Expression of the chemokine receptor CCR5 in psoriasis and results of a randomized placebo controlled trial with a CCR5 inhibitor

Several reports have indicated that the chemokine receptor CCR5 and its ligands, especially CCL5 (formerly known as RANTES), may play a role in the pathogenesis of psoriasis. The purpose of this investigation was to examine the expression of CCR5 and its ligands in chronic plaque psoriasis and to evaluate the clinical and immunohistochemical effect of a CCR5 receptor inhibitor. Immunohistochemical analysis showed low but significant increased total numbers of CCR5 positive cells in epidermis and dermis of lesional skin in comparison to non-lesional skin. However, relative expression of CCR5 proportional to the cells observed revealed that the difference between lesional and non-lesional skin was only statistically significant in the epidermis for CD3 positive cells and in the dermis for CD68 positive cells. Quantification of mRNA by reverse transcriptase-polymerase chain reaction only showed an increased expression of CCL5 (RANTES) in lesional skin. A randomized placebo-controlled clinical trial in 32 psoriasis patients revealed no significant clinical effect and no changes at the immunohistochemical level comparing patients treated with placebo or a CCR5 inhibitor SCH351125. We conclude that although CCR5 expression is increased in psoriatic lesions, this receptor does not play a crucial role in the pathogenesis of psoriasis.
Introduction
Psoriasis is a chronic skin disease affecting approximately 2–3% of the population worldwide. Despite its common occurrence, the exact pathogenesis of psoriasis remains unclear and adaptations to the pathogenesis of this inflammatory disease are continuing [4, 9, 31, 35, 40, 52]. Although the specific effector cells responsible for the inflammatory process in psoriasis are not known, reported beneficial effects of specific T cell targeted therapies, such as cyclosporine A, DAB389IL-2 and alefacept support a central role for T cells in the pathogenesis of psoriasis [3, 5, 10, 13, 15, 24, 25]. Further investigation on the immunophenotype and cytokine secretion patterns of T cells have indicated that specifically Th1-cells are involved in psoriasis [2, 32, 46].
In the many aspects encompassing T-cell homeostasis, the trafficking of T cells from blood to tissues is thought to be relevant in chronic inflammatory diseases such as psoriasis. Key factors in this migration are chemo-attractant cytokine molecules known as chemokines and their receptors [30, 33, 37, 38, 57, 61, 62]. The predominant chemokine receptors expressed on Th1-cells are CCR5 and CXCR3 [7, 9, 29, 30, 33, 34, 47, 50]. Besides its preferential expression on Th1 cells, CCR5 is also expressed on monocytes, macrophages, natural killer and dendritic cells: all thought to be significant elements in the pathogenesis of psoriasis [8, 11, 12, 18, 26, 27, 36, 39, 41, 43, 54].
The ligands of CCR5 [CCL3, CCL4 and CCL5 (formerly known as MIP1α, MIP1β and RANTES, respectively)] are highly expressed by keratinocytes in psoriatic tissue [19, 22, 42, 48]. Furthermore, it has been demonstrated that the proinflammatory cytokines IFN-γ and TNF-α can induce the expression of these chemokines [19, 22] and that treatment of psoriasis resulted in a significant decrease of CCL5, as well as a reduction of CCR5+ T cells in the skin [19, 20, 58].
Several animal models resembling psoriasis have been developed, yet, none of these models imitates psoriasis completely, hence limiting their utility [51]. Investigations by Mack et al. [36] showed a different expression pattern of CCR5 in mice and humans. Additionally, different expression of a single amino acid in the CCR5 molecule between rhesus macaques and humans resulted in a different response to inhibitors of the receptor in the species [6]. Therefore, research on the expression of CCR5 in psoriasis, as well as clinical efficacy of a CCR5 inhibitor, is limited to humans. So far, the data available on the expression of CCR5 in psoriatic skin in humans are not univocal (varying from high to minimal) and were obtained with divergent methods in investigations in which CCR5 was never the main focus [20, 27, 49, 58, 59].
The primary purpose of this study was to determine the expression of CCR5 and its ligands in chronic plaque psoriasis in situ compared to non-lesional skin, through analysis by immunohistochemistry and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). In order to examine the possibility that CCR5 plays a functional role in the pathogenesis of psoriasis, we also analyzed clinical and immunohistochemical data obtained from lesional and non-lesional skin biopsies of psoriasis patients before and after treatment with a CCR5 inhibitor.
Materials and methods
Study design and patients
Lesional and non-lesional skin biopsies were obtained from nine patients with moderate to severe chronic plaque psoriasis, defined by the psoriasis area severity index (PASI) ≥ 8. These skin biopsies were analyzed by manual quantification of immunohistochemical double-staining and quantitative RT-PCR. In order to get insight in the possibility of a functional role of CCR5 in the pathogenesis of psoriasis, 34 patients, including the previous 9 patients, participated in an 8 week, randomized, placebo-controlled, parallel group, multi-centre, double-blind clinical trial in which patients received either 50 mg twice daily of the CCR5 inhibitor SCH351125 (23 patients) or matched placebo (11 patients) orally for 28 days, followed by a follow-up period of 4 weeks. During the follow-up period patients were only allowed to use emollients as treatment and on day 56 vital signs, PASI and blood were assessed. Patients were included at the dermatology outpatient departments of four academic hospitals from April 2004 to December 2004. At baseline and the last day of treatment (day 28), lesional biopsies were taken to evaluate the immunohistochemical effect of the CCR5 inhibitor. For this immunohistochemical evaluation, single-stained sections were analyzed with digital image analysis, semi-quantitative analysis (SQA) and confocal scanning microscopy, and double-stained sections on baseline and day 28 were analyzed by manual quantification. To evaluate the clinical effect of the CCR5 inhibitor the PASI was assessed at baseline, day 28 and day 56.
In all patients, psoriasis was diagnosed at least 12 months prior to enrolment and patients were not allowed to use systemic psoriasis treatment or phototherapy within 4 weeks of study entry. Only emollients were allowed as topical treatment. All other topical anti-psoriasis therapy (e.g. corticosteroids, vitamin D derivates, etc.) had to be stopped 2 weeks before study entry. The protocol was reviewed and approved by the medical ethical committees of all participating centres and all patients gave their written informed consent before enrolment. The study was conducted according to the Declaration of Helsinki principles and is registered at the ISRCT register (http://www.controlled-trials.com/ISCRT14986467).
Biopsies
Four-millimeter biopsies were taken from the inside border of a target psoriatic plaque, preferentially from a non-sun-exposed area. Lesional biopsies from each patient were obtained from the same target lesion, separated by at least 1 cm. The biopsy samples were randomly coded, snap-frozen in Tissue-Tek OCT compound (Sakura Finetek Europe, Zoeterwoude, The Netherlands) by immersion in liquid nitrogen and stored at −80°C until processing. Five-micrometer cryostat sections were cut and mounted on glass slides (Star Frost adhesive slides, Knittelgläser, Braunschweig, Germany), before being stored at −80°C until immunohistochemical staining. For each staining three sections of each biopsy were analyzed to minimize random variation.
Immunohistochemistry
After fixation of the slides, the endogenous peroxide activity was inhibited with 0.1% sodium azide and 0.3% hydrogen peroxide in Tris-buffered saline (TBS), before incubating for 15 min with 10% normal goat serum in TBS. Next, the sections were incubated for 1 h at room temperature (overnight at 4°C in the case of CCR5) with the primary antibody in 1% bovine serum albumin (BSA; Sigma-Chemical Co, St. Louis, MO, USA) in TBS. The following mouse anti-human monoclonal antibodies were used: FITC-conjugated anti-CD3 (BD Pharmingen, San Jose, CA, USA), FITC-conjugated-anti CD68 (clone EBM11; Dako), anti-human neutrophil elastase (Dako, Glostrup, Denmark), anti-cytokeratin 8.12 (keratin 16; Sigma, Saint Louis, MO, USA), anti-CD161 (NK-T cells; BD Pharmingen) and anti-CCR5 (CD195; BD Pharmingen). After rinsing with TBS, sections were further incubated with biotin-conjugated goat anti-mouse antibody (Dako) or, in case of CD3 and CD68 staining, with rabbit anti-FITC (Dako) in 10% normal human serum (NHS) in TBS for 30 min. Following a wash step with TBS, sections were subsequently incubated with horseradish peroxidase (HPR)-conjugated streptavidin (Dako) or, in case of CD3 staining, with HRP-conjugated goat anti-rabbit antibody (Dako), in 1% BSA in TBS for 30 min. In case of CCR5 staining an amplification step was performed with the TSA biotin system (Perkin Elmer, Boston, MA, USA). Sections were counterstained with Mayer’s haematoxylin (Merck, Darmstadt, Germany) and mounted in Kaiser’s glycerol gelatine (Merck). Skin sections were double stained with anti-CCR5 together with anti-CD3 or macrophage marker anti-CD68. The double stained sections were manually counted by two independent observers blinded for order, patient and clinical data. Using a 0.5 × 0.5 mm ocular grid and at 200× magnification, single red (CCR5+), single blue (CD3+ or CD68+), and purple double-positive cells were counted in the entire section. The epidermal and dermal regions were separately counted. The results are expressed as the number of double-positive cells/mm2.
