 54 after the treatment according to Kaplan–Meier analysis (Fig. 1a). Cumulative hazards of the discontinuation during the 54-week infliximab therapy were different among the causes of discontinuation; discontinuation due to adverse events, inefficiency, remission and other causes (such as change of hospitals/clinics and economic reasons), were 0.093, 0.081, 0.007 and 0.063, respectively (Fig. 1b). Although the cause of the discontinuation was similar among three institutes, adverse events were higher in Center 1 than in the other two institutes, and remission and other causes including economic problems of the patient were greater in Center 3 than in the other two (data not shown). In 100 patients who terminated infliximab treatment, stratified Cox regression was performed to analyze factors associated with the discontinuation of the infusion. Male, older age and RF-negativity were significantly associated with the discontinuation of infliximab due to adverse reactions, whereas there was no significant factor responsible for the discontinuation due to maintained remission or a lack of efficacy (Table 2).
Fig. 1Continuation of the infliximab therapy in RA patients for 54 weeks. a Survival rate of RA patients treated with infliximab (n = 410, total and three institutes) during the 54-week therapy. b Cumulative hazards of the discontinuation of infliximab therapy by week 54 of the treatmentTable 2Results from a Cox regression analysis performed to examine the factors related to the discontinuation of infliximab therapyVariableAE (n = 38)Inefficacy (n = 33)Remission (n = 3)Coef.HRCLCUPCoef.HRCLCUPCoef.HRCLCUPAge0.0371.0381.0061.0700.020−0.0140.9870.9571.0170.38−0.0210.9800.8881.0810.68Gender−0.9400.3910.1770.8630.0200.1601.1730.3523.9100.79–––––RA duration0.0181.0180.9831.0540.3200.0051.0050.9641.0460.83−0.1140.8930.6221.2800.54RF (±)−0.9490.3870.1710.8780.0231.5454.6860.63534.5730.13−1.1500.3170.0234.2910.39MTX dose0.0961.1010.9201.3180.290−0.1420.8670.7091.0600.160.3391.4030.7992.4650.24PSL dose−0.0660.9360.8481.0330.1900.0031.0030.9081.1090.95−0.6360.5290.1871.5000.23DAS (0 week)0.2051.2270.8901.6920.2100.1131.1200.8151.5370.49−0.4740.6230.1642.3670.49Coef. coefficient, HR hazard ratio,   CL 95% confidence lower limit of HR, CU 95% confidence upper limit of HR
Efficacy of infliximab therapy
The average DAS28-CRP before starting infliximab was 5.5 ± 1.1, and this decreased to 3.4 ± 1.2, 3.2 ± 1.4, 3.1 ± 1.3 at weeks 2, 22 and 54, respectively, after the infliximab therapy (Fig. 2). Disease characteristics at baseline and after 2, 22 and 54 weeks of the inflilximab therapy were as follows: tender joints count (10.5, 4.2, 3.2 and 2.9), swollen joints count (10.6, 4.2, 2.7 and 2.3), GH (63, 34, 33 and 33 mm), and CRP (3.3, 1.0, 1.5 and 1.1 mg/dl) (Fig. 3). Before starting infliximab, the proportions of patients showing low, moderate, and high disease activity were 1.0, 9.0 and 90.0%, respectively. At week 22, patients in remission (defined as DAS28-CRP <2.3) and those showing low (<2.7), moderate (2.7–4.1), and high disease activity (>4.1) had changed to 27.8, 12.0, 32.4 and 27.8%, respectively, and at week 54 patients in remission and those showing low, moderate, and high disease activity were 27.6, 11.7, 34.4 and 26.3%, respectively (Fig. 4). Thus, approximately 27–28% of the patients satisfied the remission criteria at week 22 and still remained at week 54 after the infliximab therapy. Also, when the responses were evaluated by the EULAR response criteria, the proportions for good, moderate, and no response to infliximab as measured by DAS28-CRP were 37.3, 43.7 and 19.0%, respectively, at week 22 and 37.0, 41.7 and 21.2%, respectively, at week 54 (Fig. 5).
Fig. 2Longitudinal analysis of DAS28 values during the 54-week study of patients using infliximab. Line in the box represents the median and the upper and lower ends of the box show the 25th and 75th percentiles of the populationFig. 3Longitudinal analysis of a SJC28, b TJC28, c CRP, and d GH values during the 54-week study of patients using infliximab. Line in the box represents the median, and the upper and lower ends of the box show the 25th and 75th percentiles of the populationFig. 4Changes in DAS28 values during the 54-week study of patients using infliximab. The ratios of patients who demonstrated high disease activity (defined as DAS28-CRP >4.1), moderate activity (2.7–4.1), low activity (<2.7) and remission (<2.3) at each observation point during the 54-week study are shownFig. 5The response to infliximab therapy during the 54-week study. The ratios of patients whose responses were evaluated by the European League Against Arthritis (EULAR) response criteria are shown
Demographic factors related to the clinical efficacy of infliximab therapy
In order to clarify demographic factors related to the clinical efficacy of infliximab therapy, we performed a multivariate analysis adjust for institutional differences. Younger age, RF-negativity and lower levels of DAS28-CRP were significantly associated with the clinical remission induced by infliximab therapy at week 54, whereas gender, duration of the disease, dose of MTX and dose of PSL did not show a significant association (Table 3). On the other hand, most of the demographic factors, except for older age, did not affect secondary insufficiency from week 22 to 54 after the infliximab therapy according to the logistic regression analysis (Table 4).
Table 3Results from the logistic regression analysis used to examine the factors related to clinical remission at weeks 22 and 54 induced by infliximab therapyVariablesWeek 22 (n = 113)Week 54 (n = 112)Coef.ORSECLCUPCoef.ORSECLCUPIntercept1.5304.6171.0340.60835.0670.1391.7705.8681.0100.81142.4450.080Center 2 vs. Center 10.1741.1900.3590.5882.4050.6290.5641.7580.3500.8863.4880.107Center 3 vs. Center 11.0722.9220.3241.5495.5100.0010.9892.6890.3251.4235.0820.002Age−0.0140.9860.0100.9671.0060.168−0.0240.9760.0100.9570.9950.014Gender0.5631.7550.3920.8133.7870.1520.1541.1660.3700.5652.4090.677RA duration−0.0070.9930.0150.9641.0230.6400.0101.0100.0140.9821.0390.491RF (±)−0.4130.6620.3390.3401.2870.224−0.7420.4760.3290.2500.9080.024MTX dose0.0421.0430.0680.9131.1930.5350.0051.0050.0660.8841.1430.936PSL dose−0.0630.9390.0350.8761.0050.070−0.0580.9440.0340.8821.0090.092DAS28-CRP−0.4640.6290.1160.5010.7890.000−0.2760.7590.1120.6090.9450.014Coef. coefficient HR, OR odds ratio, SE standard error, CL 95% confidence lower limit of OR, CU 95% confidence upper limit of ORTable 4Results from the logistic regression analysis used to examine the factors related to the secondary inefficiency from week 22 to 54 during infliximab therapyVariablesWeek 54–22 >0.6 (n = 85)Week 54–22 >1.2 (n = 47)Coef.ORSECLCUPCoef.ORSECLCUP(Intercept)−1.4610.2321.0790.0281.9230.176−4.3310.0131.4390.0010.2210.003Center 2 vs. Center 1−0.0080.9920.3500.4991.9700.9810.0741.0770.4400.4552.5490.866Center 3 vs. Center 10.0711.0730.3330.5592.0600.8310.0541.0560.4260.4582.4330.899Age0.0191.0190.0110.9981.0420.0790.0321.0320.0141.0041.0620.028Gender−0.2830.7530.3580.3731.5200.4290.2501.2840.5110.4713.5000.625RA duration−0.0290.9720.0170.9411.0040.083−0.0200.9800.0200.9421.0190.313RF (±)−0.2330.7920.3660.3871.6220.5240.1641.1780.5120.4323.2120.749MTX dose−0.0010.9990.0690.8731.1440.9900.0391.0400.0860.8791.2300.649PSL dose−0.0150.9850.0360.9181.0570.6820.0191.0190.0450.9341.1120.676DAS28-CRP (0 week)−0.0330.9670.1180.7671.2190.779−0.0110.9890.1480.7391.3230.941Coef. coefficient HR, OR odds ratio, SE standard error, CL 95% confidence lower limit of OR, CU 95% confidence upper limit of OR
Discussion
The RECONFIRM-2 study was designed to fully evaluate the effect of infliximab used in combination with MTX on the clinical results during a 54-week study period in DMARD-resistant RA patients of a RA management group in Japan. The safety profile of infliximab therapy for a total of 5,000 cases was investigated in Japan using an all-case registered post-marketing surveillance system, and the entire profile of adverse events related to infliximab therapy was clearly identified [14]. The assessment of efficacy, however, was based only on the physician’s general evaluation and not on quantitative measures such as EULAR criteria. In this report, therefore, efficacy data based on DAS28-CRP and EULAR improvement criteria were intensively assessed. The average DAS28-CRP before starting infliximab was 5.5, it decreased to 3.2 at week 22 and it remained steady at 3.1 by week 54 after the infliximab therapy. At week 22 after the therapy, about 28% and 40% of patients satisfied the remission and low disease activity criteria, respectively, and these effects remained (28 and 39%, respectively) by week 54. Also, about 37% of the patients exhibited good response according to EULAR criteria continuously from week 22 to  54. These results reconfirmed that the clinical efficacy of infliximab at week 22 was maintained until week 54 in most of the patients treated with infliximab. Thus, appropriate treatment with infliximab and MTX in this study group could minimize secondary insufficiency of the therapy for at least one year.
