Isodicentric Y chromosome with SRY duplication in a female with complete gonadal dysgenesis

Abstract

Background

Sexual differentiation and development rely upon many genetic and environmental factors and any disruption of these can lead to Differences/Disorders of Sex Development (DSDs). DSDs are a diverse group of heterogeneous rare diseases that can be categorized into three main groups. 46,XY, complete gonadal dysgenesis is a subgroup of 46,XY, DSD that appears as female phenotype and external genitalia characterized by primary amenorrhea at adolescent.

Case presentation

Here, we present a 14-year-old female patient with structural rearrangements of the Y chromosome including SRY gene duplication. These chromosomes were characterized by karyotyping, Oligo-array comparative genomic hybridization (CGH), metaphase fluorescence in situ hybridization (FISH), and exome sequencing (ES). The mechanism(s) by which these rearrangements could have occurred is discussed.

Conclusions

46,X, idic(Y)(p11.32→q11.22:: q11.22→p11.32)] in this proband led to a large copy-number change including 24.5 Mb gain on the Yp11.32q11.223 region and a 33 Mb loss on the Yq11.223q11.23 region.

Background

Sexual differentiation is initiated at the time of conception once the ovum is fertilized by a sperm cell that carries either an X or Y-chromosome [1]. Following this, a variety of activating and repressing factors precisely coordinate to manage the pathways responsible for normal sex development [2]. In an embryo with a 46,XY karyotype testis formation is determined by genetic networks and testis determining factors (TDFs) that facilitate testis development and suppress ovarian differentiation [1]. The SRY gene, found on the short arm of the Y chromosome, is crucial for initiating this process. Any disruption in this complex mechanism can prevent proper testis formation, leading to a variety of disorders/differences of sex development (DSD) [3]. Some professionals substitute the word ‘disorders’ with ‘differences’ to prevent negative connotations including the stigma of ‘disorder’ [4].

DSDs are rare congenital conditions characterized by discordance among chromosomal, phenotypic, and anatomical sex [3]. Although there are numerous modes of classification by molecular genetics [5], DSD is typically categorized into three main groups: 46, XX DSD, 46,XY DSD, and sex chromosome DSD [2]. Certain DSD types are monogenic, deriving from single-gene mutations, whereas others result from multiple gene alterations [6]. The potential link between DSD and environmental chemicals is worth considering [5]. A multidisciplinary team within specialized medical facilities must oversee all instances of DSD [1].

46,XY DSDs occur due to incomplete intrauterine virilization, leading to varying degrees of virilization of external genitalia; which may also involve the presence or absence of Müllerian structures (6, 7). Determined by the specific condition, testicular tissue may not be present, partially or completely dysgenic, or even appear normal when examined histologically [7]. There is limited data on the incidence and prevalence of 46,XY DSD, and estimations vary among ethnic groups with the highest incidence in the southern African population [2, 6]. However, as the Danish Cytogenetic Central Registry reports, the prevalence of XY females is 6.4 per 100,000 live-born females [2]. Although genitalia that are sufficiently atypical to require any investigations during early infancy are rarer with a birth prevalence of about 1 in 1,200 in-term infants [8].

The term “46,XY complete gonadal dysgenesis” has replaced the term “46,XY female” [9]. 46,XY gonadal dysgenesis results from deletions or point mutations in SRY gene, duplication of DSS locus on the X chromosome, or mutations in autosomal genes [10]. 46.XY gonadal dysgenesis characterizes as high LH and FSH, female phenotype that may be accompanied by primary amenorrhea, female external genitalia, Streak Gonads with the presence of mullerian ducts and absence/hypoplasia of wolffian ducts [11].

Key indicators of DSD may be observed during prenatal ultrasound, atypical genital development in newborns, bilateral inguinal hernias in children, atypical secondary sex characteristics in adolescents, or infertility in adults [6]. Diagnosing 46, XY DSD primarily relies on clinical evaluations, frequently identified when investigating conditions like primary amenorrhea or delayed puberty. In these cases, medical professionals may notice the absence of menstrual periods or delayed onset of puberty, prompting further investigation into the underlying causes [12]. Additionally, if an older sibling is diagnosed with DSD, younger siblings might be evaluated as a precaution. Another example that triggers evaluation is when prenatal diagnostic tests reveal a mismatch with the expected karyotype, suggesting the need for a deeper exploration into the child’s genetic and phenotypic presentation [1].

