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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2007-0505
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The Journal of Clinical Endocrinology & Metabolism Vol. 92, No. 8 3305-3313
Copyright © 2007 by The Endocrine Society

Isolated 46,XY Gonadal Dysgenesis in Two Sisters Caused by a Xp21.2 Interstitial Duplication Containing the DAX1 Gene

Michela Barbaro1, Mikael Oscarson1, Jacqueline Schoumans, Johan Staaf, Sten A. Ivarsson and Anna Wedell

Department of Molecular Medicine and Surgery (M.B., M.O., J.Sc., A.W.), Karolinska Institutet, 17176 Stockholm, Sweden; Department of Oncology (J.St.), Lund University, SE-22184 Lund, Sweden; and Department of Paediatrics (S.A.I.), Malmö University Hospital, Lund University, SE-20502 Malmö, Sweden

Address all correspondence and requests for reprints to: Michela Barbaro, Karolinska Institutet, Department of Molecular Medicine and Surgery, CMM L8:02, Karolinska University Hospital, 17176 Stockholm, Sweden. E-mail: Michela.barbaro{at}ki.se.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Context: Testis development is a tightly regulated process that requires an efficient and coordinated spatiotemporal action of many factors, and it has been shown that several genes involved in gonadal development exert a dosage effect. Chromosomal imbalances have been reported in several patients presenting with gonadal dysgenesis as part of severe dysmorphic phenotypes.

Results: We screened for submicroscopic DNA copy number variations in two sisters with an apparent normal 46,XY karyotype and female external genitalia due to gonadal dysgenesis, and in which mutations in known candidate genes had been excluded. By high-resolution tiling bacterial artificial chromosome array comparative genome hybridization, a submicroscopic duplication at Xp21.2 containing DAX1 (NR0B1) was identified. Using fluorescence in situ hybridization, multiple ligation probe amplification, and PCR, the rearrangement was further characterized. This revealed a 637-kb tandem duplication that in addition to DAX1 includes the four MAGEB genes, the hypothetical gene CXorf21, GK, and part of the MAP3K7IP3 gene. Sequencing and analysis of the breakpoint boundaries and duplication junction suggest that the duplication originated through a coupled homologous and nonhomologous recombination process.

Conclusions: This represents the first duplication on Xp21.2 identified in patients with isolated gonadal dysgenesis because all previously described XY subjects with Xp21 duplications presented with gonadal dysgenesis as part of a more complex phenotype, including mental retardation and/or malformations. Thus, our data support DAX1 as a dosage sensitive gene responsible for gonadal dysgenesis and highlight the importance of considering DAX1 locus duplications in the evaluation of all cases of 46,XY gonadal dysgenesis.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
GONAD FORMATION AND sex determination are the first steps in sex development that subsequently influence the differentiation of the bipotential internal and external genitalia into the typical male or female pattern. The gonad forms as a bipotential structure during early embryogenesis and differentiates into an ovary in the presence of a 46,XX karyotype or a testis in case of a 46,XY karyotype with an intact SRY gene. The subsequent testicular production of testosterone and anti-Müllerian hormone (AMH) leads to the development of male internal and external genitalia. In the absence of androgens, the bipotential genitalia will develop toward a female pattern (1).

In case of gonadal dysgenesis in 46,XY subjects, testis development is impaired, and the absent or reduced production of testosterone and AMH will lead to the formation of female external genitalia or ambiguous genitalia. Testis development is a tightly regulated process that requires a coordinated spatiotemporal action of many factors, such as SF-1, WT1, DHH, SOX9, and SRY. Mutations in these genes can result in 46,XY gonadal dysgenesis, isolated or associated with extragonadal abnormalities depending on the gene. In 46,XY patients with isolated gonadal dysgenesis, the first candidate gene that is evaluated is usually SRY because mutations in the other genes typically cause defects in additional organ systems. However, SRY mutations are identified in only 15% of the cases.

