Identification of an SRY-negative 46,XX infertility male with a heterozygous deletion downstream of SOX3 gene

Shengfang Qin,Xueyan Wang,Jin Wang

doi:10.1186/s13039-022-00580-7

Abstract

BackgroundA male individual with a karyotype of 46,XX is very rare. We explored the genetic aetiology of an infertility male with a kayrotype of 46,XX and SRY negative.MethodsThe peripheral blood sample was collected from the patient and subjected to a few genetic testing, including chromosomal karyotyping, azoospermia factor (AZF) deletion, short tandem repeat (STR) analysis for AMELX, AMELY and SRY, fluorescence in situ hybridization (FISH) with specific probes for CSP 18/CSP X/CSP Y/SRY, chromosomal microarray analysis (CMA) for genomic copy number variations(CNVs), whole-genome analysis(WGA) for genomic SNV&InDel mutation, and X chromosome inactivation (XCI) analysis.ResultsThe patient had a karyotype of 46,XX. AZF analysis showed that he missed the AZF region (including a, b and c) and SRY gene. STR assay revealed he possessed the AMELX in the X chromosome, but he had no the AMELY and SRY in the Y chromosome. FISH analysis with CSP X/CSP Y/SRY showed only two X centromeric signals, but none Y chromosome and SRY. The above results of the karyotype, FISH and STR analysis did not suggest a Y chromosome chimerism existed in the patient's peripheral blood. The result of the CMA indicated a heterozygous deletion with an approximate size of 867 kb in Xq27.1 (hg19: chrX: 138,612,879–139,480,163 bp), located at 104 kb downstream of SOX3 gene, including F9, CXorf66, MCF2 and ATP11C. WGA also displayed the above deletion fragment but did not present known pathogenic or likely pathogenic SNV&InDel mutation responsible for sex determination and development. XCI assay showed that he had about 75% of the X chromosome inactivated.ConclusionsAlthough the pathogenicity of 46,XX male patients with SRY negative remains unclear, SOX3 expression of the acquired function may be associated with partial testis differentiation of these patients. Therefore, the CNVs analysis of the SOX3 gene and its regulatory region should be performed routinely for these patients.

Highlights

A male individual with a karyotype of 46,XX is very rare
Neither AMELY and SRY fluorescence peaks were detected in the short tandem repeat (STR) analysis results, nor Y chromosome and SRY fluorescence signals were shown in the fluorescence in situ hybridization (FISH) result
Results of chromosomal microarray analysis (CMA) analysis Taking 46,XX female genomes as a standard reference, the patient’s Xq27.1 had about 867 Kb of heterozygotic deletion, and the deletion region was located at 104 Kb downstream of the SRY-box transcription factor 3 (SOX3) gene, including F9, CXorf66, MCF2, ATP11C four protein-coding genes, as shown in Fig. 4a and b

Summary

Methods

Subject A 31-years-old patient, heigh 166 cm and weigh 52.5 kg, went to our clinic due to primary infertility. Specimen preparation and DNA extraction 5 mL of venous blood was collected from patients with heparin sodium and EDTA-Na2 anticoagulant tubes, respectively, and ready for use. Chromosome karyotype analysis Lymphocytes of heparin sodium anticoagulated blood were cultured, harvested, and prepared for microscope slides before Giemsa staining according to conventional cell culture methods. Fluorescence in situ hybridization (FISH) analysis Lymphocytes in EDTA-Na2 anticoagulant blood were isolated by lymphocyte separation solution and hybridized by CSP 18/CSP X/CSP Y probe (Jin Pujia corp.), using the same method as previously reported [15]. The labelled patient sample was mixed with the reference sample and co-hybridized to SurePrint G3 CGH + SNP (180 K) chip. X chromosome inactivation detection The sample DNA of undigested and digested by HpaII, which methylation-sensitive restriction enzyme, was amplified by androgen receptor (AR) gene-specific primers and capillary electrophoresis subsequently. XCI ratio was calculated according to formula (d1/u1)/(d1/u1 + d2/u2), and XCI bias was confirmed if the XCI ratio > 70% [22, 23]

Results

Conclusions

Background

Discussion