RNF213 was strongly associated with moyamoya disease in the Japanese ... R4810K in RNF213 in 2,508 participants from East and Southeast Asian coun-.
N eurol M ed Chir (Tokyo) 52, 299- 303, 2012
Distribution of Moyamoya Disease Susceptibility Polymorphism p.R4810K in RNF213 in East and Southeast Asian Populations Wanyang LIU,l Toshiaki HITOMI,l Hatasu KOBAYASHI, l Kouji H. HARADA, l and Akio KOIZUMI l lDepartment of H eal th and En vironmental Sci ences, Graduate School of M edicine Kyoto Un iv ersity, Kyoto, Kyoto
Abstract Moyamoya disease is an idiopathic vascular disorder of the intracranial arteries. Ring finger 213 (RNF213) was previously identified as the strongest susceptibility gene for moyamoya disease in East Asian people by a genome-wide linkage analysis and exome analysis. The coding variant p .R4810K in RNF213 was strongly associated with moyamoya disease in the Japanese (odds r ati o: 338.94, p = 1.05 x 10 - 1 0 0 ) and Korean (odds ratio: 135 .63 , p = 7.59 x 10 - 2 7 ) populations, and much less strongly associated in the Chinese population (odds ratio: 14.70, p = 2.63 X 10 - 5 ) . Th e present study investigated th e distribution of variant p.R4810K in RNF213 in 2,508 participants from East and Southeast Asian countries using a TaqMan probe. p .R4810K was detected at an allele frequen cy of about 1.00% in 4 of 11 investigated lo cations in China. In contrast , p .R4810K was detected homogeneously at relatively high fr equencies of 1.00-1.72% in all investigated locations in Kor ea and Japan, including Okinawa. p.R4810K was not detected in Southeast Asian populations. The population susceptible to moyamoya disease was estimated to be 16 .16 million people in East Asian countries, including 11.41 million Chinese, 1.36 million Korean, and 3.39 million Japanese people. The number of patients with moyamoya disease, which was esti m ated at approximately one per 300 carriers of p .R4810K, was considered to be 53 ,800 in East Asian populations. Key wo rds:
m oyamoya d isease ,
p .R4810K,
RNF2 13,
Introduction
December 5, 201 1;
Accepted
So utheast As ia n
det ermined . Th e ring fi nger 213 (RNF213) gene was finally ide ntifie d as a suscepti bility gene for mo yam oya disease by th e co nve ntiona l ri gor ou s po sitional clonin g appr oac h, exome ana lysis, a nd fu nc tiona l ana lys is u sin g zebrafi sh ." Over all , 74.5% of East Asian pat ients with mo yam oya d isease ca r ry th e rare founder va ria n t p.R4810K of RNF 213. Ho w ever , a gra d ient of pr evalen ce was ob ser ved in pat ients with mo yamoya disease: 90% in Jap anese, 79% in Kor ean , and 23% in Chinese patient populations. On th e other hand, no gra die nt of pr evalen ce was ob ser ved in th ese three gene ra l populations. In total , 2.4% of th e East Asian gene ra l populat ion ca rr ies p.R4810K . Th e minor allele fr equen cies of p.R4810K w er e 1.4 %, 1.3%, and 1.0% for th e Jap an ese, Kor ean , and Chinese in gene ra l populations, resp ectively.?' Th ese simila r allele freque ncies of p.R4810K in th ese three East Asian gene ra l populations do not explai n th e low er fr equen cy of Chinese pati ents with mo yamoya di sease com pa re d w ith Jap an ese and Kor ean pati ents w ith mo yamoya
Mo yamoya disease (Mendelian Inheritan ce in Man [MIM] number 607151) is a rar e idiopa thic progr essive disorder cha racte ri ze d by occlusive lesions in th e su prac linoid inte rnal ca ro ti d arte ry and its main br an ch es in th e ci rcle of Willis. Th e reduction in blood flow in th e regions affec te d by th e occlusive lesions is com pe nsate d by th e d evelopment of a fin e vascula r netw ork th at resembles " puffs of smoke" ("moyamoya " in [ap an esel.v-v" Mo yamoya disease occurs with th e high est pr evalen ces in East As ia n cou ntries suc h as Jap an , Kor ea , and Ch in a, es pecially com pa re d with other cou ntries w orl dw ide .2 •7 •10 ) We pr eviou sly ide n tifie d a susceptibility locus for fam ilia l moyam oya disease on 17q25 .3,11jSignificant ass ociati ons with genes in this region had been reported .t-f but ri gorou s id entificat ion had not been Recei ved 2012
