Acute success and short-term follow-up of catheter ablation of isthmus-dependent atrial flutter ; a comparison of 8 mm tip radiofrequency and cryothermy catheters Objectives To compare the acute success and short-term follow-up of ablation of atrial flutter using 8 mm tip radiofrequency -LRB- RF -RRB- and cryocatheters . Introduction Atrial flutter is a common arrhythmia -LSB- 1 -RSB- , difficult to suppress with medication -LSB- 2 -RSB- , and is associated with significant symptoms . Since it was first proposed -LSB- 3 -- 5 -RSB- , ablation for atrial flutter has increasingly been used for its therapy , especially since induction of bidirectional cavo-tricuspid isthmus block was shown to be associated with better immediate outcomes and lower recurrence rates -LSB- 6 -- 11 -RSB- . Failure to successfully ablate atrial flutter in the long-term may be due to particular anatomic problems -LSB- 12 -- 14 -RSB- , poor catheter stability in this region , and incorrect interpretation of isthmus block . Important developments in the field of atrial flutter ablation have been a better understanding of the anatomy of the isthmus , refinements of the definition of bidirectional isthmus block , and the arrival of new catheter technology . Numerous studies have compared different energy types , different catheter tip sizes and different energy settings -LSB- 15 -- 19 -RSB- , as well as the use of advanced cardiac mapping systems -LSB- 20 -RSB- . The preferred tools now are 8 mm , or irrigated tip catheters -LSB- 21 -RSB- . From a recent meta-analysis it would appear that these two technologies are equally effective with acute success quoted at 84 % and 85 % for the primary catheter chosen before changeover , whereafter final success rates of up to 99 % can be achieved -LSB- 22 -RSB- . Not all studies have this high success rate and the preponderance of studies on ablation of atrial flutter suggest that these high success rates are not repeated in all centers -LSB- 23 -RSB- . Also of importance is a clinical recurrence rate , in the face of acute success , of at least 5 to 12 % when using radiofrequency -LSB- 19 , 24 , 25 -RSB- . Disadvantages of radiofrequency -LRB- RF -RRB- energy include pain , overheating with `` popping '' , char formation and risk to coronary arteries . Cryoablation is a relatively recent addition to the transvenous ablation armamentarium and has been shown to be comparable to RF for some arrhythmias . It may have some advantages over RF , especially as regards discomfort during ablations -LSB- 17 , 26 -RSB- . The development of 6 and 8 mm tip cryocatheters has increased the interest in this technology for atrial flutter ablation . As far as we are aware there have been no randomized comparative studies published comparing 8 mm tip cryocatheters with 8 mm tip RF catheters . Methods Patient population Consecutive patients with ongoing symptoms and documented atrial flutter with or without fibrillation were included . At least one recent episode of atrial flutter -LRB- within the last 6 months -RRB- was documented on a 12 lead ECG and was suggestive for isthmus dependency . Patients with drug-induced flutter -LRB- and prior AF -RRB- could be included , and had their drug therapy continued after ablation . Patients were excluded if they had undergone a previous flutter ablation , if thrombus was present in the atria , or after previous cardiac surgery for valvular or congenital heart disease . Baseline investigations included a standard echocardiogram , a simple questionnaire asking about clinical wellbeing and a subjective assessment of the arrhythmia burden both in terms of duration and frequency . The study was approved by the ethics committee of our institution . All patients signed written informed consent . Assessment before ablation Antiarrhythmic drugs , except for AV nodal slowing agents , and amiodarone , were discontinued at least five half-lives prior to ablation . Patients were studied in the fasting , post-absorptive state . A coronary sinus -LRB- CS -RRB- catheter was inserted through the left subclavian vein , and a multipolar circular right atrial catheter with alternating 2 -- 10 -- 2 mm interelectrode distance was positioned from the right groin , with the tip positioned immediately lateral to the planned position of the isthmus line and anterior to the crista terminalis . Heparin 100 U/kg was given and a further 5 U/kg given if the procedure lasted longer than 180 min . If the patient was in sinus rhythm , isthmus conduction was confirmed by pacing . If flutter was present , entrainment was performed to confirm isthmus dependence , and the patient left in flutter . If AF was present , the patient was cardioverted after a transoesophageal echocardiogram , and then isthmus conduction confirmed . Absence of isthmus conduction or non-isthmus dependence was not seen in the selected patients . No induction of arrhythmia was attempted if patients had sinus rhythm . Radiological assessment of the right atrial isthmus was made in a right anterior oblique -LRB- RAO -RRB- 30 ° , and a left anterior oblique -LRB- LAO -RRB- 45 ° view -LRB- each with 40 cc at 18 cc/s -RRB- . Angiograms were acquired digitally to allow for post-hoc analysis . The treating physician was able to view the angiographic findings to optimize the planned ablation line . The isthmus length was assessed from the inferior hinge point of the tricuspid valve to the IVC at the end of atrial diastole -LRB- the frame before opening of the tricuspid valve -RRB- -LSB- 14 -RSB- . Morphology was assessed visually as to the presence of a Eustachian valve or a recess , as well as to the general shape i.e. flat or concave . Ablation procedure The catheters were a 9Fr 8 mm tip catheter -LRB- FreezorMax , Cryocath Technologies Inc , Kirkland , Canada -RRB- with a cryoconsole for the cryoablation group , and a 7Fr 8 mm tip single sensor catheter -LRB- EPT Blazer II , Boston Scientific , Natick , MA , USA -RRB- with an EPT-1000XP generator for the radiofrequency group . A large curve was initially selected in both groups , with change out of catheter curve during the study only as necessary . Applications of − 75 ° , for 4 min were given with cryothermy , and applications of 60 Watt , for 60 s , targeted at 60 °C for RF . Lines were made with discrete applications between the tricuspid valve and the inferior vena cava at an approximately 6 o'clock position in LAO 45 ° , unless otherwise dictated . If termination of atrial flutter occurred , or if the patient was in sinus rhythm , continuous pacing from the proximal coronary sinus was employed to continually assess isthmus conduction . After the first line , a new assessment of conduction was performed . If conduction over the isthmus remained present , gaps were sought . If there was still isthmus conduction , a slightly more medial or lateral line was made . In no patient was an attempt made to perform a septal ablation line . Final assessment of acute block was confirmed after 30 min waiting . The end point for successful ablation was induction of complete bidirectional isthmus block , defined as the presence of reversal of activation on the lateral and septal wall when pacing the CS os and low lateral RA , the presence of widely split potentials along the isthmus line , by activation mapping across the isthmus , and by differential pacing . All 4 were required before calling the ablation successful . In the case where bidirectional block was not achieved , ablation was stopped when no large , sharp signals could be identified over a broad area of the isthmus . As pain perception was assessed , sedation was standardized . Before venous puncture 5 mg of diazepam was given intravenously , and repeated at the patient 's request . Fentanyl 50 μg intravenously was given when the patient requested pain control and the physician considered this necessary . This was repeated as needed . Dosages of both diazepam and fentanyl were recorded . In the initial 40 patients creatine kinase -LRB- CK -RRB- and CK-MB were taken before the procedure , 2 and 24 h after the start of the procedure . For the final 22 patients the laboratory had changed the measurement to CK mass . We then modified the protocol to measure CK-MB mass , Troponin T , and Myoglobin at 4 and 24 h after the start of the procedure . No crossover , other than in catheter curve , was allowed in an attempt to remove any possible bias . Change over to an irrigated tip ablation catheter was also not allowed . In patients in whom no block could be induced , a repeat procedure was scheduled not earlier than 6 weeks after the initial ablation , at the physicians ' discretion . The choice of energy source at that time was at the physicians ' discretion . Patients were