Associations of a SHROOM3 variant with mild renal impairment and depressive symptoms in a Chinese Han population

Background: To explore the associations of several genetic variants identied in the genome-wide association studies (GWAS) of European ancestry with mild renal impairment glomerular ltration rate (GFR) in Chinese Han population. Methods: Data of 1788 community-dwelling elders from the baseline survey of the ageing arm of the Rugao Longevity and Ageing Study was used. Plasma creatinine based GFR was estimated using the eGFR-EPI equations. Results: Of the 10 common polymorphisms identied in GWAS of the European ancestry, rs17319721 located in the rst intron of the SHROOM3, was associated with GFR. A allele was associated with both decreased GFR level and greater odds of mild renal impairment (OR 1.12, 95% CI 1.01-1.23, p=0.029) dened by GFR<90 mL/min/1.73 m 2 after adjusting for multiple confounds of chronic kidney disease. In addition, compared with rs17319721-GG genotype, AA was associated with both higher depressive score and greater risk of depression prevalence, showing a pleiotropic effects of rs17319721. However, we did not found signicant association of GFR levels with another 42 common polymorphisms that was previously reported to be associated with the traditional risk factors of kidney diseases. Conclusions: SHROOM3-rs17319721 is associated with GFR levels, kidney impairment, and depressive symptoms in a Chinese population.


Background
Glomerular ltration rate (GFR)-de ned chronic kidney disease (CKD) is a complex disease with a heritability of 30-70% [1,2]. Understanding genetic predisposition to CKD is one approach to uncover underlying pathophysiological mechanisms for improved classi cation and targeted therapies. Over the past decade, genome-wide association studies (GWAS), a main population-based strategy to screen genetic risk factors, have identi ed a batch of CKD loci mainly in individuals of European ancestry [3,4].

Study population
Baseline survey data of the ageing arm of Rugao longevity Ageing Study (RuLAS) was used in this study.

Genotyping
Genomic DNA was extracted from EDTA anticogulated peripheral blood using a standard method. The aforementioned polymorphisms were genotyped. Primei online 3 (Version 0.040) and Oligo (Version 6.31) software were used to design speci c primers. For each sample, genomic DNA (10 ng) was ampli ed and puri ed by three-round multiplex PCR following the recommendations of the manufacturer [19] and then puri ed PCR products mix was processed on a OneTouch 2 instrument and enriched on a OneTouch 2 ES station. Then the oligonucleotide mix was sequenced on a 318 chip using the Ion Torrent PGM and the Ion PGMTM Sequencing 200 Kit v2 according to the manufacturer's instructions. Original sequencing reads were exported to FASTQ les, the index and adapter sequence were trimed out by using cutadapt software, and BWA v0.7.12 was then used to align the targeted sequence to the SNP reference sequences (NCBI, dbSNP build 142) to generate SAM le. By using samtools, the sam le was transferred to mpileup le, and SNP locus were identi ed according to ligase detection reaction (LDR). All the genotyping success rates of these loci were >95%. To assess reproducibility, 5% of samples were analyzed in duplicate and the genotypes were 100% concordant for these samples.  [20]. Since the GFR of most participants is greater than 60 mL/min/1.73 m 2 , we categorized them into mild renal impairment group (GFR<90 mL/min/1.73 m 2 group, category G1), or normal group (GFR≥90 mL/min/1.73 m 2 group) [21].
Depressive symptoms were assessed using the 15-item version of the Geriatric Depression Scale (GDS-15) with a score of 0-15. A score of ≥ 5 was considered depression symptoms. The cut-off score of ≥5 has a sensitivity of 0.97 and a speci city of 0.95 [22,23].

