How to cite this article: Herman GE, Butter E, Enrile B, Pastore M, Prior TW, Sommer A. 2006. Increasing knowledge of PTEN germline mutations: Two additional patients with autism and macrocephaly. Am J Med Genet Part A 143A:589–593.
Increasing knowledge of PTEN germline mutations: Two additional patients with autism and macrocephaly†
Article first published online: 7 FEB 2007
Copyright © 2007 Wiley-Liss, Inc.
American Journal of Medical Genetics Part A
Volume 143A, Issue 6, pages 589–593, 15 March 2007
How to Cite
Herman, G. E., Butter, E., Enrile, B., Pastore, M., Prior, T. W. and Sommer, A. (2007), Increasing knowledge of PTEN germline mutations: Two additional patients with autism and macrocephaly. Am. J. Med. Genet., 143A: 589–593. doi: 10.1002/ajmg.a.31619
- Issue published online: 22 FEB 2007
- Article first published online: 7 FEB 2007
- Manuscript Accepted: 9 NOV 2006
- Manuscript Received: 26 JUL 2006
Recently, Butler et al. [2005; J Med Genet 42:318–321] reported the presence of heterozygous germline mutations in the PTEN tumor suppressor gene in three children with autism and macrocephaly. Here, we report the presence of PTEN mutations in two additional unrelated children with macrocephaly and autism. Our findings extend those of Butler et al. and suggest that PTEN gene sequencing should be included in the genetic evaluation of this subset of autistic individuals. © 2007 Wiley-Liss, Inc.
“Autism spectrum disorders” (ASDs) are a heterogeneous and common group of developmental disorders. Affected infants and children demonstrate severe and persistent impairments in expressive and receptive language and social interaction skills that are evident before age 3, and a restricted range of interests and stereotypic behaviors. The Centers for Disease Control now estimates the prevalence of ASDs (including autism, Asperger syndrome, and pervasive developmental delay not otherwise specified (PDD-NOS)) as approximately 5.6 per 1,000 children within the United States [MMWR, 2006].
Although the causes of most ASD cases are currently unknown, there are many scientific studies that demonstrate strong heritability and genetic components to the causes of autism. These include familial aggregation and increased recurrence risks for siblings (2–8%, 10–60 times the general population risk); twin studies with concordance in monozygotic twins of 60–90% and of 0–10% in dizygotic twins; and a sex preference, with a male/female ratio of 3–4:1 [Muhle et al., 2004; Spence, 2004; Wassink et al., 2004; MMWR, 2006]. There are several single gene disorders (e.g., fragile X syndrome, Rett syndrome, untreated PKU, and tuberous sclerosis) and chromosome abnormalities (duplication of 15q11 containing the Prader–Willi/Angelman region) in which autistic manifestations are prominent and common [reviewed in Cohen et al., 2005]. However, known genetic causes account for ≤10% of cases. It is estimated that 3–15 to >100 genes, in addition to environmental effects, contribute to “disease” susceptibility.
Several large collaborative studies in the United States and other countries are ongoing with goals of identifying these genes [Geschwind et al., 2001; IMGSAC, 2001; Cantor et al., 2005; McCauley et al., 2005; Ylisaukko-Oja et al., 2006], although results, to date, have been disappointing and/or inconclusive. Such research has been hampered, in part, by a lack of comprehensive clinical phenotyping or performance of appropriate and comprehensive genetic testing of affected individuals to rule out known causes.
Several recent reports have addressed the appropriate genetic evaluation and diagnostic yield in children with proven or suspected ASDs [Challman et al., 2003; Abdul-Rahman and Hudgins, 2006; Battaglia and Bonaglia, 2006; Battaglia and Carey, 2006], including a recent report in which the authors identified heterozygous mutations in the PTEN tumor suppressor gene in three patients with macrocephaly and autism [Butler et al., 2005].
Heterozygous germline mutations in the human PTEN (phosphatase and tensin homolog, deleted on chromosome 10) gene were first described in Cowden syndrome (CS; MIM #158350), a rare autosomal dominant syndrome characterized by multiple hamartomas and increased cancer risk, particularly for breast and non-medullary thyroid tumors [Nelen et al., 1997; Eng et al., 2001; Zbuk et al., 2006]. Subsequently, based on the findings of macrocephaly, lipomas, and/or mental retardation in subsets of patients with CS, heterozygous mutations in PTEN were discovered in patients with Bannayan–Riley–Ruvalcaba syndrome (BRRS; MIM #153480), Proteus syndrome (PS; MIM #176920), and Proteus-like disorders [reviewed in Eng, 2003; Pilarski and Eng, 2004; Zbuk et al., 2006]. Recent data suggest that mutations in PTEN are identified in at least 80% of individuals fulfilling criteria for CS, 60% of BRRS patients, and 20% and 50% of Proteus and Proteus-like patients, respectively [Eng, 2003; Zbuk et al., 2006]. Because of phenotypic overlap among these disorders, they have collectively been termed the PTEN hamartoma tumor syndrome (PHTS).
