IMPE Abstracts

Introduction: We have previously reported strong association of cryptorchidism (CO) and hypospadias (HS) with a specific "AGATC" haplotype within a > 34 kb tight linkage disequilibrium (LD) block spanning five SNPs designated SNPs10–14 in the 3' region of ESR1 (JCEM 2006; Hum Reprod 208). Here, we report that a microdeletion (DESR1) identified from the specific haplotype constitutes a widely spread susceptibility factor for the development of CO and HS. Haplotype analysis of ESR1 in Italian boys We performed haplotype analysis in 243 Italian boys consisting of 80 boys with CO, 13 boys with HS, and 150 control boys (CBs). Consequently, we revealed a tight LD block encompassing SNPs10–14, the four major haplotypes, and the significant association of CO/HS with the "AGATC" haplotype, as shown in Japanese boys. Identification of a DESR1 from the specific haplotype region We revealed DESR1 by whole genome sequencing for the "AGATC" haplotype region. This microdeletion was mediated by a 5 bp microhomology in both Japanese and Italian boys, and contained a CTCF binding site (BS) as the sole candidate functional sequence. Association studies using ∆ESR1 We performed association studies between CO/HS and DESR1 in previously reported and newly recruited 415 Japanese boys consisting of 149 boys with CO, 141 boys with HS, and 125 CBs, as well as the 243 Italian boys. Consequently, DESR1 was found to be strongly associated with CO/HS in both Japanese and Italian boys. The DESR1 resided on the AGATC haplotype almost exclusively and predominantly in Japanese and Italian boys, respectively. Gene expression studies using MCF-7 cells We created MCF-7 cells with a homozygous deletion spanning the ∆ESR1 sequence (MCF-7-∆ESR1) and those with a homozygous deletion encompassing the CTCF-BS (MCF-7-∆CTCF-BS) by the CRSPR-Cas9 system. Both MCF-7-∆ESR1 and MCF-7-∆CTCF-BS cells showed increased ESR1 expression with and without estradiol, diethylstilbestrol, and bisphenol-A loads.

Discussion: The results suggest that ∆ESR1, especially the CTCF-BS, constitutes the susceptibility factor for the development of CO/HS. Notably, the fusion point structure indicates that ∆ESR1 occurred in a founder with the originally rare "AGATC" haplotype constituted by the combination of minor alleles, and has spread in the world on the "AGATC" haplotype because of elevated fitness due to an increased ESR1 expression. At present, however, ∆ESR1 has become a susceptibility factor for CO/HS because of the presence of estrogenic environmental endocrine disruptors.