all questioned with regard to pain perception using a visual analogue score , where patients are shown a line from 0 to 10 , where 0 is no pain , and 10 is the highest pain level imaginable , and were asked to point to the position on the line where their pain level during ablation was . Follow-up All patients received an event-recorder for the first 6 weeks after the procedure and were requested to send at least daily strips as well as strips made during symptoms . Patients visited the outpatient clinic 6 weeks after the procedure . After this period all patients were asked to report symptoms and if these were present were given a further event monitor until documentation of symptoms was obtained . If at all possible a 12 lead ECG was also obtained . A second assessment with a questionnaire was performed after 3 months , again asking a question about general clinical well being and also symptom burden in regard to duration and frequency . Clinical files were followed up after 9 months . Statistical analysis For patients in whom another ablation was performed -LRB- AVNRT in two , and pulmonary vein ablation in six others -RRB- procedure and fluoroscopy times were limited to the flutter approach , which was done first , including 30 min waiting time . Biomarker assessment was not done in these patients . Continuous variables are expressed as mean ± standard deviation , with medians as necessary . Parametric and non-parametric tests were used where appropriate . A p-value of < 0.05 was considered significant . Results Patient data 62 patients were included as planned , with clinical characteristics as outlined in Table 1 . There were no significant differences in any of the parameters between the group assigned to radiofrequency -LRB- RF group -RRB- versus that assigned to cryotherapy -LRB- cryo group -RRB- . The large number of patients with prior AF is due to the fact that we had initially taken a decision to perform isthmus ablation first in all patients with AF who had shown typical atrial flutter on any 12-lead ECG , prior to performing a left atrial procedure , initially in a separate procedure and only later in the same session . Table 1Demographics AllCryoRFp valueNumber623230Age -LRB- years ± SD -RRB- 56 ± 1055 ± 1156 ± 9NSMale/female27/527 / 528/2NSAtrial fibrillation history47 -LRB- 76 % -RRB- 25 -LRB- 78 % -RRB- 22 -LRB- 73 % -RRB- NSCryo Cryoablation ; NS not significant ; RF radiofrequency ; SD standard deviation Angiographic data Right atrial angiography was not performed in 4 patients because of mild renal dysfunction or allergy to contrast material . The angiogram was of insufficient quality in eight others . The mean isthmus length was 35.2 ± 14.6 mm and its topography was assessed as being flat or only mildly concave in 28 , markedly concave in 19 and showed a pit or aneurysm in 10 . A clear Eustachian valve was seen on six angiograms . Ablation data Assessment of acute results showed bidirectional isthmus block , using the criteria mentioned , in 47 of 62 patients -LRB- 76 % -RRB- . This was in 25 of 30 patients -LRB- 83 % -RRB- of the RF group and in 22 of 32 -LRB- 69 % -RRB- of the cryo group -LRB- NS -RRB- . In 1 patient in the RF group the procedure was terminated because of recurrent AF with early recurrence and inability to assess isthmus block . This patient was taken as a failure which was confirmed at the time of a subsequent AF ablation . Procedural data for all patients are given in Table 2 . Table 2Procedure data and recurrent arrhythmias AllCryoRFp valueNumber623230Application number22 ± 1318 ± 1025 ± 160.05 Ablation time -LRB- s -RRB- 2,742 ± 1,9303,792 ± 1,9001,459 ± 950 < 0.001 Acute success47 -LRB- 76 % -RRB- 22 -LRB- 69 % -RRB- 25 -LRB- 83 % -RRB- NSSingle line47 -LRB- 76 % -RRB- 25 -LRB- 78 % -RRB- 22 -LRB- 73 % -RRB- NS2 lines9 -LRB- 15 % -RRB- 2 -LRB- 6 % -RRB- 7 -LRB- 24 % -RRB- 0.0733 lines4 -LRB- 6 % -RRB- 3 -LRB- 9 % -RRB- 1 -LRB- 3 % -RRB- NSReversal of block during application7 -LRB- 11 % -RRB- 6 -LRB- 19 % -RRB- 1 -LRB- 3 % -RRB- 0,091 Reversal of block during 30 min6 -LRB- 10 % -RRB- 1 -LRB- 3 % -RRB- 5 -LRB- 7 % -RRB- NSIsthmus length -LRB- mm -RRB- 35 ± 1535 ± 1736 ± 11NSSheath usage7 -LRB- 11 % -RRB- 0 -LRB- 0 % -RRB- 7 -LRB- 23 % -RRB- < 0.005 Recurrent arrhythmiasFlutter -LRB- typical -RRB- After success1 -LRB- 2 % -RRB- 0 -LRB- 0 % -RRB- 1 -LRB- 4 % -RRB- NS After failure11 -LRB- 73 % -RRB- 7 -LRB- 70 % -RRB- 4 -LRB- 50 % -RRB- NSFlutter -LRB- atypical -RRB- 1 -LRB- 2 % -RRB- 0 -LRB- 0 % -RRB- 1 -LRB- 3 % -RRB- NSAtrial tachycardia5 -LRB- 8 % -RRB- 2 -LRB- 6 % -RRB- 3 -LRB- 10 % -RRB- NSAtrial fibrillation28 -LRB- 45 % -RRB- 13 -LRB- 41 % -RRB- 15 -LRB- 50 % -RRB- NSThe numbers are given with the standard deviation.Cryo Cryoablation ; NS not significant ; RF radiofrequency In the successful patients the number of applications to ensure block in the whole group was 17 ± 11 . It was 23 ± 13 in the RF group and 12 ± 6 in the cryo group -LRB- p < 0.005 -RRB- . Total ablation time was 1,283 ± 777 s and 2,905 ± 1,245 s -LRB- p = 0.0001 -RRB- . In those in whom bidirectional block could not be achieved , the total number of applications was 33 ± 14 . For RF and cryo the values were 39 ± 21 applications with a total time of 2,724 ± 1,102 s vs 29 ± 8 applications with a time of 5,873 ± 1,337 s -LRB- p < 0.03 and < 0.0011 , respectively -RRB- . In 22 of 30 in the RF group and 25 of 32 in the cryo group , a single line at approximately 6 o'clock was drawn . In the RF group two lines were drawn in seven , and three in one . In the cryo group two lines were made in four , and three lines in three . The need to draw more than one line was associated with failure of the procedure in four of eight in the RF group and five of seven in the cryo group -LRB- NS -RRB- . Short lived block , either recurring during the application or immediately thereafter , occurred in one patient in the RF group and in six in the cryo group , with a trend to statistical significance -LRB- p = 0.091 -RRB- , with it being a predictor of failure if it occurred more than three times . In five patients in the RF group and one patient in the cryo group , conduction recurred later during a waiting period -LRB- with a median of 15 min -RRB- requiring further applications -LRB- NS -RRB- . In only one patient in the RF group was late recurrence associated with failure to induce bidirectional block . In no patient in whom bidirectional block was present at the end of the 30 min waiting period did isoprenaline change this . The average power applied in the RF group was 52 ± 6 W . In the RF group the signal was significantly diminished at the end of each application whether isthmus block was present or not , while there tended to be preservation of signals on the cryoablation catheter after ablation across the isthmus until block occurred . Only then were low voltage signals seen . Failures were not significantly associated with length of the isthmus -LRB- 39.2 ± 23.5 vs 34 ± 9.0 mm in success , although there was a trend to this -LRB- p = 0.12 -RRB- There was no significant difference in anatomy between the two groups . Procedure data and complications The overall procedure time was 160 ± 49 min , with no difference between 170 ± 48 min in the RF group and 151 ± 49 min in the cryo group . Overall fluoroscopy times were 28 ± 14 min , with a difference between 33 ± 15 min in the RF group and 23 ± 11 min in the cryo group -LRB- p < 0.02 -RRB- . Change of catheter curve occurred in one patient in the RF group from large to standard curve . An SL1 sheath -LRB- Daig , Minnetonka , MN , USA -RRB- was used in seven patients in the RF group for stability , while no sheath usage occurred in the cryo group -LRB- p = 0.005 -RRB- . During the procedure , six patients required cardioversion for induced AF , four in the RF group and two in the cryo group -LRB- NS -RRB- . There were two small pericardial effusions seen on echocardiography without further significance -LRB- one in each group -RRB- . For the initial 40 patients there was a significantly higher peak CK and CK-MB after cryo -LRB- Table 3 -RRB- . This remained so after 24 h. For the last 22 patients we observed the same for CK-MB mass , but not for Troponin T -LRB- Table 3 -RRB- . Table 3Biomarkers after catheter ablation AllCryoRFp valueProcedure valuesCK -LRB- U/l -RRB- 141 ± 96184 ± 10296 ± 600.02CK-MB -LRB- U/l -RRB- 27 ± 1636 ± 1718 ± 80.011CK-MB mass -LRB- μg/l -RRB- 18 ± 2133 ± 244.4 ± 1.20.004 Troponin T -LRB- μg/l -RRB- 0.49 ± 0.320.54 ± 0.380.39 ± 0.27 NSValues after 24 hrsCK -LRB- U/l -RRB- 264 ± 245289 ± 173136 ± 740.022CK-MB -LRB- U/l -RRB- 37 ± 2851 ± 3018 ± 60.011CK-MB mass -LRB- μg/l -RRB- 8.4 ± 7.312 ± 105.16 ± 3.30.07 Troponin T -LRB- μg/l -RRB- 0.44 ± 0.300.54 ± 0.360.38 ± 0.29 NSThe numbers are given with the standard deviation.CK Creatine kinase ; Cryo cryoablation ; NS not significant ; RF radiofrequency In assessing the level of comfort during the procedure and the pain experienced by patients , similar numbers from both groups assessed sedation as adequate -LRB- 67 % for RF and 63 % for cryo -RRB- . The pain scores given at the end of the procedure were not significantly different -LRB- 42.9 ± 24.0 for RF and 43.7 ± 15.8 for cryo -RRB- . Diazepam was given as standard at the beginning of the procedure and as necessary thereafter for discomfort related to having to lie still for protracted periods . The usage of diazepam was statistically similar in both groups -LRB- 7.4 ± 3.4 mg in the RF group and 8.0 ± 3.1 mg in the cryo group -RRB- . However , a significantly higher usage of fentanyl in the RF group was observed -LRB- 70.0 ± 44.9 μg vs 10.0 ± 22.1 μg ; p < 0.01 -RRB- . Follow-up results Patients were followed up for between 90 and 411 days -LRB- 138 ± 81 days , median 90 days -RRB- , which was similar in both groups -LRB- p = ns -RRB- . A total of 6 patients were taking no antiarrhythmic medication prior to ablation and this increased to 13 post isthmus ablation . Recurrent arrhythmias were frequent in both groups of patients . ECG documented recurrent flutter occurred only in one patient from the RF group . A further procedure confirmed recovery of isthmus conduction . This recurrence was seen at 14 months post ablation , whereas he had previously had monthly episodes of flutter . One other patient had symptoms with an apparently non-isthmus dependent flutter documented on ECG on day 1 post ablation and not since , and elected not to have a further procedure . We can not exclude asymptomatic arrhythmias but at least during the first 6 weeks , patients sent in daily event monitor transmissions , which one would hope would have captured at least some asymptomatic recurrences , particularly of atrial flutter . In 11 of 15 patients in whom the initial procedure failed , a redo procedure was performed after the elected period of 6 weeks because of documented recurrent flutter . The redo procedure required a small number of point touch ups in five patients -LRB- three in the RF group and two in the cryo group -RRB- , while more extensive diffuse isthmus applications were required in six -LRB- one in the RF group and five in the cryo group -RRB- . In those undergoing repeat ablation , success was achieved in all and during follow up no recurrent atrial flutter was noted . Previously undocumented atrial tachycardias were also seen in both groups -LRB- three in the RF group , and two in the cryo group -RRB- . ECG documented AF recurrence occurred in 28 patients , at a similar rate in both groups -LRB- 15 in the RF group and 13 in the cryo group -RRB- . The likelihood of asymptomatic recurrences is probably higher for atrial fibrillation but was not the primary focus of this study . Discussion Acute success This study showed an acute success rate of 83 % for an 8 mm tip RF catheter vs. 69 % for an 8 mm tip cryocatheter . This non significant difference is clearly concerning . However , the cryoablation group required significantly less applications to achieve success , with a similar procedural duration , significantly lower fluoroscopy time , and with a much lower requirement for analgesia with fentanyl . Arrhythmia recurrences in the initially successful patients were similar with a very low flutter recurrence rate . The acute success rate with 8 mm tip RF compares favorably with that found in the meta-analysis of Da Costa et al. -LSB- 22 -RSB- . However , the acute results for cryotherapy are lower than in previously published studies . The shorter fluoroscopy time using cryotherapy is related to cryoadherence during applications -LSB- 26 -RSB- . With the catheter firmly attached , no fluoroscopy to check position is required . This is also reflected in the fact that no long sheaths were necessary . A lower requirement for analgesia has previously been described and this study confirms this finding -LSB- 17 , 26 -RSB- . Difficulties during ablation Recurrence during or shortly after ablation occurring more commonly in the cryo group probably relates to a reversible cooling effect at the periphery of the ice ball which recovers during or shortly after termination of ablation , while the central lesion acutely formed is more permanent . The more common recurrence during the waiting period after RF ablation probably relates to a longer reversal time of the acute RF effects -LRB- either edema or temperature effect -RRB- . Cryoablation for flutter Cryoablation may have some advantages over RF in addition to those mentioned above such as less thrombogenicity -LSB- 27 -RSB- , and maintenance of tissue architecture with homogenous well delineated lesions -LSB- 28 -RSB- . A 10Fr , 6.5 mm tip catheter showed acute success of 94 -- 100 % and 6 month recurrence rates of 0 -- 25 % -LSB- 17 , 29 , 30 -RSB- . A system with 7Fr , 6 mm tip catheters showed success in 87 % to 88 % using a septal line , without symptomatic recurrences , but with resumption of isthmus conduction at repeat study at 6 months in 30 -- 35 % -LSB- 31 , 32 -RSB- . Using a 9Fr , 8 mm tip increased success rates to100 % with symptomatic recurrence of 0 % to 10 % and recurrent conduction at 1 to 3 months in 19 % to 32 % -LSB- 32 -- 34 -RSB- . There is some discussion as to whether 3 , 4 or 8 min of ablation are needed for adequate lesion formation -LSB- 33 , 35 -RSB- . Higher peak CK levels , confirmed by the high CK-mass in the last patients , suggest that the damage caused by cryotherapy may be more extensive despite the lower number of applications . Several explanations are possible . The first is that the CK levels may be underestimated after RF , which denatures proteins in another way than cryotherapy . Troponin T levels are more accurate in estimation of myocardial damage , but they also tended to be higher , be it not significantly . This may suggest that lesions are equal with both approaches . Future developments Newer data suggest that ablation per point guided by the maximal voltage may be a useful technique , but this has not been confirmed for cryotherapy -LSB- 36 -RSB- . Study limitations This study was a single centre , largely single operator study involving a relatively limited number of patients with a high population of AF patients , making follow-up rather complicated . In present practice and later in this series , patients with the combination are often treated in one session , which still leaves physicians the choice whether cryo should be used for flutter , following its use for the pulmonary veins -LSB- 37 -RSB- . We only studied the acute and short-term success , while flutter recurrence may happen at more than 4 months of follow-up . While we attempted to exclude asymptomatic recurrences especially in the first 6 weeks , we can not exclude the possibility of these , especially of AF and also late after the ablation . Isthmus conduction recovery is also possible in asymptomatic cases . In the RF group we limited the power to 60 W to ensure safety and this may be too conservative . Further , in our practice routine use of long sheaths was discouraged . At the time of this study use of an 8 mm tip with RF was considered as effective as irrigated tip ablation , so we elected to use this approach for the RF group . More recent studies have shown higher success rates than we obtained and have also suggested alternatives in the approach . These include the use of irrigated tip catheters and long sheaths as well as other technical issues and newer techniques such as maximal voltage guided ablation . A higher success rate for RF would clearly have been even more prejudicial to cryotherapy in this study . We were also extremely critical in our assessment of isthmus conduction , using multiple criteria . Angiograms were also performed in fixed views and not based on other catheter positions as has been suggested by others -LSB- 14 -RSB- . Conclusion Acute success with cryotherapy for atrial flutter ablation , while non-statistically less effective in this study , requires fewer applications and is associated with a significantly lower requirement for pain relief . While our acute results for both cryotherapy and RF may not be as high as those in some comparable studies the recurrence rate was only 2.5 % in the RF group , with no clinical recurrence in the cryo group . While cryotherapy can not perhaps be advocated as first line therapy , it may be useful in certain circumstances . Certainly , if cryoablation is performed for AF , and if the isthmus needs to be ablated , cryotherapy might be used as well -LSB- 37 -RSB- .

