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Disruption of contactin 4 in three subjects with autism spectrum disorder.

Roohi J, Montagna C, Tegay DH, Palmer LE, DeVincent C, Pomeroy JC, Christian SL, Nowak N, Hatchwell E - J. Med. Genet. (2008)

Bottom Line: These variations were fluorescence in situ hybridisation (FISH) validated and the end points further delineated using a custom fine tiling oligonucleotide array.CNTN4 plays an essential role in the formation, maintenance, and plasticity of neuronal networks.Disruption of this gene is known to cause developmental delay and mental retardation.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Autism spectrum disorder (ASD) is a developmental disorder of the central nervous system of largely unknown aetiology. The prevalence of the syndrome underscores the need for biological markers and a clearer understanding of pathogenesis. For these reasons, a genetic study of idiopathic ASD was undertaken.

Methods and results: Array based comparative genomic hybridisation identified a paternally inherited chromosome 3 copy number variation (CNV) in three

Subjects: a deletion in two siblings and a duplication in a third, unrelated individual. These variations were fluorescence in situ hybridisation (FISH) validated and the end points further delineated using a custom fine tiling oligonucleotide array. Polymerase chain reaction (PCR) products unique to the rearrangements were amplified and sequence analysis revealed the variations to have resulted from Alu Y mediated unequal recombinations interrupting contactin 4 (CNTN4).

Conclusion: CNTN4 plays an essential role in the formation, maintenance, and plasticity of neuronal networks. Disruption of this gene is known to cause developmental delay and mental retardation. This report suggests that mutations affecting CNTN4 function may be relevant to ASD pathogenesis.

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Related in: MedlinePlus

Fluorescence in situ hybridisation (FISH) validation of chr3p26.3 copy number variations (CNVs). (A) Validation of chr3p26.3 microduplication. FISH analysis confirmed the chr3p26.3 microduplication in subject 1 and his father (FISH images from subject 1 pictured). Panel order from left to right: composite image, left flanking clone (yellow), right flanking clone (green), clone within the CNV (red). (B) Validation of chr3p26.3 microdeletion. FISH analysis confirmed the chr3p26.3 microdeletion in 2B, 2C and their father (FISH images from subject 2C pictured). Panel order from left to right: composite image, left flanking clone (yellow), right flanking clone (green), clone within the CNV (red). (C) Normal chromosome 3. (D) Chromosome 3 with duplication. Comparison of red signal intensity in (C) versus (D) demonstrates a duplication. (E) Ideogram of clone locations. Three bacterial artificial chromosomes (BACs) were selected for each CNV: a left clone flanking the CNV pseudocoloured yellow (RP11-204C23), a right clone flanking the CNV pseudocoloured green (RP11-95E11), and clone within the CNV pseudocoloured red (RP11-129K1).
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jmg-46-03-0176-f01: Fluorescence in situ hybridisation (FISH) validation of chr3p26.3 copy number variations (CNVs). (A) Validation of chr3p26.3 microduplication. FISH analysis confirmed the chr3p26.3 microduplication in subject 1 and his father (FISH images from subject 1 pictured). Panel order from left to right: composite image, left flanking clone (yellow), right flanking clone (green), clone within the CNV (red). (B) Validation of chr3p26.3 microdeletion. FISH analysis confirmed the chr3p26.3 microdeletion in 2B, 2C and their father (FISH images from subject 2C pictured). Panel order from left to right: composite image, left flanking clone (yellow), right flanking clone (green), clone within the CNV (red). (C) Normal chromosome 3. (D) Chromosome 3 with duplication. Comparison of red signal intensity in (C) versus (D) demonstrates a duplication. (E) Ideogram of clone locations. Three bacterial artificial chromosomes (BACs) were selected for each CNV: a left clone flanking the CNV pseudocoloured yellow (RP11-204C23), a right clone flanking the CNV pseudocoloured green (RP11-95E11), and clone within the CNV pseudocoloured red (RP11-129K1).

Mentions: Genomic DNA from all subjects (and parents, in select cases) was hybridised onto tiling path bacterial artificial chromosome (BAC) arrays for copy number analysis as previously described.10 Image analysis was performed using the BlueFuse package (BlueGnome, Cambridge, UK). A subgrid loss corrected log2 ratio of the background subtracted test/control was calculated for each clone. Fluorescence in situ hybridisation (FISH) validation of genomic DNA CNVs was performed with Phi29 DNA polymerase amplified BAC clones as previously described.11 Three BAC clones were selected for each FISH experiment: (1) a left clone flanking the CNV (proximal to the centromere) was labelled with Biotin (Roche, Nutley, New Jersey, USA) detected with Streptavidin Cy5 (Jackson Immunoresearch Laboratories, West Grove, Pennsylvania, USA) and pseudocoloured in yellow; (2) a right clone flanking the CNV (distal to the centromere) was labelled with Spectrum Green dNTP (1.6 nmole, Vysis, Abbott Molecular Inc, Des Plaines, Illinois, USA) and pseudocoloured in green; and (3) clones within the change were labelled with Spectrum Orange dNTP (1.6 nmole, Vysis) and pseudocoloured in red (fig 1). In all, seven different clones from within the CNVs were tested (tables 1 and 2). Metaphase chromosomes from each subject were derived from normal B lymphocytes as previously described (www.riedlab.nci.nih.gov/protocols.asp). After hybridisation, slides were washed three times in 50% formamide and 2XSSC at 45°C and subsequently in 1XSSC at 45°C. Biotin labelled flanking clones were detected by incubating hybridised slides with a 1:200 dilution of Streptavidin Cy5 Ab. Slides were stained with DAPI and mounted with antifade (phenylene diamine).