In patients with complex and/or large chromosomal rearrangements, abnormal genital phenotypes are sometimes seen as part of a complex syndrome, suggesting the presence of additional genes with a dosage effect involved in gonadal development. Examples include 9p deletion (2), 7p duplication (3), 1p duplication containing WNT4 (4), and duplications, including Xp21 (5). In most reports the rearrangements involve large chromosome segments that were visible by conventional karyotyping, proving hard to pinpoint which genes are responsible for each specific feature included in the syndrome. Because many genes involved in gonadal development are dosage sensitive, we used array based comparative genomic hybridization (CGH) to investigate two sisters with isolated gonadal dysgenesis, an apparently normal 46,XY karyotype, and without mutations in the known candidate genes.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Patient description

We studied two Iranian sisters who moved to Sweden in 1988. Apart from gonadal dysgenesis, both sisters were healthy with normal psychomotor development and have studied at the university level. Informed consent was obtained.

Sister 1 was born in 1981 and presented with amenorrhea at 15 yr of age. Pubertal stage was Tanner B4, PH3–4. There was no clitoromegaly or fusion of labia majora. Thyroid values were normal, prolactin was 3.2 µg/liter (normal value 2.0–20.0), SHBG 3.2 mg/liter (1.6–7.0), dehydroepiandrosterone 3.7 µmol/liter (1.0–12.0), androstenedione 2.9 nmol/liter (1.0–8.0), testosterone 3.9 nmol/liter (children 0.3–3.0; women 0.3–3.0; postmenopausal women 0.3–2.5), estradiol 121 pmol/liter (children <100; early follicular phase 100–250; preovulation phase 700-2000; luteal phase 150–800; postmenopausal <100), and dihydrotestosterone 0.17 nmol/liter (0.10–0.79). During the LHRH test, FSH increased from 108 to 145 IU/liter (prepubertal <4.0; fertile age 1.0–13.0; midcycle peak 4.0–30.0; menopausal 22.0–190.0), and LH from 38 to 120 IU/liter. Chromosomal analysis revealed a 46,XY karyotype. Sonographic examination showed a uterus of 12 ml, and no ovaries could be identified. Gonadectomy was performed, and chromosomal analysis of both gonads revealed a 46,XY karyotype. Histological examination revealed normal fallopian tubes and streak gonads bilaterally. The left gonad contained a gonadoblastoma.

The patient’s younger sister also had a 46,XY karyotype. She was born in 1986 and was prepubertal; at 10 yr and 10 months, she presented with estradiol less than 50 pmol/liter, testosterone 1.1 nmol/liter, and dihydrotestosterone 0.31 nmol/liter. There was no virilization of external genitalia. Sonography indicated a uterine volume of 1 ml; no ovaries could be identified. Gonadectomy was performed, and both gonads had a 46,XY karyotype. Histological examination revealed normal fallopian tubes and streak gonads with gonadoblastoma bilaterally.

The coding region and exon/intron boundaries of the SRY, SF1, WT1, SOX9, and DHH genes were sequenced without detection of any mutations.

Array CGH experiments

High-resolution CGH arrays with complete genome coverage containing 33,370 bacterial artificial chromosome (BAC) clones produced by the Swegene DNA Microarray Resource Centre, Department of Oncology, Lund University, Sweden (http://swegene.onk.lu.se), were used as previously described (6). A male reference DNA was used (Promega, Madison, WI), and color-reverse experiments were performed. Identification of individual spots on scanned arrays was performed with Gene Pix Pro 6.0 (Axon Instruments, Molecular Devices Corp., Sunnyvale, CA), and the quantified data matrix was loaded into BioArray Software Environment (7). To reduce noise, data from the two duplicates were merged. The automatic breakpoint identification tools CGH plotter (8) and Gain and Loss Analysis of DNA (GLAD) (9) were applied after smoothing with a sliding window over three clones to identify gains and losses throughout the genome using a log2 (ratio) threshold of ±0.25.