On the other hand, the discontinuation of infliximab was observed in approximately 24% of the patients during the 54-week period. This rate of discontinuation of the therapy was comparable to that obtained in other countries: 26.7% in the Anti-Tumor Necrosis Factor Trial in Rheumatoid Arthritis with Concomitant Therapy (ATTRACT) international study carried out in the USA and the EU, 34.6% in a German domestic study and 26% in a French group study during the first one-year period [7, 15, 16]. Cumulative hazards differed among causes of discontinuation; discontinuations due to adverse events, inefficiency, remission and other causes (including changes of hospital/clinic and economic problems) were 0.093, 0.081, 0.007 and 0.063, respectively. Male gender, older age and RF-negativity were significantly correlated with discontinuation of infliximab due to adverse reactions, including infusion reactions (N = 7), toxicoderma (4), bacterial pneumonia (3), pneumocystis jirovecii pneumonia (3), but more than two-thirds of the withdrawals due to adverse events were observed within the first 14 weeks after the therapy.
In this study, to clarify how predisposing factors from the demographic characteristics of RA patients were related to the clinical efficacy of infliximab therapy, a multivariate analysis using a logistic regression was performed. In this study, multiple variables including sex, age, duration of disease, stage, class, positive/negative RF, concomitant MTX dose, concomitant PSL dose, and initial levels of CRP, TJC, SJC and GH were assessed. It is worth noting that, among multiple variables, younger age, RF-negativity and lower levels of DAS28-CRP at the baseline were significantly correlated with the clinical remission induced by infliximab therapy at week 54. These results imply that the timely use of MTX and infliximab can be strongly recommended for younger RA patients that show RF-negativity in order to efficiently achieve clinical remission. On the other hand, male gender, older age and RF-negativity were significantly correlated with discontinuation of infliximab due to adverse reactions, whereas there was no significant factor responsible for discontinuation as a result of inefficacy, and only older age affected secondary insufficiency from week 22 to 54 after the infliximab therapy. Although it is intriguing that male gender predisposes for discontinuation as a result of adverse events, these results provide the first information that can be used to facilitate the more efficacious use of infliximab and MTX in the daily practice of rheumatologists.
Taken together, this REOCNFIRM-2 study reconfirms the clinical efficacy of treatment with infliximab and MTX in Japanese RA patients using the DAS28-CRP and EULAR response criteria. Among 410 patients with active RA, approximately 28% and 39% of the patients satisfied the remission and low disease activity criteria, and good response according to the EULAR criteria was achieved in 37% of the patients treated with infliximab plus MTX during the 54-week study period. The clinical efficacy of infliximab was maintained from week 22 to 54 after the treatment, and secondary insufficiency after week 22 was marginal after the appropriate treatment in this study group. Several demographic factors, including male gender, RF-negativity and lower scores of DAS28-CRP, were significant predisposing factors for remission. The promising effectiveness of infliximab at improving measures of disease activity in RA patients has led to this therapy becoming one of the key advances in the management of RA. Thus, this study is important because it provides obvious and invaluable evidence concerning the efficacy of the combinational use of infliximab and MTX, and can guide the real clinical use of infliximab in the future.

infliximab
rheumatoid arthritis
eular response
retrospective study


From evidence based bioethics to evidence based social policies

In this issue, Norwegian authors demonstrate that causes of early expulsion out the workforce are rooted in childhood. They reconstruct individual biographies in administrative databases linked by an unique national identification number, looking forward 15 years in early adulthood and looking back 20 years till birth with close to negligible loss to follow up. Evidence based bioethics suggest that it is better to live in a country that allows reconstructing biographies in administrative databases then in countries that forbid access by restrictive legislation based on privacy considerations. The benefits of gained knowledge from existing and accessible information are tangible, particularly for the weak and the poor, while the harms of theoretical privacy invasion have not yet materialised. The study shows once again that disadvantage runs in families. Low parental education, parental disability and unstable marital unions predict early disability pensions and premature expulsion out gainful employment. The effect of low parental education is mediated by low education of the index person. However, in a feast of descriptive studies of socio-economic causes of ill health we still face a famine of evaluative intervention studies. An evidence based social policy should be based on effective interventions that are able to break the vicious circles of disability handed down from generation to generation.
Natural selection is for biology what the human life course is for epidemiology: an overarching framework needed to understand the occurrence of disease in the biography of the individual person [1]. The life course of human beings as a history of health and disease starts long before conception, in the genes and life course of their parents. The odds at facing a successful life are entirely different if conception started with the rape of a young teenager by an HIV positive warrior in a horrifying African civil war or as the consequence of the deep desire to raise a child among a healthy and wealthy European loving couple. To expect a life in good health, healthy parents have to provide their offspring with good genes, food, shelter, love, an upbringing and an education.
However, a general problem of human life course epidemiology is that the human life course is a lot longer than the scientific career of epidemiologists. While there is a scope for ambitious programmes of prospective research spanning multiple generations, such programmes will nevertheless run into questions not addressed by the original design. In this issue, the Norwegian study of Gravseth et al. show how existing data from administrative databases can be put to good use to understand disabling processes originating in youth [2].
Evidence based bioethics
As in other Scandinavian countries, several databases (here the medical birth registry, benefit and income registries in the National Insurance Administration, the education register of Statistics Norway, and the Central Population Register) can be linked by a unique national identification numbers of the child and the parents. In many European countries, such linkage is considered a breach of personal autonomy through invasion of privacy. Bioethical theory is very concerned with the potential of abuse of knowledge, but seems to neglect the potential of harm by lack of available but inaccessible knowledge [3]. Gravseth et al. shows how linked administrative databases can yield important insights in the human life course, supporting policy decisions protecting the poor and the weak. Bioethical theory mistrusts human nature, suggesting that the risk of abuse of private information from linked administrative databases is high. This has never been confirmed by empirical evidence. Bioethics should consider the possibility that untested theories supported by unproved assumptions may look good in theory, but in practice waste healthy life. Medicine knows a large array of such theories. Although critical of parenting experts, we too put our first babies to sleep on their front, likely following advice that started by the celebrated parenting expert, Dr Spock. In 1970, a statistically significantly increased risk of cot death for front sleeping compared with back sleeping (pooled odds ratio 2.93; 95% confidence interval 1.15, 7.47) could already be identified from the then available studies [4]. Preferring the cool theory of babies developing their brain and body better by looking up to spot their mother over the neglected harsh evidence has caused the death of 50,000 babies worldwide, this after 1970 only [4]. Where empirical investigation is possible, bioethical theory needs corroboration by observable evidence [5]. If evidence of harms and benefits is available, as with the long-standing practice of data linkage in the Scandinavian countries, observable evidence of benefits should have precedence over theoretical considerations based on unproved assumptions.
The use of administrative databases
The use of administrative databases can be very tantalising: they carry a lot of information, but what one wants is not available or not reliable [6]. However, as in all study designs, its’ use depends on one’s aims. Flagging persons in a population register and being informed by a mortality register of their death will obtain survival figures at low costs. Death is one of those few health states even epidemiologists do not argue about. Follow up is always close to 100%.
An important principle of administrative databases is that they don’t follow patients but money. As soon as money is at stake, administrative registers become highly reliable to count processes amenable to financing, and loss to follow-up is close to negligible. Eligibility for benefits may change over periods, but the fact of being reimbursed is coded extremely well. One of the strong characteristics of administrative databases is their completeness, even over very long periods. In the article of Gravseth et al., early disability pension registered in 1988–2003 is a highly reliable proxy of definitive expulsion from paid work. Looking back to the medical birth registry in 1967–1976 and a limited number of parental characteristics between ages 0 and 18 of the child, the authors have explored the consequences of a limited numbers of determinants in youth for later expulsion out of work.
Shaping the life course
Losing the capability to do paid work between the ages 20 and 35 is a highly relevant health outcome, as defined by Sen’s capability approach [7]. Being unable to work at young adult age is a heavy burden of disability. In terms of attributable risks, education is by far the most important determinant, explaining 57% of the risk of early disability. Here, the administrative data show their inherent weakness: they contain little information of the process leading to low levels of education, be it personal factors related to the health of the child or social factors related to its’ family and living situation. However, the stratified analysis suggests that low education of the child finds it roots in low education of parents. It is worrying that even in a country as Norway, with one of the highest human development indices in the world, disadvantage as expressed by low education of parents and parental disability is still handed down from generation to generation. Last but not least, the relative importance of maternal marital status shows the importance of stable marital unions to raise healthy children. With low European fertility and postponement of first pregnancy after the age of 30, policy makers are tempted to try to push women into earlier childbirth. This policy may be ill advised, if it leads to more children of less well educated younger mothers in less stable unions.
Moving to evidence based policy
Gravseth et al. show, by using linked administrative databases, that causes of early disability in young adulthood are rooted in childhood and in parental disadvantage. A consequence is that interventions targeting the adults at risk will likely add more to the problems than to the solutions. The horns of the dilemma to give or to withhold a disability pension are terribly sharp. An early disability pension is a social death sentence, crippling the individual by removing incentives to seek work and declaring him or her definitively incurable. But those who are effectively crippled won’t be helped by the misery of repeated frustrations in a labour market in which they cannot compete. Studies carefully designed to identify determinants of irreversible disability are needed, inclusive randomised controlled experiments evaluating pension decisions as important health care interventions.
I opened this editorial by making the strong comparison between the little African girl, pregnant after being raped and the highly educated European women in her 30s. However, even in Norway, disadvantage is still a disease that runs in families. We have now a descriptive feast of the lifelong health consequences of social disadvantage and low education. However, the evidence supporting effective interventions is remaining an evaluative famine [8]. Successful interventions need to target the vicious circles of disadvantage that are handed down from generation to generation. We need theories and models that identify potentially effective interventions and study designs able to collect the evidence that supports the effectiveness. The transgenerational aspects of life course epidemiology may be a hard nut to crack in evaluative research. But as good health starts with a good education, good education will always be a worthy proxy.