Disruption of contactin 4 in three subjects with autism spectrum disorder.

Roohi J, Montagna C, Tegay DH, Palmer LE, DeVincent C, Pomeroy JC, Christian SL, Nowak N, Hatchwell E - J. Med. Genet. (2008)

Fluorescence in situ hybridisation (FISH) validation of chr3p26.3 copy number variations (CNVs). (A) Validation of chr3p26.3 microduplication. FISH analysis confirmed the chr3p26.3 microduplication in subject 1 and his father (FISH images from subject 1 pictured). Panel order from left to right: composite image, left flanking clone (yellow), right flanking clone (green), clone within the CNV (red). (B) Validation of chr3p26.3 microdeletion. FISH analysis confirmed the chr3p26.3 microdeletion in 2B, 2C and their father (FISH images from subject 2C pictured). Panel order from left to right: composite image, left flanking clone (yellow), right flanking clone (green), clone within the CNV (red). (C) Normal chromosome 3. (D) Chromosome 3 with duplication. Comparison of red signal intensity in (C) versus (D) demonstrates a duplication. (E) Ideogram of clone locations. Three bacterial artificial chromosomes (BACs) were selected for each CNV: a left clone flanking the CNV pseudocoloured yellow (RP11-204C23), a right clone flanking the CNV pseudocoloured green (RP11-95E11), and clone within the CNV pseudocoloured red (RP11-129K1).
© Copyright Policy - openaccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2643049&req=5

jmg-46-03-0176-f01: Fluorescence in situ hybridisation (FISH) validation of chr3p26.3 copy number variations (CNVs). (A) Validation of chr3p26.3 microduplication. FISH analysis confirmed the chr3p26.3 microduplication in subject 1 and his father (FISH images from subject 1 pictured). Panel order from left to right: composite image, left flanking clone (yellow), right flanking clone (green), clone within the CNV (red). (B) Validation of chr3p26.3 microdeletion. FISH analysis confirmed the chr3p26.3 microdeletion in 2B, 2C and their father (FISH images from subject 2C pictured). Panel order from left to right: composite image, left flanking clone (yellow), right flanking clone (green), clone within the CNV (red). (C) Normal chromosome 3. (D) Chromosome 3 with duplication. Comparison of red signal intensity in (C) versus (D) demonstrates a duplication. (E) Ideogram of clone locations. Three bacterial artificial chromosomes (BACs) were selected for each CNV: a left clone flanking the CNV pseudocoloured yellow (RP11-204C23), a right clone flanking the CNV pseudocoloured green (RP11-95E11), and clone within the CNV pseudocoloured red (RP11-129K1).
Mentions: Genomic DNA from all subjects (and parents, in select cases) was hybridised onto tiling path bacterial artificial chromosome (BAC) arrays for copy number analysis as previously described.10 Image analysis was performed using the BlueFuse package (BlueGnome, Cambridge, UK). A subgrid loss corrected log2 ratio of the background subtracted test/control was calculated for each clone. Fluorescence in situ hybridisation (FISH) validation of genomic DNA CNVs was performed with Phi29 DNA polymerase amplified BAC clones as previously described.11 Three BAC clones were selected for each FISH experiment: (1) a left clone flanking the CNV (proximal to the centromere) was labelled with Biotin (Roche, Nutley, New Jersey, USA) detected with Streptavidin Cy5 (Jackson Immunoresearch Laboratories, West Grove, Pennsylvania, USA) and pseudocoloured in yellow; (2) a right clone flanking the CNV (distal to the centromere) was labelled with Spectrum Green dNTP (1.6 nmole, Vysis, Abbott Molecular Inc, Des Plaines, Illinois, USA) and pseudocoloured in green; and (3) clones within the change were labelled with Spectrum Orange dNTP (1.6 nmole, Vysis) and pseudocoloured in red (fig 1). In all, seven different clones from within the CNVs were tested (tables 1 and 2). Metaphase chromosomes from each subject were derived from normal B lymphocytes as previously described (www.riedlab.nci.nih.gov/protocols.asp). After hybridisation, slides were washed three times in 50% formamide and 2XSSC at 45°C and subsequently in 1XSSC at 45°C. Biotin labelled flanking clones were detected by incubating hybridised slides with a 1:200 dilution of Streptavidin Cy5 Ab. Slides were stained with DAPI and mounted with antifade (phenylene diamine).

Bottom Line: These variations were fluorescence in situ hybridisation (FISH) validated and the end points further delineated using a custom fine tiling oligonucleotide array.CNTN4 plays an essential role in the formation, maintenance, and plasticity of neuronal networks.Disruption of this gene is known to cause developmental delay and mental retardation.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Autism spectrum disorder (ASD) is a developmental disorder of the central nervous system of largely unknown aetiology. The prevalence of the syndrome underscores the need for biological markers and a clearer understanding of pathogenesis. For these reasons, a genetic study of idiopathic ASD was undertaken.

Methods and results: Array based comparative genomic hybridisation identified a paternally inherited chromosome 3 copy number variation (CNV) in three

Subjects: a deletion in two siblings and a duplication in a third, unrelated individual. These variations were fluorescence in situ hybridisation (FISH) validated and the end points further delineated using a custom fine tiling oligonucleotide array. Polymerase chain reaction (PCR) products unique to the rearrangements were amplified and sequence analysis revealed the variations to have resulted from Alu Y mediated unequal recombinations interrupting contactin 4 (CNTN4).

Conclusion: CNTN4 plays an essential role in the formation, maintenance, and plasticity of neuronal networks. Disruption of this gene is known to cause developmental delay and mental retardation. This report suggests that mutations affecting CNTN4 function may be relevant to ASD pathogenesis.

Show MeSH
Related in: MedlinePlus