SUMO1 polymorphisms are associated with non-syndromic cleft lip with or without cleft palate

Biochemical and Biophysical Research Communications 377 (2008) 1265–1268

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SUMO1 polymorphisms are associated with non-syndromic cleft lip with or without cleft palate Tao Song a, Guolin Li a, Guangping Jing a, Xiaohui Jiao a,*, Jinna Shi b, Bing Zhang a, Li Wang b, Xiangmei Ye c, Fenglin Cao c a

Department of Oral Maxillofacial Surgery, School of Stomatology, Harbin Medical University, Harbin, China Department of Periodontology, School of Stomatology, Harbin Medical University, China c Central laboratory, the First Affiliated Hospital, Harbin Medical University, China b

a r t i c l e

i n f o

Article history: Received 20 October 2008 Available online 5 November 2008

Keywords: SUMO1 Non-syndromic cleft Cleft lip Cleft palate SNP Association study

a b s t r a c t Small ubiquitin-like modifier 1 (SUMO1) haploinsufficiency results in cleft lip and palate in animal models. However, no studies have linked SUMO1 to non-syndromic cleft lip with or without cleft palate (NSCLP) in humans. In the present study, we investigated the potential association between SUMO1 single nucleotide polymorphisms (SNPs) and risk for human NSCLP. From 181 patients and 162 healthy controls, we found statistically significant correlations between a 4-SNP SUMO1 haplotype and NSCLP. These data are the first to suggest a role for SUMO1 gene variation in human NSCLP development. Ó 2008 Elsevier Inc. All rights reserved.

Non-syndromic cleft lip with or without cleft palate (NSCLP) is a common birth defect, affecting 1 in 500 to 1 in 2000 newborns, depending on geographical origin. Rates are highest in Asian and Native American populations and lowest in people of African descent [1]. The defect requires complex, multi-disciplinary interventions and has lifelong implications for affected individuals. NSCLP is multifactorial and heterogeneous in origin. Environmental factors such as maternal cigarette smoking and specific dietary deficiencies early in pregnancy may increase cleft lip, with or without cleft palate (CL/P), risk but there are strong genetic components as well [2]. Current estimates support that 3 to 14 multiplicatively interacting genes may contribute to NSCLP development [3,4]. Among these, IRF6 gene polymorphisms were strongly associated with NSCLP, accounting for 12% of CL/P cases [5]. Additional studies support minor roles for MSX1, MSX2, PVRL1, FGF, and SKI mutations in NSCLP etiology [6–8]. A previous study of interstitial deletions and balanced translocations implicated human 2q32–q33 as a craniofacial dysmorphology locus [9–11]. Meta-analysis of 13 genome scans revealed multiple novel CL/P gene candidates with loci on 2q32–35 [12]. Notably, 2q32–q33 was one of only three genomic regions for which haploinsufficiency was significantly associated with isolated cleft palate [9]. * Corresponding author. Fax: +86 451 53625108. E-mail address: [email protected] (X. Jiao). 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.10.138

The 2q33-localized gene SUMO1 recently emerged as an NSCLP candidate gene [13]. A patient with cleft lip and palate was described with a balanced reciprocal translocation involving chromosome 2q that interrupted SUMO1 [13]. Moreover, animal studies have confirmed that SUMO1 haploinsufficiency results in cleft lip and palate [13]. However, no association studies have investigated SUMO1 in human NSCLP pathogenesis. Here, we selected four SUMO1 gene SNPs from Chinese Han from the Beijing (CHB) HapMap data, to investigate a potential association between markers in SUMO1 and human NSCLP risk. Methods Samples. All subjects were Han Chinese in origin. Hundred and eighty-one NSCLP patients (98 females and 83 males, age range 1–29 years) and 162 phenotypically normal individuals (87 females and 75 males, age range 4–35 years) were recruited between 2006 and 2008 from the Affiliated Stomatology Hospital of Harbin Medical University and the Second Affiliated Hospital of Harbin Medical University. Informed consent was obtained from all subjects and the Clinical Research Ethics Committee of Harbin Medical University approved the study. Marker selection and genotyping. Four tag SNPs (rs6761131, rs6717252, rs4675272, and rs7580433) in SUMO1 were selected using the HapMap Project for association studies. The locations of these four SNPs within the SUMO1 gene are shown in Fig. 1.

