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Complex genomic rearrangements at the PLP1 locus include triplication and quadruplication.

Beck CR, Carvalho CM, Banser L, Gambin T, Stubbolo D, Yuan B, Sperle K, McCahan SM, Henneke M, Seeman P, Garbern JY, Hobson GM, Lupski JR - PLoS Genet. (2015)

Bottom Line: We investigated 17 unrelated PMD subjects with copy number gains at the PLP1 locus including triplication and quadruplication of specific genomic intervals-16/17 were found to have a DUP-TRP/INV-DUP rearrangement product.An IR distal to PLP1 facilitates DUP-TRP/INV-DUP formation as well as an inversion structural variation found frequently amongst normal individuals.Moreover, we provide evidence that quadruplication and potentially higher order amplification of a genomic interval can occur in a manner consistent with rolling circle amplification as predicted by the microhomology-mediated break induced replication (MMBIR) model.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, United States of America.

ABSTRACT
Inverted repeats (IRs) can facilitate structural variation as crucibles of genomic rearrangement. Complex duplication-inverted triplication-duplication (DUP-TRP/INV-DUP) rearrangements that contain breakpoint junctions within IRs have been recently associated with both MECP2 duplication syndrome (MIM#300260) and Pelizaeus-Merzbacher disease (PMD, MIM#312080). We investigated 17 unrelated PMD subjects with copy number gains at the PLP1 locus including triplication and quadruplication of specific genomic intervals-16/17 were found to have a DUP-TRP/INV-DUP rearrangement product. An IR distal to PLP1 facilitates DUP-TRP/INV-DUP formation as well as an inversion structural variation found frequently amongst normal individuals. We show that a homology-or homeology-driven replicative mechanism of DNA repair can apparently mediate template switches within stretches of microhomology. Moreover, we provide evidence that quadruplication and potentially higher order amplification of a genomic interval can occur in a manner consistent with rolling circle amplification as predicted by the microhomology-mediated break induced replication (MMBIR) model.

