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Exon deletions and intragenic insertions are not rare in ataxia with oculomotor apraxia 2.

Bernard V, Minnerop M, Bürk K, Kreuz F, Gillessen-Kaesbach G, Zühlke C - BMC Med. Genet. (2009)

Bottom Line: Dosage analyses and breakpoint localisation yielded a 1.3 kb LINE1 insertion in exon 12 (patient P1) and a 6.1 kb deletion between intron 11 and intron 14 (patient P2) in addition to the heterozygous nonsense mutation R1606X.Patient P3 was compound heterozygous for a 4 bp deletion in exon 10 and a 20.7 kb deletion between intron 10 and 15.This deletion was present in a homozygous state in patient P4.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut für Humangenetik, Universität zu Lübeck, Lübeck, Germany. Veronica.Bernard@uk-sh.de

ABSTRACT

Background: The autosomal recessively inherited ataxia with oculomotor apraxia 2 (AOA2) is a neurodegenerative disorder characterized by juvenile or adolescent age of onset, gait ataxia, cerebellar atrophy, axonal sensorimotor neuropathy, oculomotor apraxia, and elevated serum AFP levels. AOA2 is caused by mutations within the senataxin gene (SETX). The majority of known mutations are nonsense, missense, and splice site mutations, as well as small deletions and insertions.

Methods: To detect mutations in patients showing a clinical phenotype consistent with AOA2, the coding region including splice sites of the SETX gene was sequenced and dosage analyses for all exons were performed on genomic DNA. The sequence of cDNA fragments of alternative transcripts isolated after RT-PCR was determined.

Results: Sequence analyses of the SETX gene in four patients revealed a heterozygous nonsense mutation or a 4 bp deletion in three cases. In another patient, PCR amplification of exon 11 to 15 dropped out. Dosage analyses and breakpoint localisation yielded a 1.3 kb LINE1 insertion in exon 12 (patient P1) and a 6.1 kb deletion between intron 11 and intron 14 (patient P2) in addition to the heterozygous nonsense mutation R1606X. Patient P3 was compound heterozygous for a 4 bp deletion in exon 10 and a 20.7 kb deletion between intron 10 and 15. This deletion was present in a homozygous state in patient P4.

Conclusion: Our findings indicate that gross mutations seem to be a frequent cause of AOA2 and reveal the importance of additional copy number analysis for routine diagnostics.

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Localisation of the Insertion and Deletion Breakpoints. (a) Long range PCR products for patient P1, his mother M1, and his father F1 separated on a 0.8% agarose gel. Marker: 100 bp DNA Ladder. Amplicon A2 represents the wildtype fragment and A1 the PCR product with the L1HS insertion. Schematic drawing shows the L1HS insertion in exon 12. Exons are indicated as boxes, introns as interrupted lines. The L1HS insertion is flanked by a 15 bp target site duplication (black boxes). (b) Sequence of the breakpoint junction in patient P2 compared to control sequence. Homologous regions are boxed. (c) Sequence alignment of the breakpoint junction in patients P3 and P4 and the control 5' and 3' regions.
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Figure 1: Localisation of the Insertion and Deletion Breakpoints. (a) Long range PCR products for patient P1, his mother M1, and his father F1 separated on a 0.8% agarose gel. Marker: 100 bp DNA Ladder. Amplicon A2 represents the wildtype fragment and A1 the PCR product with the L1HS insertion. Schematic drawing shows the L1HS insertion in exon 12. Exons are indicated as boxes, introns as interrupted lines. The L1HS insertion is flanked by a 15 bp target site duplication (black boxes). (b) Sequence of the breakpoint junction in patient P2 compared to control sequence. Homologous regions are boxed. (c) Sequence alignment of the breakpoint junction in patients P3 and P4 and the control 5' and 3' regions.

Mentions: In patient P1, long-range PCR on genomic DNA using primers flanking exon 12 revealed the 354 bp wildtype fragment and an additional ~1.6 kb PCR product (P1, Figure 1a). The same pattern could be observed in the paternal DNA (F1), whereas the mother (M1) showed the wildtype fragment. Sequencing of the 1.6 kb PCR product indicated a 1.3 kb insertion within exon 12 (c.5401_5402ins1280bp). The insertion consists of a 5' truncated L1HS element. The first part of the 5' truncated L1HS element is orientated in antisense with respect to the disrupted gene, whereas the second part is directed in sense. The insert is flanked by a 15 bp duplicated region. This insertion detected by long-range PCR escaped routine sequencing due to its size.


