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Disruption of a novel member of a sodium/hydrogen exchanger family and DOCK3 is associated with an attention deficit hyperactivity disorder-like phenotype
  1. M G de Silva1,
  2. K Elliott1,*,
  3. H-H Dahl1,2,
  4. E Fitzpatrick1,
  5. S Wilcox1,,
  6. M Delatycki1,2,3,
  7. R Williamson1,2,
  8. D Efron1,4,
  9. M Lynch1,
  10. S Forrest1,2,
  1. 1Murdoch Children’s Research Institute, Royal Children’s Hospital, Parkville, Victoria, Australia, and the Cooperative Research Centre for Discovery of Genes for Common Human Diseases, Richmond, Victoria, Australia
  2. 2Department of Paediatrics, University of Melbourne, Parkville, Victoria, Australia
  3. 3Genetic Health Services Victoria, Royal Children’s Hospital, Parkville, Victoria, Australia, and Department of Paediatrics, Monash University, Clayton, Victoria, Australia
  4. 4Centre for Community Child Health, Royal Children’s Hospital, Parkville, Victoria, Australia
  1. Correspondence to:
 Dr M de Silva
 The Murdoch Childrens Research Institute, 10th Floor, Royal Children’s Hospital, Flemington Road, Parkville, Victoria 3052, Australia; desilvmicryptic.rch.unimelb.edu.au

Abstract

Background: Attention deficit hyperactivity disorder (ADHD) is a complex condition with high heritability. However, both biochemical investigations and association and linkage studies have failed to define fully the underlying genetic factors associated with ADHD. We have identified a family co-segregating an early onset behavioural/developmental condition, with features of ADHD and intellectual disability, with a pericentric inversion of chromosome 3, 46N inv(3)(p14:q21).

Methods: We hypothesised that the inversion breakpoints affect a gene or genes that cause the observed phenotype. Large genomic clones (P1 derived/yeast/bacterial artificial chromosomes) were assembled into contigs across the two inversion breakpoints using molecular and bioinformatic technologies. Restriction fragments crossing the junctions were identified by Southern analysis and these fragments were amplified using inverse PCR.

Results: The amplification products were subsequently sequenced to reveal that the breakpoints lay within an intron of the dedicator of cytokinesis 3 (DOCK3) gene at the p arm breakpoint, and an intron of a novel member of the solute carrier family 9 (sodium/hydrogen exchanger) isoform 9 (SLC9A9) at the q arm. Both genes are expressed in the brain, but neither of the genes has previously been implicated in developmental or behavioural disorders.

Conclusion: These two disrupted genes are candidates for involvement in the pathway leading to the neuropsychological condition in this family.

  • ADHD
  • chromosome inversion
  • gene discovery
  • impulsivity
  • physical mapping
  • ADHD, attention deficit hyperactivity disorder
  • BAC, bacterial artificial chromosome
  • FITC, fluorescein isothiocyanate
  • FISH, fluorescence in situ hybridisation
  • PAC, P1-derived artificial chromosome
  • PCR, polymerase chain reaction
  • STS, sequence tagged site
  • YAC, yeast artificial chromosome
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Supplementary materials


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    • [View PDF] - Figure 2 Identification of the p arm breakpoint including the gene structure and predicted amino acid sequence of the DOCK3 gene broken by the p arm breakpoint of the chromosome 3 inversion.
      (A) Fluorescent in situ hybridisation of the BAC clone RP11-3f4 to a metaphase spread of a lymphoblast cell from the proband. The red signal indicates the marker probe used to define the q arm of the normal and inverted chromosomes 3. The green signal shows RP11-3f4 hybridising to the p arm of the normal chromosome 3 (white arrow), a small signal remaining in the normal position on the anomalous chromosome (green arrow) and a larger signal flipping across to the q arm (green arrowhead).
      (B) Southern blot analysis of genomic DNA from the proband using probe (i) from figure 1A. The arrows indicate the abnormal restriction fragments arising from the chromosome rearrangement. P, proband DNA; C, DNA from unaffected controls.
      (C) The DOCK3 gene mRNA sequence is shown with the corresponding predicted protein sequence. The numbering of the nucleotide sequence begins at the ATG start site. The exons are highlighted in alternating grey and aqua and the breakpoint is indicated by a red bar. The mRNA sequence predicts for a soluble protein of 2030 amino acids and a molecular weight of 233.1 kDa. The protein has an SH3 domain indicated in bold italics.
    • [View PDF] - Figure 4 Confirmation of the 3q breakpoint region and the gene structure and predicted amino acid sequence of the SLC9A9 gene disrupted at the q arm.
      (A) Fluorescent in situ hybridisation of the BAC clone RP11-56b20 to a metaphase spread of a lymphoblast cell from the proband. The red signal indicates the marker probe used to define the q arm of the normal and inverted chromosomes 3. The green signal shows the test probe hybridising to the q arm of the normal chromosome 3 (white arrow), and equivalent signals remaining in the normal position on the anomalous chromosome (green arrow) and flipping across to the p arm (green arrowhead).
      (B) Southern blot analysis of genomic DNA from the proband using probe (ii) from fig 1D.
      (C) The SLC9A9 gene mRNA sequence is depicted along with the corresponding predicted protein sequence. The numbering of the nucleotide sequence begins at the ATG start site. The exons are highlighted in alternating grey and red and the breakpoint is indicated by a red bar. The mRNA sequence predicts for a protein with ten transmembrane domains indicated in bold italics. The putative protein has 645 amino acids and a molecular weight of 72.6 kDa.

Footnotes

  • Current Address: Australian Genome Research Facility, Walter and Eliza Hall Institute, Parkville, Victoria, Australia

  • * Current Address: International Diabetes Institute, Caulfield, Victoria, Australia

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