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HapMap contains genetic diversity information from 1 million SNPs in four human populations, thereby providing a rich resource for association study design. SNPs were selected using minor allele frequencies greater than 0.05 in the CHB population. DNA was extracted from peripheral blood leukocytes. All genotyping experiments were performed using ligase detection reactions (LDR). The target DNA sequences were amplified by multiplex PCR, then treated with 1 ll of Proteinase K (20 mg/ml), heated at 70 °C for 10 min and quenched at 94 °C for 15 min. Ligation was performed in a final volume of 20 ll containing 20 mM Tris–HCl (pH 7.6), 25 mM potassium acetate, 10 mM magnesium acetate, 10 mM DTT, 1 mM NAD, 0.1% Triton X-100, 10 ll of Multiplex PCR product, 1 pmol of each discriminating oligo, 1 pmol of each common probe and 0.5 ll of 40 U/ll Taq DNA ligase (New England Biolabs, USA). The LDR was performed using 40 cycles of 94 °C for 30 s and 63 °C for 4 min. The fluorescent products of LDR were differentiated using an ABI sequencer 377. Statistical analysis. All allele and genotype frequencies, Hardy– Weinberg equilibrium, pair-wised linkage disequilibrium and haplotype analysis were conducted online using http://bioinfo. iconcologia.net/snpstats, a web-based association study application. Association studies are based on linear or logistic regression according to response variable, single SNP: multiple inheritance models and analysis of interactions [14]. Results All four SNPs were in Hardy–Weinberg equilibrium and no significant differences were observed in allele distributions of the four SNPs between NSCLP patients and controls (Table 1). Odds ratios were calculated as estimates of the relative risk for NSCLP by comparing genotype frequency of NSCLP patients with unaffected controls (Table 2). Odds ratios for GA and GA + AA genotype of rs7580433 were ORGA = 0.53 (95% CI: 0.30–0.94) and OR GA+AA = 0.56 (95% CI: 0.32–0.98), respectively compared to the GG genotype. For other SNP markers, we found no significant discrepancies of genotype. Strong pairwise linkage disequilibrium was found be-

tween four SNPs tested (all |D0 | > 0.85) (Table 3). We therefore performed a haplotype analysis for the four variants (Table 4). The five most common haplotypes accounted for approximately 94% of the total. Data of haplotypes with a frequency lower than 5% were not shown. A significant difference was observed for one haplotype (p = 0.0003), which showed an odds ratio of 0.14 (95% CI: 0.05– 0.40). Discussion The aim of this study was to investigate the association between human SUMO1 polymorphisms and NSCLP. Four SNPs located in SUMO1 were selected using CHB population HapMap data and utilized to type a sample of 343 individual subjects. The SNP marker rs7580433 (between alleles G and A [G > A]) showed statistical significance for GA and GA + AA genotype frequencies among all 181 patients and 162 healthy control subjects (ORGA = 0.53, 95% CI: 0.30–0.94 and ORGA+AA = 0.56, 95% CI: 0.32– 0.98), suggesting rs7580433 may be associated with NSCLP. Given its location within the SUMO1 gene, it is unlikely that SNP rs7580433 creates a functional change, but it could be a marker for other unknown SUMO1 polymorphisms. Since genetic variation resulting in SUMO1 haploinsufficiency may contribute to human cleft lip and palate; SNP rs7580433 may be an excellent candidate for an NSCLP association study within the Chinese population. Haplotype analyses further confirmed the role of SUMO1 in NSCLP development. In our study, differences in haplotype frequencies existed between cases and controls with a global p value < 0.0001. The 4-SNP haplotype T-A-G-G was statistically significant (p = 0.0003; OR = 0.14, 95% CI: 0.05–0.40); the frequency of haplotype T-A-G-G in the total set of 181 NSCLP patients (1.17%) was less than that in the total set of 162 controls (10.9%), indicating that haplotype T-A-G-G was statistically related to NSCLP as a protective haplotype. Together these results indicate SUMO1 is significantly associated with NSCLP in the Chinese population. To our knowledge, this is the first molecular epidemiology study suggesting a possible association between SUMO1 gene

Fig. 1. Position of SNPs within the SUMO1 gene. Rectangles indicate SUMO1 exons. Red, untranslated (UTR); light blue, coding exons. Note that all SNPs used in this study are located in intronic regions.

Table 1 Primary information for four TagSNPs of SUMO1 gene. dbSNP ID

Base change

Case/control

MAF for case

MAF for control

MAFa in database

p Value for HWE

rs6761131 rs6717252 rs4675272 rs7580433

C>T T>A G>C G>A

181/162 177/157 181/162 181/162

0.09 0.34 0.44 0.07

0.07 0.32 0.44 0.11

0.10 0.27 0.35 0.08

1.00 1.00 1.00 0.70

a

MAF (minor allele frequene) in HapMap database for CHB population.

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T. Song et al. / Biochemical and Biophysical Research Communications 377 (2008) 1265–1268 Table 2 Adjusted OR (95%CI) for the association between SNPs in SUMO1 and NSCLP. SNP

p Valueb

Genotype a

wt/wt

wt/var

var/var

var carrier

rs6761131

Case/control OR (95%CI)

147/139 1.00 (Ref)

34/23 1.40 (0.78–2.49)

— —

34/23 1.40 (0.78–2.49)

0.25

rs6717252

Case/control OR (95%CI)

77/72 1.00 (Ref)

79/69 1.07 (0.68–1.69)

21/16 1.23 (0.59–2.54)

100/85 1.10 (0.71–1.69)

0.85

rs4675272

Case/control OR (95%CI)

57/50 1.00 (Ref)

88/81 0.95 (0.59–1.55)

36/31 1.02 (0.55–1.88)

124/112 0.97 (0.61–1.53)

0.97

rs7580433

Case/control OR (95%CI)

156/126 1.00 (Ref)

23/35 0.53 (0.30–0.94)

2/1 1.62 (0.14–18.02)

25/36 0.56 (0.32–0.98)

0.083

a b

wt, wild-type; var, variant. p Value by v2 test.