O O O O O O B I O O B I O O O O O O O B O B O O O O O O O O O O O O O B I O O O O B O O O O O O O B I O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

arrhythmia
catheter ablation
atrial flutter
atrial fibrillation
radiofrequency
cryothermy


Far field scattering pattern of differently structured butterfly scales The angular and spectral reflectance of single scales of five different butterfly species was measured and related to the scale anatomy . The scales of the pierids Pieris rapae and Delias nigrina scatter white light randomly , in close agreement with Lambert 's cosine law , which can be well understood from the randomly organized beads on the scale crossribs . The reflectance of the iridescent blue scales of Morpho aega is determined by multilayer structures in the scale ridges , causing diffraction in approximately a plane . The purple scales in the dorsal wing tips of the male Colotis regina act similarly as the Morpho scale in the blue , due to multilayers in the ridges , but the scattering in the red occurs as in the Pieris scale , because the scales contain beads with pigment that does not absorb in the red wavelength range . The green -- yellow scales of Urania fulgens backscatter light in a narrow spatial angle , because of a multilayer structure in the scale body . Introduction Many butterflies have vivid , colourful wing patterns , created by rows of partly overlapping scales with intricate spatial structures that scatter incident light . The scattered light can interfere coherently or incoherently . When the scale structures have spatial periodicity , the interference of coherent light waves often results in striking iridescences . The displayed colours are then called structural or physical . Without periodicity , light scattering is incoherent or random , resulting in white scales , unless they contain pigment that selectively absorbs in a certain wavelength range . In the latter case a pigmentary or chemical colour results -LRB- Fox and Vevers 1960 ; Vukusic and Sambles 2003 ; Kinoshita and Yoshioka 2005 -RRB- . Many butterfly species feature structural as well as pigmentary colours -LRB- Rutowski et al. 2005 ; Yoshioka and Kinoshita 2006a -RRB- . Each butterfly wing scale is the cuticular product of a single cell , with a rather flat , unstructured lower scale lamina and a highly structured upper lamina , typically consisting of longitudinal ridges connected by crossribs -LRB- Ghiradella 1998 -RRB- . The fine structure of butterfly scales is highly variable -LRB- Vukusic et al. 2000 -RRB- . For instance , the crossribs of the white scales of pierids are adorned with granules -LRB- Yagi 1954 ; Giraldo and Stavenga 2007 ; Morehouse et al. 2007 -RRB- , the ridges of Morpho scales and many male Pieridae are elaborated into multilayer structures -LRB- Ghiradella et al. 1972 ; Vukusic et al. 1999 ; Kinoshita et al. 2002 -RRB- , and many papilionids and lycaenids have scales with photonic crystal properties -LRB- Vukusic et al. 2002 ; Vukusic and Sambles 2003 ; Kertesz et al. 2006 -RRB- . The scales reflect only part of the incident light flux , and the remaining part is transmitted unless it is absorbed by pigment . Because the scales are arranged in layers on both sides of the wing , incident light suffers reflection and transmission in each layer of the scale stacks , and therefore the wing reflectance is not solely due to backscattering by the top layer , the cover scales , but it is the cumulative effect of the scale stacks on both wing sides . Yoshioka and Kinoshita -LRB- 2006b -RRB- investigated this phenomenon in Morpho wings , and to explain the reflectance spectra of intact wings from the spectra of individual scales they used a simplified scale stack model , assuming that normally incident light rays are also reflected and transmitted normally . Stavenga et al. -LRB- 2006 -RRB- developed a more general model for the reflectance of butterfly wings to interpret reflectance measurements performed on intact as well as partly or completely denuded wings of the small white butterfly , Pieris rapae . The basic assumption of the latter model , that the scales scatter randomly , was however not specifically demonstrated . Knowledge of the spatial and spectral reflectance characteristics of single scales is essential to further develop our understanding of the coloration principles applied by butterflies . In the present study we investigate the scattering properties of wing scales of a variety of butterflies , and we correlate the scattering diagrams with the electron micrographs of the scale structure . We show that white scales of pierid butterflies are approximately random scatterers . Iridescent scales , with multilayer structures , exhibit directional reflection . Whereas some butterfly species appear to have scales with dominant iridescence , other species combine iridescence and scattering properties . Materials and methods Animals We investigated the scales of a variety of butterflies . The small white , P. rapae , was obtained from a continuous culture maintained by Dr J. J. van Loon , Entomology Department , University of Wageningen -LRB- the Netherlands -RRB- . The black jezebel , Delias nigrina , was captured near Bateman 's Bay , Australia . The Morpho aega was purchased . The purple tip , Colotis regina , was received from the butterfly collection of the Royal Museum for Central Africa , Brussels -LRB- Belgium ; curator Dr U. Dall ' Asta -RRB- . The moth Urania fulgens was a gift from Dr Marta Wolff , Entomology group , University of Antioquia -LRB- Medellín , Colombia -RRB- . Angular distribution of scattering by single scales Single scales were isolated by gently pressing the wing of a butterfly on a glass plate . Subsequently , an isolated scale was glued to the tip of a glass micropipette -LRB- tip diameter ca. 5 μm -RRB- . The micropipette was then mounted on a micromanipulator in the optical setup shown in Fig. 1a . The scale was always hanging with the longitudinal ridges of the scale oriented vertically . Light from a xenon lamp was focused on a pinhole with diameter 50 μm , which was subsequently imaged on the scale . The light beam , which had an aperture of < 10 ° , passed a small hole in a screen before it reached the scale . The scale reflected -LRB- that is , back-scattered -RRB- part of the incident light . The angular spread of the reflected light was documented by photographing the light distribution at the white backside of the screen -LRB- Fig. 1a -RRB- . We used a Nikon Coolpix 990 -LRB- Fig. 2 -RRB- and an Olympus DP70 -LRB- Fig. 6 -RRB- . The digital images were processed with MatLab -LRB- Fig. 6 -RRB- . Fig. 1Diagram of the optical system used for measuring the angular distribution of the scale reflectance . Light from a light source was focused on a pinhole , which was imaged on the scale . a A white screen with a small hole was placed in between the imaging lens and the scale . The light reflected by the scale caused a light pattern on the screen , which was photographed . b The light reflected by the scale was collected by a lightguide , which relayed the captured light to a spectrometer . The lightguide was mounted on a motorized stage rotating in the horizontal plane -LRB- top view drawing -RRB- Fig. 2Angular and spectral reflectance of isolated butterfly scales . a -- d White scale of the dorsal forewing of a male Pieris rapae . e -- h White scale from the dorsal forewing of a male black jezebel , Delias nigrina . i -- l Blue scale of a Morpho aega . m -- p Purple scale from the tip of the dorsal forewing of a male purple tip , Colotis regina . q -- t Orange -- white scale of Urania fulgens . First column : scanning electron microscopy of scale cross-sections made by cutting pieces of wing with a razor blade . Bars 1 μm . Second column : photographs of the angular distribution of light reflected by a scale and subsequently reflected by a white screen , where the scale