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Related in: MedlinePlus

CGRs at the PLP1 locus use A1a and A1b repeats.A) Southern scheme from Fig. 1 applied to a DUP-TRP/INV-DUP rearrangement results in either two copies of a 25 kb band and one of a 29 kb band if the rearrangement occurred on H1 (shown at the top), or the reciprocal copy dosage if the rearrangement occurred on H2 (bottom). Colors for LCRs are as in Fig. 1. B) Digested DNA from 2 control individuals (NA15510 and NA10851) and two PMD/SPG2 patients (BAB1290 and BAB1612/P374, respectively). Southern below depicts control individuals have expected, gender appropriate 2 and 1 copies, and affected males have three copies, with dosage of 2:1 H2:H1, indicating the rearrangement likely occurred on H2 (indicated within the black square for each patient- data for this blot and panels C and D are quantitated in S4 Table). C) BAB2389 and BAB3698 may also have rearrangements on the inverted allele (H2, indicated at the top of the image). BAB3698 is depicted with his carrier mother, sister (BAB3700 and BAB3699, indicated by a dot in a circle), and non-carrier grandmother (BAB4179). The grandfather was unaffected and unavailable for study. D) P250, P298, and P558 all likely contain rearrangements on H1 (~1:2 ratio of H2:H1) and P500, P518, and P642 contain rearrangements on H2. All rearrangement progenitor haplotypes are indicated for the patients above the Southern blot. E) The reference genomic structure of H1 is shown (inner A2 and A3 repeats are unlabeled). The qPCR primer pairs amplify a unique region outside of the A1a LCR (in red), inside of both A1a and A1b LCRs (in black) or from the A1a LCR to a unique region outside (red/black pair below). These will give rise to one copy (red pair and red/black pair) or two copies (black pair) in a non-rearranged X chromosome in a male individual. DUP-TRP/INV-DUP (on right) in an H1 haplotype will give rise to four copies amplified by the black pair (2x normal control) and three copies by the red pair and red/black pair (3X normal control) (S7 Fig). F) Analysis of Jct1 has successfully cloned one breakpoint in BAB1612/P374. The structure of the LCR-mediated rearrangement on H2 is depicted at the top (A1a and A1b are simplified to “A” and “B” and inner A2 and A3 repeats are unlabeled). Overlapping clones for each region of the two LCRs were generated (numbered 1–4, S8 Fig), and results for BAB1612/P374 were obtained for section 1 clones that both contain and lack the breakpoint. Multiple clones from this region are depicted along with the reference sequences for LCRs A1a and A1b below. The breakpoint from individual BAB1612/P374 occurred in stretch of 24bp of microhomology (bracket-denoted region).
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pgen.1005050.g003: CGRs at the PLP1 locus use A1a and A1b repeats.A) Southern scheme from Fig. 1 applied to a DUP-TRP/INV-DUP rearrangement results in either two copies of a 25 kb band and one of a 29 kb band if the rearrangement occurred on H1 (shown at the top), or the reciprocal copy dosage if the rearrangement occurred on H2 (bottom). Colors for LCRs are as in Fig. 1. B) Digested DNA from 2 control individuals (NA15510 and NA10851) and two PMD/SPG2 patients (BAB1290 and BAB1612/P374, respectively). Southern below depicts control individuals have expected, gender appropriate 2 and 1 copies, and affected males have three copies, with dosage of 2:1 H2:H1, indicating the rearrangement likely occurred on H2 (indicated within the black square for each patient- data for this blot and panels C and D are quantitated in S4 Table). C) BAB2389 and BAB3698 may also have rearrangements on the inverted allele (H2, indicated at the top of the image). BAB3698 is depicted with his carrier mother, sister (BAB3700 and BAB3699, indicated by a dot in a circle), and non-carrier grandmother (BAB4179). The grandfather was unaffected and unavailable for study. D) P250, P298, and P558 all likely contain rearrangements on H1 (~1:2 ratio of H2:H1) and P500, P518, and P642 contain rearrangements on H2. All rearrangement progenitor haplotypes are indicated for the patients above the Southern blot. E) The reference genomic structure of H1 is shown (inner A2 and A3 repeats are unlabeled). The qPCR primer pairs amplify a unique region outside of the A1a LCR (in red), inside of both A1a and A1b LCRs (in black) or from the A1a LCR to a unique region outside (red/black pair below). These will give rise to one copy (red pair and red/black pair) or two copies (black pair) in a non-rearranged X chromosome in a male individual. DUP-TRP/INV-DUP (on right) in an H1 haplotype will give rise to four copies amplified by the black pair (2x normal control) and three copies by the red pair and red/black pair (3X normal control) (S7 Fig). F) Analysis of Jct1 has successfully cloned one breakpoint in BAB1612/P374. The structure of the LCR-mediated rearrangement on H2 is depicted at the top (A1a and A1b are simplified to “A” and “B” and inner A2 and A3 repeats are unlabeled). Overlapping clones for each region of the two LCRs were generated (numbered 1–4, S8 Fig), and results for BAB1612/P374 were obtained for section 1 clones that both contain and lack the breakpoint. Multiple clones from this region are depicted along with the reference sequences for LCRs A1a and A1b below. The breakpoint from individual BAB1612/P374 occurred in stretch of 24bp of microhomology (bracket-denoted region).

Mentions: Patients with presumed DUP-TRP/INV-DUP rearrangements with sufficient DNA available were subjected to Southern blotting (10/16 total) to examine whether the same repeats involved in the common inversion polymorphism are also involved in the CGR and to investigate on which structural haplotype the rearrangement occurred. The Southern scheme in Fig. 1C was used to analyze patient DNAs; however, in a male with PMD caused by DUP-TRP/INV-DUP involving the A1a and A1b repeats, the Southern blot does not reflect the normal copy number of one allele of the X chromosome (either H1 or H2) (Fig. 3A, S4 Table). Instead, the rearrangement gives rise to two copies of the original haplotype plus an additional “flipped” haplotype in an affected individual with DUP-TRP/INV-DUP leading to PMD, similar to the observation described for the MECP2 locus [1]. This assay can presumably distinguish the SV haplotype on which the genomic rearrangement occurred. A representative gel and labeled blot are shown in Fig. 3B, with the dosage of the bands indicating that subjects BAB1290 and BAB1612/P374 both carried the inversion H2 structural haplotype prior to the rearrangement.


