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RESEARCH REPORTS |
1 Department of Orthodontics, School of Dentistry, Showa University, 2-1-1 Kitasenzoku, Outa-ku, Tokyo, 145-8515, Japan;
2 Division of Genetic Diagnosis, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan; and
3 Department of Orthodontics, Pusan National University, 1-10, Ami-dong, Seo-gu, Pusan, 602-739, Korea;
* corresponding author, ituro{at}ims.u-tokyo.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: Mandibular prognathism • linkage analysis • Korean • Japanese
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Microsatellite Genotyping
Genomic DNA was isolated from whole blood cells with the use of a QIAamp DNA Blood Kit (QIAGEN GmbH, Hilden, Germany) or from buccal cells with the use of a BuccalAmp DNA Extraction Kit (Epicentre Technologies, Madison, WI, USA). PCR amplifications were performed according to a standard protocol. Multiplex fluorescent genotyping of microsatellite markers was performed with Linkage Mapping Set version 2.5 (Applied Biosystems, Tokyo, Japan), which covers the entire genome. Because several markers were not polymorphic in Japanese (Ikari et al., 2001), a set of 47 markers obtained from online information (GDB: http://gdb.org/) was added to the original set to fill in the gaps (Onda et al., 2001). An extra sequence was attached to the 5' end of the reverse primer to promote the non-templated addition of adenine so that accurate genotyping could be achieved (Brownstein et al., 1996). Genome scan was performed with a total of 405 microsatellite markers, having an average heterozygosity of 0.76 (minimum: 0.60) and an average interval of 8.8 cM (maximum: 20.7 cM). Marker positions (in Kosambi centiMorgans) were obtained from the Marshfield Medical Research Foundation (Broman et al., 1998). For chromosome 1, which demonstrated evidence of linkage when the framework marker set was used, 31 microsatellite markers were added for dense mapping that covered the initial linkage region with < 5 cM.
To evaluate differences in allelic frequency between two populations, we compared the differences in allelic frequencies of microsatellite markers in the linkage region of chromosome 1, D1S2864, D1S234, and D1S233, between Korean and Japanese probands (33 each) by Monte-Carlo approximation of Fisher’s exact test, using permutation of datasets (Weir, 1996).
Affected Sib-pair Linkage Analysis
Because the mode of inheritance of MP is uncertain in the family set, we applied different non-parametric linkage methods: GENEHUNTER-PLUS (version 1.3)/GENEHUNTER (version 2.1) (Kruglyak et al., 1996; Kong and Cox, 1997) for multi-point analysis, and the SIBPAL program from the S.A.G.E. package (version 3.1) for single-point analysis (SAGE, Department of Epidemiology and Biostatistics, Case Western Reserve University, Cleveland, OH, USA)). We performed multi-point analysis of the data from a genome-wide scan by weighting each family equally by GENEHUNTER-PLUS, a modified version of GENEHUNTER. GENEHUNTER-PLUS, compared with the original program, assumes a linear model for risk and thus provides more accurate calculations of the variance. Results are reported with a maximum Zlr score. GENEHUNTER (version 2.1) was utilized for estimates of the mean proportion of alleles shared IBD (identical by descent). The SIBPAL program estimated the mean ratio (
) of alleles shared IBD among affected sibling-pairs at each microsatellite marker. The obtained was tested against the null hypothesis of no linkage ( = 0.5). The statistic has a standard normal distribution under the null hypothesis, and because the alternative hypothesis of linkage is given when IBD sharing is over 50%, the test is one-sided. Accordingly, accurate P values can be obtained by use of a one-sided t test like that implemented in the SIBPAL program. Allele frequencies of microsatellite markers as references were calculated in 64 unrelated Japanese subjects.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). By analysis of all 90 affected sibling-pairs, three chromosomal loci provided evidence of linkage: 1p36 (Zlr = 2.42 near marker D1S234), 6q25 (Zlr = 2.23 near marker D6S305), and 19p13 (Zlr = 1.93 near marker D19S884). In general, Japanese and Korean sibling-pairs showed similar patterns of linkage, although several discrepancies were observed. For example, Korean sibling-pairs showed linkage at chromosome 4, whereas Japanese-specific linkage was observed at chromosomes 9 and 10. These discrepancies might be caused by genetic heterogeneity between the two populations; however, type 1 error due to the small sample size most likely explains the discrepancies. Based on the evidence of linkage observed at a 47.5-cM interval of chromosome 1, a dense linkage mapping of chromosome 1 was performed by increasing microsatellite markers. Thirty-one markers that cover the initial linkage region, with < 5-cM intervals, were added, and the result of linkage with a total of 62 markers is shown (Fig. 2). In dense mapping, the linkage region was observed within a 10-cM region, with the highest linkage with D1S234 (maximum Zlr = 2.51) on 1p36. The best IBD sharing, of 68.5% (Z0 = 0.123, Z1= 0.392, Z2= 0.485), was observed at D1S234. The alternative linkage test with SIBPAL that