RNA analysis
RNA was extracted from frozen skin biopsies using the RNeasy mini kit (Qiagen). RNA quantity was assessed by OD at 260 nm and RNA quality was analyzed by measuring the ratio of 28s and 18s rRNA using the Agilent 2100 bioanalyzer.
Quantitative PCR
Taqman primers and probes were designed with Primer Express software (ABI), and purchased from ABI. The sequences of the human primers and probes are available upon request. For the human skin tissue, quantitative PCR was carried out with an ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA). The PCR reactions were prepared using the components from the Invitrogen Platinum Quantitative RT-PCR One-Step kit and assembled according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). The final concentrations of the primers and probe in the PCR reactions were 200 and 100 nM, respectively. The RT-PCR reactions for each gene were performed in a single 384-well plate. Separate plates of the same RNAs were used to quantitate 18S RNA as an internal control for RNA quality, and a primer/probe set for the CD4 promotor was used to check the RNAs for genomic contamination. The PCR data was quantitated based on a standard curve generated using fourfold serial dilutions of the target genes. The fourfold dilutions began at 0.25 ng, and eight dilutions were used to generate the standard curve. This procedure provides an absolute quantitation of the amount of CCR5 mRNA in a given tissue. Data were analyzed using Sequence Detection Systems software version 1.7 (Applied Biosystems, Foster City, CA, USA).
Digital image analysis
Single stained sections were randomly coded and analyzed by computer-assisted image analysis as described previously in detail [23]. In short, images were acquired and analyzed using Syndia algorithm on a Qwin based analysis system (Leica, Cambridge, UK). Twenty high power fields per section were analyzed. Positive staining of cellular markers was expressed as positive cells/mm2.
Semi-quantitative analysis
For keratinocyte expression of K16 keratin, a semi-quantitative score was done by two independent observers, blinded for order, patient and clinical data, with a standard binocular light microscope (Olympus) at 200× magnification. The semi-quantitative score ranged from 0 to 4+. A score of 0 represented no expression, while a score of 4 represented abundant expression in all layers of the epidermis.
Confocal scanning microscope
Cryosections of 5 μm on silanized slides were fixed with acetone and dried at room temperature, and stored at −80°C. Sections were incubated in PBS-3% BSA for 30 min and washed with PBS before all steps. Next, monoclonal anti-human CCR-5 antibody (R&D Systems, MAB183, clone 45549.11), Texas red immunoglobulins diluted in PBS-3% BSA (1:100) was applied, followed by application of the primary monoclonal specific antibodies CD3 (clone SK7; BD Biosciences), CD4 (clone SK3; BD Biosciences), CD8 (clone DK25, DakoCytomation) or CD68 (clone KP1, DakoCytomation). Next, FITC-conjugated affinipure rabbit anti-mouse IgG (Jackson, A = 492, E = 520) diluted in PSB-3% BSA (1:200) was applied and sections were mounted in fluorescent mounting medium (DakoCytomation). Of each double stained slide three pictures were taken with an image definition of 1024/1024 pixels at a magnification of 25×.
Sample size calculation
The randomized placebo controlled clinical trial was targeted to randomize a total of 30 subjects (20 on active treatment and 10 on placebo). With this sample size, the trials would be able to detect a difference of 38% in the response rate from the placebo group assuming a 0% response rate in the placebo group with 80% power at an alpha level of 0.05 (two-sided test).
Randomization
Randomization was stratified by sites. Each site was assigned a fixed number of subjects numbers; e.g. site 1 would get numbers 1–9 and so on. Once the physician of the study site would enroll a subject, the subject would be assigned the next available subject number assigned to the site, starting with the bottom of the list; e.g. the first subject enrolled in site 1 would get number 1, the second subject would get number 2, and so on. Treatments would be assigned in an active to placebo ratio of 2:1 according to a computer generated randomization schedule. No stratification based on age, sex or other characteristics was performed. Throughout the study both patient and treating physician were blinded to the group assignment.
Statistical analysis
We used SPSS 12.0.1 for Windows (SPSS, Chicago, IL, USA) for the statistical analysis of clinical data. The Mann–Whitney test was used to compare lesional and non-lesional skin at baseline and to compare skin biopsies before and after treatment. All statistical tests were two-sided: P values less than 0.05 were considered significant. The results are expressed as median ± standard error of the mean. To evaluate the clinical effect of treatment with a CCR5 inhibitor, an intention to treat analysis was performed. Quantitative PCR data analysis was performed by two-sided t test as implemented by Graphpad Prizm (version 4.0 Graphpad Software, San Diego, CA, USA). A P value less than 0.05 was considered significant.
Results
Comparison of CCR5 expression in lesional versus non-lesional psoriatic skin
The expression of CCR5 in T cells (CD3) and macrophages (CD68) in lesional and non-lesional skin of nine psoriasis patients was compared at baseline. We observed a clear expression of CCR5 which was primarily present in the dermis. In absolute numbers approximately half of the T cells and half of the macrophages co-expressed CCR5 (Fig. 1). The CD3+CCR5+ and CD68+CCR5+ double positive cells showed a low but statistically significant increased expression of CCR5 in epidermis and dermis of lesional skin in comparison to non-lesional skin, as shown in Fig. 1. Focussing on the expression of CCR5 as the percentage of all T cells or macrophages present in the sections, the difference between lesional and non-lesional skin was only statistically significant in the epidermis for CD3+ cells (P < 0.05) and in the dermis for CD68+ cells (P < 0,001).
Fig. 1Immunohistochemical analysis of CCR5 in lesional versus non-lesional skin. Data are shown as mean ± SD; ns non-significant, * P < 0.05, ** P < 0.01, *** P < 0.001
Quantitative RT-PCR analysis indicated no increased expression of mRNA for CCR5 and CCR5-ligand CCL4 (MIP-1β) in lesional skin (Fig. 2), only the expression of CCR5-ligand CCL5 (RANTES) and IL-8 was significantly increased in lesional skin (P < 0.0001 and P < 0.05). The well-known enhanced expression of IL-8 in psoriatic skin was included as positive control.
Fig. 2mRNA analysis of CCR5 in lesional psoriatic skin in comparison with non-lesional skin. IL-8 is used as a control marker. * P < 0.0001, ** P < 0.05
Lack of clinical efficacy of SCH351125: a CCR5 inhibitor
In total 34 patients were randomized, as shown in Fig. 3. The demographical data of the patients are shown in Table 1. With regards to co-morbidities, one patient had hypercholesterolemia and one had obesity in the placebo group. In the SCH351125 group one patient also had obesity and two had a history of hypertension. After treatment with the CCR5 inhibitor there was no change in mean PASI in the SCH351125 group (n = 23) [15.5 ± 3.8 at baseline, 15.4 ± 7.4 at day 28 (Fig. 4a)]. Three of the patients treated with SCH351125 (13%) attained an improvement of 50% or more compared to baseline (PASI 50 responders), showing improvements of 67, 77 and 69%. In the placebo group (n = 9) the mean PASI slightly decreased (14.2 ± 4.7 at baseline, 12.9 ± 3.7 at day 28). None of the patients treated with placebo showed an improvement of more than 50%. All changes observed were not statistically significant. In the follow-up period no changes in mean PASI were seen in either treatment groups.