Fluorescence in situ hybridization (FISH)

To investigate the genomic location of the extra copy of the DAX1 locus, FISH analysis was performed using the BAC clone RP11–662D2 (Children’s Hospital Oakland Research Institute BACPAC Resource Center) on metaphase nuclei prepared from Epstein-Barr virus-immortalized lymphocytes. For identification of X and Y chromosomes, a mix of two cosmid clones (LLNOYC03M15D10 and LLNOYC03M34F5) covering the SHOX gene were used. Probes were labeled by random priming with the BioPrime Array CGH Genomic Labeling Module kit (Invitrogen, Carlsbad, CA) and Cy5-dUTPs (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK) or fluorescein-12-dUTP (Roche Diagnostics GmbH, Mannheim, Germany). Slides were counterstained with 4',6-diamidino-2-phenylindole, analyzed on a Zeiss Axioplan 2 epifluorescence microscope (Carl Zeiss, Göttingen, Germany), and images were captured using a cooled charge-coupled device camera (Sensys Photometrics, München, Germany) and the SmartCapture 2 software (DigitalScientific Ltd., Cambridge, UK).

Multiplex ligation-dependent probe amplification (MLPA)

Synthetic 5' and 3' half-probes targeting unique sequences in the Xp21 locus either within genes or in intergenic sequences (IGSs) as well as the control genes CLDN16, ALB, and RB1 were designed according to Stern et al. (10) (Table 1Go). The probe mixes (Table 2Go) contained each half-probe at a final concentration of 4 nM. The EK1 reagent kit was obtained from MRC Holland (Amsterdam, The Netherlands). After 35 cycles of PCR amplification, PCR products were separated using an ABI 3100 genetic analyzer (Applied Biosystems, Warrington, UK). Trace data were analyzed using the GeneMapper v3.7 software (Applied Biosystems). The peak shape corresponding to the albumin probe showed rather high variability across the samples and was, therefore, excluded from the analysis. For each sample the peak areas corresponding to each probe were first normalized to the average of the peak areas of the two remaining control probes CLDN16 and RB1, and then normalized to the average peak area in five male controls.


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  		TABLE 1. Probes used for MLPA

 

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  		TABLE 2. Confirmation and fine mapping of the Xp21.2 duplication using MLPA mix A and B

 
PCR amplification and sequencing of the duplication junction

Forward primers were designed in the telomeric breakpoint region (located between the MLPA probes MAGEB2 5' and MAGEB2 ex2), and reverse primers were designed in the centromeric breakpoint region [between MAP3K7IP3 ex8 and MAP3K7IP3 ex7, exon numbered according to Jin et al. (11)]. The DyNAzyme EXT PCR Kit (Finnzyme, Espoo, Finland) was used to amplify the junction breakpoint using various combinations of these primers. Successful amplicons were directly sequenced. The primers 5'-CCCTCTGGGCCATCCTCTTTAGAA-5' and 5'-GAAGAAATTGAAGGGGTGCCTCAAC-3' were used together with internal control primers designed within the AR gene (5'-CGACTACCGCATCATCACAG-3' and 5'-CAGCTGAGTCATCCTCGTCC-3') to analyze the patients’ mother and four control DNA.

Bioinformatics and sequence analysis

Reference genomic sequence was obtained from the University of California Santa Cruz Genome Browser using the March 2006 human assembly (http://www.genome.ucsc.edu). A multiple sequence alignment was generated using ClustalW (http://www.ebi.ac.uk/clustalw/) (12), and sequence similarities between the telomeric and centromeric breakpoints were evaluated using the Basic Local Alignment Search Tool (http://www.ncbi.nlm.nih.gov/blast/) (13). Breakpoint regions were analyzed using RepeatMasker (http://www.repeatmasker.org/), for palindrome sequences using Palindrome (http://bioweb.pasteur.fr/seqanal/interfaces/palindrome.html) and for DNA sequence motifs that are known to be associated with chromosomal rearrangements using the DNA Pattern Find algorithm (http://bioinformatics.org/sms/dna_pattern.html) (14). Pipmaker (http://pipmaker.bx.psu.edu/pipmaker/) (15) was used to investigate a 1.3-Mb region around the duplication for low-copy repeat (LCR) sequences.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
Identification of a chromosome duplication using array CGH

Genomic DNA from sister 1 was analyzed using array CGH. Eight gains/losses, including three or more clones, were identified by both the CGH plotter and GLAD breakpoint identification softwares, and four additional gains/losses were identified using GLAD. Eleven of the copy number variations reported in the database of genomic variants (http://projects.tcag.ca/variation/) and were, therefore, excluded. The only remaining aberration was an approximate 700-kb duplication at Xp21.2 (Fig. 1Go) that contains the DAX1 (NR0B1) gene.


