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De novo and rare inherited mutations implicate the transcriptional coregulator TCF20/SPBP in autism spectrum disorder.

Babbs C, Lloyd D, Pagnamenta AT, Twigg SR, Green J, McGowan SJ, Mirza G, Naples R, Sharma VP, Volpi EV, Buckle VJ, Wall SA, Knight SJ, International Molecular Genetic Study of Autism Consortium (IMGSAC)Parr JR, Wilkie AO - J. Med. Genet. (2014)

Bottom Line: IMGSAC family screening identified a de novo missense mutation of TCF20 in a single case and significant association of a different missense mutation of TCF20 with ASD in three further families.We did not identify a significant association of TNRC6B mutations with ASD.This study provides the first evidence that mutations in TCF20 are also associated with ASD.

View Article: PubMed Central - PubMed

Affiliation: Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK NIHR Biomedical Research Centre, Oxford, UK.

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Structure of the chromosome 22 rearrangement deduced from fluorescence in situ hybridization (FISH) analysis and DNA sequencing. (A–C) Representative FISH analysis and diagrammatic interpretation of structure of the rearranged chromosome (der22), shown in more detail with positions of breakpoints in (D). (A) Signals from RP11-241G19 (green), which spans breakpoint A, and the more distal RP11-49A20 (red) are adjacent on the normal chromosome 22 (arrowhead) but a split green signal is seen near the opposite end of the der22 (arrow). (B) Clones W12-1927K3 (red) and W12-1574G19 (green), which lie on either side of breakpoint B, showing hybridisation together on the normal chromosome 22 (arrowhead) and at opposite ends of the der22 (arrow). C. Single signal with W12-1570N6 on normal chromosome 22 (arrowhead), but split signal on derived 22 (arrow) indicating position of breakpoint C. (D) Ideograms of wt and derived chromosome 22. The order of BAC and fosmid clones employed in figure parts A–C is shown, together with the locations of breakpoints A–C. The 2 Mb region between breakpoints A and B is shown in light red (orientation on the derived chromosome is uncertain). Breakpoint D on the satellite short arm was not further characterised. Below left, map of the 65 kb region that includes breakpoint C, showing the positions and orientations of genes. The Southern blot analysis shows an apparent breakpoint in the patient sample (P) compared with the control (C), localising the breakpoint to the indicated segment (double-ended arrows) of ∼4 kb. Below right, the DNA sequence chromatogram spanning the breakpoints A and B is shown above an alignment of this sequence with the normal sequences at the telomeric and centromeric ends of breakpoints. Arrows indicate positions and numbering of the last intact bases on either side of the translocated region.
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JMEDGENET2014102582F2: Structure of the chromosome 22 rearrangement deduced from fluorescence in situ hybridization (FISH) analysis and DNA sequencing. (A–C) Representative FISH analysis and diagrammatic interpretation of structure of the rearranged chromosome (der22), shown in more detail with positions of breakpoints in (D). (A) Signals from RP11-241G19 (green), which spans breakpoint A, and the more distal RP11-49A20 (red) are adjacent on the normal chromosome 22 (arrowhead) but a split green signal is seen near the opposite end of the der22 (arrow). (B) Clones W12-1927K3 (red) and W12-1574G19 (green), which lie on either side of breakpoint B, showing hybridisation together on the normal chromosome 22 (arrowhead) and at opposite ends of the der22 (arrow). C. Single signal with W12-1570N6 on normal chromosome 22 (arrowhead), but split signal on derived 22 (arrow) indicating position of breakpoint C. (D) Ideograms of wt and derived chromosome 22. The order of BAC and fosmid clones employed in figure parts A–C is shown, together with the locations of breakpoints A–C. The 2 Mb region between breakpoints A and B is shown in light red (orientation on the derived chromosome is uncertain). Breakpoint D on the satellite short arm was not further characterised. Below left, map of the 65 kb region that includes breakpoint C, showing the positions and orientations of genes. The Southern blot analysis shows an apparent breakpoint in the patient sample (P) compared with the control (C), localising the breakpoint to the indicated segment (double-ended arrows) of ∼4 kb. Below right, the DNA sequence chromatogram spanning the breakpoints A and B is shown above an alignment of this sequence with the normal sequences at the telomeric and centromeric ends of breakpoints. Arrows indicate positions and numbering of the last intact bases on either side of the translocated region.

