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Keywords:

  • Surfactant protein-D;
  • inflammatory bowel disease;
  • allele-specific expression;
  • genetic association

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of Interest Statement
  9. References

Surfactant protein-D (SP-D) is expressed on mucosal surfaces and functions in the innate immune response to microorganisms. We studied the genetic association of the two nonsynonymous SP-D single nucleotide polymorphisms (SNPs) rs721917 and rs2243639 in 256 inflammatory bowel disease (IBD) cases (123 CD and 133 UC) and 376 unrelated healthy individuals from an IBD population from Central Pennsylvania. Case-control analysis revealed a significant association of rs2243639 with susceptibility to Crohn's disease (CD) (p= 0.0036), but not ulcerative colitis (UC) (p= 0.883), and no association of rs721917 with CD (p= 0.328) or UC (p= 0.218). Using intestinal tissues from 19 individuals heterozygous for each SNP, we compared allelic expression of these two SNPs between diseased and matched normal tissues. rs2243639 exhibited balanced biallelic (BB) expression; while rs721917 exhibited differential allelic expression (BB 37%, imbalanced biallelic [IB] 45%, and dominant monoallelic [DM] 18%). Comparison of allelic expression pattern between diseased and matched normal tissues, 13 of 19 individuals (14 UC, 5 CD) showed a similar pattern. The six patients exhibiting a different pattern were all UC patients. The results suggest that differential allelic expression may affect penetrance of the SNP rs721917 disease-susceptibility allele in IBD. The potential impact of SP-D monoallelic expression on incomplete penetrance is discussed.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of Interest Statement
  9. References

Surfactant protein-D (SP-D) belongs to the collagen-containing C-type (calcium dependent) lectins called collectins. SP-D is mainly produced by alveolar type II cells in the lung, where it is involved in pulmonary immunity and initiates a wide range of defense mechanisms against microorganisms, including direct opsonization, neutralization, agglutination, complement activation, and enhanced phagocytosis (Kishore et al., 2006). SP-D has further been implicated in clearance of apoptotic and necrotic cells (Clark et al., 2002) and has direct bactericidal effects (Wu et al., 2003). Other functions include the control of pulmonary inflammation (Wright, 2005), TH-cell polarization (Madan et al., 2001), and suppression of lipid peroxidation (Bridges et al., 2000). Thus, SP-D plays an important role in linking adaptive and innate immune cell functions in the first line of the host defense and is thereby important in human health and disease (Kishore et al., 2005). However, SP-D is not restricted to the lung, but is instead widely distributed on mucosal surfaces of various tissues (Madsen et al., 2000), including the reproductive and gastrointestinal tract, and is most likely involved in extrapulmonary host defenses. In the porcine intestine, SP-D has been localized to epithelial cells of the intestinal glands (crypts of Lieberkuhn) in the duodenum, jejunum, and ileum (Soerensen et al., 2005), where it exerts a scavenger function by promoting uptake of pathogenic bacteria by these cells (Hogenkamp et al., 2007).

The pathogenesis of inflammatory bowel disease (IBD), consisting of two main subtypes, Crohn's disease (CD) and ulcerative colitis (UC), is believed to be a result of both genetic predisposition and environmental influences (Russell et al., 2004; Elson et al., 2005; Schreiber et al., 2005). Currently over 50 IBD-associated genes/loci have been identified (Lees & Satsangi, 2009). In addition, SP-D has recently been associated with IBD in a Japanese IBD population. Of the five known SNPs of the human SP-D gene, the 2-allele haplotype GG of an intronic SNP rs911887 and the nonsynonymous SNP rs2243639 reached statistical significance for susceptibility to both IBD and UC (Tanaka et al., 2009). Subset analysis in association with early onset UC further suggested that SP-D could be a disease-modifier gene as well as a disease- susceptibility gene. Interestingly, random monoallelic and/or heterogeneous allele expression occurring in genes involved in immunity (Ohlsson et al., 1998) has also been described for SP-D in the large intestine and other tissues in the rat (Lin & Floros, 2002). Given the role of SP-D in innate immunity and the observation that variable or heterogeneous allele expression occurs, we further examined the genetic association of SP-D nonsynonymous variants with IBD in a population of central Pennsylvania, and studied the effect of allele expression on incomplete penetrance in disease phenotype. The two genetic variants analyzed were rs721917 (C/T Met11Thr) in exon 2 and rs2243639 (G/A Ala160Thr) in exon 5. The study population consisted of 256 IBD patients and 376 unrelated healthy controls from Central Pennsylvania.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of Interest Statement
  9. References

Study Samples

IBD patients

A total of 256 IBD cases were studied, including: (1) 131 individuals with IBD (80 CD patients, 51 UC patients) from 72 families with familial IBD history and (2) 125 sporadic IBD patients (43 CD patients, 82 UC patients) from the Milton S. Hershey Medical Center from Central Pennsylvania. Samples were obtained from our IBD familial registry established in 1999. Peripheral blood was collected from study participants and used to derive B cell lines by Epstein Barr virus (EBV) transformation. Intestinal tissues were obtained at the time of surgery. IBD diagnosis was made using standard clinical, radiological, and endoscopic/histopathological procedures.

Controls

Peripheral blood samples from 376 unrelated healthy individuals from the Milton S. Hershey Medical Center were used as controls.

