46,XX sex reversal with partial duplication of chromosome arm 22q

We present a case of 46,XX sex reversal in the absence of SRY but with partial duplication of chromosome 22q. The subject had multiple congenital anomalies but nearly complete masculinization of the external genitalia. Our case along with a previous case supports the existence of a gene on chromosome 22q that can trigger testis determination in the absence of SRY. We proposed that overexpression of the SOX10 gene at 22q13 might be the cause of sex reversal. We investigated 13 additional subjects with SRY-negative 46,XX sex reversal for microduplication of chromosome arm 22q in the region of SOX10 gene, but could not find evidence for it.     

XX male sex reversal with genital abnormalities associated with a de novo SOX3 gene duplication

Differentiation of the bipotential gonad into testis is initiated by the Y chromosome-linked gene SRY (Sex-determining Region Y) through upregulation of its autosomal direct target gene SOX9 (Sry-related HMG box-containing gene 9). Sequence and chromosome homology studies have shown that SRY most probably evolved from SOX3, which in humans is located at Xq27.1. Mutations causing SOX3 loss-of-function do not affect the sex determination in mice or humans. However, transgenic mouse studies have shown that ectopic expression of Sox3 in the bipotential gonad results in upregulation of Sox9, resulting in testicular induction and XX male sex reversal. However, the mechanism by which these rearrangements cause sex reversal and the frequency with which they are associated with disorders of sex development remains unclear. Rearrangements of the SOX3 locus were identified recently in three cases of human XX male sex reversal. We report on a case of XX male sex reversal associated with a novel de novo duplication of the SOX3 gene. These data provide additional evidence that SOX3 gain-of-function in the XX bipotential gonad causes XX male sex reversal and further support the hypothesis that SOX3 is the evolutionary antecedent of SRY.     

性反转综合征患者SRY、SOX9基因检测

目的探讨性反转综合征发生与性别分化相关基因SRY、SOX9之间的关系。方法分析了4例XX男性性反转及1例XY女性性反转患者临床表现、染色体核型，并采用聚合酶链反应（PCR）对SRY、SOX9基因进行了检测。结果5例性反转中，3例XX男性性反转及1例XY女性性反转患者SRY阳性，1例XX男性性反转患者SRY阴性。5例患者均检测到SOX9基因。结论SRY易位是导致性反转发生的重要原因之一，某些性反转的发生可能与SOX9基因异常有关，SOX9的计量效应关系有待研究。     

8例性发育异常患者SRY基因分析

目的对8例性发育异常患者进行细胞遗传学及分子遗传学检查以探讨性别发育异常与SRY基因关系.方法用PY3.4,X着丝粒,SRY特异探针进行荧光原位杂交,用于分析性发育异常病人Y染色体及SRY基因异位情况.聚合酶链反应(PCR)扩增SRY基因,直接测序检测SRY基因突变.结果 2例46,XX男性,1例46,XY女性,1例45,X/46,XY嵌合体及1例46,X,t(Y;Y)(p11;q11)男性患者SRY基因均为阳性,直接测序未发现SRY基因阳性患者该基因突变.剩余1例46,XX男性,1例46,XY男性及1例46,XY女性患者SRY基因为阴性.FISH技术证实2例46,XX且SRY基因阳性的男性患者SRY基因易位至X染色体短臂末端.结论 SRY基因是人类性别决定的主导基因,但尚有其他基因参与性别分化.     

SRY-negative 46,XX male with normal genitals, complete masculinization and infertility

XX maleness is a rare syndrome with a frequency of 1 in 20,000-25,000 males. XX males exist in different clinical categories with ambiguous genitalia or partially to fully mature male genitalia, in combination with complete or incomplete masculinization. In this study, we report a case of SRY-negative XX male with complete masculinization but infertility. The patient had fully mature male genitalia with descended but small testes and no signs of undervirilization. PCR analysis for SRY, ZFY, Amelogenin, AZFa, AZFb, AZFc genes, a pair of primers from heterochromatic region and six Y-STRs showed the absence of any Y-chromosome-derived material. Absence of SRY gene was confirmed by three independent PCRs for each of two sets of primers covering an increasing length of the gene. Sequence analysis of the coding regions of SOX9 and DAX1 genes did not reveal any mutation. Real-time PCR assay revealed normal copy number for SOX9 gene. Microsatellite analysis showed no evidence of 17q (SOX9 gene) or 22q duplication. Genotyping with X-STRs ruled out the possibility of any deletion on X chromosome. Development of the male phenotype in the absence of SRY probably resulted from the loss of function mutation in some unknown sex-determining gene, which normally inhibits the male pathway, or from a gain of function mutation in a gene downstream to SRY in male pathway.     

