Unveiling the 46,XY disorder of sex development patients family with a novel mutation in the GATA4 gene

Background GATA-binding protein 4 (GATA4) is the critical regulator in gonadal development and its mutation has been reported related with 46,XY disorder of sex development (DSD). Here, we found the two Chinese cases with 46,XY DSD carried the GATA4 mutation. Physical examinations, B-ultrasound and Karyotype analysis were performed and conrmed the two patients with 46,XY DSD. Results Sequencing were performed and the heterozygous mutation p.Gly375Arg in GATA4 gene was identied in the 2 cases with 46,XY DSD. Their mother was identied carrying the p.Gly375Arg mutation in GATA4 protein. However, their father and litter sister without 46,XY DSD didn’t be found carrying the p.Gly375Arg mutation in GATA4 gene.

Unveiling the 46,XY disorder of sex development patients family with a novel mutation in the GATA4 gene Liwei

Introduction
The phenotype of 46,XY DSD is well characterized by normal 46, XY karyotype, testes, male accessory ducts and predominantly female external genitalia [1][2][3]. At present, the gonadal anomalies occur about 1 in 4500 infants [4]. A variety of genetic factors that affect sex development during gonadal differentiation have been identi ed and the mutation of these genetic factors will block gonadal differentiation and lead to DSD occurrence [5,6]. Despite the considerable advances in our learning of the genetic mechanisms of gonad development, the underlying etiological factor in the majority of 46,XY DSD cases still cannot be explained.
To date, an increasing number of causative genes have been identi ed for 46,XY DSD, including GATAbinding protein 4 (GATA4) [7][8][9][10][11]. GATA4 is an evolutionarily conserved zinc nger transcription factor gene that plays an essential role in early development of many organs in the embryonic stage, including the skin, brain, gonads, liver, hematopoietic, cardiovascular and urogenital systems [12,13]. GATA4 variants have been identi ed not only in patients with congenital heart disease [14] but also in those with 46,XY DSD due to impaired testis formation [4,7,11,15].
Here, we report a heterozygous GATA4 mutation p.Gly375Arg (c.1123G > A) in a 7yearold proband and her 5-year-old sister with 46,XY DSD. The heterozygous p.Gly375Arg mutation in GATA4 gene is inherited from their mother. The p.Gly375Arg mutation in GATA4 gene has not been reported in previous research.
Our identi cation of the p.Gly375Arg mutation in GATA4 gene from 46,XY DSD cases may provide insight of the domain function of GATA4 gene into gonadal development and DSD occurrence.

Clinical characteristics of the family
The proband II-1 was treated for a right inguinal hernia and found a testicular-like tissue. Thence, she was referred to our hospital. Physical examination revealed a female vulva with a blind-ending vagina. Color Doppler Ultrasound (Voluson E8, GE, Salzburg, Austria) showed that she had no a uterus and ovaries, but detected testicular tissues in the right groin and in the left abdominal cavity ( Figure.1a). Ultrasonic examination found no cardiac abnormalities. Her karyotype was a 46, XY with no apparent anomalies in the chromosome number or structure ( Figure.1b).
The case II-2 was referred to our hospital due to her sister's symptom. Physical examination found that she had a female vulva but with a blind-ending vagina. Ultrasound found no a uterus and ovaries, but p.Gly375Arg (c.1123G > A) mutation identi ed in the GATA4 gene The genomic DNA was extracted from the peripheral blood sample of the proband. Chip capture highthroughput sequencing by BGI corporation and alignment to the ESP data, QIANREN data and EXAC data found that the proband II-1 carried a heterozygous missense mutations (C.1123G > A; p.Gly375Arg) in GATA4 gene. Then, we extracted the genomic DNA from the peripheral blood samples of the other family members and ampli ed the GATA4 gene by PCR. Sanger sequencing was performed for the family members by Fulgent Technologies Inc.. We con rmed the heterozygous missense mutations (C.1123G > A; p.Gly375Arg) in GATA4 gene of the proband II-1 (Fig. 3a) and found that the case II-2 (Fig. 3b) and her mother I-2 (Fig. 4b) also carried the heterozygous missense mutations (C.1123G > A; p.Gly375Arg) in GATA4 gene. The father I-1 (Fig. 4a) and the proband's little sister II-3 (Fig. 4c) didn't be found carrying the heterozygous missense mutations (C.1123G > A; p.Gly375Arg) in GATA4 gene. This mutation has not been previously found in the databases of the human genome, including EPS6500, 1000 genomes, GnomAD, ExAC, and their own sequencing databases of BGI corporation. These results indicated that the heterozygous missense mutations (C.1123G > A; p.Gly375Arg) in GATA4 gene may lead to 46,XY DSD occurrence.