Ethical Considerations

All human tissues described above were approved by the Human Subjects Protection Offices of The Pennsylvania State University College of Medicine, and were undertaken with the understanding and written consent of each subject.

Genomic DNA and RNA Isolation

Genomic DNA from B cell lines and peripheral blood was isolated using a QIAamp DNA Blood Kit (Qiagen Inc., Valencia, CA), and DNA from intestinal tissues was isolated with a QIAamp DNA Mini Kit according to the manufacturer's instruction. DNA concentrations were measured using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technology, Wilmington, DE).

Total RNA was extracted from 38 diseased and nondiseased adjacent intestinal tissues from IBD patients using the RNeasy Mini Kit according to the manufacture's instruction (Qiagen Inc.). cDNA was synthesized from 1 μg of total RNA using the Superscript III First Strand Synthesis Kit (Invitrogen, Carlsbad, CA).

Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

cDNA was used as template to amplify a 484-bp fragment of SP-D using primers SPD18s (5′-CTCCAGGCTGCTTTCTCTCAG-3′) and SPD26r (5′-TGGCAGCATGAGGGTCTAAG-3′), as well as β-actin (266-bp fragment) with primers Actin7s (5′-TGTGGATCAGCAAGCAGGAG-3′) and Actin8r (5′-GTGAACTTTGGGGGATGCTC-3′). PCR reactions were prepared as previously described (Lin et al., 2009), and amplifications were performed with the following PCR profile: 95 °C for 2 min, 35 cycles of 95 °C for 30 s, 58 °C for 1 min, and 72 °C for 1 min, and then 72 °C for 4 min.

Northern Analysis

Total mRNA (3.5 μg) was separated on 1% agarose gel containing formaldehyde (0.22 M) and transferred to positively charged nylon transfer membrane (GeneScreen Plus, Perkin Elmer Life Science, Boston, MA) and fixed under a UV Stratalinker 2400 (Stratagene, La Jolla, CA). The procedure for Northern analysis was described before (Lin et al., 2001). Briefly, the blot was hybridized with 32P-ATP-labeled SP-D antisense oligonucleotide probe. The probe was prepared with the KinaseMax kit (Ambion, Austin, TX) and hybridization was performed in Ultrahyb solution according to the manufacturer's instructions (Ambion). The blot was then exposed to Kodak XAR5 film at −80°C for 5 min for lung RNA or 24 h for intestine RNA.

Analysis of Genotypes and Allele Expression with PCR-Based RFLP

Genotypes and allele expression of the two genetic variants located in exon regions of the human SP-D gene, rs721917 (C/T Met11Thr) in exon 2 and rs2243639 (G/A Ala160Thr) in exon 5 (Tanaka et al., 2009), were determined using PCR-based restriction fragment length polymorphism (RFLP) and converted RFLP methods (Lin et al., 1996).

For genotype analysis, an 11-kb DNA fragment of the SP-D gene was amplified with primers SPD30s and SPD26r (SPD30s 5′-TCACCTCTAGAAGCTGAGCCAAGCC-3′, SPD26r 5′-TGGCAGCATGAGGGTCTAAG-3′) using Elongase Enzyme Mix (Invitrogen) according to the manufacturer's instructions with the following PCR profile: 95 °C for 2 min, 10 cycles of 95 °C for 30 s, 58 °C 1 for min, and 68 °C for 10 min, followed by 20 cycles of 95 °C for 30 s, 62 °C for 1 min, and 68 °C for 12 min, and then 68 °C for 20 min. One microliter of the PCR products was used as template to further amplify a 102-bp DNA fragment containing rs721917 with primers SPD31s (5′-CTCCTCTCTGCACTGGTCAT-3′) and SPD20r (5′-ACCAGGGTGCAAGCACTGCG-3′), and a 164-bp DNA fragment containing rs2243639 with primers SPD34s (5′-AGCGTGGAGTCCCTGGAAGC-3′) and SPD35r (5′-AGATTCTCTCCATGTTCCCAG-3′). The PCR profile consisted of 95 °C for 2 min, 5 cycles of 95 °C for 30 s, 50 °C for 1 min, and 72 °C for 1 min followed by 30 cycles of 95 °C for 30 s, 58 °C for 1 min, and 72 °C for 1 min, and a final extension for 4 min at 72 °C.

For allele expression, 1-μl cDNA was used as template to amplify a 102-bp fragment containing rs721917 with primers SPD31s and SPD20r, and a 118-bp fragment containing rs2243639 with primers SPD34s and SPD23r (5′-TCTCCAGGAATGCCTTTG-3′). All PCR reactions were prepared as previously described (Lin et al., 2009).

During PCR amplification, SNPs rs721917 and rs2243639 were converted to Fsp I and Hha I restriction enzyme recognition sites, respectively. PCR products were digested with Fsp I (for rs721917) and Hha I (for rs2243639) according to the manufacturer's instructions (New England Biolabs, Ipswich, MA). The digested PCR products were separated on 8% polyacrylamide gel electrophoresis (PAGE) and visualized by ethidium bromide staining. Genotypes were scored according to the gel pattern of allelic products following digestion (Fig. 1).

image

Figure 1. Genotype analysis of SP-D SNPs rs721917 and rs2243639. PCR primers used for genotyping were SPD31s and SPD20r for rs721917, and SPD34s and SPD35r for rs2243639. The primer sequences and PCR amplification conditions were described in Section “Materials and Methods.” The allelic products of C and T for rs721917 in Panel A, and the allelic products of A and G for rs2243639 in Panel B are indicated by arrows. Individual samples are indicated below the gels. The 20-bp fragments from the T allele of rs721917 and the G allele of rs2243639 in the restriction enzyme digested PCR products have run off of the gels. M is a 1-kb DNA ladder. C is a negative control of PCR using dH2O instead of DNA. Number 1–16 is the number of the 16 individual samples.