databases
disability
pensions
epidemiology
norway
risk factors
educational status
socioeconomic factors


The effect of moderate alcohol consumption on adiponectin oligomers and muscle oxidative capacity: a human intervention study

Aims/hypothesis The aim of this study was to investigate whether moderate alcohol consumption increases plasma high molecular weight (HMW) adiponectin and/or muscle oxidative capacity.
Introduction
Moderate alcohol consumption is associated with a decreased risk of type 2 diabetes compared with abstention [1], which could be explained by improved insulin sensitivity [2]. The underlying mechanism for this is not clear, but several pathways may be involved. First, moderate alcohol consumption increases plasma adiponectin concentrations, which could precede changes in insulin sensitivity [2]. Adiponectin improves insulin sensitivity by increasing muscle fat oxidation and/or decreasing intramyocellular triacylglycerols (IMTGs) [3]. Adiponectin is present in plasma as a trimer, hexamer or high molecular weight (HMW) form, the latter possibly being the most relevant in the aetiology of insulin resistance [4]. Second, moderate alcohol consumption acutely affects energy expenditure, diet-induced thermogenesis, lipolysis and lipid oxidation [5]. These changes may result from acetate production from ethanol, which is converted to acetyl-CoA mainly in peripheral tissue such as muscle [5]. Such acute changes could cumulatively affect oxidative capacity and insulin sensitivity. This is the first human study to investigate whether chronic moderate alcohol consumption affects plasma adiponectin oligomers and muscle oxidative capacity.
Materials and methods
In a randomised, controlled, crossover trial, conducted at TNO Quality of Life, 19 healthy, moderate-drinking, lean (BMI 18–25 kg/m2, n = 11) and overweight (BMI ≥27 kg/m2, n = 8) men (aged 18–40 years), consumed 100 ml whisky (32 g alcohol per day; Famous Grouse Scotch Whisky, 40% vol, Perth, Scotland) or mineral water (Spa Reine, Spa, Belgium) daily for 4 weeks. For the last 7 days of each treatment period the diet was fully controlled, and treatments were separated by a 2 day wash-out period. This sample size was sufficient to detect ∼15% difference of β-3-hydroxyacyl-CoA dehydrogenase (β-HAD) and citrate synthase activity in this crossover trial and correlation coefficients of 0.55 or higher with 80% power and accepting an alpha of 0.05. At the end of each treatment period, muscle biopsies from the vastus lateralis muscle were taken to assess β-HAD, cytochrome c oxidase and citrate synthase activity [6], IMTG [7] and succinate dehydrogenase activity [8]. Fasting blood samples were collected and an OGTT was performed to calculate whole-body insulin sensitivity. Subjects gave written informed consent and the University Medical Center Utrecht Medical Ethics Committee approved the protocol. The study was conducted according to the Declaration of Helsinki (2000) and the International Conference on Harmonisation Guidelines for Good Clinical Practice. Data were analysed using the SAS statistical software package (SAS/STAT version 8, SAS Institute, Cary, NC, USA). Treatment effects were assessed by ANOVA, using general linear modelling, with BMI, period, treatment and treatment order included in the model. Two-sided p values below 0.05 were considered statistically significant.
Results
Compliance to treatments was good as assessed by measurements of urinary ethyl glucuronide every 5 days, showing one negative sample during the whisky-drinking and no positive samples during the water-drinking period. Another indication was the 11% increase (p < 0.001) of serum HDL-cholesterol after whisky compared with water consumption. Table 1 shows results on insulin sensitivity, adiponectin and muscle enzyme activities. Insulin sensitivity was not changed after moderate alcohol consumption, but plasma adiponectin concentrations increased from 7.9 ± 0.2 mg/l after water to 9.0 ± 0.2 mg/l (means±SEM) after whisky consumption. Moderate alcohol consumption tended to increase HMW and medium molecular weight (MMW) adiponectin, but not low molecular weight (LMW) adiponectin. Differences in insulin sensitivity and adiponectin and its oligomers tended to be more pronounced among lean than overweight men (Table 2). Muscle oxidative capacity measured by β-HAD, cytochrome c oxidase and citrate synthase activity did not differ between treatments. A borderline significant interaction between treatment and BMI was observed for cytochrome c oxidase (p = 0.072) and citrate synthase (p = 0.102) activity. Moderate alcohol consumption tended to increase cytochrome c oxidase (+23.7%; 95% CI −3.9 to +51.2%, p = 0.08) and citrate synthase (+24.7%; 95% CI −7.7 to +57.2%, p = 0.11) activity among lean, but not overweight men (Table 2). Succinate dehydrogenase activity in mixed muscle fibres decreased (p = 0.03) by 15% after moderate alcohol consumption. IMTGs did not differ, despite a 15–20% difference between both treatments. These results were not different for lean and overweight men (Table 2). After whisky consumption, HMW correlated positively with activities of citrate synthase (r = 0.64, p = 0.004), cytochrome c oxidase (r = 0.59, p = 0.009) and β-HAD (r = 0.46, p = 0.056). Activities of citrate synthase (r = 0.44, p = 0.07), cytochrome c oxidase (r = 0.32, p = 0.20) and β-HAD (r = 0.44, p = 0.07) also tended to correlate with MMW adiponectin, but not LMW adiponectin. A similar pattern was observed for correlations of adiponectin oligomers with HDL-cholesterol (HMW: r = 0.55, p = 0.014; MMW: r = 0.55, p = 0.014; LMW: r = 0.21, p = 0.38).
Table 1Insulin sensitivity index, adiponectin oligomer concentrations, HbA1c and enzyme activities after 4 weeks of consumption of whisky or mineral water in 19 lean or overweight men Mineral waterWhisky% Changep valueInsulin sensitivity indexa10.6 ± 1.39.6 ± 1.3−9.40.61Adiponectin (mg/l)7.9 ± 0.29.0 ± 0.212.50.0008 HMW0.7 ± 0.11.1 ± 0.157.1 0.074 MMW4.0 ± 0.24.5 ± 0.212.50.065 LMW3.2 ± 0.33.5 ± 0.39.40.442HbA1c (%)4.9 ± 0.024.8 ± 0.02−2.00.023Muscle β-HAD (mol/μg protein)4.22 ± 0.484.37 ± 0.493.60.827Muscle citrate synthase (mol/μg protein)0.66 ± 0.050.75 ± 0.0513.60.262Muscle cytochrome c oxidase (mol/μg protein)1.48 ± 0.121.67 ± 0.1212.80.262Intramyocellular triacylglycerols (AU) Type 10.027 ± 0.0040.022 ± 0.004−18.50.339 Type 20.010 ± 0.0020.008 ± 0.002−20.00.489 Mixed0.019 ± 0.0030.016 ± 0.002−15.8 0.429Succinate dehydrogenase activity (AU) Type 10.084 ± 0.0050.075 ± 0.004−10.70.122 Type 20.057 ± 0.0030.052 ± 0.003−8.80.221 Mixed0.073 ± 0.0030.062 ± 0.003−15.10.028Data are presented as means±SEM or %AU Arbitrary unitsaInsulin sensitivity according to Matsuda and DeFronzo [15]Table 2Insulin sensitivity index, adiponectin oligomer concentrations, HbA1c and enzyme activities (means±SEM) after 4 weeks of consumption of whisky or water in lean and overweight subjects Lean groupOverweight groupWaterWhisky% Changep valueWaterWhisky% Changep valueInsulin sensitivity indexa13.3 ± 1.913.0 ± 1.9−2.30.937.8 ± 1.64.6 ± 1.6−41.0 0.21Adiponectin (mg/l)8.1 ± 0.39.4 ± 0.316.10.017.7 ± 0.28.5 ± 0.210.40.08 HMW0.8 ± 1.21.2 ± 0.250.00.120.8 ± 0.10.8 ± 0.100.62 MMW4.2 ± 0.34.8 ± 0.314.30.133.9 ± 0.24.1 ± 0.25.10.52 LMW3.2 ± 0.43.4 ± 0.46.30.693.1 ± 0.43.5 ± 0.412.90.50HbA1c (%)4.9 ± 0.024.8 ± 0.02−2.00.044.9 ± 0.024.9 ± 0.0200.53Muscle β-HAD (mol/μg protein)4.67 ± 0.505.10 ± 0.459.20.553.46 ± 0.903.69 ± 1.016.70.87Muscle citrate synthase (mol/μg protein)0.73 ± 0.080.92 ± 0.0726.00.110.60 ± 0.050.54 ± 0.06−10.00.51Muscle cytochrome c oxidase (mol/μg protein)1.72 ± 0.152.12 ± 0.1423.30.081.33 ± 0.131.13 ± 0.15−15.00.36Intramyocellular triacylglycerols (AU) Type 10.022 ± 0.0060.016 ± 0.005−27.30.500.032 ± 0.0060.028 ± 0.006−12.50.61 Type 20.006 ± 0.0020.008 ± 0.00233.30.410.013 ± 0.0030.007 ± 0.003−46.20.14 Mixed0.014 ± 0.0040.013 ± 0.003−7.10.860.024 ± 0.0040.019 ± 0.004−20.80.40Succinate dehydrogenase activity (AU) Type 10.086 ± 0.0050.080 ± 0.005−7.00.400.084 ± 0.0090.069 ± 0.008−17.90.29 Type 20.060 ± 0.0050.055 ± 0.005−8.30.500.054 ± 0.0050.048 ± 0.004−11.10.34 Mixed0.076 ± 0.0050.066 ± 0.005−13.20.160.069 ± 0.0050.057 ± 0.004−17.40.15Data are presented as means±SEM or %AU Arbitrary unitsaInsulin sensitivity according to Matsuda and DeFronzo [15]
Discussion
This study showed that moderate alcohol consumption increases adiponectin concentrations, consistent with previous reports [2]. We have now observed that the alcohol-induced increase of adiponectin may be oligomer specific. Moderate alcohol consumption increased particularly HMW adiponectin, MMW adiponectin to a lesser extent, but not LMW adiponectin. These results are in line with those of Bobbert et al. [9] showing a similar pattern of changes in adiponectin oligomers after moderate weight reduction. Studies with thiazolidinedione or rigorous weight reduction show similar, but more pronounced results [10]. HDL-cholesterol concentrations correlated particularly with HMW adiponectin, as previously shown [9]. We have now also observed that HMW and MMW adiponectin are correlated with markers of muscle oxidative capacity, in line with reports of increased fat oxidation after adiponectin infusion [3] and a recent study showing that adiponectin increases muscle oxidative capacity [11]. Our results tend to confirm these findings and show that these relationships may be specific to HMW adiponectin. Altogether, this could provide an underlying mechanism for the proposed importance of HMW adiponectin in the aetiology of insulin resistance [4].
