Family-Based Association Study Showing that Immunoglobulin A Nephropathy Is Associated with the Polymorphisms 2093C and 2180T in the 3' Untranslated Region of the Megsin Gene
You-Ji Li*,
Yong Du*,
Cai-Xia Li,
Hui Guo,
Joseph C.K. Leung,
Man F. Lam,
Niansheng Yang*,
Fengxian Huang*,
Yun Chen,
Ji-Qian Fang,
Patrick H. Maxwell¶,
Kar N. Lai and
Yiming Wang
*Department of Nephrology, 1st Affiliated Hospital, Department of Medical Statistics, and Department of Medical Genetics, Zhongshan Medical College, Sun Yat-Sen University, Guangzhou, People’s Republic of China; Department of Medicine, Queen Marry Hospital, The University of Hong Kong, Hong Kong; and ¶Department of Medicine, Hammersmith Hospital, Imperial College, London, United Kingdom.
Correspondence to Prof. Yiming Wang, Dept of Medical Genetics, Zhongshan Medical College, 74 Zhongshan Road II, Guangzhou 510089, PR China. Phone: 86-20-87333136; Fax: 86-20-87335785; E-mail: ywzhong{at}hotmail.com
ABSTRACT. Immunoglobulin A nephropathy (IgAN) is consideredto be a multifactorial disease with genetic and environmentalfactors contributing to its pathogenesis. The genes involvedin susceptibility and progression of the disease have not yetbeen clearly elucidated. Megsin (SERPINB7) is an important candidategene, predominantly expressed in glomerular mesangium and upregulatedin IgAN. To investigate the potential role of this and othergenes in IgAN, patients with biopsy-proven IgAN were recruited,as were family members, for a family-based association study.The genotypes of the polymorphisms C2093T and C2180T withinthe 3' untranslated region of the gene were determined by polymerasechain reaction-restriction fragment length polymorphism anddirect sequencing. The results were analyzed by transmissiondisequilibrium test (TDT) and haplotype relative risk (HRR).TDT analyses revealed that Megsin 2093C and 2180T alleles weresignificantly more transmitted from heterozygous parents topatients than expected (C2093T: 127 trios, P = 0.034, C2180T:100 trios, P = 0.002). Extended TDT showed increased cotransmissionof the 2093C and 2180T alleles (232 families, P < 0.001).HRR revealed that the 2093C and 2180T alleles were more oftentransmitted to patients (P = 0.014, <0.001, respectively).Genetic variation in Megsin confers susceptibility to IgAN.
IgA nephropathy (IgAN) is the most common primary glomerulardisease (1–3). The prevalence is probably higher amongSoutheast Asians (3–5), although the exact incidence isunknown and available data are likely to be heavily influencedby differing criteria for nephrological assessment and renalbiopsy (6). The etiology is considered to be multifactorial.Evidence for a genetic contribution has come from the observationthat some families are multiply affected in an apparently autosomaldominant fashion, although this is uncommon. Furthermore, alinked locus has been demonstrated in a number of families (7).Epidemiologic studies suggest a significant difference in theincidence/prevalence among different ethnic groups and supportsignificant familial aggregation (6). Data from mouse studies,including the outbred strain ddY (8) and knockout and transgenicapproaches (9–11), have shown that specific genetic alterationsin a model organism give rise to a phenotype that shares featureswith the human disease.
Megsin, also known as SERPINB7, serine (or cysteine) proteinaseinhibitor, clade B (ovalbumin), member 7 (http://bioinfo.weizmann.ac.il,GenBank ID AF027866), is a predominantly mesangial expressedgene and belongs to the serpin (serine proteinase inhibitor)superfamily (12). Immunohistochemistry and in situ hybridizationstudies have shown that it is upregulated in IgAN when comparedwith normal renal tissue and several other forms of glomerulonephritis.The upregulation coincides with mesangial proliferation andextracellular matrix expansion (12–14). Megsin is thereforea good candidate for involvement in human IgAN. However, a previouscase-control study of a polymorphism, C2093T, did not demonstratea significant association (15).
Here we report the assembly of a DNA collection in China suitablefor family-based analysis and investigate whether there is evidencefor association of disease with transmission of two polymorphismsof Megsin, C2093T and C2180T, located in the 3' untranslatedregion (UTR) of the gene.
Patients, Families, Normal Control Subjects
The patients were recruited from centers in Guangzhou and HongKong, China. After informed consent was obtained, peripheralblood was drawn into EDTA tubes. Clinical data were also recorded(age, gender, duration of observation, proteinuria, serum creatinine,BP). The parents of the patients were also recruited, as wellas unaffected siblings if one or both of the parents were unavailable.Informed consent was obtained from all family members. Bloodsamples of the patients, and blood or saliva of the parentsor siblings, were collected in EDTA tubes or on 4-mm filterpapers in the case of saliva collection. Blood samples fromgender- and age-matched unrelated normal subjects were alsoobtained on a voluntary basis from Guangdong province.