gives accurate P-value showed positive evidence of linkage with D1S234 (P = 0.0012) (Table). The allelic distributions of D1S2864, D1S234, and D1S233 demonstrated that there was no significant difference in the frequency of the microsatellite markers between the Korean and Japanese probands (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, 2). It is well-recognized that ethnicity is a risk factor for MP—i.e., the highest incidence has been observed in Asian populations, the lowest in Caucasian populations. Koreans and Japanese are considered to share a common background of population history, as might be expected from the close geographical location of the two countries. Phylogenetic analysis revealed that the closest genetic affinity between Japanese and Koreans was observed (Horai et al., 1996; Tokunaga et al., 1996). Very similar allelic frequencies of vast SNPs (single-nucleotide polymorphisms) were also observed (Lee et al., 2003). Indeed, the allelic frequency differences of microsatellite markers—D1S2864, D1S234, and D1S233—assessed by Monte-Carlo approximation of Fisher’s exact test showed no statistical significance. Therefore, it is fairly reasonable to assume that the same etiological background for MP exists in these two populations. Accordingly, we did not investigate whether population-specific linkage to MP is largely due to lack of power of linkage analysis to detect linkage with such a small number of families. For example, we observed inflated evidence of linkage at chromosome 10p in Japanese subjects (maximum Zlr = 2.53), although no supportive evidence of linkage was obtained in Korean subjects or in the combined populations. This observation appears not to reflect a true linkage in Japanese, but, rather, to be a false-positive result. Because the relative power and accuracy of linkage tests vary with the methods used, we applied two different programs: SIBPAL from the S.A.G.E. package and GENEHUNTER-PLUS. SIBPAL calculates the excess allele sharing by comparison with the null hypothesis under no linkage by t statistics. Accurate P-values can thus be obtained. GENEHUNTER-PLUS is a likelihood method of calculating LOD-type score, which is generally a more powerful linkage test than the probability test. We obtained three loci with nominal evidence of linkage to MP on chromosomes 1p36 (Zlr = 2.51), 6q25 (Zlr = 2.23), and 19p13.2 (Zlr = 1.93). Although the Zlr score cannot be directly compared with the LOD score, the observed evidence of linkage would not meet the genome-wide screen criterion of ‘suggestive linkage’ proposed by Lander and Kruglyak (1995). The reasons for this might be attributed to heterogeneity in the susceptibility of MP or a low contribution of genetic factors for MP. Therefore, large-scale studies of well-defined families, including other ethnicities, are warranted to confirm the evidence of linkage to MP.
The peak Zlr scores at 1p36 were near D1S234 (Fig. 1). The 1p36 locus harbors positional candidate genes of interest, such as alkaline phosphatase (liver/bone/kidney), heparan sulfate proteoglycan 2 (perlecan), and matrilin-1 (cartilage matrix protein). Serum bone alkaline phosphatase has been considered to be a marker for bone formation, because bone alkaline phosphatase levels are increased when the growth rate accelerates (Tobiume et al., 1997). Perlecan, a large heparan sulfate proteoglycan, is present in the basement membrane and other extracellular matrices. Several in vitro studies have suggested multiple functions of perlecan in cell growth/differentiation and tissue organization. Recent studies of gene knockout mice with craniofacial abnormalities and human diseases revealed critical in vivo roles of perlecan in cartilage development and neuromuscular junction activity (Arikawa-Hirasawa et al., 1999, 2001). Matrilin-1 is a cartilage-specific homotrimer localizing in the growth plate of long bones, and is transcribed exclusively in the post-proliferative chondrocytes of the zone of maturation (Huang et al., 1999; Hansson et al., 2001). Accordingly, these positional candidate genes need to be screened for molecular variants by an allelic association study with a case-control design, and then the molecular variants that are implicated in the etiology of MP could be identified.
In summary, we performed a genome-wide linkage analysis with 90 MP sibling-pairs from an Asian population, and mapped three chromosomal loci, including 1p36, 6q25, and 19p13.2. The replication of these results with an independent dataset should facilitate the positional cloning of the gene or genes that influence the development of MP. Identification of the genes contributing to MP susceptibility will be very useful for establishing an understanding of the mechanisms underlying and the protecting outcomes for MP, and for the design of targeted intervention strategies to prevent MP more effectively.
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ACKNOWLEDGMENTS |
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Received February 23, 2004; Last revision December 8, 2004; Accepted December 12, 2004
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REFERENCES |
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| Journal of Dental Research ® | Critical Reviews (1990-2004) |