Figure 1
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  		FIG. 1. Array CGH results. A, Array CGH plot for the X chromosome. The X-axis corresponds to the genomic position in megabases of DNA, and the Y-axis shows the Log2 ratio of signal intensity (sample vs. reference DNA). Clones are represented as dots. Green dots correspond to duplicated clones in the Xp21.2 region. Along the X-axis lies a schematic representation of the X chromosome. Dashed lines indicate the Log2 ratio limits of ±0.25. B, Representation from the University of California Santa Cruz database of the locus involved in the duplication. Tiled BAC clones present on the array are represented as blue lines, and duplicated clones are shaded with dark blue lines.

 
Analysis of the duplication using FISH

FISH analysis was performed to establish the genomic location of the extra copy of the duplication. On metaphases from both sisters, only one signal was seen for the clone RP11–662D2 covering the DAX1 gene, and the intensity was stronger compared with the normal male and female control metaphases (Fig. 2Go), indicating an interstitial duplication on Xp21.


Figure 2
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  		FIG. 2. FISH analysis. Metaphase nuclei of sister 1 and a normal male and female. Arrows indicate the pink signal for the clone RP11–662D2 containing DAX1, and the green signal for the cosmids LLNOYC03M15D10 and LLNOYC03M34F5 covering the SHOX gene.

 
Fine mapping of breakpoints and sequencing of the duplication junction

MLPA probe mix A (Table 2Go) containing short synthetic oligonucleotides designed to hybridize within and outside the duplicated region was used not only to confirm the duplication but also to narrow down the breakpoint regions (Fig. 3AGo). This showed that the telomeric breakpoint was located within the MAGEB locus, and the centromeric breakpoint was close to the MAP3K7IP3 gene. The MLPA probe mix B (Table 2Go) was used to narrow down further the breakpoint locations, which showed that the breakpoints were located in intron 1 of the MAGEB2 gene and in intron 7 of the MAP3K7IP3 gene, respectively. Using primers located in these regions, we could amplify across the duplication junction using PCR (Fig. 3BGo). The junction sequence was compared with the genomic reference sequence (Fig. 3CGo), which confirmed the location of the breakpoints 637-kb apart and was consistent with a tandem head-to-tail duplication [g.chrX:30,145,030_30,781,736dup(NCBI Build 36.1)]. An identical structure was observed in both sisters, and PCR amplification also showed that the healthy mother is a carrier (Fig. 4Go). The duplication junction results in a fusion of the MAGEB2 and MAP3K7IP3 genes, with the two genes being encoded on opposite strands. The MAP3K7IP3 gene is disrupted at the junction, and is missing both the promoter region and the N terminally encoded CUE domain (16). Although the complete open reading frame of the MAGEB2 gene was included in the duplicated region, most likely, no gene expression occurs from this gene copy because the regulatory region and the first noncoding exon are missing.


Figure 3
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  		FIG. 3. Xp21.2 duplication fine mapping and analysis of the duplication junction sequence. A, Schematic representation of the MLPA analysis. Vertical red lines indicate the position of the probe pairs. In the first line, the probes in mix A for the first MLPA reaction are shown, and in the second line, the probes in mix B for further fine mapping of the breakpoints are shown. Vertical black arrows indicate the regions of the breakpoints. B, Representation of the tandem head-to-tail interstitial duplication. Black arrows indicate location and orientation of the primers used to amplify and sequence the breakpoint junction. C, The telomeric and centromeric breakpoint sequences were aligned with the duplication junction sequence. A CLUSTALW multiple sequence alignment of 25-bp proximal and distal sequence flanking the junction is shown. The 2-bp microhomology at the junction is boxed. DNA sequence motifs within 10 bp from the junction are shown as follows: topoisomerase I consensus cleavage sites are underlined (some of these are also complementary to DNA polymerase {alpha}-pause site core sequences), arrows indicate Ig heavy chain class switch repeats, the double arrowhead indicates a DNA polymerase {alpha}/ß frameshift hotspot, a 9-bp tract of polypyrimidines is indicated with a gray box, and palindrome sequences are indicated with dashed lines.

