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Clinical Hormonal and Cytogenetic Evaluation of 46xx Males and Review of the Literature

Ditulis Kamis, 21 April 2022 Tulis Komentar Edit
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Molecular cytogenetic assay and genetic counseling: a case report of eight 46,Twenty males and a literature review

  • Hongguo Zhang1,ii,
  • Qi Xi1,2,
  • Yuting Jiangane,2,
  • Leilei Lii,2,
  • Ruizhi Liu1,2 &
  • Ruixue Wang ORCID: orcid.org/0000-0002-8772-4954 i,2

Molecular Cytogenetics volume 12, Article number:44 (2019) Cite this article

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Abstract

Background

46,XX male syndrome is a rare disorder that unremarkably causes infertility. This study was established to identify the genetic causes of this condition in a series of 46,Twenty males through the combined application of cytogenetic and molecular genetic techniques.

Case presentation

We identified eight azoospermic 46,XX males who underwent infertility-related consultations at our centre. They all presented normal male person phenotypes. In vii of the 8 46,Xx males (87.five%), translocation of the SRY factor to the terminal short arm of the X chromosome was clearly involved in their status, which illustrated that this translocation is the chief machinery of 46,20 sex activity reversal, in line with previous reports. Still, one patient presented a homozygous DAX1 mutation (c.498G > A, p.R166R), which was non previously reported in SRY-negative Xx males.

Conclusions

Nosotros proposed that this synonymous DAX1 mutation in example 8 might not be associated with the activation of the male person sexual activity-determining pathway, and the male phenotype in this example might be regulated by some unidentified genetic or environmental factors. Hence, the detection of genetic variations associated with sex reversal in critical sex-determining genes should exist recommended for SRY-negative Xx males. Only later comprehensive cytogenetic and molecular genetic analyses tin can genetic counseling exist offered to 46,XX males.

Background

46,Twenty male syndrome, also called de la Chapelle syndrome, is a genetic disorder encountered infrequently in a clinical context [i]. This syndrome is found in approximately one in xx,000–25,000 males, who showroom a male phenotype merely a 46,Twenty karyotype [2]. These patients have three chief phenotypic manifestations: (1) classic XX males with infertility presenting normal male person internal and external genitalia; (ii) Twenty males with ambiguous genitalia presenting apparently external genital ambiguities at nativity, such as hypospadias, micropenis, or hyperclitoridy; and (iii) Twenty true hermaphrodites presenting internal or external genital ambiguities at birth [iii,4,five].

Molecular findings on the presence of the sex-determining region Y (SRY) gene can exist used to divide Twenty males into SRY-positive and -negative groups [6]. About 90% of XX males have the SRY gene, which plays a critical part in encoding the testis-determining factor (TDF) [seven]. These SRY-positive XX individuals present normal genitalia and a male phenotype at birth [4]. Withal, in the SRY-negative 46,XX males, external genital ambiguities of unlike degrees are presented [five].

According to a literature review, several genes such as SRY, SOX9, DAX1, and WNT4 are associated with sexual practice reversal. Herein, we draw eight azoospermic 46,XX males presenting a normal male phenotype and masculinization. Nosotros also perform a literature review to investigate the correlation between DAX1 mutation and XX males, aiming to explain the genetic crusade of SRY-negative XX males.

Instance presentation

Participants and clinical data

From 2015 to 2017, eight Chinese males underwent consultations for infertility at the Center for Reproductive Medicine and Eye for Prenatal Diagnosis of the Start Infirmary of Jilin University considering of no pregnancy resulting from regular unprotected coitus. The results of physical and routine clinical examinations were listed in Table ane. All patients were finally diagnosed with azoospermia based on routine semen test [8]. The Ethics Commission of the Beginning Infirmary of Jilin University approved our study protocol (No. 2016–422) and all patients provided written informed consent to participate in this study.

Table ane Summary of the concrete and clinical examinations of our patients

Full size table

Methods

Karyotype analysis

Conventional G-banding techniques were practical on the cultured peripheral blood cells for chromosomal karyotyping. We described all of the chromosomal karyotypes according to the ISCN 2016 nomenclature [9].