Genetic techniques like microarray and next generation sequencing (NGS) have uncovered new genetic variants in patients with DSD [2]. The main methods for detecting this chromosomal anomaly are the combined use of chromosomal karyotyping and fluorescence in situ hybridization (FISH). Furthermore, the use of chromosomal microarray (CMA) is crucial for identifying chromosomal microscopic imbalances, providing more detailed insights into the potential causes of abnormalities. In this report, we described a 46,XY DSD female with a derivative Y chromosome 46,X, idic(Y)(p11.32→q11.22:: q11.22→p11.32)] identified by standard cytogenetic analysis. To delineate this der(Y), FISH and SNP array characterization was performed. In addition to that, exome sequencing (ES) was performed to characterize the aCGH results of Yp11.32q11.22 direct duplication and Yq11.223q11.23 deletion and exclude any other point mutation like SOX9.

Case presentation

A 14-year-old girl was introduced to our clinic due to primary amenorrhea. She presented with typical female physical characteristics, including a feminine appearance, voice, and external genitalia with a stature of 152.5 Cm and a weight of 43.88 kg body mass index (BMI) 18.8. However, she had small vulva and sparse pubic hair. She was born after a full-term pregnancy to unrelated parents. The karyotypes of both the parents and the sister were determined to be normal. Therefore, it can be concluded that this genetic rearrangement (46,XY Female) is an occurrence rather than an inherited trait. Blood serum analysis revealed elevated levels of LH (43.2 mIU/mL), FSH (180.1 mIU/mL) and free Testosterone (1.58 pg/mL) for her age.

The findings from the magnetic resonance imaging (MRI) of the abdomen and pelvis, which was performed with and without contrast, are as follows: The uterus was visualized and measured approximately 40 *6*10 mm. The vagina was also observed and appeared to be present. No discrete ovarian tissue or evidence of testicular tissue was identified within the examined area. All other abdominal or retroperitoneal organs appeared normal.

Patients with 46,XY DSD are often advised to undergo prophylactic gonadectomy because of the elevated risk of developing gonadal malignancy [12]. Thus, under general anesthesia, a bilateral gonadectomy was performed. The examination revealed that the patient had two underdeveloped fallopian tubes, an undifferentiated tubular uterus, and two strict gonads measuring 3 × 20 mm in the area where the ovaries could have been located. No testicular tissue was found.

Methods

Ethical compliance, sample collection and initial chromosomal analysis

This study was conducted in accordance with the Declaration of Helsinki. Participants provided informed and voluntary consent for the publication of their information and images, and signed a written consent form. For individuals under the age of 18, consent was also obtained from their legal guardians.

Peripheral blood samples were collected from the patient using heparinized vacutainers, amounting to 2 milliliters. Following standardized protocols, chromosomal analysis of peripheral blood lymphocytes was conducted. Chromosome preparation employed conventional G-banding techniques, achieving a resolution of 500 to 700 bands, thereby facilitating the identification of chromosomal abnormalities relevant to genetic disorders.

Fluorescent in situ hybridization

Next, fluorescent in situ hybridization (FISH) was performed using ZYTOVISION prenatal probes on 200 peripheral blood cells. Dual-color probes were specifically utilized to target the centromeric regions of chromosomes X and Y (CEN X/Yq12). The Xp11.1-q11.1 region was signaled in red while the DYZ1 region was highlighted in green, allowing for the identification of sex-linked genetic conditions and the assessment of chromosomal duplications or deletions.

Oligo-array CGH analysis

Whole exome oligo-array comparative genomic hybridization (CGH) was conducted utilizing the SurePrint G3 ISCA V2 8 × 60 K mega-array platform, featuring 60,000 spots with an average probe spacing of 60 Kb, enhancing the density of approximately 500 established disease target regions. The patient’s sample was hybridized against a male reference sample, labeled 8268. The fragmented genomic DNA from the patient’s coding domains was amplified prior to sequencing, with custom-designed probes targeting known pathogenic variants in both coding and non-coding regions of the nuclear DNA and mitochondrial genome.