Fig. 3Flow chart of randomized, placebo controlled clinical trial. SCH351125 CCR5 ligand inhibitor, SAE serious adverse event, AE adverse eventTable 1Demographical data patientsRandomized clinical trialPlaceboSCH351125Number1123Male:female7:418:5Age (years)a41.8 (10.2)49.4 (14.3)Duration of skin disease (years)a20.6 (9.8)19.8 (11.6)Baseline PASIa14.9 (4.7)15.7 (4.3)aMean (±SD)SCH351125 CCR5 ligand inhibitor, PASI psoriasis area and severity index, BSA body surface areaFig. 4Clinical and immunohistochemical respons after treatment with a CCR5 receptor inhibitor. In a randomized placebo controlled clinical trial 34 psoriasis patients were randomized for treatment with a CCR5 receptor inhibitor (SCH351125) or placebo for 28 days. Clinical efficacy was measured by psoriasis area and severity index (PASI) (a). Lesional skin biopsies were taken from all patients at baseline and day 28 to evaluate immunohistochemical effect. Immunohistochemical single staining of CCR5 of a patient treated with SCH351125 at baseline (b) and day 28 (c). Immunohistochemical markers in relation to clinical response (d). PASI 50 non-responder, improvement of PASI of less than 50%; PASI 50 responder, improvement of PASI of 50 or more percent; data are shown as mean ± SD; ns non-significant; 1P = 0.05
In the treatment group four patients discontinued. One patient developed an erythrodermic eruption after 4 days of treatment, which was considered by the site physician as a serious adverse event (SAE). Two patients discontinued due to adverse events (AEs): one developed shingles in the n.trigeminus area of the right side of his face after 8 days of treatment and one patient discontinued due to hair loss after 21 days of treatment. One patient discontinued due to non-compliance. In the placebo group two patients discontinued due to AEs: both exacerbation of their psoriasis after 2 weeks of treatment.
CCR5 expression before and after treatment with SCH351125
Immunohistochemical analysis of lesional tissue samples from the SCH351125 group and the placebo group revealed no statistically different expression of CCR5 between baseline and day 28 in both treatment groups, as illustrated by Fig. 4b, c. When focusing on the markers CD3, CD68, CD161, elastase and K16 in relation to the clinical response, no statistically significant difference after 28 days of treatment with either SCH351125 or placebo was found (Fig. 4d), except for elastase and dermal CCR5+CD3+ cells, which were statistically significantly lowered in the three PASI 50 responders treated with SCH351125. Additional data obtained by confocal scanning microscopy corresponded with the digital image and SQA (data not shown).
Discussion
The primary objective of this study was to explore the possibility of involvement of CCR5 in the pathogenesis of chronic plaque psoriasis. Therefore we determined the expression of CCR5 in situ at the protein and mRNA level by immunohistochemical analysis and, quantitative RT-PCR, respectively. The total number of single positive (CCR5+) and double positive (CCR5+CD3+ and CCR5+CD68+) cells in lesional psoriatic skin significantly outnumbered those in non-lesional skin. However, when expressed as percentage of CD3 or CD68 cells, the difference between lesional versus non-lesional expression was less clear. With the latter approach we found that the proportion of CCR5 expression was significantly higher in the epidermal CD3+ cells and dermal CD68+ cells only, when comparing lesional skin to non-lesional skin.
Analysis of CCR5 mRNA expression demonstrated a slight, though not significant, increased expression of CCR5 in lesional psoriatic skin, perhaps due to the small numbers of patients. In line with earlier observations we found that the mRNA expression for RANTES [19, 48] and IL-8 [1, 17, 21, 45, 53] was significantly higher expressed in the lesional samples. However, in contrast to previous research [42] the expression of MIP1β was not increased in lesional psoriatic skin.
In summary, our results do not provide a clear answer to our objective of determining whether the percentage of CCR5 expressing cells is similar in lesional and non-lesional skin, or if this percentage is increased in lesional skin.
To assess any possible functional participation of CCR5 in the development or maintenance of psoriatic plaques we investigated the clinical response to treatment with a CCR5 inhibitor as well as the effect of this drug on the inflamed skin in situ. The randomized placebo-controlled clinical trial revealed no significant clinical effect and changes at the immunohistochemical level between patients treated with placebo or the CCR5 inhibitor. The specific type and dose of the CCR5 inhibitor used in this clinical trial has proven its efficacy previously in vitro, in vivo and clinical studies with other diseases in which CCR5 is known to play a pivotal or significant role [14, 44, 55, 56, 60]. However, only three patients treated with the CCR5 inhibitor demonstrated a clinical improvement of 50% or more. It cannot be excluded that this low and not statistically significant number of patients is due to a spontaneous improvement, reflecting the unpredictability of psoriasis. Surprisingly, the immunohistochemical markers analyzed in the skin biopsies of the so-called PASI 50 responders, did only partially correspond with the clinical response. Of all immunohistochemical markers only elastase and dermal CCR5+CD3+ showed a significant decline after 28 days of treatment. Notably, the expression of CD3, a marker known to correspond well with the clinical severity as measured by PASI, was increased after treatment with SCH351125 in two out of the three responding patients (Fig. 4d). In addition, their baseline expression of CD3 was lower in comparison with the other patients while their PASI was similar. This inconsistency could be due to the low number of patients or suggests an individual difference in CD3 kinetics.
The increased expression of CCR5 observed by immunohistochemistry and the increased mRNA expression of CCR5-ligand RANTES in lesional psoriatic skin may suggest an involvement of this receptor and ligand in psoriasis. However, our clinical trial with an effective CCR5 inhibitor unequivocally demonstrated that this is not the case. Previous research has shown increased expression of several chemokines and chemokine receptors in psoriasis, [16, 21, 27, 28, 49] indicating that multiple receptors may participate in regulating T cell recruitment to the inflamed skin. Furthermore, RANTES is known to also bind with CCR1 and CCR3, whereas MIP1β is solely connected to CCR5. Given this complexity of interactions between chemokines and chemokine receptors, it is not unlikely that blocking a single chemokine receptor (i.e. CCR5) would have been insufficient in diminishing the inflammatory process.
According to Homey [26], chemokine antagonistic approaches to impede with the inflammatory process may perhaps be preventive rather than therapeutic. Chemokines and their receptors play an essential role in the trafficking of T cells to all kinds of tissue, including the skin. Yet, once leucocytes have entered the target organ and underwent activation processes, impairment of recruitment of pathogenic T cells is likely to be less effective in reducing the clinical symptoms. When combined with a successful eradicative treatment of the T cells, chemokine antagonists could perhaps be promising candidates for prevention of acute flares, prolongation of lesion-free interval and therefore provide optimized long-term management of patients suffering from chronically relapsing inflammatory skin disease such as psoriasis. So, although CCR5 does not seem to be a key chemokine receptor in the pathogenesis of psoriasis, further efforts are needed to unravel the complete set of chemokines and chemokine receptors significant in the recruitment of inflammatory cells in psoriasis, and may help to identify crucial molecules, as demonstrated previously by TNF in various immune mediated inflammatory diseases.


Current erosion indices—flawed or valid? Summary

The problem of erosive tooth wear appears increasingly to be encountered by clinicians and researchers. An adequate way of defining and recording erosive tooth wear is essential in order to assess the extent of this clinical phenomenon, both on an individual level and in the population, and for the adequate provision of preventive and therapeutic measures. Well-established erosion indices have been used in most of these studies, although in many cases modifications have been made to suit the different research aims. This use of different indices is one reason why it still cannot be claimed that there is enough current knowledge on this clinical phenomenon. This article summarises the proceedings of a workshop to discuss the topic of dental erosion indices. The result of the workshop is the proposal for a new scoring system (Basic Erosive Wear Examination, BEWE) designed for use both within the research field and for dental clinicians, with the aims of standardising assessment of erosion for international comparisons, raising awareness and providing guidelines for treatment of erosive tooth wear in dental practice.
Introduction
At the 1995 International Life Sciences Institute—Europe Workshop on Dental Erosion, amongst the recommendations made were the development of a clinical index for assessment of progression of dental erosion and the validation of a proposed erosion index for large-scale epidemiological surveys. One decade later, it appears that these recommendations are still relevant in that such tools have not been validated. Several different indices for the clinical diagnosis of erosive tooth wear are still in use by researchers and clinicians around the world and research on dental erosion carried out over the past decade indicates a lack of consensus. This problem applies both in relation to the prevalence, the distribution, as well as the progression of erosive tooth wear, its early detection and in the evaluation of the efficacy of preventive/therapeutic measures. Although both patients and dental clinicians are becoming increasingly aware of the erosive component of tooth wear, there is still a need to raise diagnostic competence through the use of a simple, standardized index that properly reflects the nature, extent and progression of the defects.
This paper summarises the result of the workshop ‘Current erosion indices—flawed or valid?’ held in Basle in 20071, the aim of which was to substantiate and critically discuss the current research tools in this field.
Anthropological aspects of tooth wear
In contrast to the dental profession, anthropologists have considered tooth wear as a normal physiological phenomenon. Skeletal material from ancient remains, such as the pre-contemporary Australian Aboriginal populations, shows clear evidence of attrition and abrasion. The most common cause of these forms of wear is believed to be food mastication, that is to say hard, fibrous material typically consumed by these populations and tooth surface loss appears to be directly related to the age of the individual.