性发育异常的细胞遗传学及分子遗传学研究

目的：对530例性发育异常患者进行细胞、分子还传学检查分析以探讨性发育不良的原因及发病机制。方法：常规外周血淋巴细胞染色体制备及显带技术。聚合酶链式反应（PCR）检测SRY基因（sex-determiningregiononY）。结果139例患者染色体核型异常,其中119例为性染色体数目异常,20例为性染色体结构异常。另有17例患者为睾丸女性化综合征。1例46,XX男性性反转综合征,1例,46．XY女性性反转综合征及正常男性对照均检测出SRY基因,结论：性染色体异常是导致性发育异常的重要原因。SRY基因的分析为研究性分化原理及性反转综合征的发病机制提供了有价值的资料。     

45,X/47,XXX/47,XX, del(Y)(p?)/46,XX mosaicism causing true hermaphroditism

Sex differentiation in humans depends on the presence of the Y-linked gene SRY, which is activated in the pre-Sertoli cells of the developing gonadal primordium to trigger testicular differentiation. Occasionally testicular formation can take place in subjects lacking a Y chromosome resulting in a 46,XX sex reversal condition. True hermaphroditism (TH) is a rare form of intersexuality characterized by the presence of testicular and ovarian tissue in the same individual. Genetic heterogeneity has been proposed as a cause of dual gonadal development in some cases and recently, hidden mosaicism was reported to cause TH in some 46,XX SRY negative patients. Here we report a TH case in which hidden mosaicism for the Y and X chromosome was detected by PCR and FISH in peripheral blood and gonadal tissue, supporting the fact that mosaicism may cause TH and that molecular analysis of gonadal tissue should be performed in all 46,XX cases.     

Two novel translocation breakpoints upstream of SOX9 define borders of the proximal and distal breakpoint cluster region in campomelic dysplasia

The semilethal skeletal malformation syndrome campomelic dysplasia (CD) with or without XY sex reversal is caused by mutations within the SOX9 gene on 17q24.3 or by chromosomal aberrations (translocations, inversions or deletions) with breakpoints outside the SOX9 coding region. The previously published CD translocation breakpoints upstream of SOX9 fall into two clusters: a proximal cluster with breakpoints between 50-300 kb and a distal cluster with breakpoints between 899-932 kb. Here, we present clinical, cytogenetic and molecular data from two novel CD translocation cases. Case 1 with karyotype 46,XY,t(1;17)(q42.1;q24.3) has characteristic symptoms of CD, including mild tibial bowing, cryptorchidism and hypospadias. By standard fluorescence in situ hybridization (FISH) and by high-resolution fiber FISH, the 17q breakpoint was mapped 375 kb from SOX9, defining the centromeric border of the proximal breakpoint cluster region. Case 2 with karyotype 46,X,t(Y;17)(q11.2;q24.3) has the acampomelic form of CD and complete XY sex reversal. By FISH and somatic cell hybrid analysis, the 17q breakpoint was mapped 789 kb from SOX9, defining the telomeric border of the distal breakpoint cluster region. We discuss the structure of the 1 Mb cis-control region upstream of SOX9 and the correlation between the position of the 14 mapped translocation breakpoints with respect to disease severity and XY sex reversal.     

合肥地区性逆转综合征患者遗传学研究

目的研究在性逆转综合征中进行皮肤细胞检测对于胚层嵌合机制的探讨及诊断的意义。方法对象是2009年7月至2010年2月在安医大一附院产诊中心就诊的性逆转患者(2例46,XX男性、1例不典型46,XY女性),进行了外周血、皮肤细胞(染色体制备、SRY基因检测)。结果两例46,XX男性外周血染色体核型和皮肤细胞核型均一致(一个46,XX男性SRY基因阳性,另一个46,XX男性SRY基因阴性);一例外周血核型为46,XY女性,皮肤细胞核型分析为45X/47XYY,表现为嵌合体。外周血及皮肤细胞SRY基因均为阴性。结论对患者皮肤细胞核型及基因进行检测可以排除嵌合体,并且对于在胚层发育方面的性分化的深入探讨有重要意义。     

Acampomelic campomelic dysplasia with de novo 5q;17q reciprocal translocation and severe phenotype.

Campomelic dysplasia (CD) is a rare skeletal malformation syndrome caused by mutations in the SRY related gene SOX9, mapped to 17q24.3-q25.1. A small proportion of cases are associated with structural rearrangements involving 17q and it has been proposed that this subgroup have a milder phenotype and better prognosis compared to those with mutations in the SOX9 gene. We report a severely affected infant with the acampomelic form of campomelic dysplasia, who died at 11 days and was found to have a de novo reciprocal translocation, 46,XX,t(5;17)(q15;q25.1). This is the second reported case of severe campomelic dysplasia associated with a structural rearrangement involving 17q and suggests that this subgroup of patients may not significantly differ from those without chromosomal rearrangements with regards to phenotype or prognosis.     

