PARK2 copy number aberrations in two children presenting with autism spectrum disorder: Further support of an association and possible evidence for a new microdeletion/microduplication syndrome

Abstract

Microdeletions of PARK2 have been reported previously in seven patients with autism spectrum disorder. There are no reports of PARK2 microduplications in this population. Presented are two patients, one with deletion and the other with duplication, both with autism spectrum disorder, though their syndromic phenotypes vary. The deletion patient is cognitively normal and ectomorphic: the duplication patient is cognitively impaired, underweight and short. Further, the microduplication patient has demonstrated adverse medication reactions to psychotropic medications active in the dopamine metabolic pathway: cyclopentolate, lisdexamfetamine, methylphenidate. These patients support an association between PARK2 mutations and autism spectrum disorder and suggest that duplications may be equally causative. It is hypothesized that the disparate patient phenotypes may represent a deletion/duplication syndrome and that the adverse medication reactions may be a pharmacogenetic phenomenon. © 2011 Wiley-Liss, Inc.

INTRODUCTION

Autism spectrum disorder (ASD) comprises a defined set of aberrant behavioral signs and symptoms that have a multitude of potential causes. Being a symptom complex rather than a specific diagnosis, patients with ASD have been found to have single gene mutations as well as cytogenetic aberrations identified by conventional analysis and fluorescence in situ hybridization (FISH). The more recent technology of cytogenomic microarray analysis (CMA) has identified an increasing number of copy number gains and losses present in patients with ASD [Cook and Scherer, 2008; Glessner et al., 2009; Colak et al., 2011] In 2009, Glessner et al. identified seven patients with ASD who had a chromosome 6 copy number loss involving the PARK2 gene region. Control patients did not show this copy number loss. To our knowledge, there are no reports of ASD with duplication of PARK2. Reported here are two cases of males with ASD who were found to have copy number aberrations involving the PARK2 gene, one with deletion and one with duplication.

CASE REPORTS

Patient #1

This was a male patient seen at 14 years, 4 months with a referral diagnosis of Asperger syndrome. He was delivered at 36 weeks to a 27-year-old G2P0-1EAb1 mother. The pregnancy was complicated by preterm labor in the third trimester. Ultrasounds were normal. Amniocentesis karyogram was normal male: 46,XY. (Testing was done because mother's previous pregnancy resulted in a fetus with 45,X.) Birth weight was 2,950 g. Birth length was 50 cm. There were no neonatal problems.

Mother had concerns from infancy. The patient had vocal cord nodules removed and had a persistently hoarse voice. He had a frenulectomy and was to have periodontal surgery. He fractured an ankle in a fall. His general health had been good. There had been no hospitalizations. He was on numerous psychotropic medications to treat attention deficit disorder and a diagnosis of Asperger syndrome: methylphenidate, duloxetine, and aripiprazole. His hearing and sight were normal. He was noted to have minimal sleep need and would not sleep without medications mirtazapine and clonazepam. He had somniloquy. His diet was varied, but consisted largely of carbohydrates and fried foods. His activity level was low.

At 14 years, 4 months of age, he was in 8th grade regular and so called Gifted and Talented classes in public school. He was anticipated to graduate early with plans to attend college. He was in a social skills class. He had been discharged from speech therapy. Other than the mother's first pregnancy, there was no family history of birth defects, cognitive impairment, or pregnancy losses.

His weight was 84 kg (≫98th centile). Height was 165 cm (50th centile). Head circumference was 56.5 cm (75th centile). (Fig. 1). He was brachycephalic. He had nystagmus on lateral and particularly lateral upward gaze. His eyes were deeply set with long, horizontal palpebral fissures. The eyelashes were long. He had upturned ear lobes. His oropharynx was normal. He had a hoarse voice. His cardiovascular and pulmonary exams were unremarkable. He had a symmetric chest and a dowager hump. Visceral exam was limited by obesity. He was a circumcised Tanner I–II male with a buried penis. The left testis was descended. The right testis was not palpable. There was no pubic hair. His extremities were symmetric with tapered fingers and short 4th metacarpals. His feet were normal as were the creasing patterns and nails. He had multiple dark nevi and striae on his trunk. He was generally hypotonic with a hoarse voice, though his articulation was good. There was no tremor or ataxia. He was cooperative with the exam, but acted younger than his stated age.