Anthropologists describe a tooth wear model where genetic factors influence tooth morphology and occlusion, and, in turn, occlusion and food consistency influence the chewing pattern. The resulting gradual tooth wear is accompanied by physiological adaptation involving bone remodelling and compensatory tooth eruption. It is postulated that from an evolutionary perspective, tooth wear was one of the main selective factors leading to changes in both tooth morphology and properties of dental tissues. Masticatory efficiency is strongly related to tooth anatomy and wear characteristics as highlighted in comparative studies of dentitions of many different species including herbivores, carnivores, primates and humans.
A basic understanding of the anthropological perspective of tooth wear is highly valuable both in the diagnosis and treatment planning of contemporary erosive tooth wear and particularly in relation to discussion about the relevance of erosive wear for oral health.
Diagnosis and risk factors
Dental erosion or erosive tooth wear is a multi-factorial condition. Tooth wear mechanisms seldom happen in isolation, each occurring with different intensity and duration to produce a multitude of wear patterns. This interplay is probably the reason for confusion when attempting to use any tooth wear index based mainly on aetiology. Diagnosis requires a thorough knowledge of both morphological patterns typical of this type of wear and of the factors that are likely to contribute to the development of erosion.
Risk factors for erosive tooth wear can be categorized into chemical, biological and behavioural factors. The two most often cited chemical parameters, pH and titratable acidity, can only partly explain the erosive potential of acidic food or drinks. Mineral content, especially the common ions (calcium and phosphate) and fluoride, as well as calcium-chelation properties that vary for different dietary acids, are also important. Saliva is considered to be one important biological factor in erosion protection, but also the acquired enamel pellicle, tooth structure and positioning in relation to soft tissues and tongue may be of particular relevance. Behavioural factors can play a role in dental erosion both during and after an acidic challenge. The manner by which dietary acids are introduced and kept in the mouth before swallowing, the timing of acidic consumption/exposure and daily work/pleasure/sport activities can all have a significant effect on the development and location of erosive tooth wear.
Individuals suffering from anorexia and bulimia nervosa, rumination, chronic alcoholism or gastro-oesophageal reflux are at risk of erosive tooth wear caused by intrinsic acid and certain occupations are also conducive to erosive tooth wear such as professional wine tasting and industrial work involving exposure to acidic vapours or dust.
Current tooth wear indices
Measurement of tooth wear has been performed over the years using a large variety of both quantitative and qualitative methods. A number of indices have been developed typically using grading or scoring designed to identify increasing severity or progression of the condition, but traditionally with a focus on only one aetiological factor. Many diagnostic indices however, do not properly reflect the morphological defects and furthermore there is little international standardisation.
The earliest published attempt to diagnose tooth wear dates to the late 1800s. This index did not presuppose aetiology and formed the foundation for the development of further indices. In the latter half of the 1900s, many other indices came about largely based on work by Eccles and Jenkins [1]. The Eccles index for dental erosion of non-industrial origin denotes three classes of lesion assigned to four tooth surfaces. A few years later, based on Eccles work, Smith and Knight [2] produced the Tooth Wear Index, a comprehensive system whereby all four visible surfaces (buccal, cervical, lingual and occlusal/incisal) of all teeth present, are scored for wear. Further modifications of these indices have been published, designed for specific purposes by researchers in order to suit their own work. However, some problems have been encountered concerning the amount of data produced and inter- and intra-examiner reliability in large-scale epidemiological surveys, as well as threshold values in different population groups. The challenge still remains to develop a simple index that can be used clinically to assess progression of wear and in epidemiological prevalence studies.
Validity of current diagnostic criteria and tools for grading
The diagnosis of tooth wear and of erosive tooth wear in particular, involves the process of identifying the condition by the lesion characteristics and by obtaining medical, nutritional, and occupational information about the patients. On an individual basis this process can be very specific compared with the case for epidemiological research where, for practical reasons, diagnosis is often restricted to lesion characteristics. Although there appears to be relatively widespread consensus as to the characteristic signs and symptoms of erosive tooth wear, dating back approximately 60 years, true validation of these clinical criteria is lacking. At that early stage, the presence of (amalgam) fillings appearing to project above the surface of the surrounding tooth was considered to be the most important clinical sign. However, hypersensitivity of the affected teeth, absence of stain and tooth wear defects with rounded margins were also mentioned. More clinically precise descriptions of the lesions have appeared in the literature over time, describing tissue loss at the gingival third of the facial surfaces that were shallow, disc-shaped, smooth, polished or with a scooped out appearance. It is currently accepted that loss of surface contour, shallow concavities on smooth surfaces, cupping and grooving on occlusal/incisal surfaces and ‘proud’ restorations are characteristic signs of erosive tooth wear, but reviewing the literature, it turns out that these criteria derived from observations in small groups and individual experience rather than from systematic research.
The results of epidemiological studies aimed at relating the occurrence of erosion lesions with known aetiological factors, are somewhat unclear, and in some cases controversial. Most of these studies are performed on random cross-sectional or cluster samples of children or adolescents, with few addressing the adult and aged population. Whether the current diagnostic criteria are valid in reflecting the effect of chronic acid exposure remains to be tested.
Comparative studies on wear-lesion characteristics aimed at differentiating between abrasive and erosive wear by examining dietary and nutritional differences have concluded that occlusal/incisal lesions need to be quantified by depth/breadth ratios in order to obtain a correct diagnosis, but that the presence of shallow defects on smooth surfaces is almost certainly a more valid criterion than occlusal cupping and incisal grooving. Except in cases of occlusal cupping in young individuals, it is suggested that the presence of occlusal cupping and incisal grooving are at best uncertain diagnostic criteria for erosive tooth wear, but that shallow defects on smooth surfaces coronal to the CEJ may be pathognomonic of erosive tooth wear.
Grading the degree of erosion in an individual is important as it reflects the net exposure to the erosive challenges and the opposing protective forces, and plays a part in the assessment of the problem and of the need for intervention for prevention of further tooth wear. The reliable diagnosis of both early enamel and dentine erosive tooth lesions is challenging and not very reproducible. In cases involving cupping of the molar cusp tips, determining whether dentine is exposed at the base of the cup can be very difficult. Histological studies would tend to indicate that even experienced clinicians underestimate dentine exposure.
Assessing tooth wear in the primary dentition provides further challenges. Primary teeth do not appear to withstand wear forces to the same degree as permanent teeth and commonly show signs of wear. This fact affects the scoring of severity and makes determination of aetiology in many cases more difficult.
Pathological or physiological erosion: is there a relationship to age?
In order to be able to diagnose erosive tooth wear as unacceptable or even pathological, it is imperative that acceptable levels of tooth wear be established. Partly due to the lack of effective methods for evaluating the amount of tooth loss, these data do not exist apart from a few epidemiological surveys. These studies, combined with experienced clinical judgment currently form the basis for a diagnosis of severity.
Defining pathological erosion has proved problematic. Authors addressing this concept have considered cases to be ‘pathological’ when excessive loss of tooth tissue has occurred affecting appearance or function, or when it causes pain. This is further complicated by attempting to predict if a tooth, at any age, would survive its current rate of wear, or, if and when interceptive treatment is required, and what level of wear is acceptable to the patient. However, an erosion lesion judged to be pathological at one age may revert to be within physiological boundaries at a later age if it remains quiescent and does not progress further during the intervening period. Thus, when defining pathology, the severity of loss, the current activity and the age of the patient must be considered.
Furthermore, the concept of age-related pathological tooth wear becomes interesting when assessed from different perspectives. State health care authorities, private insurance companies, industry, dental clinicians and patients will all have differing interpretations of what constitutes ‘pathological’ tooth wear.
Requirements and demands on an erosion index
Indices have been formulated based on varying criteria, either to satisfy a particular purpose/condition or to enhance reproducibility. Among the factors considered in the development of currently existing indices are clinical versus epidemiological purposes, children versus adults, permanent versus primary teeth, full versus partial mouth scoring, exclusion/inclusion of dentine exposure and/or occlusal cupping and incisal grooving, etc. None of these indices has been found to describe dental erosion in such a way that they can be regarded as the ‘gold standard’ for measurement of erosion.
A simple and standardized index that is suitable for assessment of erosion should ideally be (1) easily applicable in general dental practice, (2) adaptable for epidemiological prevalence studies, (3) suitable for monitoring erosive lesion activities such as progression or arrestment of lesions, (4) easily reproducible under varying conditions for examination such as with/without magnification devices, ambient light, and hydration state of the tooth surface (dry/wet), (5) capable of reflecting net exposure of an affected individual to the erosive challenge, (6) capable of indicating the need for treatment, and (7) should serve for both children and adult as well as permanent and primary teeth.