AZF microdeletion analysis

Microdeletions in the AZF region were detected using polymerase chain reaction (PCR), as previously described in accordance with the recommendations of the European University of Andrology and the European Molecular Genetics Quality Network. Specific sequence-tagged sites (STSs) were mapped in the AZF region, including SY84 and SY86 for AZFa; SY27, SY134, and SY143 for AZFb; SY157, SY254, and SY255 for AZFc; and SY152 for AZFd [10].

Fluorescence in situ hybridization analysis (FISH)

FISH specific for the Y chromosome was performed on metaphase slides for the patients to further confirm the existence of SRY through the standard operating protocol (Cytocell Technologies, Cambridge, UK). The detecting probes were equally follows: red-labeled sex-determining region Y (SRY) probe with two nonoverlapping probes, dark-green-labeled probe for a heterochromatic region (DYZ1) in Yq12, and blue-labeled probe for the X centromere (DXZ1).

Sanger sequencing

Some genes had been shown to be critically involved in sex differentiation, such as SOX9, DAX1, and WNT4 [eleven, 12]. Sequencing was performed to discover mutations in these genes on an ABI 3730xl DNA analyzer (Applied Biosystems) by BGI (Beijing, China) for the SRY-negative patients [13].

Results

The results of cytogenetic Thousand-banding and AZF microdeletion analyses were listed in Table 2. All of the 46,XX males reported hither exhibited AZFa+b + c microdeletion. FISH confirmed the presence of a translocated SRY region located on the distal tip of the short arm of the X chromosome in seven patients (cases 1–7, Fig. ane). For example eight, the PCR assay demonstrated the absenteeism of the SRY gene; as such, Sanger sequencing was performed on three key genes (SOX9, WNT4, and DAX1) associated with sexual activity reversal. No mutations were discovered in the coding regions of SOX9 and WNT4. Withal, a homozygous variant in exon 1 of DAX1 (c.498G > A, p.R166R) was detected (Fig. 2), which was not previously reported in SRY-negative 46,XX males. Only cases 1 to 3 chose artificial insemination with donor sperm, according to the assisted reproductive technology follow-up outcomes.

Table 2 Sequence-tagged site deletions and chromosomal analysis

Full size table

Fig. 1
figure 1

FISH demonstrated that the SRY region was located on the distal tip of the brusque arm of the chromosome X. a:case ane. b:instance 2. c: case 3. d: example 4. east: example five. f: case half-dozen. m: instance vii. Cerise arrows indicated SRY indicate (cerise), and blue arrows indicated 10 centromere (blueish)

Full size image

Fig. 2
figure 2

Sanger sequencing analysis of DAX1 gene for example 8: a synonymous mutation of DAX1 (c.498G > A, p.R166R)

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Discussion and conclusions

In this paper, nosotros reported eight azoospermic cases of 46,XX males with normal male person genitalia and no apparent abnormalities. All subjects presented 46,XX karyotypes and AZFa+b + c microdeletion. Amidst them, seven cases were SRY-positive (87.v%), with the SRY gene being translocated to the brusk end of the X chromosome, while the 8th case was SRY-negative (12.five%) with a synonymous mutation of DAX1 (c.498G > A, p.R166R).

The first case of 46,20 male syndrome was reported in 1964 [1]. The clinical manifestations are mainly characterized by a normal phenotype in newborns, simply delayed puberty, gynecomastia, or infertility in adolescents. In addition, hypospadias, cryptorchidism, and severely ambiguous ballocks tin likewise be observed [fourteen]. The majority of Twenty males were found to be SRY-positive, presenting sterility with normal male genitalia and SRY translocation to the X chromosome or autosomes. Withal, reports on SRY-negative Xx males accept been express, with these cases unremarkably presenting infertility with immature/ambiguous to normal genitalia, incomplete testicular development or ovotestis, and varying degrees of masculinization [xv].