 

Figure 4
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  		FIG. 4. Maternal DNA analysis. Results of the PCR across the breakpoint. The band corresponding to the duplication junction (DJ) is present only in the two sisters (S1, S2) and their mother (M), while the band corresponding to a fragment of the AR gene, used as internal control of the PCR, is present also in the healthy controls (C1, C2, C3, and C4). L, 100-bp DNA Ladder (In Vitro Sweden AB, Stockholm, Sweden).

 
During the duplication process, either the telomeric or centromeric boundary between the single-copy and duplicated sequence must have been generated. These boundaries were sequenced, but no differences compared with the genomic reference sequence were observed, suggesting that an error-free homologous recombination mechanism was responsible for the formation of one end of the duplication.

Bioinformatics analysis of the breakpoints

Alignment of the junction sequence and the genomic reference sequences for the telomeric and centromeric breakpoint regions revealed minimal sequence identity between the two breakpoint regions, and only a microhomology of 2 bp was observed (Fig. 3CGo). However, analysis of 2-kb sequence flanking the breakpoint regions did identify similarity between an approximate 280-bp region containing an AluSx element that embedded the centromeric breakpoint and an AluY element 90-bp upstream of the telomeric breakpoint.

We also identified a 9-bp palindrome flanking the telomeric breakpoint and a 9-bp polypyrimidine tract adjacent to the centromeric breakpoint (Fig. 3CGo). Finally, the breakpoint regions were analyzed for 37 sequence motifs known to be associated with chromosomal rearrangements, and potentially involved in DNA cleavage and recombination (14). This revealed several topoisomerase I consensus cleavage sites flanking both breakpoints, as well as consensus sites for Ig heavy chain class switch repeats and a DNA polymerase {alpha}/ß frameshift hotspot close to the telomeric breakpoint. Using the Pipmaker software, a 1.3-Mb sequence, including the duplicated region, was analyzed for LCRs that potentially could be involved in the duplication event. Apart from the repeat structure corresponding to the MAGEB locus region, no LCRs could, however, be detected.


   Discussion
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 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
Duplications of chromosomal regions containing Xp21 are known to be associated with XY sex reversal; 17 of the 18 patients reported so far (17, 18) carried duplications or translocations that were detectable by conventional karyotyping, and all patients presented sex reversal as part of a more complex phenotype that includes dysmorphic features and/or mental retardation. By comparing patients with different chromosomal rearrangements on Xp, Bardoni et al. (5) identified a 160-kb minimal common region denoted dosage sensitive sex reversal that, if duplicated, causes sex reversal. This region contains the MAGEB genes and the DAX1 gene (19).

DAX1 is the candidate for sex reversal for many reasons. Its expression during embryonic development in the mouse is compatible with a role both in sex determination and in adrenal and hypothalamic function (20). XY mice transgenic for Dax1 show delayed testis development and sex reversal if the transgene is tested against weak alleles of Sry (21). The importance of DAX1 overexpression for sex reversal is also supported by the fact that XY patients with 1p duplications, including WNT4, show an abnormal gonadal phenotype; WNT4 is a signaling molecule that up-regulates DAX1 expression (4). Furthermore, several functional properties of DAX1 are consistent with its ability to inhibit gonadal development if present at a higher dose. DAX1 belongs to the nuclear receptor (NR) superfamily but has an atypical domain structure compared with other NRs, and no ligand has been identified. DAX1 can repress transcription by binding to DNA hairpin structures (e.g. in the promoter of steroidogenic acute regulator protein gene) (22). Notably, DAX1 has also been reported to inhibit steroidogenic factor 1 (SF1) (NR5A1), causing reduction of steroidogenic enzymes and AMH expression. It has been shown that SF1 haploinsufficiency causes gonadal deficiency, suggesting that DAX1 overexpression might cause gonadal dysgenesis through inhibition of SF1-mediated transcription. DAX1 has also inhibited several other NRs such as the androgen receptor, progesterone receptor, estrogen receptors {alpha} and ß, liver receptor homolog-1, and nur77 (23, 24). Furthermore, DAX1 has been shown to bind to RNA in polyribosomes or polyadenylated RNA, and it has been suggested that DAX1 is a shuttling RNA binding protein with a posttranscriptional regulatory role (25). Thus, DAX1 seems not only to act as a transcriptional repressor of steroidogenesis but also to have a broader functional role during the embryonic development and adult function of the hypothalamic-pituitary-adrenal-gonadal axis (26). Although the aforementioned data support DAX1 as the gene responsible for sex reversal, a direct proof in patients is still missing because there so far are no reports of a single DAX1 duplication in 46,XY patients with isolated gonadal dysgenesis.