Figure 1. Patient #1 with PARK2 deletion.

Patient #2

This was a male patient first seen at 10 years, 7 months with a referral diagnosis of Asperger syndrome and cognitive impairment. He delivered at 41 weeks to 31-year-old G3P102SAb1 mother. The pregnancy was uncomplicated. Ultrasounds were reported to have been normal. Birth weight was 3,835 g. Birth length was 53.3 cm. Concern was raised within the first year of life because of poor growth and delayed developmental milestones. He was diagnosed with hypotonia at 2 years of age. His general health was good. There was one overnight hospitalization at age 4 because of dehydration. He was on psychotropic medications to treat a diagnosis of autism spectrum disorder. He was also using melatonin because of a sleep disorder. He had chronic constipation managed with an osmotic laxative. Hearing was normal. He had strabismus and ambylopia with hyperopia in the contralateral eye. He had been in glasses since age 3.

There is a history of adverse reactions to medications. At age 7 years, 2 months he had an adverse reaction to routine use of cyclopentolate 2% eye drops consisting of transient systemic neurologic compromise. On lisdexamfetamine he complained of chest pain that resolved with discontinuation of the medication. On methylphenidate he demonstrated twitching and eye blinking that likewise resolved when the medication use was suspended.

Early developmental milestones were delayed: he walked independently at 18 months. His first words were at 30 months. He was globally delayed with particular problems in social and language skills. He had received a social promotion into 5th grade, which he was repeating. He was mainstreamed with an assistant and time out of the classroom for services. He generally performed better in a smaller classroom with more attention.

Family history was significant for a paternal first cousin with some sort of cognitive impairment. The mother was not examined directly, but reported no neurologic problems.

On exam at 10 years, 7 months, the patient was 20.5 kg (<5th centile). Height was 130 cm (<5th centile). Head circumference was 52.5 cm (20th centile). (Fig. 2). He had relative macrocephaly and a triangular face. His gaze was conjugate. His eyes were deeply set with upslanting palpebral fissures. His ears were large and low set with small lobes. His nose was large with a prominent tip. The midface was flattened. He had a normal philtrum with exaggerated Cupid's bow and patulous lips. The palate was high and he had delayed dentition. His cardiovascular and pulmonary exams were unremarkable. There were no abdominal masses or organomegaly. He was a circumcised Tanner I male with bilaterally descended testes. His extremities showed a generalized decrease in subcutaneous tissue and were long. His feet were relatively small. He had normal creasing patterns, digits, and nails. There were no unusual skin findings. He was generally hypotonic. Finger-to-nose procedure showed no tremor, but inability to touch the target. There was no ataxia. He was distractable and required re-direction, though this improved with subsequent exams. He was noted to prefer exam room toys geared toward younger children.

Figure 2. Patient #2 with intragenic PARK2 duplication.

MATERIALS AND METHODS

Conventional Cytogenetic Analysis

Dividing cells were harvested from peripheral blood cultures stimulated with phytohemagluttinin. Trypsin G-banding was performed using standard methods [Barch et al., 1997]. Briefly, metaphase cells were obtained by colcemid arrest followed by hypotonic treatment with pre-warmed 0.075 M KCl. They were then fixed and washed in freshly made modified Carnoy's fixative (3:1 absolute methanol/glacial acetic acid), dropped onto pre-cleaned wet microscope slides and air-dried. Cytogenetic abnormalities were classified according to the International System for Human Cytogenetic Nomenclature [Shaffer et al., 2009].

Cytogenomic Microarray Analysis

Oligonucleotide-based cytogenomic microarray analysis was performed with DNA extracted from peripheral blood using a 135k-feature whole genome microarray according to the manufacturer's protocol (Roche Nimblegen, Inc; Madison, Wisconsin). Data interpretation was performed using the Genoglyphix software (Signature Genomics; Spokane, Washington).