Statistical analysis
Characteristics were presented as the mean ± standard deviation (SD) or the percentage. The deviation from Hardy-Weinberg expectation for the genetic variants was tested by a chi-square statistic.
Comparisons of continuous variables were tested using a Student's t test. The effects of genetic variants on GFR level were examined using ACNOVA, adjusting for multiple covariates. Logistic regression models were used to estimate odds ratios (ORs) and 95% con dence interval (95% CI) for renal impairment and depressive symptoms, adjusting for multiple confounding factors. A p-value of less than 0.05 (two-tailed) was considered to be statistically signi cant. All data analysis was done by SPSS 19.0 software (SPSS Inc., Chicago, IL, USA). Table 1 summarizes the characteristics of the 1788 individuals included in this study. The mean age of the study participants was 75.36±3.91 years, and 53.6% was female. The GFR levels of the renal impairment group and normal group were 80.93±9.84 and 95.10±4.01, respectively. GFR decrease group was older, had less females, less coupled individuals and more smokers, with a higher TG level and a lower HDL-C level ( Table 1). The genotype distributions of the studied polymorphisms ranged from 5.1% to 47.8% (Table 2 and Supplemental Table 2).

Results
The association analysis results of the 10 selected SNPs identi ed in the GWAS of the European ancestry was presented in Table 2. Signi cant difference of GFR levels was observed across three DAB2-rs11959928 genotypes and a boardline signi cant difference of GFR levels was observed across three SHROOM3-rs17319721 genotypes. However, since the GFR level of the heterozygote of 11959928 is the highest among three genotypes, therefore, we did not further analyze this variant.
SHROOM3-rs17319721 was associated with both GFR levels and odds of renal impairment de ned by GFR<90 mL/min/1.73 m 2 , after adjusting for multiple confounds of CKD including adjusted for age, sex, education, marriage, smoking, alcohol drinking, life satisfaction, BMI, SBP, SBP, glucose, TG, HDL, and LDL levels (  (Table 4).
We did not found signi cant association of GFR levels with 42 common polymorphisms that was previously reported associated with hypertension, diabetes, dyslipidemia, or obesity, which is the established risk factors of CKD (Supplement Table2).

Discussion
Previously, SHROOM3-rs17319721 was found associated with GFR in the European ancestry. In the present study, we found that SHROOM3-rs17319721 is associated with GFR levels and kidney impairment in another race-Chinese Han population. In addition, for the rst time, we found that rs17319721 was associated with depressive symptoms. However, we did not found signi cant association of GFR levels with another 42 common polymorphisms that was previously associated with the traditional risk factors of kidney diseases.
Population studies of the SHROOM3-rs17319721 Encoded by SHROOM3, shroom3 is an actin binding protein which regulate cell morphology by coordinating the assembly of cytoskeleton [24]. In 2009, Kottgen et al conducted the rst GWAS of GFR in Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) consortium found that the minor A allele of the SHROOM3-rs17319721 was associated with an decreased eGFR cre level and increased risk of CKD [6]. The association of rs17319721 with eGFR cre was also found in another larger GWAS conducted in European ancestry populations [5] and replicated in the clinical epidemiology followup studies conducted in European populations [25,26,4]. In the present study, we replicated this association in Chinese population.
However, rs17319721-GFR association was not detected among participants of African ancestry [9], suggesting that allelic heterogeneity exists across ethnicities. For SHROOM3-rs17319721, the MAF is indeed quite different across different ethnic groups, ranging from 43.9% in Europeans, to 21.7% in Africans, and 10.4% in Chinese Han population [8].
Although SHROOM3 rs17319721-A allele decreases GFR and increases the risk of kidney impairment, it was associated with lower albuminuria [27]. The pleiotropic and contradiction association of this gene with GFR and albuminuria may be due to different etiology between them (albuminuria was associated with more smoking, diabetes and elevated triglycerides [28]), different genetic correlations between them [29], and the less coexist of them in early kidney disease [27,30].
At personalized treatment researches, SHROOM3-rs17319721 was found to in uence allograft injury. The presence of the SHROOM3 risk A allele in the donor correlated with increased allograft brosis and with reduced GFR at 12 months after transplant [31]. In 189 Chinese patients, Yan et al found that rs17319721-A allele carriers had a signi cantly higher GFR levels than GG genotype from month 1 to month 6 after transplantation [32]. In allografts of US patients, rs17319721 AA was associated with reduced albuminuria by 2 years after transplant [33].
Possible mechanisms link SHROOM3-rs17319721 to CKD SHROOM3 interacts with FYN via SH3-binding domain to regulate FYN activation and downstream signaling, and maintenances podocyte actin cytoskeleton and phenotype [33]. Rs17319721 is located in the rst intron of the high conserved region of the SHROOM3 gene. Rs17319721 A allele is associated with an increased expression of the SHROOM3 than GG genotype. The sequence containing rs17319721-A allele is a TCF7L2 (transcription factor 7-like 2) dependent enhancer that increases SHROOM3 transcription, and then promotes pro brotic gene expression by facilitating TGF-β1/SMAD3 signaling pathway [31]. In podocypes, AA preserves interaction with FYN, activates FYN kinase (Y418 phosphorylation), and stabilizes actin cytoskeleton [33]. In addition, rs17319721-A allele results in an elevated expression of a shorter isoform lacking the PDZ domain [34].