DNA Extraction
Genomic DNA was extracted with QIAamp DNA blood Maxi kits accordingto the manufacturer’s instructions. DNA was extractedfrom saliva with chelex-100 as described by Ohhashi et al. (16).
Genotyping of Megsin C2093T and C2180T
C2093T was genotyped by PCR amplification of a 256-bp fragmentin the 3' UTR of the gene with a 9700 thermocycler (Eppendorf5331, Germany). The primer sequences were: sense: 5'-TTG TTGACC TAT GAA GAT TTT AGA G-3', antisense: 5'-AAA CTT ATA AACTAC ACA GCA TAT GA-3'. The reaction mixture contained l x PCRbuffer, l.5 mmol/L MgCl2, 200 mmol/L deoxynucleotide triphosphates(dNTPs), l unit TaqDNA polymerase (MBI), 10 pmol of each primerand 100 to 200 ng genomic DNA. The PCR amplification reactionconsisted of a cycle at 95°C for 10 min, followed by 35cycles of denaturation at 94°C for 15 s, annealing at 51°Cfor 15 s, and extension at 72°C for 30 s. A final extensionwas performed at 72°C for 5 min. The PCR products were digestedwith restriction endonuclease HaeIII (New England Biolabs) andelectrophoresed on 2% agarose gel containing ethidium bromide(1.5 µg/ml). The 2093C allele produces 114- and 142-bpfragments; the T allele has no digestion site, as describedpreviously (15). The genotyping results were also confirmedby direct sequencing in randomly selected samples.
C2180T was genotyped by PCR amplification with the same primersdescribed above or the following primers: sense: 5'-TCT TTTAAC TGT TGG CAG TTG TT-3' and antisense: 5'-CAA AGA AAG CCCTAG TTG TCC-3'. The PCR products were sequenced on an ABI 3100Genetic Analyzer with a BigDye Terminator v3.1 Cycle SequencingKit (Applied Biosystems).
Statistical Analyses Family-Based Studies.
The TRANSMIT program was used for the transmission disequilibriumtest (TDT) analysis (17). Two TDT methods were used: the classicalTDT of transmission from heterozygous parents in complete trios(patients plus both parents) (18), and the extended TDT, whichused information from all families (complete trios regardlessof the heterozygosity status of the parents, plus single-parentfamilies) (17). Haplotype relative risk (HRR) was analyzed asdescribed elsewhere (19,20).
Case-Control Analysis. 2 analysis was used when comparing allele frequencies betweenthe groups. Hardy-Weinberg equilibrium was tested by a 2 testwith 1 degree of freedom.
Species Comparisons.
We aligned the sequence of human with rat and mouse by usingmultizMm3Rn2, available at http://genome.ucsc.edu. The sequencesof cow, sheep, and horse were obtained from GenBank and alignedwith human sequence by the pairs alignment tool BioEdit, availableat http://www.ncbi.nlm.nih.gov/.
A total of 423 patients with IgAN were recruited over an initial5-yr period. IgAN was diagnosed by World Health Organizationcriteria (21), with a renal biopsy performed to assess mesangialIgA deposition via immunofluorescence. Recruitment requiredthat patients were younger than 60 yr old, and did not havesignificant hepatic disease or Henoch Schonlein purpura. Theclinical data of the patients at the time of renal biopsy arelisted in Table 1.
Table 1. Clinical data of the patients at time of renal biopsya
A case-control analysis of 423 patients suggested that the 2093Callele frequency was significantly higher among the patientsthan controls (P = 0.006) (Table 2). Although this would beconsistent with an effect of the 2093C allele on susceptibilityand/or manifestations of IgAN, an important limitation of suchcomparisons is population stratification between the affectedsubjects and the control population. Our study was designedto address this issue by examining whether there was distortionfrom the predicted chance transmission of parental alleles.TDT analysis of this genetic variant could be undertaken for127 complete trios; in the other 28 complete trios, both ofthe parents were homozygotes (Tables 3 and 4). The TDT showedthat the C allele was significantly more transmitted to patientsfrom their parents than would be expected with a P value of0.034 (Table 4). An extended TDT analysis, including the 28trios where the parents were homozygous and 83 families in whichonly one parent was available, gave a similar result (P = 0.032)(Tables 3 and 4). The HRR showed that the C allele is more oftentransmitted to patients (P = 0.014) (Table 5).
Table 5. HRR analyses of C2093T and C2180T transmitted and nontransmitted alleles
To further examine the role of genetic variation in the Megsin3' UTR, we examined a second polymorphism. TDT analysis of theC2180T polymorphism could be undertaken for 100 complete triosin which at least one of the parents was heterozygous, and anextended TDT method including all complete trios and 90 single-parentfamilies gave a total of 241 families (Tables 3 and 6). BothTDT analyses showed that the 2180T alleles were significantlymore transmitted to patients from their parents than would beexpected (P = 0.002 and P < 0.001, respectively) (Table 6).The HRR showed that the 2180T allele is more often transmittedto patients (P < 0.001) (Table 5).