The mechanism of 46,XX male person syndrome in SRY-positive cases could exist summarized as involving cross-over errors in the pseudoautosomal regions of the sex chromosome during paternal meiosis [xvi]. However, the machinery in SRY-negative 46,20 males has remained unclear. Several possibilities were proposed to explain these cases: mosaicism for SRY in hidden gonads, the inhibition of male pathways resulting from mutations of autosomal or X-linked genes, and mutations of other sex activity-determining genes downstream of SRY [14, xv].

The sex of individuals is known to be adamant by the SRY cistron in most mammals, but the existence of SRY-negative males demonstrates the involvement of other genes in determining maleness in the absence of SRY. Mutations of some critical genes, such as SOX9, DAX1, and WNT4, accept been proven to exist associated with sex reversal [eleven, 12]. DAX1 (dosage-sensitive sex reversal adrenal hypoplasia built disquisitional region of the X chromosome gene 1), also called NR0B1, is located on chromosome Xp21.three-p21.2 (ΟMIM#300473). It contains 2 exons and 1 intron, which encode an orphan nuclear receptor. DAX1 is widely expressed in the adrenals, hypothalamus, pituitary, and testis, playing critical roles in testicular and ovarian development. Mutations of DAX-1 are usually associated with primary adrenal insufficiency or congenital adrenal hypoplasia (CAH) and hypogonadotropic hypogonadism (MIM #300200) [17, 18]. DAX1 was initially recognized as a dosage-sensitive ovarian-determining gene. An increased number of copies of DAX1 could pb to high expression of its encoded proteins, which would result in sex reversal [eleven]. Farther research revealed that DAX1 was actually necessary for testis differentiation and spermatogenesis [19, twenty]. Zenteno et al. [21] assumed that SRY-negative XX males with normal genitalia were homozygous for deletions or loss-of-part mutations in dosage-sensitive sex reversal. In addition, Domenice et al. [12] proposed that the loss of function of the DAX1 factor might foreclose its repressive upshot on masculinizing genes and thus determine testicular development in XX individuals, which probably explained the presence of 46,XX sex activity reversal. In add-on, Dangle et al. [22] reported a 46,Twenty SRY-negative case with a heterozygous deletion encompassing DAX1.

In example 8 presented here, Sanger sequencing of the coding regions of the DAX1 gene showed a synonymous mutation of this gene (c.498G > A, p.R166R), which was too described in other reports. For example, Mou et al. [23] reported a series of patients with secretory azoospermia and fertility who presented synonymous mutation (c.498 G > A) in DAX1. In addition, Achermann et al. [24] described the aforementioned DAX1 mutation in patients with hypogonadotropic hypogonadism or pubertal delay. Moreover, patients with CAH could too present DAX1 mutation (c.498 Thousand > A), in those with illness onset in either infancy or adulthood [25, 26]. Xu et al. [27] reported a 3-year-old boy who was diagnosed with X-linked CAH, with three novel mutations detected in DAX1: a missense mutation (c.376G > A, p.Val126Met), a synonymous mutation (c.498G > A, p.Arg166Arg), and a nonsense mutation (c.1225C > T, p.Gln409X). Currently, the mechanisms triggering testis development in SRY-negative 46,Twenty males remain unknown. Overexpression of the DAX1 cistron could crusade female-to-male sexual activity reversal [28], but this was not analyzed in case eight, as nosotros failed to investigate probably subconscious gonadal mosaicism for SRY or mutations in autosomes, or other functional mutations of unknown sex-determining genes (east.k., SF1, RSPO1, SOX3, SOX10, ROCK1, and DMRT) [14]. Considering the pathogenicity of the polymorphism as recorded in the ClinVar database, the potential risk of CAH in case 8 should be considered, also the sex reversal. Despite the DAX1 mutation detected in case 8 not previously being reported in SRY-negative 46,XX males, the potential association between DAX1 mutation and SRY-negative 46,Xx males nevertheless requires further investigation. We too speculated that other unidentified genetic or ecology factors might play critical roles in regulating sex determination and gonad sexual practice differentiation.

