Disruption at the PTCHD1 Locus on Xp22.11 in Autism spectrum disorder and intellectual disability

Abstract

Autism is a common neurodevelopmental disorder with a complex mode of inheritance. It is one of the most highly heritable of the complex disorders, although the underlying genetic factors remain largely unknown. Here, we report mutations in the X-chromosome PTCHD1 (patched-related) gene in seven families with autism spectrum disorder (ASD) and in three families with intellectual disability. A 167-kilobase microdeletion spanning exon 1 was found in two brothers, one with ASD and the other with a learning disability and ASD features; a 90-kilobase microdeletion spanning the entire gene was found in three males with intellectual disability in a second family. In 900 probands with ASD and 208 male probands with intellectual disability, we identified seven different missense changes (in eight male probands) that were inherited from unaffected mothers and not found in controls. Two of the ASD individuals with missense changes also carried a de novo deletion at another ASD susceptibility locus (DPYD and DPP6), suggesting complex genetic contributions. In additional males with ASD, we identified deletions in the 5' flanking region of PTCHD1 that disrupted a complex noncoding RNA and potential regulatory elements; equivalent changes were not found in male control individuals. Thus, our systematic screen of PTCHD1 and its 5' flanking regions suggests that this locus is involved in ~1% of individuals with ASD and intellectual disability.

Fig. 1

Detailed genomic organization of the PTCHD1 locus. The known genes, predicted CpG islands (>300 bp), predicted promoters (ElDorado Suite from Genomatix) and conserved sequences (>75% identity with chicken, >90% identity with opossum or 100% identity with dog or horse) are shown. Putative non-coding RNA transcripts PTCHD1AS1 (from cDNA clone IMAGE:1560626; BX115199) and PTCHD1AS2 (cDNA clone BRSTN2000219; DA355362) from human, mouse and rat genomes are also shown, with the transcripts assembled from RT-PCR and 5’ RACE (PTCHD1AS3) (see Supplementary Material). The dotted line between the two exons in transcript PTCHD1AS1 indicates that this is a putative exon, identified through clone sequencing. This exon is putative because, although this location represents its best genomic hit, it only partially matches the 5’ end of the clone sequence. Black boxes within the spliced transcripts indicate homologous exons between the sequences. White bars with black borders indicate CNV losses within this locus that have been identified in patients with ASD and controls. Cross-hatched or grey bars indicate CNV losses identified in patients with ADHD and ID, respectively. Colored lines within these bars indicate overlap with exons of known transcripts (blue) or ncRNA (red). The breakpoints of the deletions for all families that are reported here were mapped by sequencing the junction (see table S2 for coordinates). Breakpoints for all CNVs in controls were mapped by using the physical positions of microarray probe fragments.

Fig. 2

Pedigrees of families. (A) Pedigrees showing PTCHD1 mutations. (B) Pedigrees showing deletions at the PTCHD1/PTCHD1AS1-3 locus. The third male in Family 18 was assessed at age 4 and had speech and language problems, but was not available for further assessment. The father in Family 19 has a broader autism phenotype (BAP) (14, 15). The proband in Family 20 (hatched) has ADHD plus BAP. A diamond symbol represents siblings who were not tested as part of the study, and with gender not indicated.

Fig. 3

Transcription analysis. (A) RT-PCR expression analysis of PTCHD1 transcript in 30 different adult tissues. The housekeeping gene G3PDH was used as a control. (B) Northern hybridization analysis of PTCHD1 showing a ~4.1Kb band in all lanes. Current RefSeq annotation of PTCHD1 describes a ~5.3Kb transcript; however, the only polyadenylation site predicted for the mRNA sequence (NM_173495) by POLYAH is at 4.379 bp. RT-PCR expression analysis of (C) PTCHD1AS1 and (D) PTCHD1AS2 expression in seven human tissues, also with G3PDH as a control. Northern analysis of the ncRNAs did not give sufficient signal for detection.

Fig. 4

Expression and functional studies. (A) Whole-mount RNA in situ hybridization showing expression of PTCHD1 in mouse embryo E9 and E14. (B) Localization of PTCHD1 protein in COS7, SK-N-SH and control cells shows that the PTCHD1-GFP protein is predominantly localized in the cell membrane. (C) PTCHD1 exerted a statistically significant inhibitory effect on endogenous Gli-dependent transcription, similar to PTCH1 and PTCH2, when transfected in Hedgehog-responsive 10T1/2 cells (PTCHD1: p= 0.0101; PTCH1: p= 0.0096; PTCH2: p= 0.0159). Statistical significance was calculated using the Student’s t-test. Absolute expression of reporter gene normalized to β-gal expression is shown. Standard error bars are shown.