In 2005, in collaboration with developmental pediatricians at our institution, we developed a guideline for referral and genetic testing of children diagnosed with an ASD. Based on the report of Butler et al. 2005, our protocol includes screening for PTEN mutations in infants and young children with autism and/or significant developmental delay and absolute or relative macrocephaly. As part of a retrospective chart review, we report here on two additional children with macrocephaly, autism, and heterozygous mutations in the human PTEN gene among 16 individuals tested over a period of 15 months. Our data confirm and extend the findings of Butler et al. 2005 and support a recommendation for genetic testing in this population.
The patient is a 27-month-old girl referred at 16 months for evaluation of macrocephaly and developmental delay. She was the 4.1 kg, 53 cm product of a 39-week gestation born by induced vaginal delivery to a 30-year-old primigravid woman. The pregnancy was complicated by Factor V Leiden requiring daily injections of enoxaprin. Decreased fetal movement was noted throughout the pregnancy. Routine fetal ultrasound imaging and results of maternal serum screening were normal. Head circumference at birth was 39 cm (>98%).
The infant was discharged at age 2 days. She has no chronic health problems, and her only hospitalization was at 23 months for an episode of rotavirus infection. She was evaluated by neurosurgery at 6 months for macrocephaly and subsequently has had a normal head CT and brain MRI. By report, she crawled at age 1 year and walked at 16 months. At 16 months, she was evaluated by a developmental specialist. At that time, her weight was 13.2 kg (97%), length 80 cm (55%), and head circumference 54 cm (>98%). She was noted to have a broad forehead with obvious macrocephaly, but no other minor anomalies. She also had mild joint hyperextensibility. Her only words at that time were “mama” and “dada,” although she had polysyllabic babbling and was very interactive. Her language age equivalent was 12 months. She could feed herself finger foods and drink from a sippy cup, but would only follow one-step commands intermittently. Results of laboratory testing including high-resolution peripheral blood karyotype, DNA for fragile X syndrome, urine for quantitative mucopolysaccharides, and FISH for subtelomeres were all normal.
She returned to the Developmental Clinic for follow-up at age 25 months. At this time, she had a vocabulary of 10–20 single words. She was considered to have manifestations of autism, with deficient eye contact, parallel play, short attention span, extreme stranger anxiety, and minimal progress in her expressive language. She also had some stereotypic behaviors, including flapping her hands when excited. A Childhood Autism Rating Scale (CARS) score was 32.5, in the mildly to moderately autistic range. At this time, PTEN exons 1–9 were sequenced, in both forward and reverse directions, and a heterozygous single base insertion was identified in exon 6 (c.519_520insT). The insertion creates a reading frameshift at codon 175 of the 403 amino acid protein and premature termination at codon 179.
The family was seen for counseling at 27 months. The girl spoke approximately 20 words, and could make her wants known by pointing or leading her parents. She was receiving occupational and physical therapy and had been enrolled in an intensive applied behavioral analysis (ABA) program. The family history was unremarkable except for the Factor V Leiden mutation in the mother and the mother's head circumference of 58 cm (98%). The father's head circumference was within the normal range. The child's weight and height were 80% and 65%, respectively. Her head circumference at this time was 55.5 cm (>98 % and 50% for an adult female). She had a round face with broad forehead, mild hypertelorism, and slightly upslanting palpebral fissures. There was midface hypoplasia with a depressed nasal bridge and short nose. She did not resemble either of her parents. She had obvious global developmental delays and became fussy and irritable during the exam. No mucocutaneous lesions were noted on examination of both parents, and results of DNA sequencing of exon 6 of the PTEN gene in both parents were normal.
At 27 months, the patient also had a psychological evaluation. On the Mullen Scales of Early Learning, she obtained an Early Learning Composite standard score of 61, reflecting mild cognitive impairments with delays across fine motor and receptive and expressive language subscales. Her visual reception (e.g., nonverbal cognitive abilities) was within the low average range. She obtained a Developmental Profile: Second Edition (DP-II) IQ Equivalence score of 88. On the Scales of Independent Behavior-Revised (SIB-R), she obtained an adaptive behavior standard score of 71, indicating borderline impairment regarding functional independence affecting social interaction, language expression, toileting, dressing, and self-care. She was not responsive to structured language or nonverbal cognitive testing. A diagnosis of autism was corroborated independently with a score of 35.5 on the CARS completed by the psychologist.