Mentions: To characterise the molecular nature of the pericentric inversion, we performed FISH using multiple BACs and fosmids (table 1). These probes were initially focused on the 22q13.1 band in which the long-arm breakpoint had been tentatively located, but several further rounds of analysis were performed as greater complexity in the rearrangement became apparent (figure 2). The observation of split signals with two fosmids localised one breakpoint (termed breakpoint A) to a ∼37 kb region (figure 2A). Further analysis by Southern blotting with single-copy probes identified breakpoint fragments, initially within a ∼15 kb EcoRI fragment, and subsequently within a 248 bp fragment bordered by StuI and AflIII restriction sites (not shown). PCR primers were designed to amplify across the breakpoint in sequentially nested amplifications with degenerate primers (see ‘Methods’). Surprisingly, DNA sequencing of this amplification product identified the sequence on the centromeric side of the break as originating from a location ∼1.9 Mb centromeric of breakpoint A (figure 2D, bottom right). These sequence data showed contiguity between nucleotides at coordinates at 40 709 620 bp (breakpoint B) and 42 634 698 bp (breakpoint A), adjacent to a short stretch of 5-nucleotide (5′-GACCT-3′) complementarity (figure 2D). Confirming the identification of breakpoint B, clones closely adjacent on either side of this location mapped to opposite arms of the der(22) (figure 2B). This result implied that a third more centromeric break on the long arm (‘breakpoint C’) must have occurred, to which the intermediate segment (B-A) had been joined. This break was localised using FISH to an ∼44.7 kb region within BAC clone W12-1570N6 (figure 2C). Analysis by Southern blotting revealed a HindIII restriction fragment that likely spanned the breakpoint (figure 2D, bottom left), locating the breakpoint to a ∼4 kb region between 39 507 139  and 39 511 083 bp. Figure 2D summarises the structure of the derivative chromosome 22 as concluded from the FISH, Southern blotting and DNA sequencing results. Breakpoint D is predicted to occur in the short arm satellite sequence of chromosome 22 and was not characterised further. Although (as demonstrated by array CGH) there has been no major gain or loss of material at the breakpoints, we found evidence of a small (∼10 kb) duplication at breakpoint A (data not shown) and this may apply to others too, most consistent with the replication-based fork stalling template switching (FoSTeS)-type mechanism for the complex chromosome rearrangement.29


De novo and rare inherited mutations implicate the transcriptional coregulator TCF20/SPBP in autism spectrum disorder.

Babbs C, Lloyd D, Pagnamenta AT, Twigg SR, Green J, McGowan SJ, Mirza G, Naples R, Sharma VP, Volpi EV, Buckle VJ, Wall SA, Knight SJ, International Molecular Genetic Study of Autism Consortium (IMGSAC)Parr JR, Wilkie AO - J. Med. Genet. (2014)

