Re: Genetic variant in the promoter of connective tissue growth factor gene confers susceptibility to nephropathy in type 1 diabetes. Wang et al., J Med Genet 2010; 47:391-397. Doi:10,1136/jmg.2009.073098

It was with great interest that we read the recent study by Wang et al. on a novel C/G single nucleotide polymorphism (SNP) at position -20 in the promoter of the connective tissue growth factor (CTGF) gene confers susceptibility to diabetic nephropathy in patients with type 1 diabetes (T1D).[1] Based on these findings we studied this SNP in our cohort of T1D to determine its association with the development of diabetic nephropathy. This SNP was genotyped in 932 European Caucasoid subjects and failed to detect the polymorphism.

Connective tissue growth factor (CTGF) is a secreted protein with a molecular weight at 36-38 kDa and plays an important role in the balance of degradation and synthesis of extracellular matrix although its physiological functions are not limited to this.[2] Several studies have demonstrated that CTGF plays a fundamental role in the histopathological changes seen in diabetic nephropathy. It has been demonstrated that low levels of glomerular CTGF are found in normal human glomeruli, but both mRNA and protein levels of CTGF increase during the early stages of diabetic nephropathy and continue to increase with disease progressionand these increases also correlate with the degree of albuminuria.[3]

The CTGF gene is located on Chromosome 6q23 and has 5 exons. Several SNPs have been identified in the promoter, introns, exons and 3'UTR regions of the gene.[1,4-6] Some of these SNPs have been studied and shown to be associated with various conditions including systemic sclerosis and cardiovascular diseases.[5-6] Addition, Wang et al. report has described a novel C/G SNP at position -20 in the promoter of the CTGF gene that is associated with nephropathy in T1D.[1] In this report, 862 subjects from the DCCT/EDIC cohort of T1D were genotyped. The frequencies of the CC, CG and GG genotypes were 62.76% (541/862), 31.90% (275/862) and 5.34% (46/862) in this cohort respectively. The frequency of GG genotype in patients with microalbuminuia (albumin excretion rate (AER) >40mg/24h) was significantly higher than patients with AER <40mg/24h, p<0.0001. The GG genotype was also shown to have greater transcriptional activity than that of the CG and CC genotypes.

A total of 739 Caucasoid patients with T1D (Female: 402; Male: 347) and with or without microvascular complications and 193 normal ethnically matched controls (Female: 101; Male: 92) were recruited in our study. Patients with T1D had an average age of 30.83 years (range: 1-76 years) and the average age of onset was 16.86 years (range: <1-52 years) with an average duration of T1D at 13.98 years (range: 0-55 years). The study was approved by the Local Research Ethical Committee and informed consent was obtained from all subjects. All patients have T1D as defined by The Expert Committee on the Diagnosis and Classification of Diabetes Mellitus.[7] Normal controls were obtained from cord blood samples following normal healthy obstetric delivery in Derriford Hospital, Plymouth.

Genomic DNA was prepared from peripheral and cord blood samples using the Nucleon II extraction kit (Scotlab, Lanarkshire, UK) following the manufacture's instruction. DNA samples were sent in 96-well plates to KBioscioences together with the SNP information published in Wang's study.[1] The sequence which covers the location of the SNP in bracket: GTATAAAAGC[C/G]TCGGGCCGCC has been checked and confirmed with the published sequence of the CTGF gene.[4,8] Genotyping for this SNP was performed using KASPar assays which are a proprietary in-house system (KBiosciences, Herts, UK). Unexpectedly, our results showed there was no SNP at the position -20 of the CTGF gene in our entire population; all subjects had the CC genotype.

We tried to understand why there were discrepancies between our findings and those of Wang et al.[1] Wang's study is the only publication regarding this SNP. There are a number of possible reasons for the discrepancy. Firstly, with respect of the ethnicity, our studied population was 100% Caucasian, Wang's subjects from the DCCT/EDIC cohort of T1D contained 96-97% of Caucasian.[9] The genetic backgrounds might be different even within Caucasian populations between different geographic locations.[5,10] but we would expect that the differences in the genetic backgrounds would cause the differences in the frequency of each genotype rather than no genetic variants at the position -20 of the CTGF gene. Secondly, sample size could be an issue, according to Wang's results: the frequencies of GG and GC were 5.34% and 31.90% respectively in their population, we should be able to detect about 39 subjects with GG and 235 subjects with GC genotypes out of our 739 subjects with T1D. Therefore, it is unlikely that our sample size was too small to allow the detection of the minor genotypes GG or GC. Thirdly, genotyping techniques may give rise to false positives or negatives. We used a highly reputable commercial genotyping facility-KBiosciences. Furthermore, our samples have been extensively genotyped including another SNP (rs9399005) in the CTGF gene that were typed in parallel to this one (Our unpublished data, 2011). Wang's study used PCR in their-own laboratory and confirmed this SNP by bi -directional sequencing. Sequences were detected on a Megabase N500 sequencer and results were analysed with sequencer software (Gene Codes Corporation, Ann Arbour, Michigan, USA). Consequently, it is unlikely that technical issues could explain the discrepancy between the sets of results. Finally, we don't think that the disparate results in this SNP between the two groups are due to the gender, age, duration of diabetes or age at onset of diabetes of patients either as the CTGF gene is not located in the X or Y chromosomes and the age, duration of diabetes and age at onset of diabetes of patients in both groups were similar. In conclusion, the reason for this discrepancy is unclear but is probably a reflection of the heterogeneity of populations. Therefore, further studies are needed to confirm this SNP from different groups or independent populations.

References: 1. Wang B, Cater RE, Jaffa MA, et al. Genetic variant in the promoter of connective tissue growth factor gene confers susceptibility to nephropathy in type 1 diabetes. J Med Genet 2010;47:391-397. 2. Mason RM. Connective tissue growth factor (CCN2), a pathogenic factor in diabetic nephropathy. What does it do? How does it do it? J Cell Commun Signal 2009;3:95-104. 3. Wahab NA, Schaefer L, Weston BS, et al. Glomerular expression of thrombospondin-1, transforming growth factor beta and connective tissue growth factor at different stages of diabetic nephropathy and their interdependent roles in mesangial response to diabetic stimuli. Diabetologia 2005;48:2650-2660. 4. Blom IE, van Diji AJ, de Weger RA, et al. Identification of human ccn2 (connective tissue growth factor) promoter polymorphisms. J Clin Pathol Mol Pathol 2001;54:192-196. 5. Granel B, Agriro L, Hachulla E, et al. Association between a CTGF gene polymorphism and system sclerosis in a French population. J Rheumatol 2010;37:351-358. 6. Cozzolino M, Biondi ML, Banfi E, et al. CCN2 (CTGF) gene polymorphism is a novel prognostic risk factor for cardiovascular outcomes in hemodialysis patients. Blood Purif 2010;30:272-276. 7. The Expert Committee on the diagnosis and classification of diabetes mellitus. Report of the Expert Committee on the diagnosis and classification of diabetes mellitus. Diabetes Care 2003;26:S5-S20. 8. Homo sapiens chromosome 6, GRCh37.p2 primary reference assembly. Retrieved on 23rd June 2011 from http://www.ncbi.nlm.nih.gov 9. The Diabetes Control and Complications Trial Research Group. The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. N Eng J Med 1993;329:977-986. 10. Rueda B, Simeon C, Hesselstrand R, et al. A large multicenter analysis of CTGF-945 promoter polymorphism does not confirm association with systemic sclerosis susceptibility or phenotype. Ann Rheum Dis 2009;68:1618 -1620.

Conflict of Interest:

None declared