The SOX9 and WNT4 genes were also sequenced in example 8. The SRY-box 9 (SOX9) gene, located in 22q13, is a widely expressed transcription factor involved in male sex determination. Normal expression of SOX9 was found to be associated with testicular differentiation. However, its overexpression or duplication might lead to 46,20 male sex reversal and testicular differentiation in the absence of SRY [29, 30]. With regard to WNT4, located in 1p36.12, it has besides been shown to play a critical role in the development of the reproductive organisation as a candidate ovary-determining gene or antitestis gene [31]. Moreover, information technology has been reported that loss-of-function mutation in WNT4 could event in partial Xx male person sex activity reversal [32]. Given their lack of clear causative mutations in this written report, SOX9 and WNT4 might not exist critical factors in SRY-negative Twenty males.

In other words, irrespective of the status as SRY-positive or -negative, 46,XX males would always nowadays infertility due to the absence of the AZFa, AZFb, and AZFc regions located on chromosome Yq11, which are involved in regulating normal spermatogenesis [fourteen].

In the nowadays written report, viii cases of 46,XX male syndrome were identified based on cytogenetic and molecular genetic analyses. One SRY-negative XX male carried a homozygous p.R166R synonymous mutation in DAX1, while the other seven SRY-positive 46,Xx individuals had SRY translocated to the final of the X chromosome. Our findings enrich the understanding of the genotype–phenotype correlation in 46,Xx males, especially in patients with SRY-negative female-to-male sexual activity reversal. The combined awarding of chromosomal analysis, AZF microdeletion evaluation, SRY detection, and sequencing of key sex-determining genes should be recommended for these patients.

Availability of data and materials

The information and material used or analysed during the current report are bachelor from the corresponding writer on reasonable asking.

Abbreviations

AZF:

Azoospermia factor

FISH:

Fluorescent in situ hybridization

ISCN 2016:

International System for Human Cytogenetic Nomenclature 2016

SRY :

Sexual practice-determining region Y

STS:

Specific sequence-tagged sites

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Acknowledgements

We express our sincere gratitude to all the staff of the Genetics Laboratory and Andrology Laboratory for their splendid piece of work. We thank Liwen Bianji, Edanz Grouping China (www.liwenbianji.cn/air conditioning), for editing the English text of a typhoon of this manuscript.

Funding

This work was supported past The National Key Research and Evolution Program of China (2016YFC1000601).

Author information

Affiliations

  1. Centre for Reproductive Medicine, Center for Prenatal Diagnosis, First Hospital, Jilin University, Changchun, 130021, Cathay

    Fagui Yue, Hongguo Zhang, Qi 11, Yuting Jiang, Leilei Li, Ruizhi Liu & Ruixue Wang

  2. Jilin Applied science Research Center for Reproductive Medicine and Genetics, Jilin University, Changchun, 130021, Red china

    Fagui Yue, Hongguo Zhang, Qi 11, Yuting Jiang, Leilei Li, Ruizhi Liu & Ruixue Wang

Contributions

FY wrote the get-go draft of the manuscript. HZ and QX nerveless the data of all the patients. YJ and LL participated in analysis and interpretation of data. RL and RW reviewed the manuscript and were involved in its critical revision before submission. All authors read and approved the last manuscript.

Corresponding writer

Correspondence to Ruixue Wang.

Ethics declarations

Ethics approval and consent to participate

This report was approved by the Ethics Committee of the Offset Hospital of Jilin University (No.2016–422). The patients provided written informed consent for participating in this report.

Consent for publication

Written informed consents were obtained from the patients for publication of this manuscript.

Competing interests

The authors declare that they accept no competing interests.

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Yue, F., Zhang, H., Xi, Q. et al. Molecular cytogenetic analysis and genetic counseling: a case written report of 8 46,Twenty males and a literature review. Mol Cytogenet 12, 44 (2019). https://doi.org/x.1186/s13039-019-0456-y

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Keywords

  • 46,XX male
  • Sex reversal
  • SRY
  • DAX1 mutation

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