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Complete androgen insensitivity syndrome caused by a deep intronic pseudoexon-activating mutation in the androgen receptor gene

View Article: PubMed Central - PubMed

ABSTRACT

Mutations in the X-linked androgen receptor (AR) gene underlie complete androgen insensitivity syndrome (CAIS), the most common cause of 46,XY sex reversal. Molecular genetic diagnosis of CAIS, however, remains uncertain in patients who show normal coding region of AR. Here, we describe a novel mechanism of AR disruption leading to CAIS in two 46,XY sisters. We analyzed whole-genome sequencing data of the patients for pathogenic variants outside the AR coding region. Patient fibroblasts from the genital area were used for AR cDNA analysis and protein quantification. Analysis of the cDNA revealed aberrant splicing of the mRNA caused by a deep intronic mutation (c.2450-118A>G) in the intron 6 of AR. The mutation creates a de novo 5′ splice site and a putative exonic splicing enhancer motif, which leads to the preferential formation of two aberrantly spliced mRNAs (predicted to include a premature stop codon). Patient fibroblasts contained no detectable AR protein. Our results show that patients with CAIS and normal AR coding region need to be examined for deep intronic mutations that can lead to pseudoexon activation.

No MeSH data available.


Related in: MedlinePlus

The PCR amplification of AR cDNA (a), the identified intronic AR mutation (b), the schematic representation of the two aberrant mRNAs caused by the deep intronic mutation (c), and the nucleotide sequences of exons 6 and 7 and intron 6 (d). (a) The AR cDNA was PCR-amplified with primers on exon 5 and the 3′ UTR and the products were visualized on a 1.5% agarose gel. Lanes T1 and T2 are amplification products of the patient samples, XX31B and XY31A are control fibroblast samples, and LNCaP is from prostatic cancer cells. (b) The sequence chromatograms showing the intronic mutation which was confirmed to be present as hemizygotic in both patients and heterozygotic in the mother, whereas it was absent in the father and the healthy sister. (c) Schematic drawing of the aberrant AR pre-mRNA splicing leading to the two longer mRNAs. The cryptic exons are marked with Ψ, and the mutation site is marked with an asterisk. (d) On the left are shown the sequence chromatograms showing the borders of the cryptic exonic sequences and the normal exons derived from sequencing of the gel-extracted aberrant PCR products. On the right are shown the nucleotide sequences of exon 6, intron 6, and exon 7, including the c.2450-118A>G mutation marked in bold and in bigger font size. For splice variant 1, the predicted donor site motif is highlighted in red. For splice variant 2, the predicted SRSF1 binding motif is highlighted in green. The intronic sequences that are spliced into the mRNA are in uppercase and highlighted in yellow (or red/green for sequences that are part of a splicing motif) and correspond to the boxes marked with Ψ in panel C. Normal exonic sequences are in uppercase and are not highlighted, intronic sequences are in lowercase.
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f1: The PCR amplification of AR cDNA (a), the identified intronic AR mutation (b), the schematic representation of the two aberrant mRNAs caused by the deep intronic mutation (c), and the nucleotide sequences of exons 6 and 7 and intron 6 (d). (a) The AR cDNA was PCR-amplified with primers on exon 5 and the 3′ UTR and the products were visualized on a 1.5% agarose gel. Lanes T1 and T2 are amplification products of the patient samples, XX31B and XY31A are control fibroblast samples, and LNCaP is from prostatic cancer cells. (b) The sequence chromatograms showing the intronic mutation which was confirmed to be present as hemizygotic in both patients and heterozygotic in the mother, whereas it was absent in the father and the healthy sister. (c) Schematic drawing of the aberrant AR pre-mRNA splicing leading to the two longer mRNAs. The cryptic exons are marked with Ψ, and the mutation site is marked with an asterisk. (d) On the left are shown the sequence chromatograms showing the borders of the cryptic exonic sequences and the normal exons derived from sequencing of the gel-extracted aberrant PCR products. On the right are shown the nucleotide sequences of exon 6, intron 6, and exon 7, including the c.2450-118A>G mutation marked in bold and in bigger font size. For splice variant 1, the predicted donor site motif is highlighted in red. For splice variant 2, the predicted SRSF1 binding motif is highlighted in green. The intronic sequences that are spliced into the mRNA are in uppercase and highlighted in yellow (or red/green for sequences that are part of a splicing motif) and correspond to the boxes marked with Ψ in panel C. Normal exonic sequences are in uppercase and are not highlighted, intronic sequences are in lowercase.

Mentions: Sanger-sequencing of the AR coding sequence from genomic DNA of the two siblings with CAIS revealed only one synonymous polymorphism [c.639G>A p.(Glu213=), rs6152, minor allele frequency 0.24 in the 1000 Genomes (www.1000genomes.org; phase 3, all variants) database]. No rare potentially pathogenic variants in the coding region or in the conserved splice sites of the gene were found. PCR-amplification of the AR cDNA from the patients’ genital fibroblasts with primers situated on exon 5 and the 3′UTR, however, revealed abnormally long products (approximately 800 and 900 bp) in comparison to controls (Fig. 1a). In addition, the normal-sized product of 683 bp was much fainter in the patient samples than in the controls (Fig. 1a). These findings suggested a splicing defect caused by a deep intronic mutation between exons 5 and 8.