Sequencing and data analysis

Sequencing was performed utilizing the Illumina Nova-Seq sequencer, configured for paired-end sequencing at a 150-base pair read length. The generated sequence reads were aligned to the human reference genome (GRCh37) employing the Burrows-Wheeler Aligner (BWA) algorithm. Variant detection was executed using the Genomic Analysis Toolkit (GATK), a robust platform for variant discovery.

Variant annotation and interpretation

Annotated variants were filtered according to the guidelines set forth by the American College of Medical Genetics (ACMG). This interpretative process involved comparisons with multiple databases, including gnomAD, the Iranome database, the 1000 Genomes Project, and NCBI ClinVar. Additionally, various lines of computational evidence were assessed regarding conservation and functional impact to derive a comprehensive understanding of the genetic variants. The analysis revealed a mean depth of coverage of 229X, achieving a Quality Threshold of 98.6% based on Consensus Coding Sequence (CCDS).

Results

The conventional cytogenetic analysis (G-banding) indicated that all 50 observed metaphase cells from the patient exhibited a 46,XY chromosome pattern. Subsequent GTG-karyotyping with a 550-band resolution on 100 metaphase spreads confirmed that the individual has a 46,X, idic(Y)(p11.32→q11.22:: q11.22→p11.32)] [100] karyotype (Fig. 1).

Fig. 1.

Karyotype of the patient found with G-banding techniques, showing 46,X, idic(Y)(p11.32→q11.22:: q11.22→p11.32)]

In a FISH study intended to eliminate the possibility of sex chromosome mosaicism, the individual under examination exhibited the presence of a single X chromosome in 100% of cells. However, the Y chromosome was undetectable, as the utilized probe was designed to target the Yq12, which, according to the array, has been shown to be absent.

The Oligo-Array CGH analysis findings demonstrated a 24.5 Mb gain on the Yp11.32q11.223 region, including the SRY gene, and a 33 Mb loss on the Yq11.223q11.23 region. These results indicate an extra copy of the Yp11.32q11.223 region and the absence of the Yq11.223q11.23 region (Fig. 2).

Fig. 2.

(A) Histogram for Copy-number variants on positions: Y:1-24594676, Y:26,066,913–59,311,250. Histogram views are shown when more than 50 features are on a single chromosome. DECIPHER (GRCh38/hg38). (B) DECIPHER: Copy-Number Variants Gains only, Pathogenic/Likely Pathogenic and Under 25 Mb for the region of Yp11.32q11.223. (C) DECIPHER: Copy-Number Variants Gains only, Pathogenic/Likely Pathogenic and Under 25 Mb for the region of Yq11.223q11.23

ES analysis revealed that the individual exhibits a copy number variation (CNV) affecting genes linked to the individual’s phenotype. The CNV analysis identified a 46,XY karyotype with a duplication of a region spanning over 24 Mb of chromosome Y. Specifically, the variant NC_000024:g.(0_2655028)_(26942247_26944569)dup resulted in a copy number gain that overlaps with the 100% coding sequence of the SRY gene and 106 other genes.

Discussion/Conclusion

Here, we disclosed a 46, XY complete gonadal dysgenesis DSD with a structural rearrangement of 46,X, idic(Y)(p11.32→q11.22:: q11.22→p11.32)] karyotype demonstrating a 24.5 Mb gain on the Yp11.32q11.223 region, including the SRY gene, and a 33 Mb loss on the Yq11.223q11.23 region. The patient had no ovaries or testis and was referred to our clinic due to primary amenorrhea. To gain a comprehensive understanding of 46,XY DSD, it is essential first to comprehend the meaning of sex determination and differentiation. Sex determination pertains to the person’s genetic sex (i.e. 46,XY), while sex differentiation involves the development of testis from embryonic gonadal tissue [3].

The formation mechanisms of such a restructured Y chromosome necessitate an examination of chromosomal behavior during meiosis, mitosis, and structural alterations. Non-allelic homologous recombination (NAHR), a process involving recombination between repetitive sequences or segmental duplications, is primarily facilitated by low-copy repeats (LCRs) with recombination hotspots, gene conversion, and apparent minimal efficient processing segments [13]. NAHR is responsible for the majority of recurrent rearrangements, characterized by shared size, clustered breakpoints, and occurrence in multiple individuals [14]. Conversely, non-recurrent rearrangements exhibit size variations among patients but may share a minimal overlapping region, potentially resulting in common clinical manifestations [15]. While LCRs do not directly mediate non-recurrent events, they may stimulate their occurrence. Additionally, some infrequent NAHRs can be mediated by highly homologous repetitive sequences such as Alu or LINE elements, accounting for a subset of non-recurrent rearrangements [16]. The Y chromosome’s abundance of palindromic and repetitive regions renders it susceptible to misalignment during meiosis, potentially leading to the formation of a dicentric Y chromosome with duplicated and deleted segments [17]. In the current case, NAHR between regions in Yp11.32 and Yq11.22 may have resulted in the observed duplication of Yp11.32q11.22 and deletion of Yq11.223q11.23.