Complex genomic rearrangements at the PLP1 locus include triplication and quadruplication.

Beck CR, Carvalho CM, Banser L, Gambin T, Stubbolo D, Yuan B, Sperle K, McCahan SM, Henneke M, Seeman P, Garbern JY, Hobson GM, Lupski JR - PLoS Genet. (2015)

CGRs at the PLP1 locus use A1a and A1b repeats.A) Southern scheme from Fig. 1 applied to a DUP-TRP/INV-DUP rearrangement results in either two copies of a 25 kb band and one of a 29 kb band if the rearrangement occurred on H1 (shown at the top), or the reciprocal copy dosage if the rearrangement occurred on H2 (bottom). Colors for LCRs are as in Fig. 1. B) Digested DNA from 2 control individuals (NA15510 and NA10851) and two PMD/SPG2 patients (BAB1290 and BAB1612/P374, respectively). Southern below depicts control individuals have expected, gender appropriate 2 and 1 copies, and affected males have three copies, with dosage of 2:1 H2:H1, indicating the rearrangement likely occurred on H2 (indicated within the black square for each patient- data for this blot and panels C and D are quantitated in S4 Table). C) BAB2389 and BAB3698 may also have rearrangements on the inverted allele (H2, indicated at the top of the image). BAB3698 is depicted with his carrier mother, sister (BAB3700 and BAB3699, indicated by a dot in a circle), and non-carrier grandmother (BAB4179). The grandfather was unaffected and unavailable for study. D) P250, P298, and P558 all likely contain rearrangements on H1 (~1:2 ratio of H2:H1) and P500, P518, and P642 contain rearrangements on H2. All rearrangement progenitor haplotypes are indicated for the patients above the Southern blot. E) The reference genomic structure of H1 is shown (inner A2 and A3 repeats are unlabeled). The qPCR primer pairs amplify a unique region outside of the A1a LCR (in red), inside of both A1a and A1b LCRs (in black) or from the A1a LCR to a unique region outside (red/black pair below). These will give rise to one copy (red pair and red/black pair) or two copies (black pair) in a non-rearranged X chromosome in a male individual. DUP-TRP/INV-DUP (on right) in an H1 haplotype will give rise to four copies amplified by the black pair (2x normal control) and three copies by the red pair and red/black pair (3X normal control) (S7 Fig). F) Analysis of Jct1 has successfully cloned one breakpoint in BAB1612/P374. The structure of the LCR-mediated rearrangement on H2 is depicted at the top (A1a and A1b are simplified to “A” and “B” and inner A2 and A3 repeats are unlabeled). Overlapping clones for each region of the two LCRs were generated (numbered 1–4, S8 Fig), and results for BAB1612/P374 were obtained for section 1 clones that both contain and lack the breakpoint. Multiple clones from this region are depicted along with the reference sequences for LCRs A1a and A1b below. The breakpoint from individual BAB1612/P374 occurred in stretch of 24bp of microhomology (bracket-denoted region).
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4352052&req=5

