Whole exome sequencing reveals novel variants associated with diminished ovarian reserve in young women

Abstract

Background: Diminished ovarian reserve is one of the most important causes of female infertility. In the etiology study of DOR, besides age, it is known that chromosomal abnormality, radiotherapy, chemotherapy and ovarian surgery can result in DOR. For young women without obvious risk factors, gene mutation should be considered as a possible cause. However, the specific molecular mechanism of DOR has not been fully elucidated. Methods: In order to explore the pathogenic variants related to DOR, twenty young women under 35 years old affected by DOR without definite factors damaging ovarian reserve were recruited as the research subjects, and five women with normal ovarian reserve were recruited as the control group. Whole exome sequencing was applied as the genomics research tool. Results: As a result, we obtained a set of mutated genes that may be related to DOR, where the missense variant on GPR84 was selected for further study. It is found that GPR84^Y370H variant promotes the expression of proinflammatory cytokines (TNF-α, IL12B, IL-1β) and chemokines (CCL2, CCL5), as well as the activation of NF-κB signaling pathway. Conclusion: In conclusion, GPR84^Y370H variant was identified though analysis for WES results of 20 DOR patients. The deleterious variant of GPR84 could be the potential molecular mechanism of non-age-related pathological DOR through its role in promoting inflammation. The findings of this study can be used as a preliminary research basis for the development of early molecular diagnosis and treatment target selection of DOR.

Keywords: female infertility; molecular etiology; ovarian reserve; point mutation; whole exome sequencing.

FIGURE 1

Framework of WES data analysis. Of the qualified variants, the variants 1) identified in normal samples; 2) MAF >5% in the 1,000 Genome database; 3) in the non-coding regions; 4) predicted as no impact were removed, and top 20 mutated variants were finally reported.

FIGURE 2

Summary of whole exome sequencing results of 20 DOR patients. (A) The percentage of SNVs and indels in filtered variants; (B) The classes of SNVs and their frequencies; (C) The frequencies of different types of variants in total samples; (D) The violin plot of the number of different types of variants in each patient; (E) The distribution of different types of variants in each patient; (F) Functional analysis of 731 genes using KEGG, Reactome, and GO database.

FIGURE 3

Top 20 mutated genes in DOR. (A) The variant characteristics of top 20 mutated genes in each patient. The twenty DOR patients included for WES were represented by “P01”-“P20”. (B) Enrichment analysis of top 20 mutated genes using Reactome pathways. (C) Protein-protein interactions of mutated genes identified using GENEMANIA database.

FIGURE 4

Characteristics of the GPR84 variant. (A) Sanger sequencing validation of the heterozygous c.1108T > C variant in the GPR84 gene. The red box indicates the variant site. (B) Amino acid sequence alignment of GPR84 among different species. The blue box indicates the mutated amino acid. Tyrosine at position 370 is 100% conserved across all species. (C) The expression of GPR84 in different tissue and organs in BALB/c mouse were measured by qRT-PCR and Western blot. (D) Location of the mutated amino acid in the GPR84 protein predicted by RoseTTAFold. The extracellular segments are shown in cyan; the cytoplasmic segments are shown in violet; the transmembrane segments are shown in yellow. The wildtype amino acid tyrosine is shown in red, and the mutated amino acid histidine is shown in blue.

FIGURE 5

GPR84^Y370H variant promotes the inflammatory response in human ovarian granulosa cells. (A, B) The construction of GPR84 (shown as GPR84^WT/WT) and GPR84^Y370H (shown as GPR84 ^WT/mut) overexpressed KGN cells (human granulosa-like tumor cell line) were verified by qRT-PCR and Western blot respectively. (C, D) The GPR84^WT/WT and GPR84^WT/mut overexpressed KGN cells were treated with LPS (0.1 μg/mL) for 2 h before stimulation with lauric acid (200 μM) for 24 h. (C) The mRNA level of inflammatory cytokines (TNF-α, IL-6, IL12B, IL-1β) and chemokines (CCL2, CCL5, CXCL1) were measured by qRT-PCR. (D) The protein level of p65 in nucleus were increased in GPR84^WT/mut group compared with GPR84^WT/WT group. Lamin A/C and α-Tubulin were used as loading controls in nucleus and cytosol respectively. Data are represented as mean ± SEM. Statistical significance was assessed by two-tailed unpaired t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.