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Quantification of Allelic Products and Classification of the Allele-Specific Expression Pattern

Allele expression was quantified by densitometry using ImageJ software (NIH, http://rsbweb.nih.gov/ij/). The ratio of the digested PCR products of allele T to allele C for rs721917 and of allele G to allele A for rs2243639 in all heterozygous individuals was used to classify the allele-specific expression pattern in each individual (Lin & Floros, 2002). If the difference between the two allelic PCR products was less than 20% (the ratio = 0.8–1.2), the allele expression was classified as balanced biallelic (BB), whereas a difference of more than 20% but within 5-fold (the ratio = 0.2–<0.8 or 1.2–≤5.0) was classified as imbalanced biallelic (IB) expression. If the difference was more than 5-fold (the ratio = <0.2 or >5.0), the allele expression was classified as dominant monoallelic (DM).

Statistical Analysis of Genetic Association

For the genetic association study, Pearson's χ2-test with one degree of freedom for allelic association was performed using Haploview v4.2 (MIT/Harvard Broad Institute http://www.broadinstitute.org/scientific-community/science/programs/medical-and-population-genetics/haploview/haploview). We also calculated genotype-based odds ratio (OR) using Fisher's exact test on the contingency tables, and the effect of the association is measured by the resulting p-values. A genotype difference was considered significant when p < 0.05. The two SNPs were analyzed for linkage disequilibrium and found to be significantly correlated (p < 0.001, D′= 0.944, Corr = 0.611). As only two SNPs in linkage disequilibrium were considered in this study, the reported p-values are not adjusted for multiple comparisons.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of Interest Statement
  9. References

Genetic Association of SP-D rs2243639 (Ala160Thr) with IBD

For genotype analysis of SP-D variants rs721917 and rs2243639, 257 IBD cases including 132 familial IBD (81 CD patients, 51 UC patients) and 125 sporadic IBD patients (43 CD patients, 82 UC patients) and 376 unrelated healthy controls were studied using PCR-based RFLP and converted RFLP methods. After digestion with Fsp I (for rs721917) and Hha I (for rs2243639), PCR products were separated on 8% PAGE and visualized by ethidium bromide staining. Genotypes were scored according to the gel pattern of allelic products after digestion: for rs721917 C allele is 102 bp, T allele is 82 and 20 bp; for rs2243639 A allele is 164 bp, G allele is 144 and 20 bp. The 20-bp fragments from T allele of rs721917 and G allele of rs2243639 in the restriction enzyme digested PCR products ran off of the gels. Figure 1 shows exemplary results for genotypes at rs721917 (Fig. 1A) and rs2243639 (Fig. 1B) using genomic DNA from 16 different and randomly chosen IBD cases.

For rs721917, the frequency of genotypes in the control samples was 13% CC, 53% CT, and 34% TT calculated from Table 1. The IBD case group exhibited a comparable genotype frequency of 15% CC, 50% CT, and 35% TT. The distribution of both alleles is very similar between cases and controls. For rs2243639, the frequency of genotypes in the control samples was 39% GG, 45% GA, and 16% AA. The IBD case group exhibited a genotype frequency of 28% GG, 55% GA, and 17% AA. The distribution of both alleles is different between cases and controls. In the CD subgroup, genotypes were presented as 23.6% GG, 53.6% GA, and 22.8% AA. The UC subgroup exhibited a genotype frequency of 32.3% GG, 56.4% GA, and 11.3% AA.

Table 1.  Genotype analysis of SP-D rs2243639 and rs721917 in CD, UC, and IBD.
GenotypesCasesControlsOR95% CIp-value
rs2243639
For CD
 AA28601.55311.149–2.0980.0036
 AG66170   
 GG29144   
For UC
 AA15601.03000.765–1.3850.8846
 AG75170   
 GG43144   
For IBD
 AA43601.25770.985–1.5900.0543
 AG141170   
 GG72144   
rs721917
For CD
 CC14500.86290.625–1.1600.3285
 CT61198   
 TT49128   
For UC   0. 
 CC25501.1961892–1.6020.2180
 CT67198   
 TT41128   
For IBD
 CC39501.01890.805–1.2890.9070
 CT128198   
 TT90128   

To further analyze a possible genetic association of these genetic variants with IBD, CD and UC, genotyping data were subjected to statistical analyses. The results indicate that rs2243639 was significantly associated with CD (OR = 1.5531, 95% CI = 1.14992–2.0983, p= 0.0036), but not with UC (OR = 1.0300, 95% CI = 0.7645–1.3845, p= 0.8836). For the association with IBD the p-value is 0.054. Although very close, it failed to reach the significance level (p < 0.05). On the other hand, genetic variation at rs721917 did not reach statistical significance for susceptibility to either IBD or subgroups CD and UC (Table 1).

