Cross-level analysis of molecular and neurobehavioral function in a prospective series of patients with germline heterozygous PTEN mutations with and without autism

Thomas W Frazier, Ritika Jaini, Robyn M Busch, Matthew Wolf, Tammy Sadler, Patricia Klaas, Antonio Y Hardan, Julian A Martinez-Agosto, Mustafa Sahin, Charis Eng, Developmental Synaptopathies Consortium, Simon K Warfield, Benoit Scherrer, Kira Dies, Rajna Filip-Dhima, Amanda Gulsrud, Ellen Hanson, Jennifer M Phillips, Thomas W Frazier, Ritika Jaini, Robyn M Busch, Matthew Wolf, Tammy Sadler, Patricia Klaas, Antonio Y Hardan, Julian A Martinez-Agosto, Mustafa Sahin, Charis Eng, Developmental Synaptopathies Consortium, Simon K Warfield, Benoit Scherrer, Kira Dies, Rajna Filip-Dhima, Amanda Gulsrud, Ellen Hanson, Jennifer M Phillips

Abstract

Background: PTEN is a well-established risk gene for autism spectrum disorder (ASD). Yet, little is known about how PTEN mutations and associated molecular processes influence neurobehavioral function in mutation carriers with (PTEN-ASD) and without ASD (PTEN no-ASD). The primary aim of the present study was to examine group differences in peripheral blood-derived PTEN pathway protein levels between PTEN-ASD, PTEN no-ASD, and idiopathic macrocephalic ASD patients (macro-ASD). Secondarily, associations between protein levels and neurobehavioral functions were examined in the full cohort.

Methods: Patients were recruited at four tertiary medical centers. Peripheral blood-derived protein levels from canonical PTEN pathways (PI3K/AKT and MAPK/ERK) were analyzed using Western blot analyses blinded to genotype and ASD status. Neurobehavioral measures included standardized assessments of global cognitive ability and multiple neurobehavioral domains. Analysis of variance models examined group differences in demographic, neurobehavioral, and protein measures. Bivariate correlations, structural models, and statistical learning procedures estimated associations between molecular and neurobehavioral variables. To complement patient data, Western blots for downstream proteins were generated to evaluate canonical PTEN pathways in the PTEN-m3m4 mouse model.

Results: Participants included 61 patients (25 PTEN-ASD, 16 PTEN no-ASD, and 20 macro-ASD). Decreased PTEN and S6 were observed in both PTEN mutation groups. Reductions in MnSOD and increases in P-S6 were observed in ASD groups. Elevated neural P-AKT/AKT and P-S6/S6 from PTEN murine models parallel our patient observations. Patient PTEN and AKT levels were independently associated with global cognitive ability, and p27 expression was associated with frontal sub-cortical functions. As a group, molecular measures added significant predictive value to several neurobehavioral domains over and above PTEN mutation status.

Limitations: Sample sizes were small, precluding within-group analyses. Protein and neurobehavioral data were limited to a single evaluation. A small number of patients were excluded with invalid protein data, and cognitively impaired patients had missing data on some assessments.

Conclusions: Several canonical PTEN pathway molecules appear to influence the presence of ASD and modify neurobehavioral function in PTEN mutation patients. Protein assays of the PTEN pathway may be useful for predicting neurobehavioral outcomes in PTEN patients. Future longitudinal analyses are needed to replicate these findings and evaluate within-group relationships between protein and neurobehavioral measures.

Trial registration: ClinicalTrials.gov Identifier NCT02461446.

Keywords: Autism spectrum disorder; Behavior; Cognition; Molecular; PTEN; Protein.

Conflict of interest statement

TWF has received funding or research support from, acted as a consultant to, received travel support from, and/or received a speaker’s honorarium from Quadrant Biosciences, Impel NeuroPharma, F. Hoffmann-La Roche AG Pharmaceuticals, the Cole Family Research Fund, Simons Foundation, Ingalls Foundation, Forest Laboratories, Ecoeos, IntegraGen, Kugona LLC, Shire Development, Bristol-Myers Squibb, Roche Pharma, National Institutes of Health, and the Brain and Behavior Research Foundation and has an investor stake in Autism EYES LLC. JAMA has no competing interests. MS reports grant support from Novartis, Roche, Pfizer, Ipsen, LAM Therapeutics, Astellas, Aucta, Bridgebio, and Quadrant Biosciences. He has served on Scientific Advisory Boards for Sage, Roche, Celgene, Aeovian, Regenxbio, and Takeda.

