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Copy number variation in patients with disorders of sex development due to 46,XY gonadal dysgenesis.

White S, Ohnesorg T, Notini A, Roeszler K, Hewitt J, Daggag H, Smith C, Turbitt E, Gustin S, van den Bergen J, Miles D, Western P, Arboleda V, Schumacher V, Gordon L, Bell K, Bengtsson H, Speed T, Hutson J, Warne G, Harley V, Koopman P, Vilain E, Sinclair A - PLoS ONE (2011)

Bottom Line: Functional analysis of potential SRY binding sites within this deleted region identified five putative enhancers, suggesting that sequences additional to the known SRY-binding TES enhancer influence human testis-specific SOX9 expression.Thirdly, we identified a small deletion immediately downstream of GATA4, supporting a role for GATA4 in gonad development in humans.These CNV analyses give new insights into the pathways involved in human gonad development and dysfunction, and suggest that rearrangements of non-coding sequences disturbing gene regulation may account for significant proportion of DSD cases.

View Article: PubMed Central - PubMed

Affiliation: Murdoch Childrens Research Institute, Royal Children's Hospital, Melbourne, Victoria, Australia.

ABSTRACT
Disorders of sex development (DSD), ranging in severity from mild genital abnormalities to complete sex reversal, represent a major concern for patients and their families. DSD are often due to disruption of the genetic programs that regulate gonad development. Although some genes have been identified in these developmental pathways, the causative mutations have not been identified in more than 50% 46,XY DSD cases. We used the Affymetrix Genome-Wide Human SNP Array 6.0 to analyse copy number variation in 23 individuals with unexplained 46,XY DSD due to gonadal dysgenesis (GD). Here we describe three discrete changes in copy number that are the likely cause of the GD. Firstly, we identified a large duplication on the X chromosome that included DAX1 (NR0B1). Secondly, we identified a rearrangement that appears to affect a novel gonad-specific regulatory region in a known testis gene, SOX9. Surprisingly this patient lacked any signs of campomelic dysplasia, suggesting that the deletion affected expression of SOX9 only in the gonad. Functional analysis of potential SRY binding sites within this deleted region identified five putative enhancers, suggesting that sequences additional to the known SRY-binding TES enhancer influence human testis-specific SOX9 expression. Thirdly, we identified a small deletion immediately downstream of GATA4, supporting a role for GATA4 in gonad development in humans. These CNV analyses give new insights into the pathways involved in human gonad development and dysfunction, and suggest that rearrangements of non-coding sequences disturbing gene regulation may account for significant proportion of DSD cases.

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Deletion (1.193 Mb) on chromosome 17, upstream of SOX9 in a patient with 46,XY GD.a) The location and extent of the 1.193 Mb deletion on chromosome 17, upstream of SOX9 identified in case 10. The numbers at the top of the figure correspond to nucleotide position based on the March 2006 human reference sequence (hg18). Also shown are structural variants within this region that are listed in the database of genomic variants. (http://projects.tcag.ca/variation/). The numbered arrows indicate the positions of the seven potential gonad specific regulatory elements (enh1–7) that were cloned into reporter constructs. The position of the orthologous sequence corresponding to the mouse TESCO sequence is indicated by an asterisk (*). b). Reporter construct analysis of SOX9 regulatory regions. Effect of selected transcription factors on luciferase activity driven by putative gonad regulatory regions (enh1–7) inserted upstream of the minimal SOX9 promoter (sox prom). Results are given as relative activation of the reporter by the expression constructs (SF1, SOX9, SRY) compared with the empty vector (pcDNA3). Data represent mean values ±SEM obtained from at least four independent experiments. Statistical analysis was performed with a 2-tailed t-test. ** p<0.005; * p<0.05.
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pone-0017793-g002: Deletion (1.193 Mb) on chromosome 17, upstream of SOX9 in a patient with 46,XY GD.a) The location and extent of the 1.193 Mb deletion on chromosome 17, upstream of SOX9 identified in case 10. The numbers at the top of the figure correspond to nucleotide position based on the March 2006 human reference sequence (hg18). Also shown are structural variants within this region that are listed in the database of genomic variants. (http://projects.tcag.ca/variation/). The numbered arrows indicate the positions of the seven potential gonad specific regulatory elements (enh1–7) that were cloned into reporter constructs. The position of the orthologous sequence corresponding to the mouse TESCO sequence is indicated by an asterisk (*). b). Reporter construct analysis of SOX9 regulatory regions. Effect of selected transcription factors on luciferase activity driven by putative gonad regulatory regions (enh1–7) inserted upstream of the minimal SOX9 promoter (sox prom). Results are given as relative activation of the reporter by the expression constructs (SF1, SOX9, SRY) compared with the empty vector (pcDNA3). Data represent mean values ±SEM obtained from at least four independent experiments. Statistical analysis was performed with a 2-tailed t-test. ** p<0.005; * p<0.05.