SHROOM3-rs17319721 variant and psychological traits
Since multiple lines of evidence are consistent with widespread pleiotropy for complex traits that many segregating variants affect multiple traits [35], we further explore whether pleiotropy effects exist for SHROOM3-rs17319721 in our cohort population. For 17319721, it was not only associated with eGFR/CKD in the general populations [5,6,36], but also associated with GFR in the diabetes patients [37] and associated with allograft brosis in renal transplant patients [31,38]. In this study, we not only found the association of SHROOM3-rs17319721with GFR, but also with depressive symptoms and risk of depression.
Genetic variant of the SHROOM3 was indeed previously related to psychological traits. In a GWAS conducted in Europeans, rs12513013 and rs12509930 of the SHROOM3 were associated with neuroticism [39]. In the participants of the New England Centenarian Study, Bae et al replicated the association of rs12509930 with neuroticism [40]. In the present study, we found that SHROOM3-rs17319721 was associated with depressive symptoms. The mechanisms that rs17319721 is simultaneously associated with kidney disease and psychological traits are unknown at this stage, but previous studies showed that kidney impairment was linked to psychological traits including lower Quality of Well-Being [41], mental health impairment [42], depression and suicidal ideation [43,44].

CKD risk factor-related loci and kidney impairment
In the present study, we also explored the association of a batch of CKD risk factor-related loci with GFR in our cohort population. Among them, GCKR (Glucokinase regulatory protein) is another pleiotropic gene, the protein of which inhibits hepatic glucokinase. Common variants in GCKR was previously associated with a variety of risk factors of CKD, including serum triglycerides, fasting glucose, C-reactive protein, and diabetes [12]. This locus was associated with GFR in the GWAS of European ancestry [5]. However, in the present study, we did not found signi cant association between the variant of this locus with GFR level and kidney impairment. In addition, another pleiotropic locus, NPPB was associated with both blood pressure [16] and GFR [45] in previous large GWA studies. However, a functional variant of NPPB, rs198389 (-381T>C), was not associated with GFR or kidney impairment in our study (Supplement Table2). Insu cient statistical power resulting from the relative small sample size of the present study may account for the insigni cant association.
Conclusions SHROOM3-rs17319721-A allele was associated with decreased GFR level and greater odds of mild renal impairment in a Chinese population. AA genotypes was also associated with higher depressive score and greater risk of depression prevalence, suggesting a pleiotropic effects of rs17319721. Since the sample size of this study is relatively small, the associations need to be validated in other larger population studies.

Declarations
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Authors' contributions XH Sun and XY Jiang were responsible for managing the participant database and retrieving follow-up information; they also contributed to article preparation, in the analyses of data and in drafting the article. J Chen, ZK Chen, and ZJ Bao supervised the ongoing research, taking part in the initiation of the study and contributing to article preparation. XH Sun, ZJ Bao and XY Jiang contributed to analyses of data and in article preparation. ZJ Bao, XY Jiang and XF Wang conceived the study, participated in its design and coordination, and helped draft the article. All authors read and approved the nal article.