In the two patients described in this report, a 637-kb duplication was identified that in addition to DAX1 includes the four MAGEB genes, CXorf21, GK, and part of the MAP3K7IP3 gene. The duplication starts 1.6-kb upstream of the translation initiation site of MAGEB2, but because the first noncoding exon as well as the regulatory region is missing this extra MAGEB2, copy is probably not transcribed. However, the transcription from the extra copies of the other three MAGEB genes might be functional, resulting in overexpression of these genes. CXorf21 encodes a hypothetical protein with a short-chain dehydrogenases/reductases domain, and GK encodes the glycerol kinase that is a key enzyme in glycerol uptake and metabolism. The product of the MAP3K7IP3 gene interacts with TGF-ß-activated protein kinase 1, which is mediating intracellular actions of proinflammatory cytokines and responses to bacterial lipopolysaccharide through the nuclear factor {kappa}B signal transduction pathway (27). This gene is only partially duplicated, but a functional copy is still present at the centromeric duplication boundary. Interestingly, deletions and mutations in the IL1RAPL1 gene (IL 1 receptor accessory protein-like 1) have been identified in patients with mental retardation (28), and this gene is located just distal to the duplication.

The duplication described in this study is the first duplication containing DAX1 identified in patients presenting sex reversal due to gonadal dysgenesis without any other anomalies. Notably, the size of the duplication is too small to be identified by conventional karyotyping. A duplication of less than 1 Mb, including DAX1, was also described by Bardoni et al. (5) after screening patients with 46,XY sex reversal by Southern blot. A direct comparison of the duplicated regions in the patients cannot be made, but the more severe phenotype in the patient described by Bardoni et al. (5), which includes facial anomalies and mental retardation in addition to gonadal dysgenesis, suggests that more genes are involved, such as IL1RAPL1 or GK. The healthy mother was shown to be the carrier of the duplication. Therefore, an extra copy of DAX1 and the surrounding genes does not seem to affect normal female gonadal development. This is consistent with earlier studies (5, 29, 30, 31, 32, 33) in which healthy mothers were reported to be carriers of duplications and translocations involving Xp21.

When a duplication occurs, there are two aspects to consider to identify the genes responsible for the phenotype: the specific genes within the duplicated region because they could exert a dosage effect when present in extra copy, and whether the duplication has caused disruption or a loss of genes that have an effect as a result of haploinsufficiency. Furthermore, the number of genes responsible for the phenotype is not necessarily dependent on the size of the duplication. In fact, no correlation was observed between the size of the duplicated fragments and the severity of the phenotype in patients with Xp21 duplications, supporting the possibility of gene interruption at the breakpoints. In most cases the breakpoints have not been characterized, and there is, therefore, limited knowledge about how many genes with a dosage effect that could influence the patient phenotype were involved. In our cases no gene had been disrupted at the breakpoint boundaries, and the genes present in an extra dose were the MAGEB genes, DAX1, CXorf21, and GK. In all patients reported so far with duplications on Xp21, the MAGEB genes are included in the duplicated region together with DAX1, so because of their specific testis expression and unclear function, their possible involvement in the phenotype cannot be excluded.

The mechanisms involved in, and DNA sequences predisposing for, submicroscopic tandem head-to-tail duplications are not well understood. In a recent report (34), the junction sequences of segmental duplications on Xq22.2, including the PLP1 gene, were analyzed in patients with Pelizaeus-Merzbacher disease. Each duplication event was unique, and no common sequence motifs involved in the duplications could be identified. However, the duplications were consistent with a model (35, 36) in which the duplication event is initiated by a double-stranded DNA break (DSB), and is followed by homologous strand invasion of a sister chromatid by a single strand of one end, from which DNA synthesis is initiated and extended downstream to the duplicated sequence. The repair is completed through nonhomologous end joining of the newly synthesized strand with the other end of the DSB.