Development of one erosion index capable of satisfying all the above conditions poses a lot of challenges and the implications of the exclusion or inclusion of certain factors and conditions may need thorough consideration. An index involving partial scoring of the dentition used for rapid screening would be more acceptable than one incorporating full mouth scoring, which is both time consuming and believed to lower the accuracy of the index. While limiting the scoring to anterior teeth (e.g. central incisors) may eliminate a considerable number of individuals with posterior located erosion, it is considered that the use of a sextants technique, which involves a full mouth examination but scoring the most severely worn surface on each sextant, would favour easy application and reproducibility. In summary, the application of a sextant method, assessing only wear defects on the coronal smooth surfaces, and estimating severity by the diameter and depth (regardless of dentine involvement) of wear, may be considered for the development of a simple and standardised index acceptable for clinical practice and epidemiological studies.
Methodological considerations
The WHO Oral Health Program (2003) [3] focuses on oral health as an integral and essential component of general health and an important factor in quality of life. To date, only the DMFT index has been assessed and recorded on an international basis. No indicator of dental erosion is currently included in any EU or WHO oral health policy, most likely due to the lack of a standardized index for measurement of dental erosion. As part of a revision of the WHO Oral Health Surveys Basic Methods, there is now an emphasis on development of methodologies and approaches for evaluating newer disease entities such as dental erosion.
Important quality characteristics of epidemiological tools include validity and reliability and sensitivity and specificity. Content validity describes whether all aspects that are relevant for the ‘construct’ or content of the index are adequately considered. The content validity is based on the subjective meaning of established experts. When comparing results using a new index with results using an existing ‘gold standard’, it can be stated that the construct validity is high if results are well correlated with each other. When a gold standard is not available, approved hypotheses concerning the construct of interest can be confirmed empirically using the new instrument or index. Reliability of an erosion index indicates how precise that index is able to record dental erosion and encompasses both inter-examiner and intra-examiner reliability. The sensitivity of an erosion index indicates its ability to detect dental erosion (compared to a gold standard) and is calculated by dividing the frequency of correct positive results by the sum of correct positive and false negative results. The specificity of an index indicates how well the index can record erosion as absent when erosion is not present and is determined by dividing the frequency of correct negative results by the sum of correct negative and false positive results.
Validation studies should be carried out on an international basis and should consider the mentioned quality criteria, most of which are relatively easy to prove. A challenge however is the question of content validity which requires research and final consensus on the construct of erosive wear.
New scoring system: the Basic Erosive Wear Examination (BEWE)
After lengthy discussions flaws in current approaches were identified and it was agreed that there is need for a validated, standardised and internationally accepted index. To initiate the development of such a tool, a universal scoring system was suggested. The new BEWE proposed in this issue records at the sextant level, based on the model of the BPE (Basic Periodontal Examination). It is designed to be a simple, reproducible and transferable scoring system that can be used with the diagnostic criteria of all existing indices. In time, use of the BEWE will generate comparable prevalence data, thus allowing ongoing consensus processes in the scientific community. Researchers within this field should, in practice, be able to transfer previous results using other indices based on other diagnostic criteria over to the BEWE index thereby allowing for improved comparisons.
Furthermore, the BEWE will help students, clinicians and general practitioners to screen for erosive tooth wear and to guide them in the decision-making process for the management of erosive tooth wear.
Future research perspectives
An international alliance between dentists, epidemiologists, statisticians and other scientists and politicians is recommended in order to develop an internationally agreed and accepted dental erosion index. Efforts should be made:
to re-evaluate existing data with the BEWE system to gain comparable data on the relevance of erosive wear for oral health and to initiate the consensus processto prove the content validity of the construct ‘dental erosion’ and to define valid diagnostic criteriato use these criteria with the BEWE system thus creating a new indexto validate this index on an international basis with the perspective to implement erosive wear data in the WHO and EU oral health data bases and erosion indicators in indicator sets for oral healthto further develop the link between the sum score of the BEWE and recommendations for prevention and treatment in the individual patient

erosive tooth wear
index
epidemiology
diagnosis
prevalence


Association and synergistic interaction between promoter variants of the DRD4 gene in Japanese schizophrenics

Recent association studies suggest that polymorphisms in the promoter and exon 1 upstream region of the dopamine D4 receptor (DRD4) gene play a functional role in the development of common psychiatric illnesses, although there are also conflicting results. In this study, we re-sequenced this region to identify all genomic variants, and tested them for association with schizophrenia. A total of 570 Japanese schizophrenic cases with matched controls were studied by genotyping all identified/validated common polymorphisms (−1106T>C, −906T>C, −809G>A, −616G>C, −521T>C, −376C>T, −291C>T and 12-bp repeat) and a known microsatellite (120-bp tandem duplication) in the upstream region. A single nucleotide polymorphism (SNP) −809G>A in the promoter region was found to be significantly associated with disease (P=0.018 and 0.032 for allelic and genotypic comparisons, respectively), although not surviving after Bonferroni correction. Logistic regression analysis showed that a combination of the four polymorphisms, −809G>A, −616G>C, −291C>T and the 12-bp repeat, conferred a susceptibility to schizophrenia. These results suggest that the upstream variants have a primary functional effect in the etiology of schizophrenia in the Japanese population.
Introduction
Disturbances in the dopamine neurotransmitter system have long been suggested to play a crucial role in the pathogenesis of schizophrenia (Prasad et al. 2002). However, involvement of dopamine-related genes in the manifestation of this disorder has been difficult to determine. The dopamine D4 receptor (DRD4) gene, located at chromosome 11p15.5 (Gelernter et al. 1992; Petronis et al. 1993), has received considerable interest because clozapine, a neuroleptic which is often effective against treatment-resistant symptoms, has a high affinity for this receptor (Sanyal and Van Tol 1997; Van Tol et al. 1991). Also, DRD4 is up-regulated in postmortem brain tissue from schizophrenic patients (Murray et al. 1995; Seeman et al. 1993; Stefanis et al. 1998; Sumiyoshi et al. 1995).
To investigate its genetic association with schizophrenia, many groups have examined polymorphisms of the DRD4 gene. Early studies focused on a 48-bp variable number of tandem repeat (VNTR) in exon 3. This tandem repeat comprises 2 (2R) to 11 (11R) 48-bp repeat units and codes for the third intracellular loop of the receptor protein (Van Tol et al. 1992). There is growing genetic evidence linking the VNTR with psychiatric traits or illnesses, such as novelty-seeking (Benjamin et al. 1996; Ebstein et al. 1996), attention-deficit hyperactivity disorder (ADHD) (Faraone et al. 2001; LaHoste et al. 1996), and schizophrenia (Hwu et al. 1998; Jonsson et al. 1996; Kaiser et al. 2000; Sanak et al. 2005; Serretti et al. 2001), but the results are still variable.
In recent years, promoter region polymorphisms of the DRD4 have also received particular attention because of their possible role in the regulation of gene transcription. To date, two polymorphisms in the promoter, T allele of the single nucleotide polymorphism (SNP) −521T>C and a 120-bp tandem duplication (1.2 kb upstream from the initiation codon), are reported to reduce transcriptional efficiency (D’Souza et al. 2004; Okuyama et al. 1999). Numerous studies have examined the association of upstream (promoter region and exon 1) polymorphisms with schizophrenia and some have shown a positive association (Okuyama et al. 1999; Xing et al. 2003), although not all were in full agreement (Hong et al. 1998; Jonsson et al. 2001; Kohn et al. 1997; Lung et al. 2006; Mitsuyasu et al. 2001; Petronis et al. 1995).
Since previous studies focused primarily on individual polymorphisms, allelic heterogeneity and/or a relatively weak effect of the variants may partly explain these inconsistencies. When haplotype construction is difficult because of a weakness or absence of linkage disequilibrium (LD) in a genomic interval, as is the case for the promoter region of DRD4, it may be important to test for a synergistic interaction between genetic variants in addition to performing single SNP analysis. Haplotype analysis is a reasonable strategy, assuming that the causative allele arises from a specific ancestral haplotype. However, this method is inadequate when there is an accumulation of multiple causative variants in a restricted genomic stretch. Additionally, haplotypic analysis is further compromised in regions where LD is decayed, because of the low statistical power to detect association due to inflated degrees of freedom. Therefore, clusters of potentially functional variants in genomic region with tenuous LD are better analyzed using a logistic regression framework that provides a powerful test of SNP etiology.
In this study, we examined the 5′ upstream region of DRD4, by first re-sequencing the region to identify all genomic variants. We then tested the association of all identified/validated common variants individually, as well as performing synergistic interaction analyses between them and schizophrenia.