SP-D mRNA Expression in the Human Intestine

SP-D has been demonstrated to be expressed in rat colon and small intestine by Northern and RT-PCR analysis and in human ileum and colon by RT-PCR and immunohistochemistry analysis (Madsen et al., 2000; Bourbon & Chailley-Heu, 2001; van Rozendaal et al., 2001). We performed Northern and RT-PCR analyses for the intestinal tissues from IBD patients. The result from Northern analysis showed that the SP-D gene is expressed in both small intestine and colon. Compared to the human lung as shown in Figure 2A, the intestine SP-D mRNA band on Northern blot with 24 h of exposure was lower than that in the lung with only 5-min exposure at −80°C, suggesting that the level of SP-D transcripts in intestinal tissues is much lower. The size of SP-D mRNA from intestinal tissues was the same as that in the lung. The results from RT-PCR also confirmed that SP-D is expressed in both small intestine and colon, and no difference was observed in the size of PCR product between SP-D mRNA from intestine and lung (Fig. 2B).

image

Figure 2. SP-D expression in human intestine from Northern and RT-PCR analysis. (Panel A) Northern analysis. Total mRNA (3.5 μg) was separated on 1% agarose gel containing formaldehyde (0.22 M) and blotted on GeneScreen Plus membrane. The blot was hybridized with a 32P-ATP-labeled SP-D antisense oligonucleotide probe. The Northern blot procedure and hybridization was described in Section “Materials and Methods.” (Panel B) RT-PCR. PCR primers for PCR amplification were SPD18s and SPD26r for SP-D, and Actin7s and Actin8r for β-actin. The PCR products were 484 bp for SP-D and 266 bp for actin. Primer sequences and PCR conditions were described in Section “Materials and Methods.” Samples are indicated below the gels. Sm int: small intestine. M is a 1-kb DNA ladder, C is a PCR-negative control.

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Allele Expression Patterns of SP-D rs2243639 and rs721917 in Human Intestinal Tissues

We used a PCR-based RFLP method to distinguish the PCR products amplified from different alleles. First we amplified cDNA fragments with SP-D SNPs rs721917 and rs2243639, and then we used the same restriction enzymes to digest the PCR products. The digested PCR products were separated by 8% PAGE. The allelic PCR products were quantitatively analyzed. For homozygous individuals, only one allelic product was shown; however, for the heterozygous individuals two allelic products should be shown. If the two alleles are not equally transcribed, the patterns of allelic product will vary. We quantified each allelic product and used the ratio of allelic products to classify the allele expression as: BB (0.8–1.2), IB (0.2–<0.8 and 1.2–<5.0) or DM (<0.2 and >5.0). The results from 38 intestinal tissues revealed that SP-D variant rs721917 showed a differential allele expression (Fig. 3A and Table 2) with 14 BB samples, 17 IB samples, and 7 DM samples. For rs2243639, only BB expression was observed in all the intestinal tissues examined (Fig. 3B).

image

Figure 3. Differential allele expression of SP-D in human intestinal tissues. cDNA (1 μl) was used as template to amplify a 102-bp fragment containing rs721917 with primers SPD31s and SPD20r, and a 118-bp fragment containing rs2243639 with primers SPD34s and SPD23r as described in Section “Materials and Methods.” PCR products (5 μl) were digested with Fsp I (for rs721917) and Hha I (for rs2243639) and then separated on 8% PAGE. The PCR products of allele C and T for rs721917 in Panel A, and of allele A and G for rs2243639 in Panel B are indicated by arrows. The 20-bp fragments from T allele of rs721917 and G allele of rs2243639 in the restriction enzyme digested PCR products have run off of the gels. All samples in Panel A were heterozygous for the rs721917, and in Panel B were heterozygous for the rs2243639. Numbers 1–16 represent the number of the individual samples. Individual samples A1–16 are for rs721917 and B1–16 for rs2243639. C is a negative PCR control.

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Table 2.  Allelic expression patterns between diseased and normal tissues.
SampleDiseaseTissue typeC/T ratioBalanced 0.8–1.2Imbalanced 0.2–<0.8 >1.2–5.0Monoallelic <0.2 and >5.0
888–46-1UCND1.451 IB 
 D5.886  M
888–113-1UCND1.552 IB 
 D2.983 IB 
888–68-1UCND1.058B  
 D3.963 IB 
888–128-1UCND1.487 IB 
 D1.971 IB 
777–35-1CDND1.572 IB 
 D1.354 IB 
777–35-1CDND1.572 IB 
 D1.354 IB 
888–44-1UCND1.508 IB 
 D1.312 IB 
888–53-1UCND1.008B  
 D1.281 IB 
888–69-1UCND1.208B  
 D1.064B  
888–50-1UCND1.201 IB 
 D0.641 IB 
132–01-1CDND0.860B  
 D1.194B  
888–83-1UCND1.156B  
 D0.935B  
777–57-1CDND0.986B  
 D0.986B  
888–90-1UCND0.145  M
 D0.959B  
777–60-1UCND0.936B  
 D0.399 IB 
145–01-1UCND0.935B  
 D0.936B  
888–52-1UCND0.154  M
 D0.487 IB 
777–14-1CDND0.095  M
 D0.137  M
888–49-1UCND0.128  M
 D0.061  M