Table 6. TDT analyses of Megsin C2180T alleles transmitted to patients
Because of the differences in the parental heterozygosity statusin the two polymorphisms, some families were only entered intoTDT analysis for one marker. We therefore also undertook TDTanalyses restricted to the 232 families that were typed forboth polymorphisms (Tables 7 and 8). This showed a significantovercotransmission of the 2093C and 2180T alleles (P = 0.0003)(Table 8).
By using both TDT and HRR analysis for complete trios, our resultsshow that the Megsin 2093C and 2180T alleles are transmittedmore frequently from heterozygous parents to affected patientsthan would be expected statistically. The same result was obtainedwhen we extended the analysis to include incomplete trios. Wealso observed an increased rate of cotransmission of the 2093Cand 2180T alleles in the families typed for both. HRR also showeda significantly increased transmission rate of the 2093C and2180T alleles to the patients. Our case-control analysis alsoshowed that the 2093C allele was present at a significantlyhigher frequency in the patient group, and conversely, the Tallele was present in more of the normal subjects.
These results indicate that in this population the Megsin 2093Cand 2180T alleles are associated with a predisposition to clinicallyovert IgAN. A previous case-control study in a different ethnicgroup that used 110 patients and 104 controls did not observeany difference in allele frequency in the two groups (15). Severalexplanations could account for this discrepancy. One possibilityis that the sample size may have been too small to detect arelatively modest effect on susceptibility, especially giventhe difficulties in matching patient and control groups in suchstudies.
Cloned by a group of Japanese scientists in 1998 (12), Megsinrapidly emerged as a candidate gene that might be associatedwith, or contributing to, various mesangial lessons. It wasfurther supported by transgenic mouse studies showing that overexpressionleads to progressive mesangial matrix expansion and an increasein the number of mesangial cells, accompanied by augmented immunecomplex, Ig, and complement deposition (11). In vitro assayshave identified that plasmin is one of its substrates and haveconfirmed its proteinase inhibitory activity (12). Glomerularmesangial cells play a key role in maintaining the normal structureand function of the glomerulus by mediating extracellular matrixremodeling and immune complex disposal. Therefore, one may speculatethat by inhibiting these plasmin/proteolytic activities, Megsinwould contribute to the pathologic processes, which eventuallylead to clinically evident IgAN.
Located on chromosome 18q21.3, close to the other serpin genecluster, the Megsin gene contains 8 exons and 7 introns, spanninga 20-kb genomic region, which encodes a predicted peptide of380 amino acids (22). The transcriptional start site is located391 bp upstream of the start codon. Its identified regulatoryregions are a promoter region located within a 4021-bp intervalin the 5' region and a protein A binding motif, CTGATTCAC, locatedat -120 to -112 (22). The two polymorphisms studied here arelocated in the 3' UTR, in which regulatory sequences have notyet been identified. Comparing the predicted orthologs in differentspecies shows heterogeneity for the equivalent nucleotides tohuman Megsin 2093 and 2180 positions. The 2093 position is Cin mouse, rat, cow and horse, but T in sheep. The 2180 positionis T in mouse, rat, and sheep, but A in horse.
Currently, how the 2093C and 2180T alleles are associated withsusceptibility is unclear, and we do not know whether the C/Tsubstitutions themselves confer the effect or whether it isdue to other variants nearby with which they are in linkagedisequilibrium, but the results suggest that genetic variationor variations of Megsin, which could be in the 3' UTR, conferssusceptibility to IgAN. Further studies will be necessary toprecisely locate the causal variation or variations and themechanism through which the susceptibility is conferred. Interestingly,we observed a 2093C allele frequency of 62.9% among our Chinesecontrol subjects. This is significantly higher than the reportedfrequency of 48.5% in a recent study in the Hungarian population(15) (P = 0.001). It is plausible that this could contributeto a higher prevalence of this disease in the Chinese population.
The study presented here represents what is to our knowledgethe first family-based association study investigating the relationshipof Megsin C2093T and C2180T with IgAN, and our findings stronglysuggest a role for variation at the Megsin 3' UTR with diseasesusceptibility. Furthermore, we have assembled a genetic resourcethat should be of considerable value in assessing the contributionof other genetic variants to this important condition.
Acknowledgments
This project was supported by the National Natural Science Foundationof China (30170434), Guangdong Provincial Natural Science Foundation(013140), and the China Medical Board, Rockefeller Foundation(98-677).
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Received for publication October 24, 2003.
Accepted for publication April 14, 2004.
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Abstract
Introduction
2 analysis was used when comparing allele frequencies between the groups. Hardy-Weinberg equilibrium was tested by a 
). The TDT showed that the C allele was significantly more transmitted to patients from their parents than would be expected with a P value of 0.034 (Table 4). An extended TDT analysis, including the 28 trios where the parents were homozygous and 83 families in which only one parent was available, gave a similar result (P = 0.032) (Tables 3 and 4