Materials and methods
Subjects
Samples from 570 unrelated cases of schizophrenia (285 men, 285 women; mean age 47.0±11.4 years), and 570 age and sex matched controls (285 men, 285 women; mean age 46.7±11.1 years) were analyzed. The diagnosis of schizophrenia was made by consultation according to DSM-IV criteria with consensus from at least two experienced psychiatrists. All available medical records were taken into consideration. Control subjects were recruited from hospital staff and volunteers who showed no evidence of present or past psychoses during brief interviews with psychiatrists. All subjects were from central Japan. The study was approved by the Ethics Committees of RIKEN, Hamamatsu University and Chiba University. All participants provided written informed consent.
Sequence analysis
The upstream region encompassing the promoter region and exon 1 of the DRD4 gene (from 1,216 bp upstream of “A” in the start codon to 293 bp downstream of this “A”) was analyzed by the direct sequencing of PCR amplification from the genomic DNA of 30 unrelated Japanese schizophrenics. Primer sequences and detailed information on the reaction conditions are available upon request. Sequencing was performed using the DYEnamic ET terminator cycle sequencing kit (Amersham Biosciences, Piscataway, N.J., USA) and the ABI PRISM 3730 Genetic Analyzer (Applied Biosystems, Foster City, Calif., USA). Polymorphisms were detected by the SEQUENCHER program (Gene Codes Corporation, Ann Arbor, Mich., USA).
Genotyping
SNPs were typed by the TaqMan system (Applied Biosystems). PCR was performed using an ABI 9700 thermocycler and fluorescent signals were analyzed by an ABI 7900 sequence detector single point measurement and SDS v2.0 software (Applied Biosystems). Conflicts or flagged alleles were resolved by re-genotyping. Two microsatellite marker loci, the 120-bp tandem duplication and the 12-bp repeat polymorphism (Fig. 1), were amplified by PCR using fluorescently labeled primers. PCR fragments were analyzed on an ABI PRISM 3730 Genetic Analyzer (Applied Biosystems). Genotypes were determined using GeneScan 3.5.2 and Genotyper 3.6 software (Applied Biosystems).
Fig. 1Genomic structure and location of polymorphic sites within DRD4. Exons are denoted by boxes, with untranslated regions in white and translated regions in black. The sizes of exons and a scale are also shown. SNPs and microsatellites are indicated by solid squares and triangles, respectively. Re-sequencing was performed on the upstream region (from 1.2 kb upstream of the initiation codon to the 3′ end of exon 1)
Statistical analyses
The allelic and genotypic distributions were tested for association by Fisher’s exact test for biallelic markers (SNPs, the 120-bp tandem duplication and the 12-bp repeat polymorphism).
Haplotypic association analysis was performed using COCAPHASE in the UNPHASED package (http://www.rfcgr.mrc.ac.uk/∼fdudbrid/software/unphased/) (Dudbridge 2003). We employed a 2- and 3-marker sliding window analysis using COCAPHASE. To estimate the degree of LD between pairs of loci, the standardized disequilibrium coefficient (D′) was calculated and haplotype blocks were defined using the Haploview program (http://www.broad.mit.edu/mpg/haploview/) (Barrett et al. 2005).
Logistic regression analysis
To test the multiple polymorphisms in the upstream region for a synergistic effect, stepwise logistic regression analysis was performed using the SPSS Software (Release 11.0J) (SPSS Japan, Tokyo, Japan). We employed a procedure of backward selection in which we started with all the markers genotyped in the present study. According to the approach of Cordell and Clayton (2002), we set dummy variables, x1 and x2, taking values x1=−1, 0 and 1, x2=−0.5, 0.5 and −0.5, for genotypes w/w, w/m and m/m, respectively (w: wild-type, m: mutant).
Results
We re-sequenced the upstream region of the DRD4 gene, and identified a total of eleven SNPs: −1106T>C, −930C>G, −906T>C, −873G>A, −809G>A, −616G>C, −603G>T, −595G>del, −521T>C, −376C>T, and −291C>T, and a 12-bp repeat polymorphism (Fig. 1). One novel variant, −930C>G (deposited into GenBank as ss61570833), was detected in only 1 out of 58 chromosomes. The remaining variants were already present in the public databases and were validated in Japanese subjects during the course of this study.
The eight markers, −1106T>C, −906T>C, −809G>A, −616G>C, −521T>C, −376C>T, −291C>T, and the 12-bp repeat polymorphism in exon 1, were selected for genotyping using the criterion of a minor allele frequency >0.05 from our sequencing data. We also typed the size of the 120-bp tandem duplication (bi-allelic polymorphism with either one or two repeat alleles). The information on markers is shown in Table 1. The 12-bp repeat polymorphism was mostly bi-allelic with the exception of four chromosomes (three from schizophrenics and one from controls) that contained a three-repeat allele.
Table 1Genotype distributions of bi-allelic polymorphisms from the upstream region of DRD4MarkerdbSNP IDPhysical positionCase or controlnGenotype frequenciesP value for allelic associationP value for genotypic association−1.2 kb120-bp repeat–625826–6260651/11/22/2Case5690.04 0.32 0.64 0.202 0.377 Control5700.06 0.34 0.61 −1106 T>Crs936460626199T/TT/CC/CCase5690.79 0.20 0.02 0.522 0.644 Control5700.76 0.22 0.02 −906 T>Crs3758653626399T/TT/CC/CCase5690.66 0.31 0.03 0.116 0.223 Control5690.62 0.34 0.05 −809 G>Ars936461626496A/AA/GG/GCase5620.03 0.33 0.64 0.018 0.032 Control5640.06 0.35 0.59 −616 G>Crs747302626689C/CC/GG/GCase5650.09 0.44 0.47 0.769 0.257 Control5680.10 0.39 0.50 −521 T>Crs1800955626784T/TT/CC/CCase5660.34 0.48 0.19 0.371 0.377 Control5690.34 0.50 0.16 −376 C>Trs916455626929T/TT/CC/CCase5610.01 0.18 0.81 0.732 0.838 Control5620.01 0.19 0.80 −291 C>Trs916457627014T/TT/CC/CCase5650.01 0.21 0.79 0.518 0.080 Control5660.02 0.19 0.79 Exon 112-bp repeatrs4646983627392–6273911/1a1/2a2/2aCase5680.02 0.25 0.73 0.512 0.248 Control5690.03 0.21 0.76 aAlleles are coded by the number of repeats. Four subjects (three from the schizophrenia and one from the control group) with three repeats are omitted from this table. Significant results are shown in bold type
Allelic and genotypic distributions of the promoter SNP −809G>A differed significantly between cases and controls (P=0.018 and 0.032 for allelic and genotypic comparisons, respectively). In the exploratory haplotype window analysis, the two-SNP haplotype [(−1106C) − (−906C)] tended to be over-represented in schizophrenia (frequencies in cases and in controls were 0.70 and 0.66, respectively, P=0.0656) (data not shown).
In LD analysis, D′ values were close to 1 in the distal [(120-bp repeat) − (−1106T>C) − (−906T>C)] and proximal [(−376C>T) − (−291C>T) − (−12-bp)] segments of the upstream region, with each defined as a haplotype block, but it did not hold for the region in between (Fig. 2). Decay of LD in this short stretch (1.2 kb) may imply a high local recombination rate. Therefore, in addition to conventional haplotype-window analysis, we employed logistic regression analysis, which can test a combinatorial effect of multiple SNPs simultaneously. A combination of four polymorphisms (−809G>A, −616G>C, −219C>T and 12-bp repeat) appeared to affect susceptibility to schizophrenia (Table 2).
Fig. 2Haplotype organization of DRD4 in Japanese. The haplotype block pattern was constructed by the Haploview program using the genotype data from both case and control samples (1,140 subjects). The number in each cell represents the LD parameter D′ (×100), blank cells mean D′=1. Each cell color is graduated relative to the strength of LD between markers, which is defined by both the D′ value and confidence bounds on D′. Strong LD was observed in the discrete distal [(120-bp repeat) − (−1106T>C) − (−906T>C)] and proximal [(−376C>T) − (−291C>T) − (12-bp repeat)] segments, but not in the intervening regionTable 2Logistic regression analysis in the upstream region of DRD4MarkerβSEWald’s χ2P valueOROR 95% CI−809G>A0.346 0.127 7.384 0.007 1.414 1.10–1.82−616G>C0.247 0.125 3.872 0.049 1.280 1.00–1.64−291C>T1.031 0.400 6.649 0.010 2.804 1.28–6.2412-bp repeat−0.725 0.274 6.988 0.008 0.484 0.28–0.83β Logistic regression coefficient in the model, SE standard error of the coefficient, Wald χ2 Wald statistic to test significance of the coefficient, OR the odds ratio, CI 95% confidence interval of the odds ratio
Discussion
In this study of the DRD4 gene, we focused on the upstream region, where associations with psychiatric phenotypes have been previously reported. However, genetic variants in this region do not show consistent association with schizophrenia. This may have been due to insufficient statistical power or inadequate interrogation of genomic variations in some studies. In addition, the differences in marker sets between studies increase the ambiguity when interpreting data. To minimize these problems, we adopted the thorough approach of re-sequencing and genotyping all validated polymorphisms.