The 38 intestinal tissues studied were 19 pairs of diseased and matched nondiseased tissues from the same organ, such as diseased colon/matched nondiseased colon, diseased terminal ileum/matched nondiseased terminal ileum. Thirteen individuals exhibited the same allelic expression pattern between diseased and normal tissues, and only six individuals exhibited different allelic expression patterns between diseased and normal tissues (Table 2). In these six individuals, five were from BB to IB, or from IB to DM, and only one exhibited a large difference from BB to DM. We have previously studied SP-D allelic expression in eight rat pedigrees with three generations. We observed that the parents from a family with high frequency of SP-D monoallelic expression among their family members exhibited high frequency of monoallelic expression, suggesting that inheritable factors may regulate allelic expression (Lin & Floros, 2002). The present results from human SP-D further support the notion that genetic factors play a role in allelic expression traits. It is very interesting to point out that all six individuals with a different allelic expression pattern were UC patients. Calculated from Table 2, 43% (6 out of 14) of UC patients exhibited SP-D allele differential expression pattern between disease and normal tissues, while all six CD patients exhibited the same pattern.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of Interest Statement
  9. References

SP-D plays an important function in host defense and regulation of inflammation. The SNP rs721917 has been shown to associate with risk of tuberculosis (Floros et al., 2000). Although genetic variants of SP-D alone failed to show a significant association of with lung diseases, such as ARDS (acute respiratory distress syndrome), COPD (chronic obstructive pulmonary disease), and IPF (idiopathic pulmonary fibrosis) (Lin et al., 2000; Guo et al., 2001; Selman et al., 2003), haplotypes of SP-D with other surfactant proteins were associated with BPD (bronchopulmonary dysplasia) and ARDS (acute RDS) (Pavlovic et al., 2006; Thomas et al., 2007). While human SP-D has been intensively studied in lung diseases (Floros & Hoover, 1998; Floros & Kala, 1998; Lin et al., 2000), it is also believed to play a role in extrapulmonary systems (Madsen et al., 2000). Recently, the genetic association of SP-D with IBD was studied in a Japanese population. Among five SP-D SNPs (two exonic, three intronic) analyzed, the minor allele G of the intronic rs911887 was associated with UC, whereas both nonsynonymous SNPs, rs721917 in exon 2 and rs2243639 in exon 5, were not associated with IBD. However, a 2-allele haplotype GG of the intronic rs911887 and exonic rs2243639 was associated with UC (Tanaka et al., 2009).

In the present study, we investigated two exonic nonsynonymous SNPs of SP-D in 257 IBD cases (124 CD and 133 UC) and 376 unrelated healthy individuals in an IBD population from Central Pennsylvania. Different from the previous published study, we found that the exonic SNP rs2243639 is significantly associated with IBD alone, and also the association is with IBD subtype CD (p= 0.0036), and not UC. Our results are consistent with the published report in the sense that SP-D has been associated with IBD. However, we found that rs2243639 alone was associated with IBD, specifically CD; whereas their IBD association was with a 2-allele haplotype comprising rs2243639 with another minor allele (rs911887), specifically for UC. In different IBD populations differences in genetic background and environmental factors may collectively contribute to differential IBD susceptibilities and/or other complexities discussed below. Differences in the association of genetic variations with IBD in different populations have been observed for several IBD-susceptibility genes. Some risk alleles are absent or fixed in a population, which implies that risk alleles identified in one population do not necessarily account for disease prevalence in all human populations (Myles et al., 2008). Indeed, risk loci for one population may not be found for a different population (Morgan et al., 2010; Nakagome et al., 2010). Therefore disease-association studies in diverse populations are required in order to determine whether different alleles are responsible for disease prevalence in different populations. Recently, a correlation between SP-D SNPs and SP-D serum protein concentrations has been demonstrated (Foreman et al., 2011). The reason that SP-D nonsynonymous SNP rs721917 shows no association with IBD may be that the amino acid change from Met>Thr at that position has no effect on protein function, or that there is incomplete penetrance of genetic information.

The penetrance of a disease-causing mutation is the proportion of individuals with the mutation exhibiting clinical symptoms. If the proportion is less than 100%, it is defined as an incompletely penetrant mutation. Incomplete penetrance likely results from genetic, epigenetic, and environmental factors, all of which affect gene function from transcription to protein activation. Figure 4 illustrates the effect of allelic expression on penetrance of a disease allele. As a result of variation in allele expression, the final gene expression products (mRNA and protein) of a heterozygous individual (TC) may consist of an equal amount of product from wild-type allele T and mutated allele C; only the product of wild-type allele T or mutated allele C; or only the dominant product of wild-type T or mutated allele C. The complete loss of heterozygosity or quantitative changes in expression of a mutated allele may lead to a different disease phenotype outcome. From the point of view of gene function, a heterozygous individual will be the same as a homozygous wild-type allele individual, or a homozygous mutated allele individual. Monoallelic expression is widely observed in human and other diploid eukaryotic organisms (Chess et al., 1994; Rajewsky, 1996; Zakharova et al., 2009). The genes exhibiting monoallelic expression include imprinted genes (Reik & Walter, 2001), X-inactivated genes (Lyon, 1986), and some autosomal genes (Chess et al., 1994). Using a genome-wide assay, it was assessed that more than 5% of genes were subjected to random monoallelic expression (Gimelbrant et al., 2007). Monoallelic expression can lead to differences in levels of gene expression. These include loss of imprinting in imprinted genes and loss of heterozygosity in autosomal genes. A role of monoallelic expression has been observed in disease susceptibility, such as for imprinted genes IGF2 and MEST in lung adenocarcinoma (Kohda et al., 2001), IGF2 in Wilms’ tumor (Ogawa et al., 1993), and GABRA5, GABRB3, and SNRPN in Angelman syndrome (Bittel et al., 2005); imprinted and autosomal genes in cancers (Barrois et al., 2001; Morison et al., 2005; Das et al., 2009); and APOE4 in Alzheimer disease (Xu et al., 2007). Recently, a decrease in allele-specific expression of the TGFBR1 gene was shown to predispose to colorectal cancer (Tomsic et al., 2010).