Fluorescence In Situ Hybridization (FISH)

FISH of a DNA probe set (RP11-965F3 and RP11-164C22; Empire Genomics; Buffalo, New York) was hybridized to metaphase cells and interphase nuclei from the peripheral blood cultures of Patient #1 according to the manufacturer's protocol. RP11-965F3 was labeled with spectrum orange and was specific for the locus of interest, the 6q26 band region at locus chr6:162,977,038–163,172,620. RP11-164C22 was labeled with spectrum green for use as an internal control, and was specific for the 6q11.1 band region at locus chr6:62,379,465–62,577,198. FISH of a DNA probe set utilizing a different probe for the locus of interest (RP11-148P13 at chr6:162,690,146–162,851,341, spectrum orange, and RP11-164C22, spectrum green; Empire Genomics) was also hybridized to metaphase and interphase nuclei from blood cultures of Patient #2 according to the manufacturer's protocol.

RESULTS

Conventional cytogenetic analysis from stimulated peripheral blood cultures of Patient #1 showed a normal male karyogram (46,XY), confirming the prenatal diagnosis from an amniotic fluid specimen. Cytogenomic microarray analysis of DNA extracted from Patient #1 showed a 118.67 kb copy number loss at locus chr6:162,962,435–163,081,102 (Fig. 3A) encompassing the PARK2 and PACRG genes. FISH analysis confirmed this copy number loss with a diminished signal (dim) for the locus of interest, when compared with the intensity of this probe signal on the normal chromosome 6 homolog as well as that of a concurrent external control (Fig. 3B). This is the expected confirmatory signal pattern since the FISH probe spanned approximately 50% of the patient's deleted region. The mother declined testing. The father was not available. Patient #1's complete karyogram was: 46,XY.ish del(6)(q26q26)(RP11-965F3 dim).arr 6q26(162,962,435-163,081,102)x1.

Figure 3. A: Cytogenomic mocroarray analysis of the 6q26 band region of Patient #1 showing a copy number loss at chr6:162,962,435–163,081,102. B: FISH analysis of a metaphase cell (left) and interphase nucleus (right) from Patient #1 demonstrating a diminished signal (dim) on one chromosome 6 homolog consistent with the presence of a copy number loss (indicated with the arrow). Locus of Interest labeled with spectrum orange. Control probe at 6q11.1 labeled with spectrum green. These FISH findings are consistent with a deletion in the 6q26 band region and the microarray results.

Conventional cytogenetic analysis from stimulated blood cultures of Patient #2 showed an abnormal male karyogram with a paracentric inversion in the short arm of one chromosome 6, 46,XY,inv(6)(p12.2p25). Cytogenomic microarray analysis of DNA extracted from Patient #2 showed a 161.21 kb copy number gain in the long arm of one chromosome 6 at locus chr6:162660383–162821588 (Fig. 4A) which was confirmed by FISH (Fig. 4B) The mother was found to also have this aberration by FISH. Patient #2's complete karyogram was: 46,XY,der(6)inv(6)(p12.2p25) dup(6)(q26q26).ish dup(6)(q26q26)(RP11-148P13 enh).arr 6q26(162,660,383-162,821,588)x3mat.

Figure 4. A: Cytogenomic microarray analysis of the 6q26 band region of Patient #2 showing a copy number gain at chr6:162,660,383–162,821,588. B: FISH analysis of a metaphase cell (left) and interphase nucleus (right) from Patient #2 demonstrating an enhanced signal (enh) on one chromosome 6 homolog consistent with the presence of a copy number gain (indicated with the arrow). Locus of Interest labeled with spectrum orange. Control probe at 6q11.1 labeled with spectrum green. These FISH findings are consistent with a tandem duplication in the 6q26 band region and the microarray results, and exclude insertion of the duplicated region of chromosome 6 into another chromosome.

DISCUSSION

There is a documented phenotype for patients with terminal 6q deletions who have been identified both by conventional cytogenetics and molecular cytogenetics, for example, FISH. This includes cognitive impairments, hypotonia, seizures and a defined facies [Bertini et al., 2006; Backx et al., 2010]. Backx et al. report a patient with 6q26 deletion resulting from a t(5;6) translocation. They provide a review of the literature cases and further identify disruption of the QKI gene (see below) as possibly contributing to the phenotype [Backx et al., 2010].