Structure of the chromosome 22 rearrangement deduced from fluorescence in situ hybridization (FISH) analysis and DNA sequencing. (A–C) Representative FISH analysis and diagrammatic interpretation of structure of the rearranged chromosome (der22), shown in more detail with positions of breakpoints in (D). (A) Signals from RP11-241G19 (green), which spans breakpoint A, and the more distal RP11-49A20 (red) are adjacent on the normal chromosome 22 (arrowhead) but a split green signal is seen near the opposite end of the der22 (arrow). (B) Clones W12-1927K3 (red) and W12-1574G19 (green), which lie on either side of breakpoint B, showing hybridisation together on the normal chromosome 22 (arrowhead) and at opposite ends of the der22 (arrow). C. Single signal with W12-1570N6 on normal chromosome 22 (arrowhead), but split signal on derived 22 (arrow) indicating position of breakpoint C. (D) Ideograms of wt and derived chromosome 22. The order of BAC and fosmid clones employed in figure parts A–C is shown, together with the locations of breakpoints A–C. The 2 Mb region between breakpoints A and B is shown in light red (orientation on the derived chromosome is uncertain). Breakpoint D on the satellite short arm was not further characterised. Below left, map of the 65 kb region that includes breakpoint C, showing the positions and orientations of genes. The Southern blot analysis shows an apparent breakpoint in the patient sample (P) compared with the control (C), localising the breakpoint to the indicated segment (double-ended arrows) of ∼4 kb. Below right, the DNA sequence chromatogram spanning the breakpoints A and B is shown above an alignment of this sequence with the normal sequences at the telomeric and centromeric ends of breakpoints. Arrows indicate positions and numbering of the last intact bases on either side of the translocated region.
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JMEDGENET2014102582F2: Structure of the chromosome 22 rearrangement deduced from fluorescence in situ hybridization (FISH) analysis and DNA sequencing. (A–C) Representative FISH analysis and diagrammatic interpretation of structure of the rearranged chromosome (der22), shown in more detail with positions of breakpoints in (D). (A) Signals from RP11-241G19 (green), which spans breakpoint A, and the more distal RP11-49A20 (red) are adjacent on the normal chromosome 22 (arrowhead) but a split green signal is seen near the opposite end of the der22 (arrow). (B) Clones W12-1927K3 (red) and W12-1574G19 (green), which lie on either side of breakpoint B, showing hybridisation together on the normal chromosome 22 (arrowhead) and at opposite ends of the der22 (arrow). C. Single signal with W12-1570N6 on normal chromosome 22 (arrowhead), but split signal on derived 22 (arrow) indicating position of breakpoint C. (D) Ideograms of wt and derived chromosome 22. The order of BAC and fosmid clones employed in figure parts A–C is shown, together with the locations of breakpoints A–C. The 2 Mb region between breakpoints A and B is shown in light red (orientation on the derived chromosome is uncertain). Breakpoint D on the satellite short arm was not further characterised. Below left, map of the 65 kb region that includes breakpoint C, showing the positions and orientations of genes. The Southern blot analysis shows an apparent breakpoint in the patient sample (P) compared with the control (C), localising the breakpoint to the indicated segment (double-ended arrows) of ∼4 kb. Below right, the DNA sequence chromatogram spanning the breakpoints A and B is shown above an alignment of this sequence with the normal sequences at the telomeric and centromeric ends of breakpoints. Arrows indicate positions and numbering of the last intact bases on either side of the translocated region.
Mentions: To characterise the molecular nature of the pericentric inversion, we performed FISH using multiple BACs and fosmids (table 1). These probes were initially focused on the 22q13.1 band in which the long-arm breakpoint had been tentatively located, but several further rounds of analysis were performed as greater complexity in the rearrangement became apparent (figure 2). The observation of split signals with two fosmids localised one breakpoint (termed breakpoint A) to a ∼37 kb region (figure 2A). Further analysis by Southern blotting with single-copy probes identified breakpoint fragments, initially within a ∼15 kb EcoRI fragment, and subsequently within a 248 bp fragment bordered by StuI and AflIII restriction sites (not shown). PCR primers were designed to amplify across the breakpoint in sequentially nested amplifications with degenerate primers (see ‘Methods’). Surprisingly, DNA sequencing of this amplification product identified the sequence on the centromeric side of the break as originating from a location ∼1.9 Mb centromeric of breakpoint A (figure 2D, bottom right). These sequence data showed contiguity between nucleotides at coordinates at 40 709 620 bp (breakpoint B) and 42 634 698 bp (breakpoint A), adjacent to a short stretch of 5-nucleotide (5′-GACCT-3′) complementarity (figure 2D). Confirming the identification of breakpoint B, clones closely adjacent on either side of this location mapped to opposite arms of the der(22) (figure 2B). This result implied that a third more centromeric break on the long arm (‘breakpoint C’) must have occurred, to which the intermediate segment (B-A) had been joined. This break was localised using FISH to an ∼44.7 kb region within BAC clone W12-1570N6 (figure 2C). Analysis by Southern blotting revealed a HindIII restriction fragment that likely spanned the breakpoint (figure 2D, bottom left), locating the breakpoint to a ∼4 kb region between 39 507 139  and 39 511 083 bp. Figure 2D summarises the structure of the derivative chromosome 22 as concluded from the FISH, Southern blotting and DNA sequencing results. Breakpoint D is predicted to occur in the short arm satellite sequence of chromosome 22 and was not characterised further. Although (as demonstrated by array CGH) there has been no major gain or loss of material at the breakpoints, we found evidence of a small (∼10 kb) duplication at breakpoint A (data not shown) and this may apply to others too, most consistent with the replication-based fork stalling template switching (FoSTeS)-type mechanism for the complex chromosome rearrangement.29

Bottom Line: IMGSAC family screening identified a de novo missense mutation of TCF20 in a single case and significant association of a different missense mutation of TCF20 with ASD in three further families.We did not identify a significant association of TNRC6B mutations with ASD.This study provides the first evidence that mutations in TCF20 are also associated with ASD.

View Article: PubMed Central - PubMed

Affiliation: Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK NIHR Biomedical Research Centre, Oxford, UK.

Show MeSH
Related in: MedlinePlus