image

Figure 4. Illustration of the effect of allelic expression on incomplete penetrance of disease phenotype. As a result of variation in allele expression, the final gene expression products (mRNA and protein) of a heterozygous individual (TC) may have an equal (balanced biallelic) amount of product from wild-type allele T and mutated allele C; only a single (monoallelic) product of wild-type allele T or mutated allele C; or only the dominant (imbalanced biallelic) product of wild-type T or mutated allele C. The complete loss of heterozygosity or quantitative changes in expression of a mutated allele may lead to a different disease phenotype outcome.

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We examined allele expression analyses of the two SP-D SNPs in intestinal tissues and observed that rs2243639 exhibited BB expression; while rs721917 exhibited a differential allele expression (BB 37%, IB 45%, and DM 18%). Interestingly, from our comparison of the allelic expression pattern between diseased and matched normal tissues, 13 of 19 individuals (14 UC, 5 CD) showed a similar expression pattern, while the other six patients exhibiting a different pattern were all UC patients. These results are consistent with our previous finding that the family background is involved in the regulation of SP-D allele expression in a rat pedigree, thereby suggesting that a genetic factor(s) may also be involved in the regulation of SP-D allele expression. Our data suggest that heterogeneous allele expression may be a factor affecting incomplete penetrance of mutations, which thus may cause disease, and may help in understanding why SP-D rs2243639 is associated, while rs721917 is not associated with CD. A recent paper (Arai et al., 2011) showed allelic expression imbalance in intestinal tissue (n= 10) of the risk haplotype of the IBD-associated gene, NKX2–3. It is thought that the expression imbalance of the risk haplotype may confer susceptibility to UC in the colonic mucosa. We conclude that SP-D rs2243639 confers susceptibility to CD (p= 0.0036), but not to UC (p= 0.883), and there is no association of rs721917 with CD (p= 0.328) or UC (p= 0.218). The present observations need to be confirmed with a large number of samples, and the mechanism of SP-D monoallelic expression on disease outcome should be investigated.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of Interest Statement
  9. References

The authors thank Tammy Lin for her aid in genotype analysis. This work is supported by a grant from the Philadelphia Health Care Trust (WAK), a feasibility research grant from Department of Surgery (ZL), a research grant from American Lung Association (ZL), and NIH HL34788 (JF).