Mentions: Secondly, a deletion of 1.193 Mb approximately 300 kb upstream of the SOX9 gene was identified in case 10 (Figure 1b). This 46,XY individual had been diagnosed with complete gonadal dysgenesis. The only other clinical features reported were a cleft palate and height at the 3rd centile. This is very unusual as rearrangements and mutations affecting SOX9 normally result in patients with campomelic dysplasia (deformity of chondrogenesis) and in some 46,XY cases, gondal dysgenesis. SOX9 is known to play a critical role in testis development, and other rearrangements upstream of this gene have been described in 46,XY DSD (or CGD) patients. The deletion described here did not cover the orthologous sequence of the recently identified TESCO element in mouse, which was proposed to act as an enhancer in regulating SOX9 expression in the gonad [14]. The orthologous human TESCO region was sequenced in the index patient, and no mutations or polymorphisms were identified (data not shown). The gonadal dysgenesis observed in this patient suggests that one or more testis-specific regulatory elements of SOX9 are contained within the 1.2 Mb deletion. In an attempt to identify such regions we performed a bioinformatic analysis of the 1.2 Mb region, searching for potential SRY/SOX9 binding sites that were conserved in human, mouse and rat. In total we identified seven such regions, hereafter referred to as enh1–7 (Figure 2a). Each of these regions was PCR amplified and cloned into a pGL4 vector containing the human SOX9 minimal promoter. Following transfection into a human Sertoli cell line, Addition of SF1, SRY or SOX9 led to a statistically higher level of enhancer activity for enh3, 4, 5, 6 and 7 for at least one of the transcription factors, with enh5 affected by all three (Figure 2b).


Copy number variation in patients with disorders of sex development due to 46,XY gonadal dysgenesis.

White S, Ohnesorg T, Notini A, Roeszler K, Hewitt J, Daggag H, Smith C, Turbitt E, Gustin S, van den Bergen J, Miles D, Western P, Arboleda V, Schumacher V, Gordon L, Bell K, Bengtsson H, Speed T, Hutson J, Warne G, Harley V, Koopman P, Vilain E, Sinclair A - PLoS ONE (2011)

Deletion (1.193 Mb) on chromosome 17, upstream of SOX9 in a patient with 46,XY GD.a) The location and extent of the 1.193 Mb deletion on chromosome 17, upstream of SOX9 identified in case 10. The numbers at the top of the figure correspond to nucleotide position based on the March 2006 human reference sequence (hg18). Also shown are structural variants within this region that are listed in the database of genomic variants. (http://projects.tcag.ca/variation/). The numbered arrows indicate the positions of the seven potential gonad specific regulatory elements (enh1–7) that were cloned into reporter constructs. The position of the orthologous sequence corresponding to the mouse TESCO sequence is indicated by an asterisk (*). b). Reporter construct analysis of SOX9 regulatory regions. Effect of selected transcription factors on luciferase activity driven by putative gonad regulatory regions (enh1–7) inserted upstream of the minimal SOX9 promoter (sox prom). Results are given as relative activation of the reporter by the expression constructs (SF1, SOX9, SRY) compared with the empty vector (pcDNA3). Data represent mean values ±SEM obtained from at least four independent experiments. Statistical analysis was performed with a 2-tailed t-test. ** p<0.005; * p<0.05.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3049794&req=5