Despite this, the alcohol-induced increase of adiponectin and specifically HMW adiponectin did not affect muscle oxidative capacity, IMTG content and insulin sensitivity. As some subtle differences were present, we cannot completely rule out the hypothesis that changes in IMTG content and/or oxidative capacity could occur. Although it did not reach significance, we observed a 15–20% lower IMTG content after whisky than water consumption, which is of similar magnitude to that observed for a weight loss and physical activity intervention [12]. Furthermore, moderate alcohol consumption tended to increase muscle citrate synthase activity among lean men, but succinate dehydrogenase activity declined after moderate alcohol consumption in mixed muscle fibres. All citric acid cycle and respiratory chain enzymes are thought to change in parallel to perturbation [13]. Our findings with citrate synthase and succinate dehydrogenase, however, are contradictory to this notion. This could simply be due to the measurement of ex vivo oxidative capacity or to chance. Alternatively, moderate alcohol consumption could differentially affect various enzymes in oxidative pathways such as the citric acid or glyoxylate cycle as suggested by Kokavec and Crowe [14]. Because acetyl-CoA from ethanol oxidation is generated independently from activation of pyruvate dehydrogenase complex, it may not affect the citric acid cycle. Alcohol may instead affect the glyoxylate cycle that bypasses part of the citric acid cycle, including succinate dehydrogenase [13, 14].
Strengths of this study are its randomised, controlled crossover design. We assessed compliance to study treatments several times throughout the study and observed no significant deviations. It is therefore unlikely that our results are confounded by diet or lifestyle. Our study was, however, limited by a slightly small sample size for certain contrasts such as analyses in subgroups of lean and overweight men or of IMTG content. Therefore these results may be somewhat preliminary and need to be confirmed with larger sample sizes. Second, because insulin sensitivity was not the primary endpoint of this study, an OGTT was used to assess insulin sensitivity. However, we used an insulin sensitivity index that correlates well with the gold standard, the hyperinsulinaemic–euglycaemic clamp technique [15]. In addition, our results on insulin sensitivity do not differ from our previous studies using the clamp technique [2]. We therefore believe that using this insulin sensitivity index has not influenced our results to a large extent.
In conclusion, this study shows that 4 weeks of daily moderate alcohol consumption increases adiponectin concentrations and particularly HMW adiponectin concentrations, but does not affect insulin sensitivity and oxidative capacity. Concentrations of HMW and MMW adiponectin were positively associated with muscle oxidative capacity.

moderate alcohol consumption
adiponectin
muscle oxidative capacity
insulin sensitivity
intramyocellular triacylglycerols
high molecular weight adiponectin


Discovery and characterization of a putrescine oxidase from Rhodococcus erythropolis NCIMB 11540

A gene encoding a putrescine oxidase (PuORh, EC 1.4.3.10) was identified from the genome of Rhodococcus erythropolis NCIMB 11540. The gene was cloned in the pBAD vector and overexpressed at high levels in Escherichia coli. The purified enzyme was shown to be a soluble dimeric flavoprotein consisting of subunits of 50 kDa and contains non-covalently bound flavin adenine dinucleotide as a cofactor. From all substrates, the highest catalytic efficiency was found with putrescine (KM = 8.2 μM, kcat = 26 s−1). PuORh accepts longer polyamines, while short diamines and monoamines strongly inhibit activity. PuORh is a reasonably thermostable enzyme with t1/2 at 50°C of 2 h. Based on the crystal structure of human monoamine oxidase B, we constructed a model structure of PuORh, which hinted to a crucial role of Glu324 for substrate binding. Mutation of this residue resulted in a drastic drop (five orders of magnitude) in catalytic efficiency. Interestingly, the mutant enzyme showed activity with monoamines, which are not accepted by wt-PuORh.
Introduction
Flavoprotein oxidases catalyze the oxidation of a wide range of compounds, while at the same time they reduce oxygen to hydrogen peroxide. They are valuable biocatalysts for the oxidative activation of biomolecules, as they usually selectively oxidize their substrate at a specific position, leaving other positions unaffected. Due to their ability to use molecular oxygen as electron acceptor, no expensive coenzymes like NAD(P)H are needed. This makes oxidases inexpensive and rather straightforward in usage compared to other redox enzymes.
By far, the best studied oxidase is glucose oxidase from Aspergillus niger. This enzyme has been applied for decades, mostly for diagnostic applications (Wilson and Turner 1992). Another example is pyranose oxidase (glucose-2-oxidase) from Peniophora gigantea, which oxidizes specifically the C2 position of glucose and has been applied for the chemoenzymatic synthesis of rare sugars and sugar-based synthons (Giffhorn et al. 2000). Human monoamine oxidases (MAO-A and MAO-B) are important for the oxidation of neurotransmitters (Abell and Kwan 2001). An engineered mutant of the homologous A. niger enzyme (MAO-N) has been applied in an elegant biocatalytic deracemization process to obtain enantiomerically pure chiral amines (Carr et al. 2005; Dunsmore et al. 2006).
So far, the most described oxidases are from eukaryotic origin, and heterologous expression of the recombinant protein in Escherichia coli for large scale enzyme production can be problematic. In a search for novel bacterial oxidases, we looked in sequenced genomes and identified genes encoding novel oxidases. By this, we discovered oxidases primarily acting on alditols (Heuts et al. 2007) and phenolic compounds (Jin et al. 2007). These oxidases, both from actinomycetes, could be highly overexpressed in E. coli. Another way of searching for novel bacterial oxidases is to screen unsequenced bacterial genome libraries directly for genes encoding enzyme activity. As actinomycetes appear to be rich in oxidases, we have chosen to explore these organisms for relevant oxidases. Using a plate-based screening protocol for oxidase activity (Alexeeva et al. 2002), we screened a genomic DNA library of the actinomycete Rhodococcus erythropolis NCIMB 11540 to find novel oxidases acting on polyols and/or amines.
In this paper, we describe the discovery and characterization of a novel flavin-containing oxidase primarily acting on putrescine. This putrescine oxidase (PuORh) shares 67% sequence identity with the enzyme from Micrococcus rubens (PuOMr; Ishizuka et al. 1993). We show that PuORh is highly overexpressed in E. coli and can be easily purified. Using a structural model, we were able to identify a glutamate residue (Glu324) that is crucial for substrate binding, and we could alter the substrate specificity by protein engineering.
Materials and methods
Chemicals
Restriction enzymes were from Roche and New England Biolabs. One-shot electrocompetent E. coli TOP10 cells and the TOPO TA Cloning Kit were purchased from Invitrogen. Plasmid isolation was performed using the Qiagen DNA purification kit. Oligonucleotides were purchased from Sigma. Nitrocellulose filters (Protran BA85 132 mm, 0.45 μm pore size) were from Schleicher and Schuell BioScience, Dassel (Germany). Deprenyl and rasagiline were a kind gift from Prof. A. Mattevi (University of Pavia, Italy). All other chemicals were of analytical grade. The genomic DNA library from R. erythropolis NCIMB 11540 was provided by DSM (Geleen, The Netherlands). Constructs were sequenced at GATC Biotech (Kostanz, Germany).
Plate-based screening method for oxidase activity
A gene library of R. erythropolis NCIMB 11540 in pZErO-2 was screened for oxidases using the plate-based oxidase activity screening method adapted from the group of Turner (Alexeeva et al. 2002). This gene library was constructed by partial digestion of genomic DNA from R. erythropolis NCIMB 11540 by Sau3A1. Fragments of 4–10 kb were isolated, ligated in pZErO-2 (cut with BamHI), and followed by transformation of E. coli DH10B. Colonies were collected from plate, stored as glycerol stock, and plasmids were isolated. The total amount of plasmids contained a mean insert size of 6.0 kb and 1% of self-ligated vector molecules.
Electrocompetent E. coli TOP10 cells were transformed with the gene library, and the transformed cells were diluted in Luria–Bertani (LB) medium to obtain single colonies on plate. The diluted cell suspensions were plated on nitrocellulose filters. The filters were placed on top of LB agar containing 0.05 mg/ml kanamycin and incubated for 48 h at 30°C. Subsequently, the nitrocellulose filters were transferred to empty petri dishes and stored at −20°C to partially lyse the cells. Each filter was submersed with 50 ml 50 mM sodium phosphate buffer pH 7.5 containing 1% (w/v) agarose, 2 U/ml Horseradish peroxidase (HRP) and a mixture of 1 mM 4-chloro-1-naphthol, 100 μM cholesterol, 10 mM xylitol, 10 mM sarcosine, 10 mM l-alanine, 10 mM d-glucose, 10 mM d-galactose, 10 mM lactose, and 10 mM d-glucosamine. Plates were incubated at room temperature and regularly checked by visual inspection for color formation in and around colonies. Positives were picked and cultivated overnight in 5 ml liquid LB medium and subjected to another screening cycle to assure that color formation was caused by single clones.