East As ia n ,
Ja nua ry 23,
299
W. Liu et 01.
300
disease. Moyamoya disease occurs in approximately one of 300 carriers in [apan." This discrepancy in the allele frequency between cases and controls in Chinese people might be attributable to selection bias in the general populations in the previous study. g) The primary aim of the present study was to estimate the population susceptible to moyamoya disease by conducting large scale screening for p.R4810K in general populations in East and Southeast Asia. The secondary aim was to estimate association of the p.R4810K variant of RNF213 in patients with moyamoya disease .
Materials and Methods A total of Z,508 unrelated pa rticipants from East Asian and Southeast Asian countries were recruited, comprising 587 Chinese, Z94 Korean, 1474 Japanese, 103 Vietnamese, and 50 Filipino people (Table 1). The Japanese participants were recruited from mainland Japan, including Hokkaido, Honshu, Shikoku, and Kyushu, and Okinawa. The Korean participants were recruited from five locations. The Chinese participants were recruited from 11 locaTable 1
tions. Blood samples were obtained from two sources. On e source was the Kyoto University Human Specimen Bank, which collected blood samples from the 1990s and ZOOOs as previously described.sv' These blood samples included Chinese, Korean, Japanese, and Philippine samples. The other source involved samples co llected in an international collaboration that included Vietnam and Seoul, South Korea. Ethical approval for this study was given by the Institutional Review Board and Ethics Committee of Kyoto University School of Medicine, Kyoto University, Japan (approval number: G140; approval date: Oct. 18, Z004), by the Institutional Review Board of Seoul National University Hospital, Seoul, South Korea (approval number: H0507-509 -153; approval date: Aug. Z4, Z005). The study participants were recruited in these institutes. Subjects who participated in this study after ZOOo were recruited by School of Medicine, Kyoto University. All subjects gave written informed consent. Subjects who donated blood samples before ZOOo were recruited by Tohoku University School of Medicine (Sendai, Miyagil, or Akita University School of Medicine (Akita, Akita), or Kyoto University School of Medicine, and
Demographic features of the participa nts in the East and Southeast Asian populations
Area East Asia
Country China
Korea
Japan
Southeast Asia
Vietnam Philippines
Location
Size of population (million)
Sex (male:female)
Dehui Huludao Beijing [inan Xian Baoji Shanghai Changsha Heping Nanning Tainan
1.00 2.82 19.61 6.81 8.47 3.72 23.02 7.04 4.60 6.66 1.88
0:50 0:50 0:50 0:50 0:94 0:48 0:50 0:50 0:46 0:50 0:49
Total
85.63
0:587
Seoul Chonan Haman Pusan [eju-do
10.46 0.58 0.06 3.60 0.53
Total
Age [yrs]" NA NA
Angiography' ,
36.4 ±11.5
0 0 0 0 0 0 0 0 0 0 0
37.4 ±11.1
0
80:25 0:29 0:46 0:49 0:65
42.2 ± 13.3 34.6 ±8.8 43.6 ±6.8 39.4 ±7.8
NA
46 0 0 0 0
15.23
80:214
40.9 ± 10.9
46
Mainland Okinawa
123.62 1.38
608:766 0:100
57.7 ±14.1 47.5 ±7.2
384 0
Total
125.00
608:866
57.0 ±14.0
384
6.50 1.66
0:103 0:50
22.8 ± 10.2
0 0
Hanoi Manila
38.4 ± 10.6
NA NA NA NA NA NA NA
NA
'Values are means ± standard deviation. " Conventional digital subtraction angiography, magnetic resonance angiography, computed tomography angiography, and others. NA: not applicable.
