Analysis of a 1-Megabase Deletion in 15Q22-Q23 in an Autistic Patient
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American Journal of Medical Genetics (Neuropsychiatric Genetics) 96:765–770 (2000) Analysis of a 1-Megabase Deletion in 15q22-q23 in an Autistic Patient: Identification of Candidate Genes for Autism and of Homologous DNA Segments in 15q22-q23 and 15q11-q13 Moyra Smith,* Pauline A. Filipek, Charles Wu, Maureen Bocian, Simin Hakim, Charlotte Modahl, and M. Anne Spence Department of Pediatrics, University of California, Irvine, California We have identified a one megabase deletion sequences or to non-coding homologous in the 15q22-15q23 region in a patient with DNA sequences. The PTPN9 gene encodes a autism, developmental delay, and mild dys- non-receptor protein tyrosine phosphatase. morphism. Genes that map within the dele- The Slp-1 [hUNC24] gene is expressed tion region and genes that are interrupted mainly in the brain. Am. J. Med. Genet. or rearranged at the deletion breakpoints (Neuropsychiatr. Genet.) 96:765–770, 2000. are candidate genes for autism. Fluroes- © 2000 Wiley-Liss, Inc. cence in situ hybridization studies in this patient revealed that part or all of the PML KEY WORDS: autism; 15q22-q23; 15q11-q13; gene is absent from one chromosome 15 and PTPN9; SLP-1[hUNC24] a BAC clone containing the D15S124 gene locus hybridizes to only one chromosome 15. BAC clones containing the PTPN9, and INTRODUCTION SLP-1[hUNC24] genes showed markedly re- duced hybridization in the 15q22-q23 region Autism is characterized by impairments in reciprocal on one chromosome 15 in the patient. These social interaction and communication, restricted and BACs also hybridize to the 15q11-q13 region stereotyped patterns of interests and activities, and the in close proximity to SNRPN and HERC2, presence of developmental abnormalities by 3 years of and in this region there is equal intensity of age [Bailey et al., 1996]. Autism most likely represents signal on the normal and on the deleted a genetically heterogeneous condition. There is evi- chromosome. There are previous reports of dence from linkage studies [Bass et al., 1998] and from deletions and duplications of the 15q11-q13 cytogenetic studies [Steffenburg et al., 1996; Schroer et region in patients with autism. Our patient al., 1998] that a locus on chromosome15q11-15q13 represents the first report of a 15q22-q23 de- plays a role in the development of autism. Schroer et al. letion. Hybridization of the PTPN9 and [1998] proposed that the GABA receptor A genes and Slp-1 Bac clones to the 15q11-q13 and the the UBE3A gene represent candidates for autism. We 15q22-q23 regions of chromosome 15 may be undertook an analysis of chromosome 15 gene loci by due to the presence of PTPN9 or SLP-1 gene fluorescence in situ hybridization (FISH) in a patient sequences or to the presence of other gene with autism, developmental delay, and mild dysmor- phology who exhibited a number of features suggestive of Angelman syndrome. The results of high-resolution karyotype analysis were normal. The Angelman region fluorescence in situ hybridization; was examined by FISH using SNRPN and GABRB3 ס Abbreviations: FISH probes, each in combination with the PML gene as a ס National Center for Biotechnical Information; PCR ס NCBI polymerase chain reaction. control probe for chromosome 15. SNRPN and Contract grant sponsor: National Institute of Child Health and GABRB3 genes mapped to the 15q11-q13 region and Human Development (NICHD); Contract grant number: HD yielded normal signals. The PML gene probe hybrid- 35458 (M.A. Spence, principal investigator); Contract grant spon- sor: Funds from the National Alliance for Autism; Contract grant ized to only one member of the chromosome 15 pair. number: 26165 (Moyra Smith, principal investigator). This finding indicated that there was a deletion in the *Correspondence to: Moyra Smith, Department of Pediatrics, chromosome 15q22-q23 region. Further studies were Medical Sciences 1, Room C214, University of California, Irvine, undertaken to define the extent of the deletion, to rule CA 92697. E-mail: [email protected] out the occurrence of uniparental disomy, and to exam- Received 26 January 2000; Accepted 13 March 2000 ine the SNRPN methylation status to determine © 2000 Wiley-Liss, Inc. 766 Smith et al. whether imprinting defects were present. This is the The following tests revealed no abnormalities: brain- first report of a deletion in the 15q22-q23 region in a stem auditory evoked potentials, computed tomogra- patient with autism. It is likely that the autistic behav- phy scan, creatine phosphokinase levels, routine high- iors in our patient are due to the absence of one copy of resolution karyotype, DNA for fragile-X syndrome, a specific gene or genes (haploinsufficiency) or to the sleep-deprived electroencephalography, free/total/acyl interruption or rearrangement of specific genes within carnitine profile, quantitative serum amino acids, lac- the breakpoint regions. Genes that map within the de- tate, pyruvate, and urine organic acids. Physical