Sequence analysis
To identify unique positive clones, plasmids were isolated and subjected to restriction analysis by EcoRI. Inserts were sequenced and open reading frames (ORFs) were identified using the ORF Finder tool of the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). A BLAST search (blastp) was performed with the protein sequence of PuORh using the BLAST function of the NCBI website (http://www.ncbi.nlm.nih.gov/BLAST).
Cloning and expression of the gene encoding PuORh
To overexpress the novel putrescine oxidase, the corresponding gene (puoRh) was amplified from the fragment of genomic DNA of R. erythropolis using the following primers: puoRh_fw: 5′-GCTCCATATGCCTACTCTCCAGAGAGATG (NdeI site shown in italics) and puoRh_rv: 5′-GCTCAAGCTTTCAGGCCTTGCTGCGGGCG (HindIII site shown in italics). The amplified gene was purified from gel and ligated between the NdeI and HindIII restriction sites of the pBAD/Myc-His vector (Invitrogen). The plasmid carrying the puoRh gene (pBADpuoRh) was transformed to CaCl2-competent E. coli TOP10 cells and spread on LB agar plates containing 50 μg/ml of ampicillin. E. coli TOP10 cells containing pBADpuoRh were tested for overexpression of the protein at 17, 30, and 37°C and at arabinose concentrations of 0, 0.00002, 0.0002, 0.002, 0.02, and 0.2% (w/v). Cell extracts and cell-free extracts of E. coli TOP10 containing pBADpuoRh were analyzed on a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel to find the best conditions for overexpression of soluble PuORh.
Enzyme purification
To obtain purified protein, E. coli TOP10 containing pBADpuoRh was cultivated for 24 h at 30°C in 1 l of terrific broth medium containing 50 μg/ml ampicillin and 0.02% (w/v) arabinose. Cells were harvested by centrifugation for 15 min at 6,000 rpm at 4°C. The supernatant was discarded, and the pellet was suspended in 40 ml of 50 mM Tris–HCl pH 7.5 and sonicated for 10 min to break the cells. The cell extract was centrifuged for 30 min at 15,000 rpm at 4°C to remove the broken cells and obtain cell-free extract. From this cell-free extract PuORh was purified using a Q-Sepharose anion exchange column. Unbound protein fractions were washed from the column with 50 mM Tris–HCl buffer pH 7.5. PuORh was eluted from the column with a 50 mM Tris–HCl buffer pH 7.5 by increasing the KCl concentration. Eluted fractions containing PuORh were combined, concentrated using an Amicon filter, and desalted using a HiPrep 26/10 Desalting Column (Amersham Biosciences).
Enzyme activity assay and determination of steady-state kinetic parameters
PuORh activity was measured at 25°C using a peroxidase-coupled assay containing 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) as the chromogenic substrate (Childs and Bardsley 1975). The H2O2 formed by PuORh can be coupled to the Horseradish-peroxidase-mediated oxidation of ABTS resulting in the formation of a green product that can be measured at 420 nm (ɛ420 = 35.7 mM−1 cm−1). The standard assay mixture with ABTS (HRP-ABTS assay) contained 50 μM putrescine, 50 mM Tris–HCl buffer, pH 8.0, 100 μM ABTS, 5 U of Horseradish peroxidase, 10 μl of enzyme dilution (appropriately diluted), and a fixed amount of substrate. The reaction was started with adding the enzyme solution. Alternatively, 0.1 mM 4-aminoantipyrine (AAP) and 1.0 mM 3,5-dichloro-2-hydroxybenzenesulfonic acid (DCHBS) were used as chromogenic substrates for HRP (Federico et al. 1997; HRP-AAP/DCHBS assay). The oxidation of these substrates results in the formation of a purple product, which can be measured at 515 nm (ɛ515 = 26 mM−1 cm−1). For the determination of kinetic parameters, PuORh activity was measured at different substrate concentrations at 25°C using the HRP-ABTS enzyme assay. The kinetic parameters of the mutant enzymes (PuORh Glu324Ala and PuORh Glu324Leu) for putrescine were determined by measuring directly the H2O2 production at 240 nm (ɛ420 = 43.6 M−1 cm−1). Data were fitted with Origin 7.0 using the Michaelis–Menten equation for enzyme kinetics.
Influence of temperature and pH on enzyme activity and stability
To determine the optimal temperature for PuORh activity, the oxidase activity was measured at temperatures between 20 and 60°C. Before adding the enzyme, the assay mixture was equilibrated for 20 min to assure the right temperature. The influence of temperature on the enzyme stability was determined by incubating 1-ml portions of the enzyme at 4, 30, 37, and 50°C. Twenty-microliter samples were taken, placed on ice after which activity was measured. In both experiments, the activity was determined with the HRP-AAP/DCHBS activity assay using 50 μM putrescine and 0.01 μM PuORh.
The pH optimum for putrescine oxidase was determined by measuring the activity at different pH values at 25°C. The following buffers were used: 50 mM Pipes buffer (pH 6.4–7.2), 50 mM Tris–HCl (pH 7.4–8.8), and 50 mM Ches buffer (pH 8.7–9.6). The enzyme activity was measured using the HRP-AAP/DCHBS assay.
Inhibition experiments
Rasagiline, deprenyl, and cyclopropylamine were tested to probe whether they were able to inhibit PuORh by the formation of a covalent adduct with the flavin adenine dinucleotide (FAD) cofactor, as is the case for monoamine oxidase B (Binda et al. 2005). For 900 μl of a 20 μM PuORh solution, an absorbance scan was recorded from 650–300 nm. After the addition of 100 μl of 1 mM inhibitor (final inhibitor concentration, 100 μM), absorbance spectra were recorded regularly in time to see if addition of the inhibitor would cause an alteration of the typical FAD spectrum of PuORh. For cyclopropylamine, also a final inhibitor concentration of 1 mM was tested as described above.
Butylamine, aminoethanol, ethylenediamine, 1,3-diaminopropane, and (2-aminoethyl)-trimethylammonium were tested as competitive inhibitors of PuORh. For this, the Michaelis constant (KM) of PuORh for putrescine was determined for three different concentrations for each of these amino compounds with the HRP-ABTS assay. Inhibition constants (KI) for each compound were calculated with the following formula:
Where [I] represents the inhibitor concentration, KM,inhibitor the apparent Michaelis constant in presence of the inhibitor and the KI the inhibition constant.
Analytical methods
All absorbance spectra were recorded in 50 mM Tris–HCl pH 8.0 at 25°C on a Perkin Elmer Lambda Bio40 spectrophotometer. From a cuvet containing 5 μM PuORh all oxygen was removed by flushing with argon and an absorbance spectrum was recorded from 650 to 300 nm. After adding 50 μM putrescine, another spectrum was recorded for the reduced enzyme. Reoxidation was monitored by collecting spectra in time after the cuvet was exposed to air. A spectrum of the unfolded enzyme was recorded by adding 0.1% SDS and heating for 5 min at 80°C.
Modeling and mutant construction
Based on the structure of MAO-B in complex with rasagiline (PDB/1S2Q; Binda et al. 2004b), a model of PuORh was made using the CHPmodels 2.0 Server. Mutants were constructed by Quick Change PCR. PuORh Glu324Ala was made by a substitution of the codon GAG for GCG using the following primers: puoRhGlu324Ala_fw, 5′-CGAGGTAGTGCAGGCGGTGTACGACAACACC, and puoRhGlu324Ala_rv, 5′-GGTGTT GTCGTACACCGCCTGCACTACCTCG (mutated codon underlined). For construction of PuORh Glu324, the codon GAG was replaced by CTG using the following primers: puoRhGlu324Leu_fw, 5′-CGAGGTAGTGCAGCTGGTGTACGACAACACC, and puoRhGlu324Leu_rv, 5′- GGTGTTGTCGTACACCAGCTGCACTACCTCG (mutated codon underlined). Both mutants were expressed in E. coli TOP 10 and cultivated and purified in the same way as wild-type PuORh.
Nucleotide sequence accession number
The nucleotide sequence of the PuORh encoding gene (puoRh) has been submitted to GenBank under accession number EU240877.
Results
Identification of a novel putrescine oxidase
To identify novel oxidases acting on alcohols and/or amines, a genomic library from the actinomycete R. erythropolis NCIMB 11540 was screened using a plate-based oxidase assay. Positive clones, expressing oxidase activity, could be identified by their purple color formation. From 55,000 transformants, 5 were observed to produce a purple color around the colony. Retransformation to E. coli TOP10 cells and repeated screening on plate resulted in three positive clones, which showed oxidase activity. Restriction analysis with EcoRI and PvuI showed a different insert for each clone indicating that these were independent clones. Sequencing of one of these clones revealed an inserted fragment of genomic DNA of 5,311 bp. On this fragment, an ORF was identified which shares 67% sequence identity (at the amino acid level) with putrescine oxidase (EC 1.4.3.10) from M. rubens, a FAD-containing amine oxidase that catalyzes the oxidative deamination of putrescine (Ishizuka et al. 1993). When we tested the cell extract of the clone containing the above-mentioned insert of 5,311 bp, we found that it was indeed active with putrescine. The other two positive clones probably contained the same gene, as for both clones by using the primers puoRh_fw and puoRh_rv a PCR product was seen on gel.
Gene cloning, overexpression, and protein purification
For overexpression, the PuORh encoding gene was cloned into the pBAD/Myc-His A vector behind the araBAD promotor (without His-tag), and the plasmid carrying the puo gene was transformed to E. coli TOP10. Several temperatures (17, 30, and 37°C) and inducing conditions (0–0.2% L-(+)-arabinose) were tested to find the optimal conditions for overexpression. The gene was well expressed at arabinose concentrations of 0.02 and 0.2% as judged by a dominant protein band of ±54 kDa on SDS-PAGE. At 17 and 30°C, the protein was mainly present in the soluble fractions while at 37°C the unsolube fractions contained most of the overexpressed protein. The overexpressed protein did not show any fluorescence under UV-light upon SDS-PAGE, which is an indication that this protein does not contain a covalently histidyl-bound flavin cofactor (Fraaije et al. 1997).