Discussion
We identi ed a new heterogeneous GATA4 variant (p.Gly375Arg) in the two Chinese cases with 46, XY DSD. No clinically recognizable cardiac feature was found in the GATA4 (p.Gly375Arg) mutation cases. Their father without 46, XY DSD symptom didn't carry the heterogeneous p.Gly375Arg in GATA4 gene. The heterogeneous p.Gly375Arg in GATA4 gene of the two DSD cases was inherited from their mother. This is the rst time to report the p.Gly375Arg mutation in GATA4 gene identi ed in the DSD cases.
GATA4 is a member of the GATA proteins family containing 442 amino acids and 7 exons, which is a tissue-and organ-speci c transcriptional regulator implicated in development and differentiation of endoderm-and mesoderm-derived tissues [12,13]. GATA4 have two zinc nger domains (ZNI and ZNII). The zinc nger ZNII region at the C-terminal requiring for the DNA recognition and binding [16], and the zinc nger ZNI region at the N-terminal contributing to the stability. Both the zinc ngers are indispensable for protein-protein interactions with other transcription factors [17][18][19]. GATA4 is expressed in adult vertebrate heart, gut epithelium, and gonads. The mutation of GATA4 gene have been identi ed prevailingly in patients with ventricular septal defect, tetralogy of Fallot, or other cardiac malformations [20][21][22][23].
GATA4 gene mutation lead to 46,XY DSD with or without the congenital heart diseases. A family with 46,XY DSD and congenital heart disease was identi ed of p.Gly221Arg mutation in the conserved Nterminal zinc nger of GATA4 since the mutation compromised the ability of the protein trans-activating the anti-Müllerian hormone (AMH) promoter and disrupted synergistic activation of the AMH promoter by GATA4 and NR5A1 [11]. The inactivation of GATA-binding motif of the AMH promoter failed to upregulate AMH expression in the developing fetal and neonate testis, suggesting that GATA4 is a positive modulator of AMH expression [24]. The p.Pro407Gln mutation in GATA4 gene were identi ed in four patients with 46,XY DSD but no heart anomaly because p.Pro407Gln mutation in GATA4 gene had markedly decreased transactivation activity of AMH promoter similar to p.Gly221Arg [7]. The p.Glu359X mutation in GATA4 gene reduced GATA4 transcriptional activity on the different gonadal promoters and have deleterious effects on gonadal development [15]. Although GATA4 gene p.Pro394Thr (c.1180C > A) [25] was detected in 46,XY DSD, there was no evidence to prove that this mutation would cause 46,XY DSD. The variant p.Gly375Arg in GATA4 gene, like p.Glu359X and p.Pro407Gln mutation locating at Cterminal far away from zinc nger ZNI and ZNII region, was identi ed with the 46,XY DSD cases. Both men and women with p.Gly375Arg mutation in GATA4 gene had no congenital heart disease, so this mutational site in GATA4 did not cause CHD.

Conclusions
Collectively, our study identi ed the heterogeneous p.Gly375Arg mutation of GATA4 protein in the two 46,XY DSD cases. However, the heterogeneous GATA4 p.Gly375Arg mutation was found in the 46,XY DSD cases without heart anomalies. Further studies are necessary to clarify whether GATA4 p.Gly375Arg variants contribute to the genetic predisposition of 46,XY DSD.

Cases
The 7-year-old proband was referred to Shahe hospital due to the right inguinal hernia. She was prepared to perform the hernia operation. The surgeon found that the tissue was like a testicular tissue and stop the operation. Then, she was transferred to our hospital. Her 5-year-old sister and 2-year-old sister were also referred to our hospital. Physical examinations were performed to check female external genitalia. Bultrasound was used to detect male or female internal genital organs, including testis, uterus, and ovaries.
Karyotype analysis was performed to test the anomalies in the chromosome number or chromosomal structure.
Written informed consent was obtained from all the family members. The study was approved by the Medical Ethical Committee of the People's Hospital of Xingtai City.

Cytogenetic Analysis
Peripheral blood lymphocytes from the 2 DSD siblings were isolated and cultured in RPMI1640 medium supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% fetal bovine serum (FBS). Each one's lymphocyte were cultured in duplicate. Phytohemagglutinin was added to promote cell division. Dividing cells were arrested at metaphase stage by Colchicine (20 µg/ml) for two hours and xed in methanol and acetic acid (3:1). Fixed cells were dropped onto glass slides and placed in an incubator at 75 °C for three hours to air-dry. Giemsa solution was used to stain the G-bands of the chromosomes.

Genetic analysis
The genomic DNA was extracted from the proband according to the manufacturer's instructions using the TIANamp Blood DNA Kit (TIANGEN, China). Chip capture high-throughput sequencing was carried out by BGI Corporation (Shenzhen, China). Sequence data were aligned to the ESP data, QIANREN data and EXAC data. Based on the patients' symptoms, the suspected genes recruited by OMIM data were analyzed. Since the heterozygous missense mutations (C.1123G > A; p.Gly375Arg) in GATA4 gene were found in the proband II-1, the genomic DNA was extracted from the other members of the family and PCR ampli ed GATA4 gene. Sanger sequencing of GATA4 gene were done for all family members by Fulgent Technologies Inc. (Beijing, China).