Table 3 Linkage disequilibrium between SUMO1 markers. rs6761131 rs6761131 rs6717252 rs4675272 rs7580433

0.0276 0.0416 0.0077

rs6717252

rs4675272

rs7580433

0.9979

0.8973 0.8183

0.9925 0.5221 0.391

0.1518 0.0325

0.0204

D0 above the diagonal, D below the diagonal.

polymorphisms and NSCLP risk. Animal model studies support an important role for SUMO1 in CL/P development. Cleft lip or palate was observed in 8.7% of heterozygous SUMO1 gene trap mice (SUMO1gt/+) or in 36% of SUMO1 gt/+, Eya1+/ double-heterozygous animals [13]. SUMO1 was expressed at embryonic day 13.5 (E13.5) by whole-mount in situ hybridization with strong expression in the upper lip, primary palate and medial edge epithelia of the secondary palate. At E14.5, expression of SUMO1 could be seen in the medial edge epithelial seam [13]. SUMO1 protein is bound to target proteins as part of a posttranslational modification system but, unlike ubiquitin, does not target proteins for degradation. It is interesting to note that several sumoylated proteins are directly associated with the human CL/P defect, including those encoded by MSX1, SATB2, and TBX22. Human and mouse mutation studies indicate that MSX1 is associated with specific human craniofacial disorders including cleft lip or palate and anodontia [15]. MSX1 is sumoylated in vivo and this modification constitutes a regulatory mechanism modulating MSX1 function during organogenesis [16]. SATB2 knockout mice have cleft palate and some SATB2 SNPs are significantly associated with NSCLP [17,18]. Importantly, SATB2 is a target for sumoylation, which is a reversible protein modification that modulates its activity as a transcription factor [19]. Temporal and spatial in situ hybridization studies using in both humans and mice showed that TBX22 is expressed primarily in the palatal shelves and tongue

during palatogenesis [20,21]. TBX22 mutations may partly contribute to NSCLP development [22]. It is clear that TBX22 is a target for SUMO1 and that this modification is required for TBX22 repressor activity [23]. SUMO1 modification has also been described for a number of other genes that are linked to craniofacial development, either by their expression pattern or because the classic mouse mutant phenotype has an orofacial cleft, including SOX9, SMAD4, EYA1 and P63 [13,24–26]. SUMO1 modification may represent a common pathway that regulates normal craniofacial development and is involved in the pathogenesis of both Mendelian and idiopathic forms of orofacial clefting. SUMO1 modification is susceptible to environmental effects that are strikingly similar to some of the risk factors described for CL/P. For cleft lip and palate, a 20–50% genetic contribution has been estimated and remaining cases were associated with a wide variety of environmental factors during early pregnancy [27]. It now seems likely that some of these factors may manifest through SUMO1 pathway disturbances. Destabilizing the normal balance of expression and activity for genes such as TBX22, MSX1, SATB2, and p63 during early pregnancy is likely to provide a high-risk environment for CL/P occurrence [23]. Elucidating the relationship among environmental factors, the SUMO1 pathway and the networks of craniofacial genes that are influenced by this posttranscriptional modification may be crucial to understanding idiopathic CL/P forms [23]. Other important studies may investigate potential signaling pathways involved in orofacial defects. Finally, identifying other SUMO1 gene polymorphisms that may be in linkage disequilibrium with our SNPs may help further solidify a role for SUMO1 in NSCLP development. Acknowledgments The authors thank the NSCLP patients and control subjects for their participation in this study. We thank the clinical staff of Department of Oral and Maxillofacial Surgery, Affiliated Stomatol-

Table 4 Associations between SUMO1common haplotypes and NSCLP. Haplotype rs6761131

rs6717252

rs4675272

rs7580433

T T C T T

A T A A T

C G G G G

G G G G A

Rare haplotypesb a b

Global haplotype association p-value: <0.0001. ‘‘Rare haplotypes” consists of haplotypes with frequency <0.05.

p Valuea

Total freq.

Case freq.

Control freq.

OR (95%CI)

0.50 0.25 0.08 0.06 0.05

0.55 0.27 0.09 0.01 0.07

0.43 0.22 0.07 0.11 0.03

1.00 0.98 1.37 0.14 1.52

(Ref) (0.66–1.45) (0.70–2.70) (0.05–0.40) (0.62–3.72)

— 0.91 0.36 0.0003 0.36

0.06

0.01

0.14

0.09 (0.03–0.26)

<0.0001

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ogy Hospital of the Harbin Medical University. This research was supported by the Smile Train and the National Science Funds of China. References

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