A follow-up psychological evaluation was completed at 3 2/12 years, one year after being enrolled in a 20–30 hr per week ABA program. Her Full Scale IQ standard score on the Stanford-Binet Intelligence Scales: Fifth Edition was 92 and within the average range. This was substantial improvement over the comparable score from the Mullen (e.g., Early Learning Composite standard score of 61) one year earlier. Her nonverbal IQ score on the Leiter International Performance Scale: Revised was also within the average range at 109. Language testing documented an average receptive and expressive vocabulary level. Gains in adaptive behavior were also evident with a standard score of 93, within the average range, on the SIB-R. However, there were remaining manifestations of autism as indicated by an Autism Diagnostic Observation Schedule (ADOS) classification of “Autism Spectrum” [Lord et al., 1989].
The patient was seen at age 4 years after an evaluation in our Developmental Disabilities Clinic. He was born at 41 weeks by induced vaginal delivery with forceps assisted vacuum extraction to a 32-year-old G2P2 mother after an uncomplicated pregnancy, weighing 3.8 kg. There were no neonatal complications, and the baby was discharged home with his mother. At 1 month, his weight was 4.5 kg (50%), length 57.8 cm (75%), and head circumference 40 cm (98%).
The baby did well until age 12 months when he developed petechiae, and at 18 months, a diagnosis of idiopathic thrombocytopenic purpura (ITP) was made. Platelet counts were performed at regular intervals and he is currently asymptomatic. The patient's mother has chronic ITP and has had a splenectomy.
The patient was also noted to have mild global developmental delays. He walked at 16 months. At his initial evaluation in the Developmental Clinic at 2½ years, he had no intelligible speech. He babbled and conveyed his needs by leading and gesturing. He also had some unusual behaviors, such as hand flapping, tactile sensitivity, and deficient social interaction and eye contact. His weight was 15.6 kg (90%), height 95 cm (80%), and head circumference 54 cm (>98%). A Developmental Profile: Second Edition quotient of 63 was obtained based on parental report and the examiner's observations. A CARS score was 38.5, in the moderately to severely autistic range. A diagnosis of autism based on DSM-IV criteria with macrocephaly was made. Evaluations at that time included chromosomes and DNA for fragile X syndrome, with normal results. An MRI demonstrated small areas of gliosis within the periventricular white matter adjacent to the posterior body and atria of the lateral ventricles that were not considered to be clinically significant.
A psychological evaluation was completed at 2 years, 9 months, validating a diagnosis of autistic disorder and mild mental retardation. On the Mullen scale, he obtained an Early Learning Composite standard score below 49, with significant delays across gross motor, fine motor, visual reception, and receptive and expressive language subscales. An adaptive behavior standard score of 54 on the SIB-R indicated significantly impaired functional independence. He was not responsive to structured language testing, and his nonverbal cognitive abilities were estimated to be below the 24-month level. A diagnosis of autism was corroborated independently with a score of 43 on the CARS, completed by the psychologist, and parent ratings resulting in standard scores of 96 and 94 on the Gilliam Autism Rating Scale (GARS). Subsequently, the patient was enrolled in physical, occupational, and speech therapies. He also receives ABA 20–26 hr each week.
The child was seen again at age 4 years, at which time his language skills had improved significantly. He was consistently using several 2-word phrases and 3–4-word simple sentences, although he also demonstrated some echolalia. He had improved socialization with other children. His only medication at this time was rivastigmine 0.4 ml twice a day. His weight was 16.7 kg (60–70%), height 102.8 cm (50%), and head circumference 55.5 cm (>98%, 50% for adult male). PTEN gene sequencing showed a single base substitution in exon 5, resulting in the nonsense mutation R130X.
The family was referred for genetic counseling; only the mother attended the appointment. Her head circumference was 56 cm. A vague maternal family history of “large heads” and several different forms of cancer in relatives over age 65 years was elicited. A maternal aunt developed ovarian cancer at age 50. No hamartomatous disorders were reported. On physical examination of the patient, macrocephaly with a high forehead was noted, but no other abnormal physical findings were identified. Subsequent sequencing of exon 5 of the PTEN gene in the father showed the same heterozygous mutation as in the patient, while in the mother and a younger developmentally normal sibling, only the wild type allele could be detected. Recent photographs of this patient and his father are shown in Figure 1.