Chromosomal breaks at fragile or structurally predisposed regions, particularly those enriched in repetitive or palindromic sequences, can facilitate the formation of derivative chromosomes [18]. A break in the proximal Yp arm (p11.32) and distal Yq arm (q11.23) may result in fusion of the broken ends, potentially creating a dicentric chromosome with the observed imbalances [17]. Non-recurrent rearrangements can also be elucidated through mechanisms such as NHEJ and replication-based mechanisms like Fork Stalling and Template Switching (FoSTeS) or Microhomology-Mediated Break-Induced Replication (MMBIR) [19]. During DNA replication, fork stalling at repetitive sequences may occur. Subsequent template switching between homologous or non-homologous regions can lead to duplications and deletions [20]. In this instance, replication fork stalling in the Yp11.32 and Yq11.22 regions, followed by template switching, could account for the duplication on Yp and deletion on Yq. Additionally, a balanced chromosomal rearrangement in a parent, such as an inversion involving Yp11.32 to Yq11.23, could result in unbalanced gametes post-meiosis. If a gamete carrying the derivative Y chromosome participated in conception, the offspring would inherit the described duplication and deletion. However, this possibility was evaluated through parental karyotyping in the current case. Postzygotic chromosomal rearrangements during early embryonic mitoses could also explain the derivative Y chromosome [21]. Breakage and reunion within repetitive regions during mitosis might lead to the observed duplication and deletion pattern. In rare instances, a paracentric inversion during meiosis could initially form a dicentric chromosome, which may subsequently stabilize by losing one centromere or forming a pseudodicentric chromosome [17]. The interplay of these mechanisms offers a comprehensive framework for understanding the formation of the described derivative Y chromosome, underscoring the complex nature of genomic rearrangements.

The 24.5 Mb gain on Yp11.32q11.22 encompasses several OMIM-annotated genes and is situated adjacent to the pseudoautosomal region 1 (PAR1), overlapping with known recombination-prone areas. The proximity to PAR1 suggests that misalignment during recombination, potentially facilitated by repetitive elements in PAR1 or adjacent Yp11.2 regions susceptible to NAHR, could have precipitated the duplication (Repping et al., 2006). This duplication affects genes within the PAR region, including SHOX, SRY, ALPHA, CSF2RA, TBL1Y, USP9Y, LWD, and DFNY2, which may contribute to the patient’s clinical presentation. Equally, the 33 Mb loss on Yq11.223q11.23 involves at least 10 OMIM-catalogued genes, notably CGY1, CSPG4P1Y, GOLGA2P2Y, DAZ4, DAZ2, DAZ3, BPY2, and TTTY3. While not directly implicated, the distal location of PAR2 on Yq suggests that adjacent regions might play a role in structural rearrangements. Of particular significance is the potential involvement of genes crucial for spermatogenesis, such as DAZ and RBMY, if the deletion extends into amplicon regions proximal to PAR2. The cumulative effect of these genomic alterations likely contributes to the complex phenotypic manifestations observed in the patient and finally cause to the 46,XY DSD phenotype [22].

In mammals, the genes SF1, WNT4, and WT1 are actively expressed in the urogenital ridge which develops into the gonads and up-regulates SRY expression [10]. SRY gene activates the expression of the downstream effector SOX9, leading to Sertoli cell formation during testis development [3, 10]. Furthermore, SRY initiates a feedback mechanism between SOX9 and FGF9, that up-regulates FGF9 and down-regulates Wnt4 and DAX1 in the male gonad. SRY may also directly regulate other factors [3].