Cytogenetic and molecular study of a premature male infant with 46,XX derived from ICSI: case report

We investigated the aetiology of the male phenotype in a premature infant derived from ICSI with a 46,XX karyotype. A karyotypically normal couple underwent ICSI because of obstructive azoospermia in the male partner. Sperm were retrieved by testicular sperm extraction (TESE), cryopreserved, and later used for ICSI. The pregnancy after ICSI ended at 20 weeks. A normal-appearing male was delivered but he did not survive. Umbilical cord blood and placenta were sampled and used for molecular and cytogenetic investigation. The 46,XX karyotype from G-banding in this male infant correlated to a balanced female comparative genomic hybridization (CGH) profile in placental tissue. No PCR amplification of SRY on the p arm of the Y chromosome was observed while fluorescence in-situ hybridization (FISH) with the SRY probe also could not detect the gene in cord blood or placental tissues. CGH and FISH, with X and Y centromeric probes, failed to detect mosaicism in the trophoblast, stroma and amnion. Skewed X-chromosome inactivation (81%) was found in the chorionic villi. The molecular and cytogenetic studies indicated a 46,XX male infant without the SRY gene or 46,XX/XY mosaicism. The possible mechanism in this SRY-negative XX male by ICSI is discussed.     

性发育异常患者的细胞分子遗传学分析

目的: 探讨性发育异常患者的细胞分子遗传学特征。方法: 应用多重连接依赖的探针扩增(MLPA)技术对3例染色体核型为46,XX的男性性反转综合征患者及1例女性假两性畸形患者父母进行SRY、CYP21A2、DSS、DAX1、WNT4、SOX9、NR5A1等性别相关基因的拷贝数筛查,并采用细菌人工染色体(BAC)克隆制备探针,以荧光原位杂交技术(FISH)进行基因定位。结果: 3例男性性反转综合征患者经MLPA基因筛查均发现存在单拷贝SRY基因,FISH技术鉴定存在2条X染色体,SRY基因易位于其中1条X染色体的短臂上;女性假两性畸形患者的母亲染色体核型为46,XX,MLPA基因筛查发现其CYP21A2-ex03杂合性缺失,CYP21A1P-ex02杂合性重复;父亲染色体核型为46,XY,MLPA基因筛查发现CYP21A2-ex01和CYP21A2-ex03杂合性缺失,CYP21A1P-ex02和CYP21A1-ex10杂合性重复。结论: 性别决定是以SRY基因为主导、其它多个基因参与的过程,对性发育异常患者进行MLPA基因筛查有利于明确病因。     

Partial disomy of Xp and the presence of SRY in a phenotypic female.

We present a study of a mentally retarded and mildly dysmorphic female in whom initial cytogenetic studies identified the karyotype 46,X, + mar. Further characterisation of the structurally abnormal chromosome by fluorescence in situ hybridisation (FISH) showed that it is composed of both X and Y chromosome material with a centromere originating from the Y chromosome. The presence of the DMD gene and the absence of the XIST gene was shown by FISH using locus specific probes. The Y segment included the SRY and ZFY genes. Based on these findings, the karyotype was defined as 46, X,der(Y)t(X;Y) (p21.1;q11). This case illustrates male to female sex reversal owing to a partial duplication of the short arm of the X chromosome in the presence of SRY.     

A rare case of 46, XX SRY-negative male with a ∼74-kb duplication in a region upstream of SOX9

The 46, XX male disorder of sex development (DSD) is a rare genetic condition. Here, we report the case of a 46, XX SRY-negative male with complete masculinization. The coding region and exon/intron boundaries of the DAX1, SOX9 and RSPO1 genes were sequenced, and no mutations were detected. Using whole genome array analysis and real-time PCR, we identified a ∼74-kb duplication in a region ∼510–584 kb upstream of SOX9 (chr17:69,533,305–69,606,825, hg19). Combined with the results of previous studies, the minimum critical region associated with gonadal development is a 67-kb region located 584–517 kb upstream of SOX9. The amplification of this region might lead to SOX9 overexpression, causing female-to-male sex reversal. Gonadal-specific enhancers in the region upstream of SOX9 may activate the SOX9 expression through long-range regulation, thus triggering testicular differentiation.     

An autosomal or X linked mutation results in true hermaphrodites and 46,XX males in the same family.