Although it is not possible to decipher the exact molecular mechanism involved in the duplication event, we identified several DNA sequence motifs and structures that can predispose to DSB or facilitate chromosomal instability and/or DNA recombination (Fig. 3CGo). Ig heavy chain class switch repeats and DNA polymerase {alpha}/ß frameshift hotspot were identified at the telomeric breakpoint. Both of these motifs have been shown to be overrepresented at translocation breakpoints (14). Multiple topoisomerase I consensus cleavage sites overlapping or located close to both breakpoints were also identified, but because of their frequent occurrence throughout the genome, the significance of this finding is unclear.

The centromeric breakpoint is flanked by a 9-bp long polypyrimidine tract, a sequence that is overrepresented at translocation breakpoints (14). Polypyrimidine sequences can adopt the triple helical H-form of DNA (37), which is partially single stranded and susceptible to a nuclease attack that can facilitate recombination (38). The telomeric breakpoint is flanked by a palindrome sequence, which can predispose for chromosomal translocations (39). Although the palindrome sequence is short, it is interesting that the breakpoint occurred at the tip of the putative hairpin loop. Both breakpoints were either overlapped or flanked by evolutionary younger Alu repeat sequences (AluSx and AluY), which usually are enriched near or within duplication junctions (40). Interestingly, the telomeric breakpoint is also located within the rapidly evolving MAGEB locus that might contain up till now unidentified sequence motifs promoting DNA recombination.

Neither the telomeric nor the centromeric breakpoint boundary sequences contained any deviation from the genomic reference sequences. Therefore, it is not possible to determine which of the boundaries was formed during the duplication event, but this suggests that an error-free homologous repair process occurred at one end of the DSB. The presence of only 2-bp sequence overlap at the duplication junction suggests that the other end of the DSB was repaired through nonhomologous end joining. Together, we believe that the duplication in our patients has occurred through a coupled homologous and nonhomologous recombination process.

In conclusion, we describe a 637-kb interstitial duplication on Xp21.2 in two sisters with isolated 46,XY gonadal dysgenesis. Our data support DAX1 duplication as the genetic cause of gonadal dysgenesis, even if a role for the MAGEB genes cannot be completely excluded. This finding stresses the importance of using methods that can detect submicroscopic duplications of the region surrounding DAX1 in the evaluation of patients with isolated 46,XY gonadal dysgenesis and not only when gonadal dysgenesis is part of a complex dysmorphic phenotype. Even if this is only the second submicroscopic DAX1 duplication reported, such events could be more frequent but have escaped detection due to the methods that have been used so far and the selection of the target patients. The lack of phenotype in the carrier mother also suggests that such duplications are more commonly spread and explain gonadal dysgenesis in additional 46,XY individuals. The development of new techniques for detection of submicroscopic copy number variations on a genome-wide basis nowadays makes it easier to identify such duplications or deletions.


   Footnotes
 
This work was supported by the Stiftelsen Frimurare Barnhuset (to M.B.), the Fredrik and Ingrid Thuring Foundation, the Magn. Bergvall Foundation, and the Swedish Society of Medicine (to M.O.), the Swedish Research Council (Grant 12198), the Novo Nordisk Foundation, The Centre of Gender Related Medicine, Karolinska Institutet, and the Stockholm County Council (to A.W.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online May 15, 2007

1 M.B. and M.O. contributed equally to this work. Back

Abbreviations: AMH, Anti-Müllerian hormone; BAC, bacterial artificial chromosome; CGH, comparative genomic hybridization; DSB, double-stranded DNA break; FISH, fluorescence in situ hybridization; GLAD, Gain and Loss Analysis of DNA; IGS, intergenic sequence; LCR, low-copy repeat; MLPA, multiplex ligation-dependent probe amplification; NR, nuclear receptor; SF1, steroidogenic factor 1.

Received March 5, 2007.

Accepted May 7, 2007.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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