pgen.1005050.g003: CGRs at the PLP1 locus use A1a and A1b repeats.A) Southern scheme from Fig. 1 applied to a DUP-TRP/INV-DUP rearrangement results in either two copies of a 25 kb band and one of a 29 kb band if the rearrangement occurred on H1 (shown at the top), or the reciprocal copy dosage if the rearrangement occurred on H2 (bottom). Colors for LCRs are as in Fig. 1. B) Digested DNA from 2 control individuals (NA15510 and NA10851) and two PMD/SPG2 patients (BAB1290 and BAB1612/P374, respectively). Southern below depicts control individuals have expected, gender appropriate 2 and 1 copies, and affected males have three copies, with dosage of 2:1 H2:H1, indicating the rearrangement likely occurred on H2 (indicated within the black square for each patient- data for this blot and panels C and D are quantitated in S4 Table). C) BAB2389 and BAB3698 may also have rearrangements on the inverted allele (H2, indicated at the top of the image). BAB3698 is depicted with his carrier mother, sister (BAB3700 and BAB3699, indicated by a dot in a circle), and non-carrier grandmother (BAB4179). The grandfather was unaffected and unavailable for study. D) P250, P298, and P558 all likely contain rearrangements on H1 (~1:2 ratio of H2:H1) and P500, P518, and P642 contain rearrangements on H2. All rearrangement progenitor haplotypes are indicated for the patients above the Southern blot. E) The reference genomic structure of H1 is shown (inner A2 and A3 repeats are unlabeled). The qPCR primer pairs amplify a unique region outside of the A1a LCR (in red), inside of both A1a and A1b LCRs (in black) or from the A1a LCR to a unique region outside (red/black pair below). These will give rise to one copy (red pair and red/black pair) or two copies (black pair) in a non-rearranged X chromosome in a male individual. DUP-TRP/INV-DUP (on right) in an H1 haplotype will give rise to four copies amplified by the black pair (2x normal control) and three copies by the red pair and red/black pair (3X normal control) (S7 Fig). F) Analysis of Jct1 has successfully cloned one breakpoint in BAB1612/P374. The structure of the LCR-mediated rearrangement on H2 is depicted at the top (A1a and A1b are simplified to “A” and “B” and inner A2 and A3 repeats are unlabeled). Overlapping clones for each region of the two LCRs were generated (numbered 1–4, S8 Fig), and results for BAB1612/P374 were obtained for section 1 clones that both contain and lack the breakpoint. Multiple clones from this region are depicted along with the reference sequences for LCRs A1a and A1b below. The breakpoint from individual BAB1612/P374 occurred in stretch of 24bp of microhomology (bracket-denoted region).
Mentions: Patients with presumed DUP-TRP/INV-DUP rearrangements with sufficient DNA available were subjected to Southern blotting (10/16 total) to examine whether the same repeats involved in the common inversion polymorphism are also involved in the CGR and to investigate on which structural haplotype the rearrangement occurred. The Southern scheme in Fig. 1C was used to analyze patient DNAs; however, in a male with PMD caused by DUP-TRP/INV-DUP involving the A1a and A1b repeats, the Southern blot does not reflect the normal copy number of one allele of the X chromosome (either H1 or H2) (Fig. 3A, S4 Table). Instead, the rearrangement gives rise to two copies of the original haplotype plus an additional “flipped” haplotype in an affected individual with DUP-TRP/INV-DUP leading to PMD, similar to the observation described for the MECP2 locus [1]. This assay can presumably distinguish the SV haplotype on which the genomic rearrangement occurred. A representative gel and labeled blot are shown in Fig. 3B, with the dosage of the bands indicating that subjects BAB1290 and BAB1612/P374 both carried the inversion H2 structural haplotype prior to the rearrangement.

Bottom Line: We investigated 17 unrelated PMD subjects with copy number gains at the PLP1 locus including triplication and quadruplication of specific genomic intervals-16/17 were found to have a DUP-TRP/INV-DUP rearrangement product.An IR distal to PLP1 facilitates DUP-TRP/INV-DUP formation as well as an inversion structural variation found frequently amongst normal individuals.Moreover, we provide evidence that quadruplication and potentially higher order amplification of a genomic interval can occur in a manner consistent with rolling circle amplification as predicted by the microhomology-mediated break induced replication (MMBIR) model.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, United States of America.

ABSTRACT
Inverted repeats (IRs) can facilitate structural variation as crucibles of genomic rearrangement. Complex duplication-inverted triplication-duplication (DUP-TRP/INV-DUP) rearrangements that contain breakpoint junctions within IRs have been recently associated with both MECP2 duplication syndrome (MIM#300260) and Pelizaeus-Merzbacher disease (PMD, MIM#312080). We investigated 17 unrelated PMD subjects with copy number gains at the PLP1 locus including triplication and quadruplication of specific genomic intervals-16/17 were found to have a DUP-TRP/INV-DUP rearrangement product. An IR distal to PLP1 facilitates DUP-TRP/INV-DUP formation as well as an inversion structural variation found frequently amongst normal individuals. We show that a homology-or homeology-driven replicative mechanism of DNA repair can apparently mediate template switches within stretches of microhomology. Moreover, we provide evidence that quadruplication and potentially higher order amplification of a genomic interval can occur in a manner consistent with rolling circle amplification as predicted by the microhomology-mediated break induced replication (MMBIR) model.

Show MeSH
Related in: MedlinePlus