Both upstream region variants, the single marker −809G>A and a multi-marker combination, displayed association with schizophrenia in this study. The SNP −521T>C, which was previously reported to show association with schizophrenia in an independent Japanese sample set (Okuyama et al. 1999), was not significant in our data set, as in other population data sets (Ambrosio et al. 2004; Jonsson et al. 2001; Segman et al. 2003). Since it is conceivable that particular combinations of multiple variants may confer an enhanced susceptibility to schizophrenia, we performed a logistic regression analysis to test all SNPs in the upstream region for association. This approach may be more appropriate than conventional haplotype analysis, when examining the upstream region of the DRD4 gene where there is evidence of LD decay in a relatively short genomic stretch and most markers do not correlate with each other. Results appear to indicate a synergistic interaction among the four promoter polymorphisms in the DRD4 gene. These results should be treated with caution since there are limitations to the present association study. Firstly, this is a case-control study, where false positive findings due to population stratification can occur. Analysis of this sample set using STRUCTURE software (Pritchard et al. 2000) detected no evidence of population stratification (Shimizu et al. 2006). To address this issue further, we also performed a transmission disequilibrium test. This testing failed to replicate the association of −809G>A in a sample set of 80 complete schizophrenic trios (data not shown). This sample size would have only a limited statistical power to detect true association. Secondly, the significance of the −809G>A polymorphism in the promoter region disappeared after Bonferroni correction for multiple testing. Thirdly, the sample size is obviously limited and so the insignificant result for −521T>C needs to be interpreted cautiously. The sample size in this study had a power of 0.59 to detect a susceptibility variant with a relatively small effect (relative risk = 1.2 and 1.44 for heterozygotes and homozygotes, respectively), when the allele frequency is 0.41. This frequency equals that of SNP −521C, whose association with schizophrenia has been previously reported (Purcell et al. 2003; Xing et al. 2003).
In conclusion, to our knowledge, this is the first study that highlights a possible combinatorial effect of promoter SNPs in the DRD4 gene. It will be interesting to determine whether this multi-marker association can be confirmed in independent data sets, where samples are well-defined in terms of clinical variables such as symptoms, age-at-onset, severity, medication type, and response to antipsychotic treatment. Furthermore, it will be important to examine the functional effects of variant combinations, to understand the mechanisms by which they increase or decrease susceptibility to schizophrenia.

dopamine d4 receptor
re-sequencing
promoter region
logistic regression analysis
linkage disequilibrium
haplotype block


Should medical students learn to develop a personal formulary?

Objective This study was performed to determine whether students who are trained in developing a personal formulary become more competent in rational prescribing than students who have only learned to use existing formularies.
Introduction
Many medical schools in developed as well as developing countries are changing their undergraduate education in pharmacotherapy from classic lecture-based learning to problem-based learning (PBL). They are moving away from the presentation of classic pharmacological knowledge on medicines towards promoting problem-solving and prescribing skills [1, 2]. The World Health Organisation (WHO) Guide to Good Prescribing and several training courses on problem-based pharmacotherapy teaching have contributed to this development [3, 4].
The WHO Guide to Good Prescribing gives medical students a normative model for therapeutic reasoning and prescribing and provides a six-step guide to the process of rational prescribing: (1) define the patient’s problem, (2) specify the therapeutic objective, (3) choose a (drug) treatment, (4) write the prescription and start the treatment, (5) give patient information and warnings, and (6) monitor the treatment. One of the key principles of the WHO approach is the division of step 3 [choose a (drug) treatment] into two steps. Step 3a, consider the suitability of a standard (p-drug) treatment for the disease in general, and step 3b, verify its suitability for the particular patient and alter the (drug) treatment if necessary. Consequently, undergraduate students are taught how to define their own p-drugs and are encouraged during their medical education and clinical activities to develop a personal formulary [5].
The WHO method emphasises that future doctors should master both steps of the drug-selection process. Selecting a p-drug trains students in pharmacological, clinical and epidemiological principles and in collecting and comparing evidence. By considering various treatment alternatives when developing their personal formulary, students are able to choose an alternative drug treatment more rationally when the drug of first choice is not suitable for a particular patient; for example, because of contraindications or side effects. In addition, by considering treatment alternatives, students are able to evaluate the information provided about new drugs during their professional career more critically.
Application of the WHO method has improved the prescribing skills of undergraduate medical students [4]. However, it is not known to what extent the p-drug concept and developing a personal formulary has contributed to this improvement. Some have argued that existing formularies and standard treatment guidelines, developed by experts, would lead to better prescribing than would a personal formulary developed by students. Furthermore, doctors are usually expected to work within the limitations of existing hospital formularies or practice guidelines. Despite the fact that teaching the p-drug concept and developing a personal formulary is a challenging and time-consuming task, we hypothesise that training in developing a personal formulary would lead to increased prescribing skills compared with using an existing formulary.
Therefore, the aim of this study was to determine whether there is a difference in prescribing skills between medical students who have been trained in creating and using a personal formulary and those who have been trained in using existing formularies only.
Materials and methods
Study design
A randomised controlled pre and posttest design with three arms was implemented (Fig. 1). All students participated in a pretest. After the pretest at each university, students were randomly divided into three equally sized groups: a personal formulary (PF) group, an existing formulary (EF) group and a control group. Students in the PF and EF groups received two different training programmes before taking the posttest. The control group received no additional training and was given only the pre- and posttests.
Fig. 1Study design: PF personal formulary group, EF existing formulary group and C control group. Clinical subjects: A  essential hypertension, B  osteoarthritis, C  acute bronchitis and D  gastroenteritis. Complexity levels: 1  straightforward case of a middle-aged adult patient, 2  patient with a contraindication to a generally accepted drug of first choice, 3  severe case and  4 case without effect of previous treatment and side effects
After the posttest, a structured questionnaire with 21 questions was administered to identify differences in the type of teaching students received prior to the study. The questions were about two major teaching issues: curriculum type; and teaching methods in pharmacology, clinical pharmacology and pharmacotherapy. Other questions were designed to verify whether the universities had carried out the study in full accordance with the standard protocol. The study was approved by the educational research committee of the VU University Medical Center (VUMC).
Study population
Six hundred and nine undergraduate medical students from eight countries were selected on a voluntary basis (Vellore in India, Yogyakarta in Indonesia, Maastricht in the Netherlands, Kazan in the Russian Federation, Bratislava in Slovakia, Cape Town in South Africa, La Laguna in Spain and Aden/Sana’a (jointly) in Yemen). All students had passed their pharmacology examinations (basic pharmacology and clinical pharmacology) and were about to start their clinical internships. Participation in the study was in addition to their normal curriculum. All students were informed in advance about the general aim of the study and the method of testing. Cross-contamination of the students in the study groups and the control group was prevented by giving the students in the control group the opportunity to participate in a training programme after the study.
Materials
For the intervention and tests, 16 written patient cases were developed using four clinical subjects (A–D), each with a different level of complexity (1–4) (Fig. 1). All cases were formulated according to a standard design:
General patient information (e.g. age, gender, allergy, occupation and pregnancy)Summary of previous and current diseases and treatments (comorbidity and comedication)Extensive description of the present history and a physical examinationDiagnosis
Each case differed in disease severity and complicating factors such as age, comorbidity and comedication, drug allergy, pregnancy and breast feeding. All three study groups received a copy of the British National Formulary (BNF) as a drug reference [6]. In addition, the PF group received the WHO Guide to Good Prescribing, whereas the EF group received an adapted version of the guide without the chapters about the p-drug concept. The EF group also received the Oxford Formulary, containing treatment advice for symptoms and diseases that occur frequently in general practice, including the four clinical subjects used in this study [7].