PuORh was purified from a 1-l culture containing 0.02% arabinose that was grown for 24 h at 30°C. Due to the high level of overexpression (200 mg), PuORh could be easily purified in one-step by anion exchange chromotography. SDS-PAGE analysis of the purified protein revealed that, besides the major protein band present at around ∼54 kDa, there is a minor band corresponding to a protein mass which is approximately 3 kDa larger (Fig. 1, lane A). It was concluded that this must be the product of the same gene with a His-tag extension, as this minor protein band could be removed by an additional purification step using a Ni-agarose column. (Fig. 1, lane B). Although in the plasmid pBADpuoRh the stop codon (TGA) was properly introduced, it is known that in some cases, this stop codon can be translated (MacBeath and Kast 1998). With the constructed plasmid, this would indeed result in a 3 kDa larger protein.
Fig. 1SDS-PAGE gel of purified recombinant PuORh. Lane A Purified PuORh. Lane B Purified PuORh after removal of His-tagged PuORh. Lane C Low molecular weight marker
Spectral and molecular properties of PuORh
PuORh shows a typical flavoprotein spectrum with absorbance maxima at 377 and 459 nm (Fig. 2). In the presence of 5% trichloroacetic acid, PuORh precipitated and was visible as a white pellet after centrifugation. The supernatant contained a yellow color, and its absorbance spectrum with maxima at 375 and 450 nm was typical for that of free FAD. This shows that the FAD cofactor in PuORh, like in PuOMr, is not covalently bound. At room temperature, the addition of 0.1% SDS did not unfold PuORh, indicating this flavoprotein is rather robust. When 50 μM putrescine was mixed with PuORh under anaerobic conditions, the FAD cofactor was completely reduced (Fig. 2, dotted line). Heating the same sample for 5 min at 80°C completely unfolded the enzyme as can be seen from the resulting free FAD UV/VIS spectrum (Fig. 2, dashed line). The reduced enzyme could be rapidly and fully reoxidized by addition of oxygen, indicating that the enzyme is a true oxidase. From the difference in absorbance between protein-bound FAD and free FAD, the molar extinction coefficient for PuORh (ɛ459) was calculated, 11.0 mM−1 cm−1. Based on the protein concentration as determined by Wadell’s method (Wolf 1983) and the FAD concentration (A459), a protein/FAD ratio of 0.5 was calculated. This is consistent with what has been described earlier for PuOMr (Desa 1972). Incubation of PuORh with additional FAD did not yield an increase of FAD incorporation or oxidase activity. Gel filtration experiments revealed that PuORh is mainly present as a dimer of ±100 kDa. This corresponds well with the theoretical monomeric mass of 49,375 Da.
Fig. 2Spectral properties of PuORh. Absorbance spectra of oxidized native (solid line) and unfolded (dashed line) PuORh are shown. After addition of 50 μM putrescine under anaerobic conditions, the native enzyme is fully reduced (dotted line)
Temperature and pH dependence of activity and stability
To investigate the effect of temperature on enzyme activity and stability, both were determined at different temperatures. The optimal temperature for enzyme activity was found to be 30°C. Temperature stability experiments revealed that PuORh is a reasonable thermostable enzyme. At 50°C, half of the activity was lost after 2 h, while at 37°C, such a degree of inactivation was reached only after 1 day. PuORh has a rather sharp pH optimum for activity with putrescine with a peak around pH 8. Below pH 6.4, no significant activity can be detected.
Substrate specificity
Several alcohols, amino alcohols, and amines were tested as substrate, and if they turned out to be converted by PuORh, the steady-state kinetic parameters were determined at pH 8.0. Enzyme activity was found with several aliphatic diamines, amino alcohols, and polyamines. From Table 1, it is clear that putrescine is by far the best substrate. With aromatic amines and amino alcohols, no activity was observed. An increase in carbon chain length of the diamine from 4 to 6 C-atoms resulted in a more than 1,000-fold drop in catalytic efficiency (kcat/KM). For diamines containing 3 or 2 C-atoms and for n-butylamine, no activity could be detected. These amines were found to strongly inhibit the enzyme (Table 2). Apparently, they bind efficiently but are not converted. Clearly, a minimum of two amino groups is required to be a substrate for PuORh, and these amino groups should be preferably 4 C-atoms apart. Polyamines like spermine and spermidine were also converted by PuORh but are poor substrates. Compared to putrescine, 4-amino-1-butanol is also a poor substrate, while 1,4-butanediol is not accepted at all. This again indicates that two amino groups are essential for efficient catalysis, and it confirms that PuORh is a true amine oxidase. Inhibition studies showed that aminoethanol is a very effective competitive inhibitor of PuORh (KI = 1.8 μM). Apparently, this amino alcohol binds very strongly in the active site, while it cannot be oxidized. When we tested 2-hydroxyputrescine, which contains an aminoethanol moiety, we found that this compound is readily oxidized. l-Ornithine was found to be a very poor substrate. Apparently, the presence of a negatively charged and relative bulky carboxylic acid group at the C1 position prevents efficient amine oxidation. Taken together, it can be concluded that PuORh has a narrow substrate specificity and is very selective for putrescine.
Table 1Steady-state kinetic parameters of PuORh at pH 8.0.ND Not determinedaValues of putrescine oxidase from Micrococcus rubens (Okada et al. 1980)Table 2Inhibition constants of competitive inhibitors of PuORhaValues of putrescine oxidase from Micrococcus rubens (Swain and Desa 1976)
Model structure of PuORh reveals a key role for Glu324 in substrate binding
PuORh shares 32% sequence identity with human monoamine oxidase B (MAO-B), which is a 59 kDa FAD-containing protein involved in the oxidation of neurotransmitters and other arylalkylamines like benzylamine and phenylethylamine (Shih et al. 1999). Based on the structure of MAO-B (Binda et al. 2004b), it was possible to construct a model for PuORh. The C-terminal membrane anchor of MAO-B is absent in the resulting model structure of PuORh. This could explain why PuORh is well expressed in the cytosol as a soluble protein in contrast to MAO-B, which is membrane associated (Binda et al. 2004a). In the model, the active site of the PuORh exhibits a similar architecture as that of MAO-B (Fig. 3). The catalytically important “aromatic cage,” present in flavin-containing amine oxidases (Li et al. 2006), is formed by His432 and Tyr395. The other two key amino acid residues, Lys296 and Trp385, involved in non-covalent FAD binding (Binda et al. 2002b) can also be found in the model of PuORh. However, there are also some striking differences. In MAO-B, FAD is covalently attached to Cys397 at the C8α position of the flavin. PuORh has an alanine residue at this position (Ala394), which precludes covalent binding of FAD. Furthermore, the active site of PuORh seems to be narrower than that of MAO-B due to the presence of Trp60 and Met173 in PuORh (Tyr60 and Cys172 in MAO-B). The model of PuORh also shows a glutamic acid residue (Glu324) pointing towards the N5 of the FAD, while MAO-B contains a tyrosine at this position (Tyr326). The presence of such a negatively charged amino acid in the active site has already been predicted for PuOMr several decades ago by Swain and Desa. They suggested the presence of a negatively charged carboxyl group in the active site, as PuOMr was found to be irreversibly inactivated by carbodiimides (Swain and Desa 1976). The presence and position of Glu324 can well explain the difference in substrate specificity between MAO-B and PuORh. PuORh prefers short aliphatic diamines, while MAO-B is active with a range of aromatic monoamines. Probably, Glu324 is involved in binding the protonated amino group of the diamine substrate, while the other amino group is positioned in the “aromatic cage” near the N5 of the flavin. Such a mode of binding for putrescine is consistent with our PuORh model. To confirm this hypothesis, we mutated Glu324 into an alanine (PuORh Glu324Ala) and a leucine residue (PuORh Glu324Leu) and found that the catalytic efficiency for putrescine drops, respectively, 100,000 and 200,000-fold (Table 3).
Fig. 3Active site residues in the structure of MAO-B and in the model of PuORh. In MAO-B, the inhibitor rasagiline is covalently bound to the flavin. In the model of PuORh, the substrate putrescine is modeled in the active siteTable 3Steady-state kinetic parameters for putrescine of wild-type PuORh and of the mutants Glu324Ala and Glu324Leu at pH 9.0. Km (μM)kcat (s−1)kcat/Km (s−1 mM−1)PuORh WT3.5 ± 0.620.7 ± 1.15900PuORh Glu324Ala110,000 ± 20,0006.1 ± 0.40.05PuORh Glu324Leu110,000 ± 20,0003.2 ± 0.20.03
The distance between Glu324 and the N5 of the isoalloxazine ring of the flavin cofactor (9 Å) may explain the specificity for the specific chain length of the substrate. To be a substrate, a minimal carbon chain length of 4 C-atoms is required. Shorter diamines can bind to Glu324 but cannot reach the flavin and therefore are strong competitive inhibitors of PuORh (Table 2). In case of longer diamines or polyamines, the position of the amino group that is oxidized is less favorable compared to the case of putrescine resulting in a decrease in catalytic efficiency. Aliphatic monoamines like n-butylamine will bind to Glu324 via the amine moiety but cannot be oxidized and therefore are also inhibitors of PuORh. Initial substrate screening using 96-wells plates for the mutants PuORh Glu324Ala and PuORh Glu324Leu revealed that both showed some activity with aromatic amines like phenyl-1-butylamine and with aliphatic monoamines (e.g., kcat/KM for n-butylamine = 0.001 s−1 mM−1 at pH 9.0 for PuORh Glu324Leu). Unfortunately, accurate determination of the kinetic parameters of the mutant enzymes is not straightforward due to their low substrate affinity, which requires the use of high concentrations of amines, causing interference with the peroxidase-based assay.