The PTEN gene encodes a ubiquitously expressed and essential protein that functions primarily as a lipid phosphatase [Maehama and Dixon, 1999], although it also has enzymatic activity against selected phosphorylated tyrosine, serine, and threonine residues in a variety of proteins. PTEN is the major enzyme involved in the removal of a phosphate moiety from the signaling molecule phosphoinositide-3,4,5-triphosphate (PIP3) and thus inhibits growth factor signaling through the phosphoinositol 3-kinase (PI3K) pathway. PTEN protein phosphatase activity is involved in the negative regulation of focal adhesion kinase (FAK) and mitogen-activated kinase (MAK) signaling, among others [Waite and Eng, 2002; Stiles et al., 2004]. The multiple enzymatic targets of PTEN help explain its critical roles as a tumor suppressor and regulator of cell proliferation, differentiation, and migration [Stiles et al., 2004; Cully et al., 2006].
Somatic mutations in PTEN have been documented in multiple tumor types, while heterozygous germline PTEN mutations are found in patients with PHTS that includes CS, BRRS, PS, and Proteus-like syndromes [Stiles et al., 2004; Zbuk et al., 2006]. Zori et al. 1998 reported on a family with the PTEN nonsense mutation R130X in which the mother had manifestations of CS and her son had BRRS with macrocephaly and “autistic behavior.” Subsequently, Goffin et al. 2001 found the heterozygous nonsense mutation Y178X in exon 6 of the PTEN gene in an autistic, macrocephalic male and his mother who carried a clinical diagnosis of CS. PTEN gene sequencing in these two families was prompted by the presence of CS in a parent. In 2001, Dasouki et al. 2001 reported a de novo heterozygous PTEN missense mutation, H93R, in a 4-year-old boy with macrocephaly, macrosomia, and autism [patient 1 in Butler et al., 2005]. Butler et al. 2005 subsequently identified the missense mutations D252G (exon 7) and F241S (exon 7) in two additional unrelated males among 17 subjects with macrocephaly and autism. Twelve of the patients studied were identified clinically, including the patient of Dasouki et al. 2001, while 6 were from the Autism Genetic Resource Exchange [AGRE, 2006].
The R130X nonsense mutation within exon 5 found in our Patient 2 occurs within the core phosphatase domain of the protein and has been found in multiple CS, BRRS, and CS–BRRS overlap families [Eng, 2003], including the family of Zori et al. 1998 (see above). Frameshift mutations, similar to that found in Patient 1, have caused CS, BRRS, and Proteus syndrome. As in the study of Butler et al. 2005, our patients were ascertained based on a diagnosis of autism and macrocephaly without BRRS or CS in patient or parents. Since many of the manifestations of PHTS often do not become apparent until later in childhood or adulthood [Zbuk et al., 2006], it is certainly possible that they could meet criteria for this diagnosis in the future.
The risk for the development of tumors in patients with PTEN mutations and clinical phenotypes other than CS has not been established [Eng, 2003; Zbuk et al., 2006]. However, it has been previously recommended that these patients undergo the same medical surveillance beginning at age 18 years as CS patients. The possibility of increased tumor risk and need for regular medical surveillance of both patients and the father of Patient 2 using published CS guidelines [Eng et al., 2001; Zbuk et al., 2006] was discussed with both families.
The identification of a causative gene in a child with autism is important. Rarely, this may allow specific treatment, such as dietary therapy in PKU. It may aid prognosis; it may alert clinicians to possible medical complications, such as the increased cancer risk and need for medical surveillance in adults with germline PTEN mutations. Finally, it may provide the ability to offer better recurrence risk counseling for the family. From a research standpoint, there is extreme variable expressivity among individuals with PHTS, including those with identical mutations [Eng, 2003; Zbuk et al., 2006]. Individuals with macrocephaly, autism, and a PTEN mutation could provide a “sensitized” cohort among which to search for modifiers or additional genes that influence the resultant clinical phenotype.
Approximately 20% of children with autism have macrocephaly, while an increased rate of head growth has been reported in up to 70% of autistic children within the first year of life [reviewed in Lainhart, 2006]. The finding of PTEN mutations in a subset of these children by our group and that of Butler et al. 2005 suggests that PTEN and the pathways in which the protein is involved are important for normal brain development. The importance of PTEN signaling pathways in the pathogenesis of autism is further emphasized by the recent report of a targeted Pten mouse model in which the gene was deleted in selected populations of differentiated neurons in the cerebral cortex and hippocampus. Homozygous deleted animals had macrocephaly, abnormal social behavior, and neuronal hypertrophy [Kwon et al., 2006].
Large prospective studies will be necessary to determine the frequency of PTEN mutations in macrocephalic autistic children and whether such mutations occur in children with ASDs with relative or no macrocephaly. Additional autism susceptibility loci may also occur in PTEN-regulated genes or pathways. Screening for PTEN mutations should be included in the genetic evaluation of children with autism and macrocephaly, since a positive result has implications for recurrence and future medical cancer risks for any affecteds or mutation carriers within the family.
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