Differences in the gonadal and reproductive tract development are known as DSDs. The case of our study, female patient with structural rearrangements of the Y chromosome, including SRY gene duplication, highlights the complex interplay of genetic, hormonal, and developmental factors in DSDs. DSDs represent a diverse spectrum of conditions that arise from disruptions in gonadal and reproductive tract development [2]. While monogenic etiologies account for many cases of DSD [23], the variability of phenotypes in individuals with similar genetic abnormalities suggests additional influences, such as modifier genes or environmental factors [24]. Advances in genetic techniques have significantly improved our understanding of the molecular mechanisms underlying DSDs, particularly through mutational and functional analyses [25].

The SRY gene plays a pivotal role in sex determination, with mutations in this gene accounting for 10–15% of cases of 46,XY DSD or gonadal dysgenesis [10, 25]. Functional impairments in SRY, such as reduced DNA-binding or nuclear transport capabilities, are well-documented causes of gonadal dysgenesis [25]. The ClinVar database currently lists approximately 100 pathogenic and likely pathogenic SRY mutations, underscoring the critical role of this gene in testis development [26]. Despite this, approximately half of individuals with 46,XY DSD do not receive a precise molecular diagnosis, reflecting significant gaps in our current knowledge [27]. Emerging technologies, such as exome sequencing, have identified new genetic loci and pathways associated with these conditions [28], offering hope for improved diagnostic precision and personalized management strategies [29].

In individuals with 45,X/46,XY mosaicism, the female phenotype and the risk of gonadal tumors are of particular concern. The presence of Y chromosome material, including the GBY (gonadoblastoma locus on the Y chromosome) region, predisposes dysgenetic gonads to neoplastic transformation [30]. Gonadoblastoma, a mixed germ cell/sex cord-stromal tumor, frequently arises in such settings and has a high rate of malignant progression, most commonly to dysgerminoma [30, 31]. Dysgerminomas, which are histologically analogous to seminomas in males, are notable for their bilaterality in about 15% of cases [30]. The pathogenesis of these tumors is influenced by the dysplastic nature of the gonads and the hormonal milieu, creating a permissive environment for tumor development [32, 33].

Prophylactic gonadectomy is widely advocated for individuals with Y chromosome material and dysgenetic gonads to mitigate the risk of gonadoblastoma and dysgerminoma [32, 34]. Early intervention is crucial to prevent malignancy and potential masculinization at puberty. Advances in molecular diagnostics, including the use of biomarkers such as OCT3/4 and chromosomal analysis, have improved the ability to monitor and detect precursor lesions in retained gonads [34]. Nevertheless, challenges remain, as many cases of DSD still lack a clear genetic diagnosis, highlighting the need for further research into the molecular basis of these disorders [29, 35].

This case emphasis the importance of a multidisciplinary approach to the diagnosis and management of DSDs. Improved genetic techniques and novel tools such as artificial intelligence and stem cell models hold promise for bridging current diagnostic gaps and enhancing personalized care [35]. Continued research into the genetic and molecular mechanisms of sex development will be essential to improving outcomes for individuals with DSDs and associated risks of gonadal tumors.

Abbreviations

DSD

Disorders of Sex Development

CGH

Comparative Genomic Hybridization

FISH

Fluorescence in situ hybridization

ES

Exome Sequencing

NGS

Next generation sequencing

TDFs

Testis Determining Factors

CMA

Chromosomal microarray

BMI

Body mass index

MRI

Magnetic resonance imaging

GRCh37

Human reference genome

BWA

Burrows-Wheeler Aligner

GATK

Genomic Analysis Tool Kit

ACMG

American Collage of Medical Genetics

MIF

Müllerian inhibiting factor

DHT

Dihydrotestosterone

NAHR

Non-allelic homologous recombination

LCRs

Low-copy repeats

PAR1

Pseudoautosomal region 1

FoSTeS

Fork Stalling and Template Switching

MMBIR

Microhomology-Mediated Break-Induced Replication

GBY

Gonadoblastoma locus on the Y chromosome

Author contributions

AS, ZY, ML and TH Conceptualization, Investigation, Methodology, Writing – original draft preparation, AS, RB, SE, and SD Resources, Visualization, Validation, Data processing, Reviewing and Editing.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Informed consent was obtained by the authors. The research ethics committees of Guilan University of medical sciences approved this study (NO: IR.GUMS.REC.1403.514). Written consent was obtained from parents.

Consent for publication

Written informed consent was obtained from the patients’ parents for publication of this case report.

Competing interests

The authors declare no competing interests.