Discussion
In an attempt to discover novel bacterial oxidases with biocatalytic potential, a putrescine oxidase from R. erythropolis NCIMB 11540 was identified. This putrescine oxidase was discovered by using a plate-based screening method for oxidase activity, which has been used before to screen for improved mutants during directed evolution experiments (Alexeeva et al. 2002). Our study shows that this method is also valuable for the discovery of novel bacterial oxidases of unsequenced genomes. Although the physiological substrate of the discovered putrescine oxidase, 1,4-diaminobutane, was not present during screening, clones expressing this oxidase could still be detected based on their oxidase activity. As polyamines like putrescine, spermine, and spermidine are present in most living cells, including E. coli (Tabor and Tabor 1984), this resulted in the formation of hydrogen peroxide and detection of positive clones.
The physiological role of putrescine oxidase in R. erythropolis is probably related to polyamine degradation (Large 1992), which is supported by the presence of a neighboring gene encoding a putative aldehyde dehydrogenase on the sequenced DNA fragment (data not shown). PuORh displays 67% sequence identity with PuOMr, which is the only bacterial putrescine oxidase that has been characterized so far. A BLAST search with the amino acid sequence of PuORh resulted in a high number of homologous (putative) flavin-containing amine oxidases, which are widely distributed among most kingdoms of life. The seven closest homologs (sequence identity >65%) can all be found in actinomycetes. These sequences all contain the active site glutamate (Glu324), involved in substrate binding, the alanine residue (Ala394), which excludes covalent FAD binding, and the residues, which limit the size of the active site cavity (Trp60 and Met173). Most likely, they represent a clade of orthologous putrescine oxidases, which are not active with monoamines, within the family of (putative) flavin-containing amine oxidases (Supplementary information, ESM 1).
PuORh shares some properties with PuOMr. Both enzymes are soluble dimeric proteins of approximately 100 kDa. Interestingly, both contain only 1 mol of non-covalently bound FAD per mole of dimer, which is rather unique among flavoproteins. The substrate specificity of PuORh is very narrow, like PuOMr, the enzyme is very specific for putrescine. Polyamines are also accepted, but aliphatic monoamines are not converted at all. In general, PuORh appears to have a higher affinity for its substrates than PuOMr (Km for putrescine = 8.2 vs 38 μM).
The narrow substrate specificity of PuOMr for di- and polyamines was already rationalized by the suggestion of the presence of an ‘anionic point’ in the active site. This anionic point, likely due to a carboxylate function, binds one positively charged amino group of the substrate, while another amino group is oxidized (Swain and Desa 1976; Okada et al. 1979). Based on sequence alignment between PuORh and PuOMr and a structural model of PuORh, we can now identify Glu344 as the anionic point in PuOMr. The presence of a carboxylate function as a manner to bind a positively charged amino group is not restricted to putrescine oxidase. In polyamine oxidase (PAO), a negatively charged ‘carboxylate ring’ can be found at one side of its substrate tunnel to guide polyamine substrate molecules into the active site (Binda et al. 1999). In addition, in PAO, two glutamate residues (Glu62 and Glu170) are present in the active site opposite to the N5 of the flavin. However, these residues have been suggested to be protonated (Binda et al. 1999). Moreover, in PAO, Glu62 and Glu170 are in close proximity to a substrate secondary amine group, which will be oxidized, while in PuORh, Glu324 interacts with the (primary) amine group, which will not be oxidized. The amine group to be oxidized in PuORh is placed between His432 and Tyr395, near the N5 of the isoalloxazine ring. This structural feature resembles the “aromatic cage” found in other flavin-containing amine oxidases, like MAO-A (De Colibus et al. 2005), MAO-B (Binda et al. 2002a), and PAO (Binda et al. 1999). For MAO-B, this “aromatic cage” has been shown to play a steric role in substrate binding and in flavin accessibility and helps to increase the substrate amine nucleophilicity (Li et al. 2006).
Polyamines like putrescine, cadaverine, spermine, and spermidine are related to cell growth and differentiation processes. Increased levels in body fluids and tissue occur in cancer patients, and therefore, polyamines represent important cancer markers (Casero and Marton 2007). Polyamines are also used to monitor food freshness, as an increased level can be found in spoiled food due to amino acid degradation (Bardócz 1995). Due to efficient heterologous expression and its catalytic properties, PuORh may develop as a valuable diagnostic enzyme for the detection of low amounts of putrescine and polyamines. Moreover, based on the structural model, enzyme redesign may also allow creation of mutants that can be used for synthetic purposes.
Electronic supplementary material
Below is the link to the electronic supplementary material.
ESM 1
Unrooted phylogenetic tree representation of PuORh homologs, which exhibit over 30% sequence identity (at the amino acid level) throughout the whole sequence. In gray, the clade of putrescine oxidases. MAO-B Human monoamine oxidase B; PuORh putrescine oxidase from Rhodococcus erythropolis NCIMB 11540; PuOMr putrescine oxidase from Micrococcus rubens. (DOC 36 kb)

putrescine oxidase
flavin
amine
activity screening


The spectrum of ATM missense variants and their contribution to contralateral breast cancer

Heterozygous carriers of ATM mutations are at increased risk of breast cancer. In this case-control study, we evaluated the significance of germline ATM missense variants to the risk of contralateral breast cancer (CBC). We have determined the spectrum and frequency of ATM missense variants in 443 breast cancer patients diagnosed before age 50, including 247 patients who subsequently developed CBC. Twenty-one per cent of the women with unilateral breast cancer and 17% of the women with CBC had at least one ATM germline missense variant, indicating no significant difference in variant frequency between these two groups. We have found that carriers of an ATM missense mutation, who were treated with radiotherapy for the first breast tumour, developed their second tumour on average in a 92-month interval compared to a 136-month mean interval for those CBC patients who neither received RT nor carried a germline variant, (p = 0.029). Our results indicate that the presence of ATM variants does not have a major impact on the overall risk of CBC. However, the combination of RT and (certain) ATM missense variants seems to accelerate tumour development.
Introduction
Homozygous or compound heterozygous germline mutations in the ATM gene cause the autosomal recessive disorder ataxia-telangiectasia (A-T). This progressive neurological childhood disease is characterized by cerebellar degeneration, immunological defects, extreme sensitivity for ionising radiation and increased risk for cancers, particularly lymphomas [1]. ATM mutations identified in A-T families can be classified in three categories; truncating mutations, mutations that lead to some expression of mutant protein that lacks kinase activity and missense mutations with reduced kinase activity (http://chromium.liacs.nk/lovd/).
Heterozygous pathogenic ATM mutation carriers, ∼0.5–1% of the general population, do not display the symptoms observed in A-T patients. Several epidemiological studies have consistently shown elevated rates of breast cancer among female blood relatives of patients with A-T [2, 3]. Thompson et al. have shown that the overall relative risk in carriers was 2.23 [95% confidence interval (CI) 1.16–4.28] compared to the general population and 4.95 (95% CI 1.9–12.9) in those younger that age 50. A large review showed that ATM mutations are more frequent in breast cancer patients selected on the basis of a family history of breast cancer than in unselected patients [4]. Besides pathogenic ATM mutations, a large number of ATM variants (common polymorphisms and unclassified variants) have been described, which were found in cancer patients as well as in the general population. It has been hypothesized that the cancer risk among ATM heterozygotes might be related to mutation type, suggesting that particularly missense mutations are associated with an increased risk [5, 6]. However, two recent studies by Thompson et al. and Renwick et al. showed that pathogenic ATM mutations that cause A-T are breast cancer susceptibility alleles [2, 7]. This argues against the hypothesis that missense rather that truncating are associated with breast cancer.
Women with breast cancer have in general a three to fourfold increased risk of developing a new primary cancer in the opposite breast [8]. The contralateral breast cancer (CBC) risk might be explained by the same genetic and hormonal factors that caused the first breast cancer. Treatment related factors, e.g. radiotherapy for primary breast cancer, may also contribute to the development of cancer in the contralateral breast [9] (our own data, manuscript under review).
To evaluate whether germline ATM missense variants are significantly associated with CBC risk (results regarding ATM truncating mutations are reported elsewhere) and whether treatment modifies this risk, we conducted a case-control study in which we assessed the ATM missense mutation spectrum and frequency in women who developed their first breast cancer before age 50, with and without a second primary breast cancer.
Methods
Patients
The consecutive breast cancer patients included in this study were all selected from the hospital tumour registries of The Netherlands Cancer Institute, Amsterdam (NKI-AVL) or The Dr. Daniel den Hoed Cancer Center/Erasmus Medical Center, Rotterdam (DDHK). Of all patients that were invited to participate we achieved an 80% response rate. The breast cancer patients were included if their (first) breast cancer was diagnosed before age 50 (n = 443). For CBC we required an interval of at least 1 year (n = 247). The unilateral breast cancer patients (UBC) patients all had to be disease-free (of a second breast cancer) for at least 5 years. The first 57 CBC patients were individually age-matched (1:3) to UBC controls. All patients had invasive breast carcinoma and were treated with surgery. Of the CBC patients 169 did and 78 did not receive radiotherapy treatment for their primary breast tumour. Average age at diagnosis for the first breast cancer in the RT group was 41.2/41.3 years (mean/median) and the non-exposed group 42.0/43.2 years (mean/median). Detailed treatment data, disease and patient characteristics were obtained from medical records and risk factor questionnaires (data not shown) [10, 11]. Patients were asked to donate a 20 ml blood sample or permission for use of paraffin-embedded tissue blocks and patients gave their written informed consent for mutation analysis. This study received approval of the Medical Ethical Committees of NKI-AVL and DDHK.