Complete androgen insensitivity syndrome caused by a deep intronic pseudoexon-activating mutation in the androgen receptor gene
The PCR amplification of AR cDNA (a), the identified intronic AR mutation (b), the schematic representation of the two aberrant mRNAs caused by the deep intronic mutation (c), and the nucleotide sequences of exons 6 and 7 and intron 6 (d). (a) The AR cDNA was PCR-amplified with primers on exon 5 and the 3′ UTR and the products were visualized on a 1.5% agarose gel. Lanes T1 and T2 are amplification products of the patient samples, XX31B and XY31A are control fibroblast samples, and LNCaP is from prostatic cancer cells. (b) The sequence chromatograms showing the intronic mutation which was confirmed to be present as hemizygotic in both patients and heterozygotic in the mother, whereas it was absent in the father and the healthy sister. (c) Schematic drawing of the aberrant AR pre-mRNA splicing leading to the two longer mRNAs. The cryptic exons are marked with Ψ, and the mutation site is marked with an asterisk. (d) On the left are shown the sequence chromatograms showing the borders of the cryptic exonic sequences and the normal exons derived from sequencing of the gel-extracted aberrant PCR products. On the right are shown the nucleotide sequences of exon 6, intron 6, and exon 7, including the c.2450-118A>G mutation marked in bold and in bigger font size. For splice variant 1, the predicted donor site motif is highlighted in red. For splice variant 2, the predicted SRSF1 binding motif is highlighted in green. The intronic sequences that are spliced into the mRNA are in uppercase and highlighted in yellow (or red/green for sequences that are part of a splicing motif) and correspond to the boxes marked with Ψ in panel C. Normal exonic sequences are in uppercase and are not highlighted, intronic sequences are in lowercase.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5016895&req=5

f1: The PCR amplification of AR cDNA (a), the identified intronic AR mutation (b), the schematic representation of the two aberrant mRNAs caused by the deep intronic mutation (c), and the nucleotide sequences of exons 6 and 7 and intron 6 (d). (a) The AR cDNA was PCR-amplified with primers on exon 5 and the 3′ UTR and the products were visualized on a 1.5% agarose gel. Lanes T1 and T2 are amplification products of the patient samples, XX31B and XY31A are control fibroblast samples, and LNCaP is from prostatic cancer cells. (b) The sequence chromatograms showing the intronic mutation which was confirmed to be present as hemizygotic in both patients and heterozygotic in the mother, whereas it was absent in the father and the healthy sister. (c) Schematic drawing of the aberrant AR pre-mRNA splicing leading to the two longer mRNAs. The cryptic exons are marked with Ψ, and the mutation site is marked with an asterisk. (d) On the left are shown the sequence chromatograms showing the borders of the cryptic exonic sequences and the normal exons derived from sequencing of the gel-extracted aberrant PCR products. On the right are shown the nucleotide sequences of exon 6, intron 6, and exon 7, including the c.2450-118A>G mutation marked in bold and in bigger font size. For splice variant 1, the predicted donor site motif is highlighted in red. For splice variant 2, the predicted SRSF1 binding motif is highlighted in green. The intronic sequences that are spliced into the mRNA are in uppercase and highlighted in yellow (or red/green for sequences that are part of a splicing motif) and correspond to the boxes marked with Ψ in panel C. Normal exonic sequences are in uppercase and are not highlighted, intronic sequences are in lowercase.
Mentions: Sanger-sequencing of the AR coding sequence from genomic DNA of the two siblings with CAIS revealed only one synonymous polymorphism [c.639G>A p.(Glu213=), rs6152, minor allele frequency 0.24 in the 1000 Genomes (www.1000genomes.org; phase 3, all variants) database]. No rare potentially pathogenic variants in the coding region or in the conserved splice sites of the gene were found. PCR-amplification of the AR cDNA from the patients’ genital fibroblasts with primers situated on exon 5 and the 3′UTR, however, revealed abnormally long products (approximately 800 and 900 bp) in comparison to controls (Fig. 1a). In addition, the normal-sized product of 683 bp was much fainter in the patient samples than in the controls (Fig. 1a). These findings suggested a splicing defect caused by a deep intronic mutation between exons 5 and 8.

View Article: PubMed Central - PubMed

ABSTRACT

Mutations in the X-linked androgen receptor (AR) gene underlie complete androgen insensitivity syndrome (CAIS), the most common cause of 46,XY sex reversal. Molecular genetic diagnosis of CAIS, however, remains uncertain in patients who show normal coding region of AR. Here, we describe a novel mechanism of AR disruption leading to CAIS in two 46,XY sisters. We analyzed whole-genome sequencing data of the patients for pathogenic variants outside the AR coding region. Patient fibroblasts from the genital area were used for AR cDNA analysis and protein quantification. Analysis of the cDNA revealed aberrant splicing of the mRNA caused by a deep intronic mutation (c.2450-118A>G) in the intron 6 of AR. The mutation creates a de novo 5′ splice site and a putative exonic splicing enhancer motif, which leads to the preferential formation of two aberrantly spliced mRNAs (predicted to include a premature stop codon). Patient fibroblasts contained no detectable AR protein. Our results show that patients with CAIS and normal AR coding region need to be examined for deep intronic mutations that can lead to pseudoexon activation.

No MeSH data available.


Related in: MedlinePlus