Exon deletions and intragenic insertions are not rare in ataxia with oculomotor apraxia 2.

Bernard V, Minnerop M, Bürk K, Kreuz F, Gillessen-Kaesbach G, Zühlke C - BMC Med. Genet. (2009)

Localisation of the Insertion and Deletion Breakpoints. (a) Long range PCR products for patient P1, his mother M1, and his father F1 separated on a 0.8% agarose gel. Marker: 100 bp DNA Ladder. Amplicon A2 represents the wildtype fragment and A1 the PCR product with the L1HS insertion. Schematic drawing shows the L1HS insertion in exon 12. Exons are indicated as boxes, introns as interrupted lines. The L1HS insertion is flanked by a 15 bp target site duplication (black boxes). (b) Sequence of the breakpoint junction in patient P2 compared to control sequence. Homologous regions are boxed. (c) Sequence alignment of the breakpoint junction in patients P3 and P4 and the control 5' and 3' regions.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2749023&req=5

Figure 1: Localisation of the Insertion and Deletion Breakpoints. (a) Long range PCR products for patient P1, his mother M1, and his father F1 separated on a 0.8% agarose gel. Marker: 100 bp DNA Ladder. Amplicon A2 represents the wildtype fragment and A1 the PCR product with the L1HS insertion. Schematic drawing shows the L1HS insertion in exon 12. Exons are indicated as boxes, introns as interrupted lines. The L1HS insertion is flanked by a 15 bp target site duplication (black boxes). (b) Sequence of the breakpoint junction in patient P2 compared to control sequence. Homologous regions are boxed. (c) Sequence alignment of the breakpoint junction in patients P3 and P4 and the control 5' and 3' regions.
Mentions: In patient P1, long-range PCR on genomic DNA using primers flanking exon 12 revealed the 354 bp wildtype fragment and an additional ~1.6 kb PCR product (P1, Figure 1a). The same pattern could be observed in the paternal DNA (F1), whereas the mother (M1) showed the wildtype fragment. Sequencing of the 1.6 kb PCR product indicated a 1.3 kb insertion within exon 12 (c.5401_5402ins1280bp). The insertion consists of a 5' truncated L1HS element. The first part of the 5' truncated L1HS element is orientated in antisense with respect to the disrupted gene, whereas the second part is directed in sense. The insert is flanked by a 15 bp duplicated region. This insertion detected by long-range PCR escaped routine sequencing due to its size.

Bottom Line: Dosage analyses and breakpoint localisation yielded a 1.3 kb LINE1 insertion in exon 12 (patient P1) and a 6.1 kb deletion between intron 11 and intron 14 (patient P2) in addition to the heterozygous nonsense mutation R1606X.Patient P3 was compound heterozygous for a 4 bp deletion in exon 10 and a 20.7 kb deletion between intron 10 and 15.This deletion was present in a homozygous state in patient P4.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut für Humangenetik, Universität zu Lübeck, Lübeck, Germany. Veronica.Bernard@uk-sh.de

ABSTRACT

Background: The autosomal recessively inherited ataxia with oculomotor apraxia 2 (AOA2) is a neurodegenerative disorder characterized by juvenile or adolescent age of onset, gait ataxia, cerebellar atrophy, axonal sensorimotor neuropathy, oculomotor apraxia, and elevated serum AFP levels. AOA2 is caused by mutations within the senataxin gene (SETX). The majority of known mutations are nonsense, missense, and splice site mutations, as well as small deletions and insertions.

Methods: To detect mutations in patients showing a clinical phenotype consistent with AOA2, the coding region including splice sites of the SETX gene was sequenced and dosage analyses for all exons were performed on genomic DNA. The sequence of cDNA fragments of alternative transcripts isolated after RT-PCR was determined.

Results: Sequence analyses of the SETX gene in four patients revealed a heterozygous nonsense mutation or a 4 bp deletion in three cases. In another patient, PCR amplification of exon 11 to 15 dropped out. Dosage analyses and breakpoint localisation yielded a 1.3 kb LINE1 insertion in exon 12 (patient P1) and a 6.1 kb deletion between intron 11 and intron 14 (patient P2) in addition to the heterozygous nonsense mutation R1606X. Patient P3 was compound heterozygous for a 4 bp deletion in exon 10 and a 20.7 kb deletion between intron 10 and 15. This deletion was present in a homozygous state in patient P4.

Conclusion: Our findings indicate that gross mutations seem to be a frequent cause of AOA2 and reveal the importance of additional copy number analysis for routine diagnostics.

Show MeSH
Related in: MedlinePlus