was illuminated with a narrow beam of white light -LRB- see Fig. 1a -RRB- . The scales were glued at the tip of a glass micropipette , which was suspended from above on a micromanipulator ; see the black shadow in the upper part of b and f . The bright spot at the right of the central black hole is due to light forward scattered by the scale . The ellipses in b -LRB- due to the slightly oblique position of the camera -RRB- represent directional angles in steps of 10 ° , from 10 ° to 60 ° , as well as that for 65 ° . The hole in the screen is ca. 8 ° . Third column : angular dependence of the reflectance in the horizontal plane , measured with the setup of Fig. 1b . Fourth column : reflectance spectra of single scales measured with a microspectrophotometer Spectrophotometry The angular dependence of the light scattering by the scale was measured with a lightguide , which was mounted on a rotating motor and connected to a spectrometer -LRB- Yoshioka and Kinoshita 2006b -RRB- . The scales were adjusted so that the scale plane was perpendicular to the light beam , as judged by the symmetrical reflection pattern with respect to the axial direction . The experiments were run using a Labview interface , which allowed the measurement of reflectance spectra in angular steps of 1 ° over a 180 ° angle -LRB- Fig. 1b -RRB- . Angular reflectance curves were calculated for a series of wavelengths with 10 nm interval by sequentially averaging the reflectance spectra over 10 nm wavelength ranges . In addition , the spectral reflectances of single scales were measured with a microspectrophotometer , consisting of a xenon light source , a Leitz Ortholux microscope , and a fiber optic spectrometer . The microscope objective was an Olympus 20 × , NA 0.46 . A white reflectance standard -LRB- Spectralon , Labsphere , North Sutton , NH , USA -RRB- served as the reference in all cases . Electron microscopy Subsequent to the microspectrophotometry , the single scales were prepared for scanning electron microscopy -LRB- SEM -RRB- . Additionally , pieces of wing were cut and put on the SEM-holder in different positions to visualize the upper surface as well as cross-section of the scales . Samples were sputtered with palladium for 5 min with 800 V and 200 mTorr -LRB- Hummer , Alexandria , VA , USA -RRB- . A Philips XL-30 scanning electron microscope with a voltage of 3 kV was used to investigate the scale anatomy . For transmission electron microscopy , wing pieces were prepared as usual . In brief , samples were embedded in agar for better handling , prefixed in 2 % glutaraldehyde in 0.1 M sodium cacodylate buffer , and postfixed in 1 % OsO4/1 .5 % K4 Fe -LRB- CN -RRB- 6 in 0.1 M cacodylate . Subsequent washing with double-distilled water and dehydration with an alcohol series that ended with 100 % , were followed by propylene oxide during 30 min and embedding in Epon . Post-microtomed samples were contrasted with uranyl acetate in methanol for 2 min and lead in water for 1 min , and were then examined with a Philips 201 transmission electron microscope . Results The white scales of the small white , P. rapae , are marked by ovoid beads that adorn the crossribs -LRB- Fig. 2a -RRB- . The angular light scattering of a single scale can be readily visualized with the setup of Fig. 1a , where a beam of white light is focused on a scale via a small hole in a screen . Figure 2b is a photograph of the screen , showing the angular distribution of the light reflected by a white scale taken from the dorsal wing of a male small white , P. rapae . The scale scatters light approximately circular-symmetrically , suggesting that the scale acts as a diffuse scatterer . The angular distribution of the scattering was quantitatively investigated with the setup of Fig. 1b , where the light reflected by the scale is measured as a function of angle in the horizontal plane . Figure 2c presents the reflectance of the scale of Fig. 2b as a function of angle for a number of wavelengths , normalized to the maximal reflectance value ; the angle is 0 ° for the normal to the scale . The scale 's reflectance spectrum for normally incident light is given in Fig. 2d . The reflectance is high in the visible wavelength range , but it is low in the ultraviolet , because of an ultraviolet-absorbing pigment , presumably leucopterin -LRB- Wijnen et al. 2007 -RRB- , which is concentrated in pigment granules , the ovoid beads -LRB- Fig. 2a -RRB- . The beads act as strong scatterers at wavelengths where the pigment absorption is negligible -LRB- Stavenga et al. 2004 ; Giraldo and Stavenga 2007 ; Morehouse et al. 2007 -RRB- . In addition to the beads , the other structures of the scale , that is the ridges and crossribs of the upper lamina of the scale as well as the lower lamina , contribute to the scattering -LRB- Fig. 2c -RRB- . Figure 2e -- h presents a similar set of data for a white scale of the dorsal forewing of a male black jezebel , D. nigrina . This case is obviously very similar to that of the white P. rapae scale . The scale anatomy with crossribs studded with beads is very similar -LRB- Fig. 2e -RRB- , the scattering is again approximately random -LRB- Fig. 2f , g -RRB- , and the reflectance spectrum is also high in the visible and low in the UV -LRB- Fig. 2h -RRB- . The strikingly blue M. aega has scales where the lamellae of the ridges form multilayers -LRB- Fig. 2i -RRB- . Different from Morpho didius , for example , which has glass cover scales and strongly pigmented ground scales -LRB- Vukusic et al. 1999 -RRB- , cover and ground scales of M. aega can not be distinguished . The scattering diagram of a scale of a M. aega is a horizontal stripe -LRB- Fig. 2j -RRB- , perpendicular to the vertically oriented scale ridges . The angular dependence of the reflectance varies strongly with the wavelength -LRB- Fig. 2k -RRB- , and the reflectance measured with normally incident light features a distinct peak in the blue -LRB- Fig. 2l -RRB- , which is due to the multilayered structure of the scale ridges -LRB- Fig. 2i ; see also Vukusic et al. 1999 ; Kinoshita et al. 2002 ; Yoshioka and Kinoshita 2004 -RRB- . A reflectance peak value of more than two results , because the scale 's scattering is highly directional and the reflectance was measured relative to the diffusely scattering white standard -LRB- Fig. 2l -RRB- . The purple scales at the dorsal wing tips of the male purple tip , C. regina , have ridges that are fine-structured similarly as in the case of M. aega -LRB- Fig. 2m -RRB- . The purple scale features a scattering diagram with a blue stripe and a red diffuse pattern -LRB- Fig. 2n -RRB- , which is a mixture of the stripe phenomenon of Fig. 2j and the diffuse patterns of Fig. 2b , f . The blue stripe is due to light backscattered by the fine-structured ridges , and the red diffuse pattern results from randomly scattered light , filtered by a pigment contained in granules below the multilayered ridges -LRB- Fig. 2m -RRB- . The blue peaking reflectance spectrum , shown in Fig. 2p , is mainly due to reflection by the ridges , and the red band , above 600 nm , is mainly caused by the light scattering pigment granules -LRB- Fig. 2p -RRB- . A green -- yellow reflecting scale of the moth U. fulgens has between the upper and lower scale laminae -LRB- Fig. 2q -RRB- an elaborate multilayer system , which yields a spatially restricted , directional scattering diagram -LRB- Fig. 2r , s -RRB- . The high amplitude of three of its reflectance spectrum is again due to the directionality of the scale reflectance -LRB- Fig. 2t -RRB- . The reflectance spectrum features a distinct band , peaking at 590 nm , with halfwidth about 120 nm , indicating that an interference reflector is involved . To evaluate whether the beaded scales of P. rapae , D. nigrina and C. regina act as diffuse scatterers , the angular reflectances of these species were normalized and compared with a Lambertian reflector -LRB- Fig. 3 -RRB- . The wavelengths selected are outside the absorption bands of the pigments , which means that the wavelengths chosen for P. rapae and D. nigrina are in the visible spectrum , but for C. regina only in the red wavelength range . The white scale of D. nigrina -LRB- Fig. 3b -RRB- well approximates a Lambertian reflector . The angular distribution patterns of the white scale of P. rapae slightly deviate from the ideal curve -LRB- Fig. 3a -RRB- , but notably the purple scale of C. regina is not a perfectly diffused red scatterer . Presumably this is due to the highly structured , multilayered ridges . Fig. 3Normalized angular reflectances measured from the scales of the three pierid butterflies , P. rapae , D. nigrina , and C. regina , compared to a Lambertian reflector -LRB- bold line -RRB- . Angular reflectance curves are shown in the wavelength range where the reflectance is high , that is , where pigment absorption is low . Four wavelengths covering the visible range are shown for P. rapae -LRB- a -RRB- and D. nigrina -LRB- b -RRB- . The scattering diagrams of D. nigrina well approximate a Lambertian reflector , but the angular dependence of the scattering by C. regina shows regular peaks , which indicates that the light scattered by the pigment granules is affected by the multilayered ridges Discussion The relationship between the optical properties of butterfly scales and their structure has been the topic of several studies -LRB- e.g. , Vukusic et al. 1999 , 2002 ; Kinoshita et al. 2002 -RRB- . Most of the previous investigations have focused on iridescent scales . Here , we have compared the reflection pattern of five differently structured single butterfly scales that scatter light coherently or incoherently , or both . We started with a simple white scale common to many species of the pierid subfamily Pierinae . Due to the characteristic beaded structure light scattering is strong , thus causing the intense white colour . We find that the white pierid scales approximate the properties of a Lambertian diffuser , at least in the wavelength range where pigment absorption is negligible -LRB- Fig. 3 -RRB- . The ridges of the scales of Morpho butterflies cause a blue colour . Melanin pigment below the multilayered ridges absorbs stray light over the whole visible wavelength range , including the ultraviolet , thus supporting the strikingly blue wing colour -LRB- Yoshioka and Kinoshita 2006a -RRB- . The pigment of the scales in the dorsal wing tips of C. regina also absorbs stray light , but not in the long-wavelength range . The remaining red light together with the blue iridescence causes the purple colour . A similar , however , short-wavelength-shifted case is formed by the yellow scales of the dorsal wings of many male butterflies of the pierid subfamily Coliadinae , where a UV and blue absorbing pigment is combined with UV iridescence -LRB- Ghiradella et al. 1972 ; Rutowski et al. 2005 -RRB- . Figure 4a is a transmission electron micrograph of a yellow cover scale from the dorsal wing of a male brimstone , Gonepteryx rhamni . The UV iridescent , yellow scales of the male brimstone have numerous beads , which contain the yellow pigment xanthopterin -LRB- Wijnen et al. 2007 -RRB- . The beads are seen in Fig. 4a as empty ovoids , but this is presumably due to the procedures for transmission electron microscopy -LRB- Morehouse et al. 2007 -RRB- . Fig. 4Transmission electron microscopy images of a cover scale of the dorsal wing of a male brimstone , Gonopteryx rhamni -LRB- a -RRB- , and of a scale of Urania fulgens -LRB- b -RRB- . Ridges are indicated by closed arrows . The brimstone scale has multilayers restricted to the ridges . The space between ridges and crossribs is studded with pigment granules -LRB- open arrow -RRB- . U. fulgens has continuous multilayers connected by numerous pillars that result in spreading of the scattering . Bar : 1 μm The scattering diagram of U. fulgens is not perfectly directional -LRB- Figs. 2r , s -RRB- , which should have been the case when the scales consisted of an ideal multilayer . Transmission electron microscopy shows that between the multilayers exist pillars -LRB- Fig. 4b -RRB- , which presumably cause the spread in the scattering diagram . Of course , the scales -- and therefore the multilayers -- are also not perfectly flat . The ridges -LRB- see Figs. 2q , 4b -RRB- will further contribute to some diffuse scattering . Knowing how light is scattered by individual scales is necessary for a proper understanding of butterfly wing coloration . As has been previously demonstrated , wing reflectance is a cumulative effect due to multiple reflection and transmission by the layers of scales on the butterfly wing -LRB- Yoshioka and Kinoshita 2006b ; Stavenga et al. 2006 -RRB- . The scales are often arranged in quite regular rows and have approximately the same orientation with respect to the wing surface , but of course there are deviations from perfect order . This will be rather irrelevant in the case of the small white , P. rapae , where scattering is approximately diffuse , but in cases where scattering is highly directional , as in Morpho butterflies , disordered scale arrangement will result in broadening of the scattering diagrams . This is illustrated in Fig. 5 , where scattering by a single scale of M. aega -LRB- Fig. 5a -RRB- is compared with scattering by an array of scales on the wing -LRB- Fig. 5c -RRB- . The far-field scattering diagram of a single scale is a narrow stripe -LRB- Fig. 5b ; see also Fig. 2j -RRB- , but that of a set of scales is a distinctly broadened stripe -LRB- Fig. 5d -RRB- . The angular extent of the scattering by the single scale in the vertical plane is < 10 ° , but that of the scale set is about 30 ° -LRB- Y , Fig. 6 -RRB- . The broadening of about 20 ° will be due to a rotational variation of the scale plane around the long axis of the scales . The tilt angle , that is the angle of the scale plane with the wing , will have a similar variation , as the scattering diagram of the scale set is about 20 ° wider than that of the single scale -LRB- X , Fig. 6 -RRB- . Further broadening of the scattering diagram will occur when reflection by the complete wings of a Morpho butterfly is considered . The dynamic changes of light scattering by a Morpho flying in a natural environment , and how the butterflies will be perceived by conspecifics and predators will be interesting themes for future research . Fig. 5Scattering by scales of Morpho aega . a Single scale about normally illuminated by a beam with about 50 μm diameter ; the outline of the scale is visible because of an additional , obliquely illuminating beam -LRB- bar : 50 μm -RRB- . b Far field scattering pattern of the single scale photographed at a white screen -LRB- see Fig. 2j -RRB- . The bright white spot is due to light scattered at the hole in the white screen and direct scattering by the scale . The interrupted circles indicate the angular directions of the scattering in steps of 10 ° . c A wing piece about normally illuminated by a beam with about 900 μm diameter ; cover and ground scales can not be distinguished -LRB- bar : 200 μm -RRB- . d Far field scattering pattern of the wing piece . The white spot is due to light scattered at the hole in the white screen and to direct scattering by the scaleFig . 6Spatial profiles of the scattering diagrams of Fig. 5b , d . The extent of the scattering in the horizontal plane -LRB- X -RRB- is much broader than the scattering in the vertical plane -LRB- Y -RRB- for both the single scale as for the intact wing piece . The angular width in the vertical plane of the scattering by the single scale is < 10 ° , but the width in the horizontal plane is about 100 ° . The vertical angular extent of the scattering by the wing piece is about 30 ° , meaning a broadening of about 20 ° with respect to the single scale , and the extent in the horizontal plane is similarly broadened