Neural Med Chir (Tokyo) 52, May, 2012
p.R4810K in RNF213 in East and Southeast Asian Populations
gave verbal informed consent. The use of blood samples donated before 2000 for genetic analysis was also approved by the Institutional Review Board and Ethics Committee of Kyoto University School of Medicine (approval number: G140; approval date: Oct. 18, 2004). Peripheral blood (10 ml) was collected from all participants. Genomic deoxyribonucleic acid (DNA) was extracted from the blood samples using a QIAamp DNA Blood Mini Kit (Qiagen, Germantown, Maryland, USA) according to the manufacturer's protocol. The quality and concentration of the extracted DNA were assessed using an Infinite w M200 PRO (TECAN, Kawasaki, Kanagawa). The DNA was stored in a freezer at - 20°C until analysis. Genotyping of p.R4810K in all participants was conducted using a Taq Man w probe (Custom Taq Mari e SNP Genotyping Assays; Applied Biosystems, Foster City, CA, USA) and a 7300/7500 Real-Time PCR System (Applied Biosystems) ac cording to the manufacturer's protocols. Briefly, the po lymerase chain reaction (PCR) amplifications were performed with 1-20 ng of purified genomic DNA, 0.1,u1 of 40 x SNP Genotyping Assay, 6.25,u1 of 2 x TaqMan ® Universal PCR Master Mix, No Table 2
301
AmpErase ® UNG, and 5.15,u1 of deoxyribonucleasefree water. The final reaction volume was 12.5 ,ul/well in 96-well plates. The standard protocol for the cycling conditions was maintenance for 10 minutes at 95°C for AmpliTaq Gold e (Applied Biosystems) enzyme activation, followed by 40 cycles of 15 seconds at 92°C for denaturation, and 1 minute at 60°C for annealing/extension. After each PCR amplification, an endpoint plate read was performed using the Rea l-Time PCR System (Applied Biosystems). The associated sequence detection system software uses the fluorescence measurements taken during the plate read to plot fluorescence (normalized reporter signal: Rn) values based on the signals from each well. The plotted fluorescence signals indicate which alleles are present in each sample.
Results The demographic features of all participants in this study are shown in Table 1. The sampled cities covered 234.02 million people. A total of 2,508 pa rticipants from three East Asian countries and two Southeast Asian countries were recruited from 1987
Geographic distribution of p. R4810K in RNF213 in East and South east Asian populatio ns Genotype of p.R4810K
Area
East Asia
Country
China
Korea
Japan
Southeast Asia
Vietnam Philippines
Location
Sample si ze
Minor allele frequency
GG
GA
50 50 49 50 92 48 49 50 46 49 49
0 0 1 0 2 0 1 0 0 1 0
0 0 0 0 0 0 0 0 0 0 0
50 50 50 50 94 48 50 50 46 50 49
0.00 0.00 1.00 0.00 1.06 0.00 1.00 0.00 0.00 1.00 0.00
Total
582
5
0
587
0.4 3
Seoul Chonan Haman Pusan [eju-do
102 28 45 48 63
3 1 1 1 2
0 0 0 0 0
105 29 46 49 65
1.4 3 1.72 1.09 1.02 1.54
Total
286
8
0
294
1.36
Mainland Okinawa
1339 98
32 2
3 0
1374 100
1.38 1.00
Total
1437
34
3
1474
1.36
103 50
0 0
0 0
103 50
0.00 0.00
Dehui Huludao Beijing [inan Xian Baoji Shanghai Changsha Heping Nanning Tainan
Hanoi Manila
Neural Med Chir (Tokyo) 52, May, 2012
AA
(%)
Total population (million)
Estimated population with p.R4810K in a country (million)
1340.00
11.41
50.00
1.36
125.00
3.39
90.50 94.00
0.00 0.00
302
W. Liu et 01.
~ D~Ui Huludao Beijing.
Jinan· Baoji, Xian
Shanghai
Chang~hal
Hepinq Nanning
"l(_'~~_
.
...