It is now well established that the differentiation of the primitive gonad into the testis during early human embryonic development depends on the presence of the SRY gene. However, the existence of total or partial sex reversal in 46,XX males with genetic mutations not linked to the Y chromosome suggests that several autosomal genes acting in association with SRY may contribute to normal development of the male phenotype. We report a family in which four related 46,XX subjects with no evidence of Y chromosome DNA sequences underwent variable degrees of male sexual differentiation. One 46,XX male had apparently normal male external genitalia whereas his brother and two cousins had various degrees of sexual ambiguity and were found to be 46,XX true hermaphrodites. The presence of male sexual development in genetic females with transmission through normal male and female parents indicates that the critical genetic defect is most likely to be an autosomal dominant mutation, the different phenotypic effects arising from variable penetrance. Other autosomal loci have been implicated in male sexual development but the genetic mechanisms involved are unknown. In this family there may be an "activating" mutation which mimics the initiating role of the SRY gene in 46,XX subjects.     

Testis development in the absence of SRY: chromosomal rearrangements at SOX9 and SOX3

Duplications in the ~2 Mb desert region upstream of SOX9 at 17q24.3 may result in familial 46,XX disorders of sex development (DSD) without any effects on the XY background. A balanced translocation with its breakpoint falling within the same region has also been described in one XX DSD subject. We analyzed, by conventional and molecular cytogenetics, 19 novel SRY-negative unrelated 46,XX subjects both familial and sporadic, with isolated DSD. One of them had a de novo reciprocal t(11;17) translocation. Two cases carried partially overlapping 17q24.3 duplications ~500 kb upstream of SOX9, both inherited from their normal fathers. Breakpoints cloning showed that both duplications were in tandem, whereas the 17q in the reciprocal translocation was broken at ~800 kb upstream of SOX9, which is not only close to a previously described 46,XX DSD translocation, but also to translocations without any effects on the gonadal development. A further XX male, ascertained because of intellectual disability, carried a de novo cryptic duplication at Xq27.1, involving SOX3. CNVs involving SOX3 or its flanking regions have been reported in four XX DSD subjects. Collectively in our cohort of 19 novel cases of SRY-negative 46,XX DSD, the duplications upstream of SOX9 account for ~10.5% of the cases, and are responsible for the disease phenotype, even when inherited from a normal father. Translocations interrupting this region may also affect the gonadal development, possibly depending on the chromatin context of the recipient chromosome. SOX3 duplications may substitute SRY in some XX subjects.     

46,XX男性和46,XX/45,X病例分析

目的探讨SRY基因在性分化和发育中的作用.方法细胞遗传学核型分析以及PCR技术检测外周血SYR基因.结果病例1的核型为46,XX,SRY( ).诊断为46,XX男性性反转综合征.病例2的核型为46,XX/45,X,SRY(-).诊断为Turner嵌合型.结论SRY基因检测比Y染色体更能预示睾丸组织的存在,是诊断性别发育异常患者的重要手段.性腺的病理取决于性腺组织的染色体核型和SRY基因.除SRY基因外,还存在多个参与性别决定和分化的基因,性分化异常表现现高度遗传异常性.     

Atypical breakpoint in a t(6;17) translocation case of acampomelic campomelic dysplasia

Campomelic dysplasia (CD) is a skeletal dysplasia characterized by Pierre Robin sequence (PRS), shortened and bowed long bones, airway instability, and the potential for sex reversal. A subtype of CD, acampomelic CD (ACD), is seen in approximately 10% of cases and preserves long bone straightness. Both syndromes are caused by alterations in SOX9, with translocations and missense mutations being overrepresented in ACD cases. We report a term infant with PRS, severe cervical spine abnormalities, eleven rib pairs, hypoplastic scapulae, and female genitalia. Chromosome analysis identified a 46,XY,t(6;17)(q25;q24) karyotype. FISH analysis with a series of BAC probes localized the translocation breakpoints to 6q27 and a region at 17q24.3 in the range of 459–379 kb upstream of SOX9. Therefore, this case extends the region classified as the proximal breakpoint cluster. In addition, the comorbidity of acampomelia, complete sex reversal, and severe spinal anomalies in our patient underscores the variability in the level of malformation in the CD/ACD family of disorders.     

Velocardiofacial syndrome in an unexplained XX male

We report the unusual finding of velocardiofacial syndrome (VCF) in an unexplained 46,XX male. A microdeletion of 22q11.2 was confirmed by fluorescence in situ hybridization (FISH) analysis. Routine G-banded chromosome analysis revealed an XX sex chromosome constitution. FISH was performed using the SRY probe and failed to detect hybridization. The sex chromosome status of the patient was further investigated by PCR testing to screen for the presence of 24 distinct loci spanning the Y chromosome. PCR screening failed to detect any apparent Y chromosome material.