Involvement of 6q in isolated autism was first reported in a linkage study by Weiss and Arking [2009]. They performed a genome-wide association study of 1,031 multiplex autism families and found suggestive linkage to 6q27 (LOD = 2.94). Glessner et al. carried out a case–control study of autism spectrum disorder using a 550k single nucleotide polymorphism (SNP) microarray. They identified three cases in their study cohort and four cases (including one sib pair) in the Autism Genetic Resource Exchange collection as having deletions specifically of PARK2. There were no such deletions in the control samples [Glessner et al., 2009].

The PARK2 gene codes for Parkin, an E3 ubiquitin-protein ligase and transcriptional repressor of p53 [Kitada et al., 1998; da Costa et al., 2009]. The gene is 1,380 kb in length with 12 exons [Kitada et al., 1998; Asakawa et al., 2001]. It is linked head-to-head with PACRG with a 198 bp interval and the genes share a common promoter [Asakawa et al., 2001; West et al., 2003]. Parkin is a cytoskeletal-associated protein that is expressed in neuronal processes and cell bodies of midbrain, basal ganglia, cerebral cortex, and cerebellum [Huynh et al., 2000].

Intracellularly, in addition to generalized presence in the cytoplasm, Parkin is associated with mitochondrial DNA, where it appears to serve in the mtDNA repair pathway and with a number of other proteins [Rothfuss et al., 2009]. This may indicate the link between Parkin and autism if the current work implicating mitochondrial abnormalities in autism spectrum disorders bears fruit [Giulivi et al., 2010]. Underexpression leads to endoplasmic reticulum accumulation of the Parkin-associated endothelin receptor-like receptor (PAEL-R) that Parkin would otherwise have ubiquinated for ligation [Imai et al., 2001]. This accumulation is presumed to cause neural cell death and decreased protection against stress-induced apoptosis [Shimura et al., 2000; Imai et al., 2002; Jiang et al., 2004]. Overexpression of PARK2 seems to be selectively protective against stress-induced apoptosis [Jiang et al., 2004].

PARK2 is mutant in autosomal recessive juvenile parkinsonism (PDJ; MIM 600116), also referred to as parkin-type juvenile Parkinson disease. The physical signs and symptoms are consistent with parkinsonism and the histologic brain abnormalities include neuronal loss and gliosis of the substantia nigra. There are no Lewy bodies as are typically seen in classic Parkinson Disease [Takahashi et al., 1994]. Various other genotypes of PARK2 have been associated with Parkinson disease in general and with early-onset adult Parkinson more specifically [Hedrich et al., 2001; Lesage et al., 2008; Wang et al., 2008]. There does not appear to be a genotype–phenotype correlation with this type of mutation [Lücking et al., 2000; Brice et al., 2007]. One study of parkinsonism showed heterozygous mutations in PARK2 in a statistically significant number of apparently unaffected individuals while being absent in affected patients. All affected patients were homozygous for a mutation or were compound heterozygotes [Kay et al., 2010]. Additionally, PARK2 mutations have been implicated in other neuropsychiatric diseases including schizophrenia [Cook and Scherer, 2008] and Alzheimer disease [Locke, 2006].

Parkinson disease is a neurodegenerative disorder resulting from loss of dopamine-producing neurons in the midbrain. The classic clinical features include resting tremor, muscular rigidity, bradykinesia, and postural instability. There are no apparent reports of whole gene deletion or duplication in Parkinson, early onset Parkinson or PDJ disease patients, though our search was likely not exhaustive. It is interesting to note that knock-out mice do not have substantia nigra degeneration [Brice et al., 2007]. Khan did note that PARK2 mutation patients may have psychiatric symptoms or abnormal behaviors prior to the onset of physical neurologic symptoms [Khan et al., 2003]. Both dementia and hallucinations have been reported in Parkinson patients [Paleacu et al., 2005].