pone-0017793-g002: Deletion (1.193 Mb) on chromosome 17, upstream of SOX9 in a patient with 46,XY GD.a) The location and extent of the 1.193 Mb deletion on chromosome 17, upstream of SOX9 identified in case 10. The numbers at the top of the figure correspond to nucleotide position based on the March 2006 human reference sequence (hg18). Also shown are structural variants within this region that are listed in the database of genomic variants. (http://projects.tcag.ca/variation/). The numbered arrows indicate the positions of the seven potential gonad specific regulatory elements (enh1–7) that were cloned into reporter constructs. The position of the orthologous sequence corresponding to the mouse TESCO sequence is indicated by an asterisk (*). b). Reporter construct analysis of SOX9 regulatory regions. Effect of selected transcription factors on luciferase activity driven by putative gonad regulatory regions (enh1–7) inserted upstream of the minimal SOX9 promoter (sox prom). Results are given as relative activation of the reporter by the expression constructs (SF1, SOX9, SRY) compared with the empty vector (pcDNA3). Data represent mean values ±SEM obtained from at least four independent experiments. Statistical analysis was performed with a 2-tailed t-test. ** p<0.005; * p<0.05.
Mentions: Secondly, a deletion of 1.193 Mb approximately 300 kb upstream of the SOX9 gene was identified in case 10 (Figure 1b). This 46,XY individual had been diagnosed with complete gonadal dysgenesis. The only other clinical features reported were a cleft palate and height at the 3rd centile. This is very unusual as rearrangements and mutations affecting SOX9 normally result in patients with campomelic dysplasia (deformity of chondrogenesis) and in some 46,XY cases, gondal dysgenesis. SOX9 is known to play a critical role in testis development, and other rearrangements upstream of this gene have been described in 46,XY DSD (or CGD) patients. The deletion described here did not cover the orthologous sequence of the recently identified TESCO element in mouse, which was proposed to act as an enhancer in regulating SOX9 expression in the gonad [14]. The orthologous human TESCO region was sequenced in the index patient, and no mutations or polymorphisms were identified (data not shown). The gonadal dysgenesis observed in this patient suggests that one or more testis-specific regulatory elements of SOX9 are contained within the 1.2 Mb deletion. In an attempt to identify such regions we performed a bioinformatic analysis of the 1.2 Mb region, searching for potential SRY/SOX9 binding sites that were conserved in human, mouse and rat. In total we identified seven such regions, hereafter referred to as enh1–7 (Figure 2a). Each of these regions was PCR amplified and cloned into a pGL4 vector containing the human SOX9 minimal promoter. Following transfection into a human Sertoli cell line, Addition of SF1, SRY or SOX9 led to a statistically higher level of enhancer activity for enh3, 4, 5, 6 and 7 for at least one of the transcription factors, with enh5 affected by all three (Figure 2b).

Bottom Line: Functional analysis of potential SRY binding sites within this deleted region identified five putative enhancers, suggesting that sequences additional to the known SRY-binding TES enhancer influence human testis-specific SOX9 expression.Thirdly, we identified a small deletion immediately downstream of GATA4, supporting a role for GATA4 in gonad development in humans.These CNV analyses give new insights into the pathways involved in human gonad development and dysfunction, and suggest that rearrangements of non-coding sequences disturbing gene regulation may account for significant proportion of DSD cases.

View Article: PubMed Central - PubMed

Affiliation: Murdoch Childrens Research Institute, Royal Children's Hospital, Melbourne, Victoria, Australia.

ABSTRACT
Disorders of sex development (DSD), ranging in severity from mild genital abnormalities to complete sex reversal, represent a major concern for patients and their families. DSD are often due to disruption of the genetic programs that regulate gonad development. Although some genes have been identified in these developmental pathways, the causative mutations have not been identified in more than 50% 46,XY DSD cases. We used the Affymetrix Genome-Wide Human SNP Array 6.0 to analyse copy number variation in 23 individuals with unexplained 46,XY DSD due to gonadal dysgenesis (GD). Here we describe three discrete changes in copy number that are the likely cause of the GD. Firstly, we identified a large duplication on the X chromosome that included DAX1 (NR0B1). Secondly, we identified a rearrangement that appears to affect a novel gonad-specific regulatory region in a known testis gene, SOX9. Surprisingly this patient lacked any signs of campomelic dysplasia, suggesting that the deletion affected expression of SOX9 only in the gonad. Functional analysis of potential SRY binding sites within this deleted region identified five putative enhancers, suggesting that sequences additional to the known SRY-binding TES enhancer influence human testis-specific SOX9 expression. Thirdly, we identified a small deletion immediately downstream of GATA4, supporting a role for GATA4 in gonad development in humans. These CNV analyses give new insights into the pathways involved in human gonad development and dysfunction, and suggest that rearrangements of non-coding sequences disturbing gene regulation may account for significant proportion of DSD cases.

Show MeSH
Related in: MedlinePlus