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of Interest Statement
  9. References
  • Arai, T., Kakuta, Y., Kinouchi, Y., Kimura, T., Negoro, K., Aihara, H., Endo, K., Shiga, H., Kanazawa, Y., Kuroha, M., Moroi, R., Nagasawa, H., Shimodaira, Y., Takahashi, S. & Shimosegawa, T. (2011) Increased expression of NKX2.3 mRNA transcribed from the risk haplotype for ulcerative colitis in the involved colonic mucosa. Hum Immunol 72, 587591.
  • Barrois, M., Eychenne, M. K., Terrier-Lacombe, M. J., Duarte, N., Dubourg, C., Douc-Rasy, S., Chompret, A., Khagad, M., Hartmann, O., Caput, D. & Benard, J. (2001) Genomic and allelic expression status of the p73 gene in human neuroblastoma. Med Pediatr Oncol 36, 4547.
  • Bittel, D. C., Kibiryeva, N., Talebizadeh, Z., Driscoll, D. J. & Butler, M. G. (2005) Microarray analysis of gene/transcript expression in Angelman syndrome: Deletion versus UPD. Genomic 85, 8591.
  • Bourbon, J. R. & Chailley-Heu, B. (2001) Surfactant proteins in the digestive tract, mesentery, and other organs: Evolutionary significance. Comp Biochem Physiol A Mol Integr Physiol 129, 151161.
  • Bridges, J. P., Davis, H. W., Damodarasamy, M., Kuroki, Y., Howles, G., Hui, D. Y. & Mccormack, F. X. (2000) Pulmonary surfactant proteins A and D are potent endogenous inhibitors of lipid peroxidation and oxidative cellular injury. J Biol Chem 275, 3884838855.
  • Chess, A., Simon, I., Cedar, H. & Axel, R. (1994) Allelic inactivation regulates olfactory receptor gene expression. Cell 78, 823834.
  • Clark, H., Palaniyar, N., Strong, P., Edmondson, J., Hawgood, S. & Reid, K. B. (2002) Surfactant protein D reduces alveolar macrophage apoptosis in vivo. J Immunol 169, 28922899.
  • Das, R., Hampton, D. D. & Jirtle, R. L. (2009) Imprinting evolution and human health. Mamm Genome 20, 563572.
  • Elson, C. O., Cong, Y., Mccracken, V. J., Dimmitt, R. A., Lorenz, R. G. & Weaver, C. T. (2005) Experimental models of inflammatory bowel disease reveal innate, adaptive, and regulatory mechanisms of host dialogue with the microbiota. Immunol Rev 206, 260276.
  • Floros, J. & Hoover, R. R. (1998) Genetics of the hydrophilic surfactant proteins A and D. Biochim Biophys Acta 1408, 312322.
  • Floros, J. & Kala, P. (1998) Surfactant proteins: Molecular genetics of neonatal pulmonary diseases. Annu Rev Physiol 60, 365384.
  • Floros, J., Lin, H. M., Garcia, A., Salazar, M. A., Guo, X., Diangelo, S., Montano, M., Luo, J., Pardo, A. & Selman, M. (2000) Surfactant protein genetic marker alleles identify a subgroup of tuberculosis in a Mexican population. J Infect Dis 182, 14731478.
  • Foreman M. G., Kong X., Demeo D. L., Pillai S. G., Hersh C. P., Bakke P., Gulsvik A., Lomas D. A., Litonjua A. A., Shapiro S. D., Tal-Singer R., Silverman E. K. (2011) Polymorphisms in surfactant protein-D are associated with chronic obstructive pulmonary disease. Am J Respir Cell Mol Biol 44, 316322.
  • Gimelbrant, A., Hutchinson, J. N., Thompson, B. R. & Chess, A. (2007) Widespread monoallelic expression on human autosomes. Science 318, 11361140.
  • Guo, X., Lin, H. M., Lin, Z., Montano, M., Sansores, R., Wang, G., Diangelo, S., Pardo, A., Selman, M. & Floros, J. (2001) Surfactant protein gene A, B, and D marker alleles in chronic obstructive pulmonary disease of a Mexican population. Eur Respir J 18, 482490.
  • Hogenkamp, A., Herias, M. V., Tooten, P. C., Veldhuizen, E. J. & Haagsman, H. P. (2007) Effects of surfactant protein D on growth, adhesion and epithelial invasion of intestinal Gram-negative bacteria. Mol Immunol 44, 35173527.
  • Kishore, U., Bernal, A. L., Kamran, M. F., Saxena, S., Singh, M., Sarma, P. U., Madan, T. & Chakraborty, T. (2005) Surfactant proteins SP-A and SP-D in human health and disease. Arch Immunol Ther Exp (Warsz) 53, 399417.
  • Kishore, U., Greenhough, T. J., Waters, P., Shrive, A. K., Ghai, R., Kamran, M. F., Bernal, A. L., Reid, K. B., Madan, T. & Chakraborty, T. (2006) Surfactant proteins SP-A and SP-D: Structure, function and receptors. Mol Immunol 43, 12931315.
  • Kohda, M., Hoshiya, H., Katoh, M., Tanaka, I., Masuda, R., Takemura, T., Fujiwara, M. & Oshimura, M. (2001) Frequent loss of imprinting of IGF2 and MEST in lung adenocarcinoma. Mol Carcinog 31, 184191.
  • Lees, C. W. & Satsangi, J. (2009) Genetics of inflammatory bowel disease: Implications for disease pathogenesis and natural history. Expert Rev Gastroenterol Hepatol 3, 513534.
  • Lin, Z., Cui, X. & Li, H. (1996) Multiplex genotype determination at a large number of gene loci. Proc Natl Acad Sci USA 93, 25822587.
  • Lin, Z., Pearson, C., Chinchilli, V., Pietschmann, S. M., Luo, J., Pison, U. & Floros, J. (2000) Polymorphisms of human SP-A, SP-B, and SP-D genes: Association of SP-B Thr131Ile with ARDS. Clin Genet 58, 181191.
  • Lin, Z., Demello, D., Phelps, D. S., Koltun, W. A., Page, M. & Floros, J. (2001) Both human SP-A1 and Sp-A2 genes are expressed in small and large intestine. Pediatr Pathol Mol Med 20, 367386.