Genomic DNA isolation
Genomic DNA was either isolated from peripheral blood lymphocytes with DNAzol (Invitrogen, Breda, The Netherlands) methods according to the manufacturer’s instructions, or from three 10-μm paraffin embedded normal tissue slides according to standard protocols [12]. For histopathological examination we used a hematoxylin-eosin stained slide.
Mutation analysis
The complete ATM Open Reading Frame (ORF) was analysed, each exon (exon 4-65) and all intron-exon boundaries were screened for germline mutations using Denaturing Gradient Gel Electrophoresis (DGGE) identifying ∼90% of all ATM mutations and polymorphisms (details from the author upon request). All aberrations were confirmed with genomic sequence analysis, performed using the ABI PRISM BigDyeTerminator Cycle Sequencing Ready Reaction Kit Version 3.1 (Applied Biosystems, Nieuwerkerk a/d yssel, The Netherlands). Sequencing products were analysed with the ABI PRISM 3700 DNA Analyzer and corresponding software.
Statistical analysis
Statistical analyses were performed using standard methods for analysis of case-control studies [13]. We compared the mutation frequency between UBC and CBC and between CBC cases previously treated with RT and cases not-treated with RT. Odds ratios (ORs) and 95% CI were calculated to evaluate the association between mutation carriers status and breast cancer risk. We have used the Mann–Whitney test to determine whether the difference between the intervals between the two breast cancers of the CBC patients was significant. All analyses were performed using SPSS 12.0 (SPSS Inc., Chicago, IL, USA).
Results and discussion
ATM germline mutations
In the present study, we have used the DGGE method to screen the complete ATM ORF to obtain insight in the ATM missense mutation spectrum in (contralateral) breast cancer patients. With DGGE we were able to confirm all the previously identified truncating mutations. A subset of the CBC patients described in this study had been screened in the past for ATM truncating mutations with the Protein Truncating Test, revealing seven ATM truncating mutations (including a non-sense mutation and small insertions and deletions; generating stop codons within a previously functional protein coding sequence causing premature termination of translation of the protein) [10]. Among all 443-breast cancer patients that were tested in this study with DGGE we detected a large number of ATM silent mutations (presumed neutral polymorphisms, data not shown and excluded from all analyses) and missense mutations (causing an amino acid substitution in the coded protein, most common ones; i.e. D1853N, not included in further analysis).
ATM missense mutation spectrum
In our study cohort we have detected 35 distinct ATM missense variants and 6 distinct truncating mutations. Several of the detected missense variants have been reported in the ATM database as being detected in A-T patients/or as polymorphisms (Table 1). None of the missense variants identified in this study are known as pathogenic A-T causing missense mutations. Seventeen of the missense variants have not been reported previously. Eleven missense variants were exclusively found in the CBC group and 10 exclusively in the UBC group. Whether this distinction in the spectrum indicates an association between particular variants and bilateral breast cancer risk cannot be concluded from the small numbers obtained in this study population. The ATM protein has several functional domains and the identified missense variants are located throughout the ORF. Potential functional implications of the newly identified unclassified variants remain to be established.
Table 1ATM missense variant and truncating mutation spectrum in contralateral and unilateral breast cancer patientsMissense variantsAmino acid changeCBC n = 247UBC n = 190Databasea or literature37C>TR13C1[10]146C>GS49C55database162T>CY54H21 [4], [14]378A>TD126E1database1009C>TR337C1Novel1132A>GS377G1Novel1229T>GV410A21 [4]1810C>TP604S1database2119T>CS707P78database2276G>A S759NNovel2336T>CM779T1Novel2414G>AR805Q2Novel2572T>CF858L43database2650C>TP884S12650C>TP884S1Novel2614C>TP872S [15]3161C>GP1054R813database3925G>AA1309T11 [16]4138C>TH1380Y1database4258C>TL1420F54database4324T>CY1442H2Novel4362A>CK1454N1database4477C>GL1493V1Novel4664T>AL1555H1Novel4722G>TL1574F1Novel5044G>TD1682Y1database5071A>CS1691R22database5557G>AbD1853N3549database5558A>TD1853V31database5741A>GD1914GNovel6067G>AG2023R1database6820G>AA2274T1database6919C>TL2307F1 [14]7446G>AM2482I1Novel7874A>GD2625G1Novel8659C>GH2887D1NovelTruncating mutationsIVS10-6T>G419X12database [10], [17]1563delAG521X1database1660delA554X1NovelIVS14 + 2T>Gdel 601-6331database2572insTF858X1Novel3115A>TR1039X1Novela http://chromium.liacs.nk/lovd/b Not included in frequency analysis
Despite the fact that ATM plays a role in breast cancer risk, the role of most distinct ATM missense variants remains unclear. Some studies tried to predict the relevance of each particular mutation on basis of co-segregation with breast cancer in families, the location in a functional domain or interference with the splicing machinery. Only a few studies present functional analysis that are necessary to assess the biological impact of unidentified variants found frequently in ATM [18–20].
ATM missense mutations and contralateral breast cancer
Twenty-one per cent of the patients carried at least one ATM germline variant (missense and truncating; Table 2). Among the patients with CBC (n = 247) we identified in total 55 ATM variants in 45 individuals (18%); 51 missense variants and 4 truncating mutations (Table 2). Eight CBC patients had multiple ATM missense variants and 2 patients carried both a missense and a truncating ATM mutation. In the women with UBC (n = 196) we identified 52 ATM variants in 46 individuals (23%); 48 missense and 4 truncating mutations. Three UBC patients carried double missense and 3 patients both a truncating and a missense variant. Although it is known from the literature that ATM missense variants might be involved in breast cancer pathogenesis, the identified 17% missense variant carriers among the CBC patients compared to the 21% missense variants among the UBC patients indicate that there is not a significantly increased risk for bilateral breast cancer among ATM missense variant carriers, OR 0.77 (95% CI 0.48–1.24).
Table 2ATM variant frequencies in all breast cancer patients diagnosed under age 50 and according to uni- or contralateral breast cancerBreast cancer patients withAll patients n = 443CBC n = 247UBC n = 196Total ATM variantsa55: 51 missense and 4 truncating52: 48 missense and 4 truncatingAt least one ATM variant91 (21%)45 (18%)46 (23%)At least one ATM missense variant85 (19%)43 (17%)42 (21%)Only one ATM truncating mutations321One truncating and one missense variant523Double missense variants1183a Not included are the most common and silent variants
Association with radiation treatment
Women at high risk for developing breast cancer may respond differently to radiation exposures associated with screening and treatment, than the general population. Candidate-genes like ATM are implicated in maintenance of genome integrity. Their involvements in breast cancer susceptibility as well as their role in DNA-damage repair signalling make them excellent candidates for a role in radiation-induced breast cancer [21]. Recently, we showed that women with a pathogenic germline mutation in a DNA repair pathway gene (e.g. BRCA1, BRCA2, CHEK2 and ATM) have an over 2-fold increased risk of developing radiation-associated breast cancer (manuscript under review). Therefore, we now investigated whether exposure to ionising radiation had a greater biological impact on certain ATM genotypes than on others.
We did not detect a significantly increased risk of developing radiation-associated CBC among missense mutation carriers. Among those 169 CBC patients who had developed a second primary breast tumour following radiotherapy for their first breast tumour we identified 19.5% ATM missense variants carriers compared to 13% among those CBC patients who did not receive RT, the OR from this case-only analysis is 1.65 [95% CI (0.77–3.55) p = 0.2]. Furthermore, we have observed that 21% of the UBC patients, who received RT but did not develop a CBC carried an ATM missense variant, compared to 19.5% of the CBC patients that received RT for their first tumour [OR 0.86 (95% CI 0.52–1.43)]. These results suggest that RT is not a strong risk factor for the development of CBC among carriers of those ATM missense variants.
It has however been shown that particular alterations in the ATM gene are associated with increased radiation sensitivity [22–24]. Gutierrez-Enriquez et al. showed that lymfoblastoid cell lines carrying the ATM variant 3161G (linked to 2572C) was associated with increased in vitro chromosomal radio-sensitivity, perhaps by interfering with ATM function in a dominant-negative manner [22]. We found this particular variant allele (3161G/2572C) exclusively in our CBC group exposed to radiotherapy (four times) and not in the non-RT-exposed CBC group. This finding supports the hypothesis that particular ATM variants might play a differential role in radiation response. Although a subset of the missense variants was only detected in the RT exposed subpopulation, individual numbers were probably too small to detect a significant effect of particular mutations associated with treatment.
We observed that CBC patients with an ATM missense variant had an mean interval between the first and second breast tumour of ∼101 months, compared to 122 months for non-carriers CBC patients (p = 0.085). Interestingly, the combination of radiation treatment and a missense variant resulted in an even shorter mean interval of a 92 months in the CBC patients compared to a 136-month interval for CBC patients who neither received RT nor carried a germline variant (p = 0.029). These data suggest that carrier-ship of an ATM missense variant may accelerate the development of a second tumour and decreases the age at onset of the second breast tumour, especially in case of exposure to RT.
The suggestion of a shorter induction period of RT-associated breast cancer in patients, who carry an ATM missense mutation, while the proportion of patients with missense variants was similar in CBC and UBC cases, might be attributable to a different spectrum of mutations in those patients who developed CBC. A big challenge in such a study remains to assess which particular missense mutations have an impact on ATM function. Large association studies, as performed by the Breast Cancer Association Consortium (coordinated by Doug Easton and Paul Pharoah, Cambridge), and functional studies are clearly necessary to determine the importance of particular variants and their contribution to the breast cancer risk.

atm
missense variants
breast cancer
radiotherapy