O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B I O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

lambertian reflector
scattering
angular reflectance

single butterfly scale
structural colour


Association

and

synergistic

interaction

between

promoter

variants

of

the

gene

in

Japanese

schizophrenics

Recent

association

studies

suggest

that

polymorphisms

in

the

promoter

and

exon

upstream

region

of

the

dopamine

receptor

-LRB-

-RRB-

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

Japanese

schizophrenic

cases

with

matched

controls

were

studied

by

genotyping

all

identified/validated

common

polymorphisms

-LRB-

C

C

A

C

C

T

T

and

repeat

-RRB-

and

a

known

microsatellite

-LRB-

tandem

duplication

-RRB-

in

the

upstream

region

A

single

nucleotide

polymorphism

-LRB-

SNP

-RRB-

A

in

the

promoter

region

was

found

to

be

significantly

associated

with

disease

-LRB-

P

and

for

allelic

and

genotypic

comparisons

respectively

-RRB-

although

not

surviving

after

Bonferroni

correction

Logistic

regression

analysis

showed

that

a

combination

of

the

four

polymorphisms

A

C

T

and

the

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

-LRB-

Prasad

et

al.

-RRB-

However

involvement

of

dopamine-related

genes

in

the

manifestation

of

this

disorder

has

been

difficult

to

determine

The

dopamine

receptor

-LRB-

-RRB-

gene

located

at

chromosome

-LRB-

Gelernter

et

al.

Petronis

et

al.

-RRB-

has

received

considerable

interest

because

clozapine

a

neuroleptic

which

is

often

effective

against

treatment-resistant

symptoms

has

a

high

affinity

for

this

receptor

-LRB-

Sanyal

and

Van

Tol

Van

Tol

et

al.

-RRB-

Also

is

up-regulated

in

postmortem

brain

tissue

from

schizophrenic

patients

-LRB-

Murray

et

al.

Seeman

et

al.

Stefanis

et

al.

Sumiyoshi

et

al.

-RRB-

To

investigate

its

genetic

association

with

schizophrenia

many

groups

have

examined

polymorphisms

of

the

gene

Early

studies

focused

on

a

variable

number

of

tandem

repeat

-LRB-

VNTR

-RRB-

in

exon

This

tandem

repeat

comprises

-LRB-

-RRB-

to

-LRB-

-RRB-

repeat

units

and

codes

for

the

third

intracellular

loop

of

the

receptor

protein

-LRB-

Van

Tol

et

al.

-RRB-

There

is

growing

genetic

evidence

linking

the

VNTR

with

psychiatric

traits

or

illnesses

such

as

novelty-seeking

-LRB-

Benjamin

et

al.

Ebstein

et

al.

-RRB-

attention-deficit

hyperactivity

disorder

-LRB-

ADHD

-RRB-

-LRB-

Faraone

et

al.

LaHoste

et

al.

-RRB-

and

schizophrenia

-LRB-

Hwu

et

al.

Jonsson

et

al.

Kaiser

et

al.

Sanak

et

al.

Serretti

et

al.

-RRB-

but

the

results

are

still

variable

In

recent

years

promoter

region

polymorphisms

of

the

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

-LRB-

SNP

-RRB-

C

and

a

tandem

duplication

-LRB-

kb

upstream

from

the

initiation

codon

-RRB-

are

reported

to

reduce

transcriptional

efficiency

-LRB-

D'Souza

et

al.

Okuyama

et

al.

-RRB-

Numerous

studies

have

examined

the

association

of

upstream

-LRB-

promoter

region

and

exon

-RRB-

polymorphisms

with

schizophrenia

and

some

have

shown

a

positive

association

-LRB-

Okuyama

et

al.

Xing

et

al.

-RRB-

although

not

all

were

in

full

agreement

-LRB-

Hong

et

al.

Jonsson

et

al.

Kohn

et

al.

Lung

et

al.

Mitsuyasu

et

al.

Petronis

et

al.

-RRB-

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

-LRB-

LD

-RRB-

in

a

genomic

interval

as

is

the

case

for

the

promoter

region

of

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

upstream

region

of

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

unrelated

cases

of

schizophrenia

-LRB-

men

women

mean

age

years

-RRB-

and

age

and

sex

matched

controls

-LRB-

men

women

mean

age

years

-RRB-

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

of

the

gene

-LRB-

from

bp

upstream

of

A

in

the

start

codon

to

bp

downstream

of

this

A

-RRB-

was

analyzed

by

the

direct

sequencing

of

PCR

amplification

from

the

genomic

DNA

of

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

-LRB-

Amersham

Biosciences

Piscataway

N.J.

USA

-RRB-

and

the

ABI

PRISM

Genetic

Analyzer

-LRB-

Applied

Biosystems

Foster

City

Calif.

USA

-RRB-

Polymorphisms

were

detected

by

the

SEQUENCHER

program

-LRB-

Gene

Codes

Corporation

Ann

Arbor

Mich.

