Identification of a novel ESR1 mutation in a Chinese PCOS woman with estrogen insensitivity in IVF treatment

Patient and hormone tests

Peripheral blood was collected from the PCOS sample with estrogen insensitivity in the Reproductive Medicine Center, Jiangxi Provincial Maternal and Child Health Hospital (Nanchang, China). Additionally, 364 unrelated female controls without PCOS were recruited from Jiangxi Maternal and Child Health Hospital. All participants provided written informed consent. This study complies with the Declaration of Helsinki and was approved by the Institutional Review Board of Jiangxi Provincial Maternal and Child Health Hospital. Before the initiation of therapy, the basal serum levels of estradiol (E2), FSH, LH, thyroid stimulating hormone (TSH), progesterone (PRGO), prolactin (PRL), testosterone (T), thyrotropin (TSH), free triiodothyronine (FT3), free thyroxine (FT4) and cancer antigen 125 (CA125), were measured by commercial kits as described previously [18]. Anti-Mullerian hormone (AMH) was measured by an automated Roche Elecsys instrument (Roche; Elecsys).

WES

Genomic DNA (gDNA) was isolated from peripheral blood leukocytes with a Blood DNA kit (OMEGA Bio-tek Inc., Doraville, GA, USA) according to the manufacturer’s instructions. The gDNA sample was quantified with a NanoDrop 2000 fluorospectrometer (Thermo Fisher Scientific, MA, USA). A total of 3 μg of DNA sample was used for high-throughput sequencing. Subsequent exome capture, library construction and high throughput sequencing were carried out at the Beijing Genomics Institute (BGI, Shenzhen, China). Prior to high-throughput sequencing with a BGISEQ-500 sequencer, exome capture was performed with a BGI Exome V4 Kit (59 Mb target region) and the sequencing libraries were constructed by the MGIEasy™ DNA Library Prep Kit V1 (BGI, Shenzhen, China, Cat No. 85–05533-00). The sample had an average coverage depth of ~ 100 x. The sequenced data were aligned to the human reference genome (GRCh37/hg19). The common variants (minor allele frequency > 0.01) found in the 1000 Genomes Project (1000G, http://www.1000genomes.org), dbSNP147 (https://www.ncbi.nlm.nih.gov/projects/SNP/) or ExAC (http://exac.broadinstitute.org/) were excluded for further analysis.

Candidate gene selection

Three online bioinformatics programs, MutationTaster (www.mutationtaster.org) [19], SIFT (http://sift.jcvi.org/) [20] and PolyPhen-2 (genetics.bwh.harvard.edu/pph2) [21], were applied to predict the potential pathogenicity of the rare variants/mutations. These programs automatically predict whether rare variants/mutations are likely pathogenic or benign.

Sanger sequencing

Sanger sequencing was used to confirm the presence of the variant. Meanwhile, gDNA was isolated from the peripheral blood samples of 364 unrelated female controls without PCOS with the Blood DNA kit (OMEGA Bio-Tek Inc., Doraville, GA, USA) according to the manufacturer’s instructions. A 302 bp PCR amplicon spanning exon 2 of the ESR1 gene (NM_001291230.1) was amplified with a pair of primers (ESR1_Forward: 5′-ttctaatgttaatggatt − 3′/ESR1_Reverse: 5′-ttcctcagtcgctttggctc-3′). PCR was carried out in 30 μl reactions containing 50 ng gDNA as the template, 1 × PCR buffer, 0.5 μM of each forward and reverse primer, 2.5 mM of MgCl2 (Takara Biotechnology), 2.5 mM dNTPs (Takara Biotechnology), and 1 U LA Taq (Takara Biotechnology). The amplification program for the PCR was performed in a Thermal Cycler 2720 (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with a three-step PCR protocol: an initial denaturation phase at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 30 sec, annealing at 53 °C for 30 sec and extension at 72 °C for 30 sec, and ending with an extension phase at 72 °C for 10 minutes. The PCR products were visualized on a 1.5% agarose gel stained with ethidium bromide. The PCR products were then purified with a DNA purification kit (Tiangen, Beijing, China) and sequenced in both directions on an ABI 3730 XL Automatic Capillary DNA Sequencer (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with a BigDye terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, Calif, USA). The sequencing data were assembled and aligned to the corresponding genomic sequence (ESR1, NM_001291230.1) with the SeqMan II program in the LaserGene (DNAStar, Madison, WI, USA).

Evolutionary conservation analysis

The ESR1 protein sequences from 18 different vertebrate species, including Homo sapiens (NP_000116), Pan troglodytes (XP_009450519), Mus musculus (NP_001289460), Rattus norvegicus (NP_036821), Ovis aries (NP_000116), Bos taurus (NP_001001443), Gallus gallus (NP_990514), Sus scrofa (NP_999385), Canis lupus familiaris (NP_001273887), Equus caballus (NP_001075241), Tupaia chinensis (NP_001304001), Mustela putorius furo (XP_004753629), Oryctolagus cuniculus (XP_008261925), Pongo abelii (XP_002817538), Coturnix japonica (NP_001310118), Alligator sinensis (XP_014375965), Ceratotherium simum simum (NP_001266182) and Xenopus tropicalis (NP_988866), were subjected to evolutionary conservation analysis. Multiple sequence alignment was carried out with Molecular Evolutionary Genetics Analysis (MEGA) software (version 7.0) developed by the laboratory of Dr. Kumar [22].

Protein structural modelling

The PDB file of ESR1 was generated by SWISS-MODEL in the ExPASy database (http://www.expasy.org) based on the protein sequences of human ESR1. With the generated PDB file, the protein structure of ESR1 was generated by DeepView Swiss-PdbViewer 4.0 software, by selecting “show dots surface”, “show backbone oxygen” and “sender in solid 3D”. Within the structure of the wild-type ESR1 protein, mutated ESR1 protein (p.A207T) was generated by changing alanine to threonine at the 207th residue.

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