.~
no'
_--
• Okinawa
Fig. 1 Locations of the sampling sites in East and Southeast Asian countries.
to 2009 . Briefly, the recruited subjects comprised 587 participants from 11 Chinese locations, 294 from five Korean locations, 1474 from five Japanese locations, 103 from one Vietnamese location, and 50 from one Philippines location. Most of the participants were females, with the exception of 608 Japanese males and 80 Korean males. Information on age was only available for the participants from certain locations. Angiography was only conducted for 384 Japanese and 46 Korean participants. The geographic distributions of p.R4810K in the different ethnicities are shown in Table 2 and Fig. 1. p.R4810K was detected in East Asian populations, but not in Southeast Asian populations. p.R4810K was detected at allele frequencies of 0.43%, 1.36%, and 1.36% in the Chinese, Korean, and Japanese populations, respectively. p.R4810K was detected at an allele frequency of about 1.00% in 4 of 11 investigated locations in China, suggesting that the distribution of p.R4810K was heterogeneous and limited to certain specific locations in China. In contrast, p.R4810K was homogeneously located throughout Korea and Japan at relatively high allele frequencies of 1.00-1.72%. p.R4810K was not detected in 49 Taiwanese, 103 Vietnamese, and 50 Filipino participants.
frequency in the Japanese or Korean populations. Therefore, our previous observation was considered to be attributable to selection bias.P' The lower prevalence of p.R4810K in the Chinese general population might be proportional to the lower carrier rate (i.e., 23%) in Chinese patients. In accordance with this observation, a single dominant polymorphism may be associated with Japanese or Korean patients, whereas various polymorphisms in RNF213 may be associated with Chinese patients. In fact, we observed five additional mutations in Chinese patients but no additional mutations in Korean or Japanese patients." In the present study, magnetic resonance angiography was only conducted for limited numbers of participants. This limitation did not affect our results because of the very low prevalences of moyamoya disease in the general populations. The estimated total numbers of carriers were 11.41 million for the Chinese, 1.36 million for the Korean, and 3.39 million for the Japanese populations. Assuming that moyamoya disease occurs in one of 300 carriers with p.R4810K,B,9) the estimated numbers of patients with moyamoya disease attributable to p.R4810K are 38,000 in the Chinese, 4,500 in the Korean, and 11,300 in the Japanese populations. Although these numbers are very approximate and minimum estimates, because only the p.R4810K variant is considered, the large number of potential sufferers suggests that more attention should be paid to moyamoya disease in East Asian countries. Differences in the clinical profiles of moyamoya disease have been recognized in China, Korea, and [apan.t-" However, the differences between the clinical profiles of Korean and Japanese patients are unlikely to be explained by genetic differences. Specific factor(s) other than the genetic factor are needed to explain the variations in the clinical phenotypes and low penetrance among carriers.s-" Therefore, unknown modifier factor(s) may also be contributory to the differences in the clinical phenotypes. Further research is needed to identify such factor(s), which may also be crucial for the development of moyamoya disease among carriers of variant p.R4810K in RNF213 .
Acknowledgments Discussion The present study confirmed the presence of similar allele frequencies of the variant p .R4810K of RNF213 in the Japanese and Korean general populations. However , expansion of the examined population revealed that the allele frequency of p.R4810K in the Chinese population was 0.43%, only one-third of the
This study was mainly supported by the Research Committee on Spontaneous Occlusion of the Circle of Willis (Moyamoya disease) (Chaired by Dr . Nobuo Hashimoto) by Science Research Grants of the Ministry of Health, Labour and Welfare of Japan (H23Nanji-Ippan-019) and partially supported by the Japan Society for the Promotion of Science for Neural Med Chir (Tokyo) 52, May, 2012
p.R4810K in RNF21 3 in East and Southeast A sian Populations
you ng scie ntists .
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Ad dress reprint requ ests to : Akio Koizumi, MD, Dep artm ent of H ealth a nd En vironm ental Sci en ces, Gr aduate School of M edi cine Kyoto Univer sity, Konoe- cho, Yosh ida , Sak yo-ku, Kyoto 606-8501, Japan . e-m ail: koizumLakio .5v@kyoto-u .a c.jp