USA

-RRB-

Genotyping

SNPs

were

typed

by

the

TaqMan

system

-LRB-

Applied

Biosystems

-RRB-

PCR

was

performed

using

an

ABI

thermocycler

and

fluorescent

signals

were

analyzed

by

an

ABI

sequence

detector

single

point

measurement

and

SDS

software

-LRB-

Applied

Biosystems

-RRB-

Conflicts

or

flagged

alleles

were

resolved

by

re-genotyping

Two

microsatellite

marker

loci

the

tandem

duplication

and

the

repeat

polymorphism

-LRB-

Fig.

-RRB-

were

amplified

by

PCR

using

fluorescently

labeled

primers

PCR

fragments

were

analyzed

on

an

ABI

PRISM

Genetic

Analyzer

-LRB-

Applied

Biosystems

-RRB-

Genotypes

were

determined

using

GeneScan

and

Genotyper

software

-LRB-

Applied

Biosystems

-RRB-

Fig.

structure

and

location

of

polymorphic

sites

within

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

-LRB-

from

kb

upstream

of

the

initiation

codon

to

the

end

of

exon

-RRB-

Statistical

analyses

The

allelic

and

genotypic

distributions

were

tested

for

association

by

Fisher

's

exact

test

for

biallelic

markers

-LRB-

SNPs

the

tandem

duplication

and

the

repeat

polymorphism

-RRB-

Haplotypic

association

analysis

was

performed

using

COCAPHASE

in

the

UNPHASED

package

-LRB-

-RRB-

-LRB-

Dudbridge

-RRB-

We

employed

a

and

sliding

window

analysis

using

COCAPHASE

To

estimate

the

degree

of

LD

between

pairs

of

loci

the

standardized

disequilibrium

coefficient

-LRB-

D

-RRB-

was

calculated

and

haplotype

blocks

were

defined

using

the

Haploview

program

-LRB-

-RRB-

-LRB-

Barrett

et

al.

-RRB-

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

-LRB-

Release

J

-RRB-

-LRB-

SPSS

Japan

Tokyo

Japan

-RRB-

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

-LRB-

-RRB-

we

set

dummy

variables

and

taking

values

and

and

for

genotypes

w/w

w/m

and

m/m

respectively

-LRB-

w

wild-type

m

mutant

-RRB-

Results

We

re-sequenced

the

upstream

region

of

the

gene

and

identified

a

total

of

eleven

SNPs

C

G

C

A

A

C

T

del

C

T

and

T

and

a

repeat

polymorphism

-LRB-

Fig.

-RRB-

One

novel

variant

G

-LRB-

deposited

into

GenBank

as

-RRB-

was

detected

in

only

out

of

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

C

C

A

C

C

T

T

and

the

repeat

polymorphism

in

exon

were

selected

for

genotyping

using

the

criterion

of

a

minor

allele

frequency

from

our

sequencing

data

We

also

typed

the

size

of

the

tandem

duplication

-LRB-

bi-allelic

polymorphism

with

either

one

or

two

repeat

alleles

-RRB-

The

information

on

markers

is

shown

in

Table

The

repeat

polymorphism

was

mostly

bi-allelic

with

the

exception

of

four

chromosomes

-LRB-

three

from

schizophrenics

and

one

from

controls

-RRB-

that

contained

a

three-repeat

allele

Table

distributions

of

bi-allelic

polymorphisms

from

the

upstream

region

of

IDPhysical

positionCase

or

controlnGenotype

frequenciesP

value

for

allelic

associationP

value

for

genotypic

association

repeat

T

T

G

G

T

C

C

Exon

aAlleles

are

coded

by

the

number

of

repeats

Four

subjects

-LRB-

three

from

the

schizophrenia

and

one

from

the

control

group

-RRB-

with

three

repeats

are

omitted

from

this

table

Significant

results

are

shown

in

bold

type

Allelic

and

genotypic

distributions

of

the

promoter

SNP

A

differed

significantly

between

cases

and

controls

-LRB-

P

and

for

allelic

and

genotypic

comparisons

respectively

-RRB-

In

the

exploratory

haplotype

window

analysis

the

two-SNP

haplotype

-LSB-

-LRB-

-RRB-

-LRB-

-RRB-

-RSB-

tended

to

be

over-represented

in

schizophrenia

-LRB-

frequencies

in

cases

and

in

controls

were

and

respectively

P

-RRB-

-LRB-

data

not

shown

-RRB-

In

LD

analysis

D

values

were

close

to

in

the

distal

-LSB-

-LRB-

repeat

-RRB-

-LRB-

C

-RRB-

-LRB-

C

-RRB-

-RSB-

and

proximal

-LSB-

-LRB-

T

-RRB-

-LRB-

T

-RRB-

-LRB-

-RRB-

-RSB-

segments

of

the

upstream

region

with

each

defined

as

a

haplotype

block

but

it

did

not

hold

for

the

region

in

between

-LRB-

Fig.

-RRB-

Decay

of

LD

in

this

short

stretch

-LRB-

kb

-RRB-

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

-LRB-

A

C

T

and

repeat

-RRB-

appeared

to

affect

susceptibility

to

schizophrenia

-LRB-

Table

-RRB-

Fig.

organization

of

in

Japanese

The

haplotype

block

pattern

was

constructed

by

the

Haploview

program

using

the

genotype

data

from

both

case

and

control

samples

-LRB-

subjects

-RRB-

The

number

in

each

cell

represents

the

LD

parameter

D

-LRB-

-RRB-

blank

cells

mean

D

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

-LSB-

-LRB-

repeat

-RRB-

-LRB-

C

-RRB-

-LRB-

C

-RRB-

-RSB-

and

proximal

-LSB-

-LRB-

T

-RRB-

-LRB-

T

-RRB-

-LRB-

repeat

-RRB-

-RSB-

segments

but

not

in

the

intervening

regionTable

regression

analysis

in

the

upstream

region

of

's

valueOROR

CI

repeat

β

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

confidence

interval

of

the

odds

ratio

Discussion

In

this

study

of

the

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

A

and

a

multi-marker

combination

displayed

association

with

schizophrenia

in

this

study

The

SNP

C

which

was

previously

reported

to

show

association

with

schizophrenia

in

an

independent

Japanese

sample

set

-LRB-

Okuyama

et

al.

-RRB-

was

not

significant

in

our

data

set

as

in

other

population

data

sets

-LRB-

Ambrosio

et

al.

Jonsson

et

al.

Segman

et

al.

-RRB-

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

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

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

-LRB-

Pritchard

et

al.

-RRB-

detected

no

evidence

of

population

stratification

-LRB-

Shimizu

et

al.

-RRB-

To

address

this

issue

further

we

also

performed

a

transmission

disequilibrium

test

This

testing

failed

to

replicate

the

association

of

A

in

a

sample

set

of

complete

schizophrenic

trios

-LRB-

data

not

shown

-RRB-

This

sample

size

would

have

only

a

limited

statistical

power

to

detect

true

association

Secondly

the

significance

of

the

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

C

needs

to

be

interpreted

cautiously

The

sample

size

in

this

study

had

a

power

of

to

detect

a

susceptibility

variant

with

a

relatively

small

effect

-LRB-

relative

risk

and

for

heterozygotes

and

homozygotes

respectively

-RRB-

when

the

allele

frequency

is

This

frequency

equals

that

of

SNP

whose

association

with

schizophrenia

has

been

previously

reported

-LRB-

Purcell

et

al.

Xing

et

al.

-RRB-

In

conclusion

to

our

knowledge

this

is

the

first

study

that

highlights

a

possible

combinatorial

effect

of

promoter

SNPs

in

the

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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