Part2:Cistanche-Common Risk Variants in NPHS1 And TNFSF15 Are Associated With Childhood Steroid-sensitive Nephrotic Syndrome

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DISCUSSION

The present Japanese GWAS with the largest sample size to date and replication in multiple continental populations identified common variants in the NPHS1 and TNFSF15 regions as new susceptibility factors for childhood SSNS. Post-GWAS analysis of the chromosome 19 locus identified a potential transcriptional mechanism by which the NPHSI risk haplotype may contribute to disease. Finally, the larger sample size empowered additional fine-mapping of the previously implicated HLA loci.1"Altogether, these findings markedly expand and improve our understanding of the genetic background of childhood SSNS. They identify nephrin, the proinflammatory cytokine tumor necrosis factor superfamily member 15(TNFSF15), and their associated molecules as new targets for biologic inquiry to better understand SSNS and potentially for therapeutic development. Finally, the association with common variants in the NPHSI locus provides another example showing that Mendelian kidney disease genes can harbor susceptibility variants for a more common multifactorial disease(SSNS).

Rare mutations in NPHS1 cause a congenital nephrotic syndrome of the Finnish type, a rare monogenic nephrotic syndrome that is steroid-resistant and has a poor renal prognosis. Surprisingly, the present study revealed that variants in NPHS1 are associated with susceptibility to SSNs.

Although NPHS1 has never been implicated in genome-wide scans for SSNS, a candidate association study between this gene's variants and SSNs in East Asian patients supports our current findings. In our present study, one of the significant SNPs in the NPHSI locus was the synonymous variant rs2285450(c.294 C>T)in exon 3. Previously, Sun et al. reported a higher frequency of the rs2285450 minor allele in Chinese sporadic nephrotic syndrome patients from Singapore than in controls(20% vs. 13%, P= 0.025)and showed that the risk allele resulted in a decreased ability to inhibit TRPC6 currents in HEK293-M1 cells via patch-clamp, which might account for susceptibility to proteinuria.27,28 This study provides independent support for rs2285450 as a risk factor for SSNS and suggests an alternative mechanism for susceptibility to nephrotic syndrome requiring further inquiry.

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Using paired human genetic and glomerular transcriptomic data, we did not observe differences in total NPHS1 expression as a function of this risk haplotype. However, RNA-seq data allowed observation of significant ASE resulting in lower NPHSI expression from the haplotype harboring the risk alleles (Supplementary Figure S11). A number of the risk variants at this locus were synonymous with exonic changes in NPHS1. We, therefore, focused more on NPHSI and did not pursue discussion of the role of KIRREL2 in potential disease mechanisms. Future work should include:(i) validating this observation in additional patients with genotyping and kidney expression data; and (ii)gaining a mechanistic understanding of how this ASE of NPHSI is contributing to or causing the association with SSNs that we observed. Potential hypotheses for dysfunction include:(i)increased burden on the cell to produce the necessary amount of nephrin from the reference chromosome, leading to cellular stress and increased susceptibility to injury; and(i) potentially dysfunctional nephrin protein produced from the risk haplotype (e.g, via differential posttranslational modifications)resulting in an abnormal glomerular filtration barrier.

Genetic polymorphisms at the 9q32 locus are linked with several autoimmune and inflammatory diseases.23,29-31 The most likely disease-causing gene within 9q32 is TNFSF15, which encodes TNFSF15. In the present study, the significant association of rs4979462 was replicated independently and further strengthened by a transethnic meta-analysis. Previously, Hitomi et al.3 identified rs4979462 as a functional variant by in vitro functional analysis using luciferase assay and electrophoretic mobility shift assay. Super-shift assay clarified that the risk allele(T)of rs4979462 generated a novel NF-1 binding site.2 In addition, several reports have shown that the risk allele of rs4979432 affects the expression level of TNFSF15 mRNA.0Further studies are required to confirm the association of TNFSF15 with SSNS in children.

In the present study, the variants in NPHSI and TNFSF15 loci were identified and replicated mainly in the East Asian (Japanese and Korean) and South Asian populations, in which a higher incidence of the disease has been reported. In contrast, the frequencies of risk alleles of candidate SNPs in NPHS1 were rare in people of European ancestry(Table 1). These findings may partially explain the epidemiologic difference among populations from the perspective of disease-associated polymorphisms. This difference is further illustrated by the absence of significant signals in our Japanese dataset in the CALHM6/FAM26F and PARMl regions, which were detected in a homogeneous cohort of European ancestry.5

With the predominant contribution of HLA-DR/DQ genes, gene sets of major histocompatibility complex class II protein complex and major histocompatibility complex class II receptor activity were strongly associated with the disease. Innate immune response was also identified as a significant gene set with a moderate effect. The innate immune system including the activation of professional cells (antigen-presenting cells and B cells/Toll-like receptors), as well as the adaptive immune system, in which HLA molecules play a crucial role, is involved in the disease process and response to treatment.3,4

Around 90% of the heritability of childhood SSNS was contributed by common variants, although variants with relatively rare allele frequencies(MAF: 0.5%-5%)were also taken into consideration. However, a large proportion of the disease heritability remains unaccounted for. Other disease-associated variants in non-HLA regions might be identified by future genome-wide studies with larger sample sizes, especially for patients of European and other non-East Asian ancestries, as disease-associated polymorphisms in non-HLA regions are still largely unknown.

In the present study, the sample sizes in replication cohorts with different ancestries have limited the power for the replication of signals with smaller effect sizes. Population stratification might exist in the replication stage due to a lack of adjustment. Moreover, batch effects might have occurred between cases and controls when allele frequencies from public databases were utilized as population-matched controls.

In summary, the discoveries here provide new SSNS loci. The NPHS1 locus, in particular, provides a provocative new concept for the disease's pathogenesis, linking and reinforcing an emerging paradigm in nephrology—in certain genes harboring Mendelian variants, more common alleles can increase the susceptibility to multifactorial, polygenic diseases, as previously demonstrated for UMOD, COL4A3, and NPHS2.26,35- Finally, the current findings again emphasize the importance of performing genomic research in diverse populations. By doing so, we discovered specific SNPs that are likely to have more impact on East and South Asian populations, while identifying genes and loci that may be important in all populations, to which we would be statistically blind if using a European discovery cohort.

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METHODS

See Supplementary Methods for additional details.

Samples

In the discovery stage,1018 Japanese patients diagnosed with childhood SSNS(onset age<18 years) were recruited. Patients with a history of steroid resistance during follow-up were excluded. Overall, 3331 Japanese healthy adults were recruited as controls. All participants provided written informed consent.

Genotyping and whole-genome imputation in the discovery stage

In the discovery stage,1018 cases and 3331 controls were genotyped using the Affymetrix Japonica array. Nineteen samples were excluded by a low call rate(<97%) during the genotype calling process. Nine controls with ambiguous sex were excluded. Then, whole-genome imputation was performed with IMPUTE4-(version 2.3.1)using a phased reference panel of 2036 healthy Japanese individuals. After whole-genome imputation, there were 22,049,786 autosomal single-nucleotide variants and short insertions and deletions with info