Intervention
The training course for the PF and EF groups lasted for 5 weeks. The intervention for both groups consisted of an introductory session and four training sessions (Fig. 1)—one per week for about 2 h. The chosen teaching time was based on earlier experiences with teaching programmes using the PF concept [4, 8]. In the introductory session, the WHO step-wise approach to prescribing drugs, was explained. However, the p-drug concept was explained only to the PF group. In each training session, two written patient cases of one clinical subject were discussed. Students completed a treatment plan form, following the six steps of the WHO model. The students prepared for the sessions by studying the treatment of the clinical subject but in different ways. The PF group was asked to make an inventory of available treatments and select a p-drug from this group (on the basis of efficacy, safety, suitability and cost), whereas the EF group was asked to study the relevant treatment guidelines of the Oxford Formulary. One facilitator per university supervised the sessions of both PF and EF groups only by guiding the discussions and clarifying aspects of the case if necessary. Facilitators were specifically instructed not to provide solutions to the patient problems but only to advise students on how to solve these themselves. All facilitators had received specific training on this teaching methodology in a research meeting at the VUMC. The control group received no additional training between the pre- and posttest but proceeded only with their routine curriculum.
Test and scoring
The pre- and posttests consisted of four written patient cases. Each case contained a different clinical problem of a different level of complexity (Fig. 1). The combination between the clinical problem and level of complexity were randomly chosen. For each case, the students had to complete a treatment plan form. They were allowed to use any reference materials but not to consult one another. Directly after the posttest, the answer sheets for both pre- and posttests were blindly scored by the facilitator in a standardised manner using a 5-point scale (0 = no answer, 1 = poor answer, 2 = arguable answer, 3 = acceptable answer and 4 = good answer). Prior consensus about the scoring system had been reached between all facilitators during the research meeting at the VUMC. The scores were sent to the researchers at the VUMC for analysis. Individual student scores were not used by the participating universities for formal student assessments.
Statistical analysis
The results were analysed in SPSS 13.0 for Windows. The scores on the six steps of the WHO model were clustered into four prescribing skills: treatment choice, prescription writing, patient information and treatment monitoring. The differences between groups were analysed by analysis of variance (ANOVA) with the university as a factor, as well as separately. For pair-wise differences between the three groups, a multiple comparison procedure was applied. Significance was accepted at a two-tailed p < 0.05. Only the results of students who finished both pre- and posttest were included in the analysis.
Results
A total of 609 students participated. Five hundred and eighty-three students (96%) completed the pre- and posttests:194/203 (95.6%) students from the PF groups, 198/203 (97.5%) from the EF groups and 191/203 (94.1%) from the C groups. The three groups did not differ in their averages for previous examination results and pretest scores. The increases in the mean scores of the PF group (0.93, 23%) and the EF group (0.78, 19%) were both significantly higher than in the C group (0.23, 6%) (Table 1). In addition, the increase in the PF group was significantly higher than in the EF group (Table 1). Within this total, the differences were significant for three of the four prescribing skills (treatment choice, patient information and monitoring treatment but not prescription writing) and for three of the four complexity levels (straightforward case, cases with a contraindication to first-line drugs and severe cases but not cases with no effect of previous treatment and side effects present) (data not shown).
Table 1Pre- and posttest scores per university NujmberPretestPosttestPFEFCPFEFCYemen730.410.350.332.98ab1.56b0.46Russia880.830.910.871.83ab1.67b1.03Indonesia751.121.201.342.45ab1.98b1.42Netherlands642.202.062.253.04b3.06b2.63cSlovakia722.272.132.123.203.122.90cIndia722.172.392.232.192.51b2.08Spain732.382.332.282.612.76b2.36South Africa662.542.482.383.022.972.81cAll Increase5831.711.621.692.64a,b 0.93 (23%)2.40b 0.78 (19%)1.92c 0.23 (6%)PF personal formulary group, EF existing formulary group, C control groupaPF > EF (p < 0.05), bPF, EF > C (p < 0.05), cC > pretest (p < 0.05)
Results of medical students from the eight universities are listed separately (Table 1). In Yemen, the Russian Federation and Indonesia, pretest scores were lower than in the other five universities. In addition, the increase in their scores was significantly higher in the PF groups than in the EF groups. The differences among the PF and EF groups were also significant for all levels of complexity and all four prescribing skills (Fig. 2). In the remaining five schools, there was no significant difference between the PF and EF groups.
Fig. 2Pre- and posttest scores for different prescribing skills of students from countries with a traditional curriculum (Yemen, the Russian Federation and Indonesia). PF personal formulary group (–), EF existing formulary group (.....), C control group (- -). Posttest scores are annotated, # = PF > EF
The results of the questionnaire about the type of teaching that students received indicated that Yemen, the Russian Federation and Indonesia had a predominantly classic curriculum. On average, the participating students from these universities had received 90 h (range 52–160 h) of pharmacology teaching by lectures, 23 h (range 3–50 h) of clinical pharmacology teaching also by lectures and no explicit pharmacotherapy teaching. These students were not familiar with PBL but were somewhat accustomed to discussion in small groups. The remaining five universities had a predominantly problem-based teaching curriculum. The participants had received an average of 43 h (range 6–100 h) of pharmacology teaching by lectures, including small group teaching; 24 h (range 6–40 h) of clinical pharmacology teaching, mainly by small group teaching; and no explicit pharmacotherapy teaching. The students from these universities were all familiar with PBL and were all accustomed to discussion in small groups. Some of these students already had some clinical experience (Slovakia and Spain). Except for the presence (or absence) of PBL, no other differences in teaching could be identified between any of the eight universities. No specific differences between universities in the way the study was performed or indications for possible bias were identified.
Discussion
The results of this study show that developing a personal formulary and using an existing formulary both increase the competence of medical students in rational prescribing. However, the overall differences between the PF group and the EF group (23% vs. 19% increase in mean score, p < 0.05) should be interpreted with caution. It is tempting to conclude that teaching students to develop a personal formulary adds to their prescribing skills. However, this effect was only visible in the universities in Yemen, the Russian Federation, and Indonesia. No significant differences between PF and EF scores were found in the universities in the Netherlands, Slovakia, Spain, India or South Africa. A possible reason could be the high pretest scores in the last five universities, which reduced the likelihood of a significant increase after a relatively short intervention period of 5 weeks. A similar effect was seen in the first multicountry study where universities with a problem-based curriculum showed less benefit from a short intervention course than those without a problem-based curriculum [4].
An important question is why these five universities had such high initial scores? The questionnaire was specifically designed for the purpose of this study, and the only explanation that emerged was that students from these five universities had received problem-based teaching and were therefore familiar with the process of solving patient problems. In the universities with lower pretest scores, classic teaching was paramount and students had less experience with solving patient problems.
This finding is in accordance with the fact that successful clinical problem solvers possess comprehensive knowledge, but the way they organise and understand their knowledge is even more critical. Modern teaching is aimed at creating both a knowledge structure and the necessary “search-and-retrieve” strategy for responding to complex diagnostic problems [9, 10]. Students who were already trained in this way via problem-based curricula seemed better able to transfer this ability to therapeutic problem solving and did not show an additional increase in such skills when they participated in our study. However, students from universities with a classic curriculum quickly assimilated the p-drug concept, and their results came very close to those of the other five universities, even after a short intervention of only 5 weeks.
The limitations of our study need to be addressed. The voluntary participation of students may have created a positive bias for the total group, but allocation to the intervention groups was randomised and therefore the comparison was unbiased. In addition, to ensure baseline comparability among the groups, previous results on university examinations were averaged for the three groups. The scoring by the medical schools was blinded, with the pre- and posttest forms of all three groups being mixed during the scoring. Cross-contamination between the study and control groups was prevented by explaining to the students the importance of not exchanging any information and by reassuring the control group that it would receive the training after finishing the study.
Therefore, taking the above-mentioned limitations and considerations into account, it can be concluded that training in developing and using a personal formulary or in the use of an existing formulary may increase the rational prescribing of medical students. However, with regard to universities with a general problem-based curriculum, training in the development of a personal formulary is probably not worth the additional effort. In these circumstances, pharmacotherapy teaching can be based on existing formularies instead of a personal formulary.
In this international study, only the short-term effects on rational prescribing of developing a personal formulary or using an existing formulary have been studied. Despite the fact that the long-term effects are unknown, it can be hypothesised that after developing a personal formulary, students may judge new drugs more effectively and be more capable of deciding whether or not to include these drugs into their (personal) formulary. Furthermore, the results may diminish the concerns ventilated by some clinical pharmacology teachers regarding the changes in the style of undergraduate medical curricula from classic to PBL teaching [11, 12].
In practice, this implies that the decision to organise a teaching programme for medical students on how to develop and use a personal formulary needs to be based on an initial assessment of both the students and the curriculum. The same may be true for postgraduate and continuing education of doctors who have been trained in the classic manner [13, 14]. A screening test, such as the pretest in this study, could perhaps be used to identify universities, students or prescribers that would benefit most from this type of training.

personal formulary
rational prescribing
prescribing
drug treatment
undergraduate medical education