The association between dopamine metabolism and autism spectrum disorders is under study. In 2004, Nieminen-von Wendt et al. noted increased dopamine function in the putamen and caudate nucleus and in the frontal cortex of Asperger syndrome patients by positron emission tomography [Nieminen-von Wendt et al., 2004]. This has been reinvestigated a number of times. One of the more recent reports from Japan notes abnormalities in both serotonin and dopamine transporter binding in adults with autism [Nakamura et al., 2010].

Patient #1's deletion comprises the 5′ ends of both PARK2 and PACRG as well as their shared promotor. Presuming that the promotor acts only in cis, this deletion leads to functional heterozygous loss of both genes in their entirety. There are no previous reports in humans of this isolated deletion; however, there is a mouse model. The quakingviable mouse has an approximately 1.1 Mb deletion encompassing the quaking gene (Qki as identified in Backx's patient), Pacrg, and Park2. The mouse phenotype includes ciliary dysfunction and central nervous system dysmyelination [Lorenzetti et al., 2004; Wilson et al., 2010].

Patient 2 has an intragenic duplication, which, as mentioned above, is similar to genotypes seen in patients with parkinsonism. More detailed molecular studies would be necessary to define the precise duplication. Of the two patients, he has the more severe phenotype in terms of both cognitive development and neuromuscular symptoms. The adverse medication reactions of patient #2 are interesting and may reflect a dyscrasia of dopamine metabolism.

Lisdexamfetamine dimesylate (trade name Vyvanse; Shire) is a pro-drug of dextroamphetamine. It is approved for treatment of Attention-Deficit/Hyperactivity Disorder (ADHD). It is a non-catecholamine (i.e., adrenergic) sympathomimetic amine, but the specific mode of therapeutic action is not known [Shire, 2007]. Methylphenidate (trade name Concerta; Ortho) is also an amphetamine approved for ADHD treatment. Tics are a recognized adverse reaction to methylphnidate [Ortho, 2010]. Amphetamines are thought to block the reuptake of dopamine and norepinephrine in presymaptic neurons while increasing their release in the synapse.

Cyclopentolate (trade name Cylcogyl; Alcon) is a common mydriasis medication used in routine ophthalmologic examination. It is an antimuscarinic, anticholinergic compound, so is presumed to block release of neurotransmitter from the presynaptic neuron. The adverse reactions experienced by the patient—ataxia, slurred speech, confusion—are known rare side effects presumably due to systemic absorption [Alcon, 2004]. It is hypothesized that the adverse reactions are pharmacogenetic in this patient because all three medications have a mechanism of action presumed to involve dopamine, and dopamine metabolism is aberrant in persons with PARK2 mutations. It is reported that dopa-induced complications can be severe in PDJ [Kitada et al., 1998; Pankratz et al., 2009].

As noted in the case report, the mother of patient 2 also carries the small duplication. She is neurologically normal by history, but has never had a detailed neurologic examination or brain imaging study. Intragenic duplications have been seen in some persons enrolled as controls in case-control studies and presumably showing no signs or symptoms of parkinsonism [Pankratz et al., 2009; Kay et al., 2010]. Also, familial copy number variants are no longer considered benign if discovered in apparently unaffected family members. As with many dominant point mutation conditions, it is recognized that there can be variable penetrance [Redon et al., 2006]. It is therefore difficult to predict the mother's risk of developing Parkinson disease, though she has been informed of the risk and advised to consult with a neurologist.

CONCLUSION

Presented are two cases which reinforce the observed relationship between PARK2 copy number aberrations and autism spectrum disorder, and the first reported case of a copy number gain (duplication) in this gene region specifically in a patient with autism spectrum disorder. One case has a promotor deletion, which implies a hemizygous activity of the gene and the related PACRG gene. His phenotype is not generally consistent with, or is milder than, that of other 6q deletion patients. There does not appear to be previous report of isolated PARK2 null mutation, though there is a mouse model. The other case represents a case of autism spectrum disorder with a mutation more in line with those seen in previous parkinsonism reports, but the child manifests a phenotype that is not parkinsonism. His adverse reactions to neuroleptic medications may indicate a specific, and identifiable, pharmacogenetic effect. His apparently unaffected mother is at unknown risk of parkinsonism in the future.