and letion region on 15q22-q23 and genes that map at the dysmorphology examinations at 7 years of age revealed deletion breakpoint region therefore represent candi- a height in the 10th percentile, weight in the 5th per- date genes for autism. We identified a series of over- centile, and head circumference at 50th percentile. The lapping BAC clones that map in the distal breakpoint patient (Fig. 1a) has blond hair, light blue lacy irises, region of the deletion in our patient. These overlapping and skin, hair, and eye color much lighter than that of BACs, which contain the sequence of two different the parents. She has a long, narrow face, slightly flat genes, PTPN9, and SLP-1, the human homolog of the occiput with no horizontal groove, minimal epicanthal Caenorhabditis elegans UNC24 gene, hybridize to the folds, slightly concave nasal base and bridge, thin up- 15q22-q23 region and to the 15q11-q13 region. This per lip, normal mouth width, wide dental spacing in finding indicates that homologous DNA sequences ex- the upper jaw, narrow anterior palate with broad lat- ist in the 15q22-q23 region, which is deleted in our eral palatine ridges, relatively prominent pointed chin, patient with autism, and in the 15q11-q13 region, mild pectus excavatum, poor muscular contours of which has been shown to be duplicated deleted or in- thighs and calves, flat nails, lax fingers, and somewhat verted in other cases of autism that have been reported transparent skin on chest and shoulders. The patient is in the literature [Bundey et al, 1994; Steffenburg et al., hypotonic, and deep tendon reflexes are brisk. She 1996; Cook et al., 1997]. walks with an immature gait, often with her arms in an uplifted flexed position. She has occasional episodes of hand flapping and intermittent episodes of excessive CLINICAL REPORT chewing and mouthing behaviors and teeth grinding. The mother was a 37-year-old gravida 3 woman, and There is no tongue protrusion, no excessive smiling, pregnancy was uneventful with no teratogenic expo- and no unprovoked outbursts of laughter. The child has sures. This infant was delivered at term by cesarean frequent infections. The results of a complete develop- section for possible fetal distress, with a birth weight of mental and neuropsychological evaluation, and a quan- 6 pounds 13 ounces. Hypotonia and severe global de- titative brain magnetic resonance imaging scan will be velopmental delay were evident by 6 to 12 months: the presented elsewhere (Filipek et al., unpublished manu- child sat at 10 months, crawled at 20 months, and script). walked at 26 to 29 months. She developed at least seven spontaneous words after 18 months, had good METHODS AND RESULTS eye contact, pointed, and waved bye-bye, before losing all of these skills by 29 months of age. Evaluation for suspected autism was carried out at 4.2 years of age, Cultured lymphoblastoid cell lines were used to pre- when she was evaluated as meeting the criteria for pare slides with metaphase spreads and interphase nu- autistic disorder by the DSM-IV, the Autism Diagnos- clei. FISH was carried out by standard techniques. tic Interview- Revised [Lord et al., 1994], and the Au- Probes for FISH included commercially available tism Diagnostic Observation Schedule-Generic [Lord et probes from Oncor, Gaithersburg, MD [GABRB3/PML al., 1989]. The Autism Diagnostic Interview algorithm and SNRPN/PML] and Vysis, Downer’s Grove, IL [SN- scores are presented in Table I. The child’s medical RPN and PML/RARA]. We isolated a series of BAC history is significant for a lack of feeding problems, clones for loci in the 15q22-q23 region [National Center excessive drooling, sleep disturbance, hypermotoric be- for Biotechnology Information (NCBI), 1999]. BAC havior, gait ataxia, or seizures. The infant was noted to clones corresponding to D15S124 and D15S160 genes have strabismus, which was treated by eye patching. were isolated by screening BAC libraries with oligo- nucleotide primers corresponding to these loci. BAC clones containing sequences corresponding to the genes TABLE I. Algorithm Scores for the Autism TLE3, PTPN9, PML, SLP1(hUNC24), CHRNA5, and Diagnostic Interview ETFA were identified by searching for genomic DNA Patient Autism clones with matching sequences in the NCBI genome Categories score cutoffa survey sequence database. BAC clones thus identified were ordered from Research Genetics, Huntsville, AL. Qualitative impairment of reciprocal social interaction 20 10 Individual BAC clones were cultured, and DNA was Impairment of communication 14 7 isolated and used for polymerase chain reaction (PCR) Repetitive behaviors and to confirm that each BAC clone was positive for the stereotyped patterns 7 3 corresponding gene sequences. Purified BAC DNA was Abnormal development labeled with spectrum green or spectrum orange (Vy- evident before 36 months 5 1 sis), using the Vysis labeling system. Labeled BAC aNote that scores at or above the cutoff indicate autism DNA was hybridized to human Cot 1 DNA to block Analysis of 1Mb Deletion in 15q22-q23 in an Autistic Patient 767 Fig.