  • Lin, Z. & Floros, J. (2002) Heterogeneous allele expression of pulmonary SP-D gene in rat large intestine and other tissues. Physiol Genomics 11, 235243.
  • Lin, Z., Poritz, L., Franke, A., Li, T.-Y., Ruether, A., Byrnes, K. A., Wang, Y., Gebhard, A. W., Macneill, C., Thomas N. J., Schreiber, S., Koltun, W. A. (2009) Genetic association of DLG5 R30Q with familial and sporadic inflammatory bowel disease in men. Dis Markers 27, 193201.
  • Lyon, M. F. (1986) X chromosomes and dosage compensation. Nature 320(6060): 313.
  • Madan, T., Kishore, U., Singh, M., Strong, P., Clark, H., Hussain, E. M., Reid, K. B. & Sarma, P. U. (2001) Surfactant proteins A and D protect mice against pulmonary hypersensitivity induced by Aspergillus fumigatus antigens and allergens. J Clin Invest 107, 467475.
  • Madsen, J., Kliem, A., Tornoe, I., Skjodt, K., Koch, C. & Holmskov, U. (2000) Localization of lung surfactant protein D on mucosal surfaces in human tissues. J Immunol 164, 58665870.
  • Morgan, A. R., Han, D. Y., Huebner, C., Lam, W. J., Fraser, A. G. & Ferguson, L. R. (2010) PTPN2 but not PTPN22 is associated with Crohn's disease in a New Zealand population. Tissue Antigens 76, 119125.
  • Morison, I. M., Ramsay, J. P. & Spencer, H. G. (2005) A census of mammalian imprinting. Trends Genet 21, 457465.
  • Myles, S., Davison, D., Barrett, J., Stoneking, M. & Timpson, N. (2008) Worldwide population differentiation at disease-associated SNPs. BMC Med Genomics 1, 22: 1–10.
  • Nakagome, S., Takeyama, Y., Mano, S., Sakisaka, S., Matsui, T., Kawamura, S. & Oota, H. (2010) Population-specific susceptibility to Crohn's disease and ulcerative colitis; dominant and recessive relative risks in the Japanese population. Ann Hum Genet 74, 126136.
  • Ogawa, O., Eccles, M. R., Szeto, J., Mcnoe, L. A., Yun, K., Maw, M. A., Smith, P. J. & Reeve, A. E. (1993) Relaxation of insulin-like growth factor II gene imprinting implicated in Wilms’ tumour. Nature 362, 749751.
  • Ohlsson, R., Tycko, B. & Sapienza, C. (1998) Monoallelic expression: ‘there can only be one’. Trends Genet 14, 435438.
  • Pavlovic, J., Papagaroufalis, C., Xanthou, M., Liu, W., Fan, R., Thomas, N. J., Apostolidou, I., Papathoma, E., Megaloyianni, E., Diangelo, S. & Floros, J. (2006) Genetic variants of surfactant proteins A, B, C, and D in bronchopulmonary dysplasia. Dis Markers 22, 277291.
  • Rajewsky, K. (1996) Clonal selection and learning in the antibody system. Nature 381, 751758.
  • Reik, W. & Walter, J. (2001) Genomic imprinting: Parental influence on the genome. Nat Rev 2, 2132.
  • Russell, R. K., Nimmo, E. R. & Satsangi, J. (2004) Molecular genetics of Crohn's disease. Curr Opin Genet Dev 14, 264270.
  • Schreiber, S., Rosenstiel, P., Albrecht, M., Hampe, J. & Krawczak, M. (2005) Genetics of Crohn disease, an archetypal inflammatory barrier disease. Nat Rev Genet 6, 376388.
  • Selman, M., Lin, H. M., Montano, M., Jenkins, A. L., Estrada, A., Lin, Z., Wang, G., Diangelo, S., Guo, X., Umstead, T. M., Lang, C. M., Pardo, A., Phelps, D. S. & Floros, J. (2003) Surfactant protein A and B genetic variants predispose to idiopathic pulmonary fibrosis. Hum Genet 113, 542550.
  • Soerensen, C. M., Nielsen, O. L., Willis, A., Heegaard, P. M. & Holmskov, U. (2005) Purification, characterization and immunolocalization of porcine surfactant protein D. Immunol 114, 7282.
  • Tanaka, M., Arimura, Y., Goto, A., Hosokawa, M., Nagaishi, K., Yamashita, K., Yamamoto, H., Sonoda, T., Nomura, M., Motoya, S., Imai, K. & Shinomura, Y. (2009) Genetic variants in surfactant, pulmonary-associated protein D (SFTPD) and Japanese susceptibility to ulcerative colitis. Inflam Bowel Dis 15, 918925.
  • Thomas, N. J., Fan, R., Diangelo, S., Hess, J. C. & Floros, J. (2007) Haplotypes of the surfactant protein genes A and D as susceptibility factors for the development of respiratory distress syndrome. Acta Paediatr 96, 985989.
  • Tomsic, J., Guda, K., Liyanarachchi, S., Hampel, H., Natale, L., Markowitz, S. D., Tanner, S. M. & de la Chapelle, A. (2010) Allele-specific expression of TGFBR1 in colon cancer patients. Carcinogenesis 31, 18001804.
  • Van Rozendaal, B. A., Van Golde, L. M. & Haagsman, H. P. (2001) Localization and functions of SP-A and SP-D at mucosal surfaces. Ped Pathol & Mol Med 20, 319339.
  • Wright, J. R. (2005) Immunoregulatory functions of surfactant proteins. Nat Rev 5, 5868.
  • Wu, H., Kuzmenko, A., Wan, S., Schaffer, L., Weiss, A., Fisher, J. H., Kim, K. S. & Mccormack, F. X. (2003) Surfactant proteins A and D inhibit the growth of Gram-negative bacteria by increasing membrane permeability. J Clin Investig 111, 15891602.
  • Xu, P. T., Li, Y. J., Qin, X. J., Kroner, C., Green-Odlum, A., Xu, H., Wang, T. Y., Schmechel, D. E., Hulette, C. M., Ervin, J., Hauser, M., Haines, J., Pericak-Vance, M. A. & Gilbert, J. R. (2007) A SAGE study of apolipoprotein E3/3, E3/4 and E4/4 allele-specific gene expression in hippocampus in Alzheimer disease. Mol Cell Neurosci 36, 313331.
  • Zakharova, I. S., Shevchenko, A. I. & Zakian, S. M. (2009) Monoallelic gene expression in mammals. Chromosoma 118, 279290.