Figures

Fig. 1
Fig. 1
Canonical PTEN pathway depicting PI3K/AKT/mTOR and MAPK/ERK signaling
Fig. 2
Fig. 2
Boxplots for PTEN, MnSOD, P-S6, and S6. *p < .05 for group comparisons. kD = kilo Dalton
Fig. 3
Fig. 3
(Left) Western blots on murine brain tissue at 6 weeks of age showing levels of PI3K pathway mediators in a model of PTEN mutation. (Right) Quantification of signal intensity for each mediator normalized to Gapdh (n = 3 each)
Fig. 4
Fig. 4
Effects of PTEN and total AKT protein levels on full-scale IQ directly and indirectly via EIF2A

References

    1. Yehia L, Ngeow J, Eng C. PTEN-opathies: from biological insights to evidence-based precision medicine. J Clin Invest. 2019;129(2):452–464. doi: 10.1172/JCI121277.
    1. van Diepen MT, Eickholt BJ. Function of PTEN during the formation and maintenance of neuronal circuits in the brain. Dev Neurosci. 2008;30(1–3):59–64. doi: 10.1159/000109852.
    1. Delatycki MB, Danks A, Churchyard A, Zhou X-P, Eng C. De novo germline PTEN mutation in a man with Lhermitte–Duclos disease which arose on the paternal chromosome and was transmitted to his child with polydactyly and Wormian bones. J Med Genet. 2003;40:e92. doi: 10.1136/jmg.40.8.e92.
    1. Eng C. PTEN: one gene, many syndromes. Hum Mutat. 2003;22(3):183–198. doi: 10.1002/humu.10257.
    1. Buxbaum JDC, Cai G, Chaste P, Nygren G, Goldsmith J, Reichert J, Anckarsäter H, et al. Mutation screening of the PTEN gene in patients with autism spectrum disorders and macrocephaly. Am J Med Genet Part B N(europsychiatric Genet) 2007;144:484–491. doi: 10.1002/ajmg.b.30493.
    1. Butler MG, Dasouki MJ, Zhou X-P, Talebizadeh Z, Brown M, Takahashi TN, et al. Subset of individuals with autism spectrum disorders and extreme macrocephaly associated with germline PTEN tumour suppressor gene mutations. J Med Genet. 2005;42:318–321. doi: 10.1136/jmg.2004.024646.
    1. Klein S, Sharifi-Hannauer P, Martinez-Agosto JA. Macrocephaly as a clinical indicator of genetic subtypes in autism. Autism Res. 2013;6(1):51–56. doi: 10.1002/aur.1266.
    1. McBride KL, Varga EA, Pastore MT, Prior TW, Manickam K, Atkin JF, et al. Confirmation study of PTEN mutations among individuals with autism or developmental delays/mental retardation and macrocephaly. Autism Res. 2010;3(3):137–141. doi: 10.1002/aur.132.
    1. Orrico A, Galli L, Buoni S, Orsi A, Vonella G, Sorrentino V. Novel PTEN mutations in neurodevelopmental disorders and macrocephaly. Clin Genet. 2009;75(2):195–198. doi: 10.1111/j.1399-0004.2008.01074.x.
    1. Varga EA, Pastore M, Prior T, Herman GE, McBride KL. The prevalence of PTEN mutations in a clinical pediatric cohort with autism spectrum disorders, developmental delay, and macrocephaly. Genet Med. 2009;11(2):111–117. doi: 10.1097/GIM.0b013e31818fd762.
    1. Hobert JA, Embacher R, Mester JL, Frazier TW, Eng C. Biochemical screening and PTEN mutation analysis in individuals with autism spectrum disorders and macrocephaly. Eur J Hum Genet. 2014;22(2):273–276. doi: 10.1038/ejhg.2013.114.
    1. Herman GE, Butter E, Enrile B, Pastore M, Prior TW, Sommer A. Increasing knowledge of PTEN germline mutations: two additional patients with autism and macrocephaly. Am J Med Genet. 2007;143A:589–593. doi: 10.1002/ajmg.a.31619.
    1. Tilot AK, Frazier TW, 2nd, Eng C. Balancing proliferation and connectivity in PTEN-associated autism spectrum disorder. Neurotherapeutics. 2015;12(3):609–619. doi: 10.1007/s13311-015-0356-8.
    1. Yuen RK, Merico D, Bookman M, Howe JL, Thiruvahindrapuram B, Patel RV, et al. Whole genome sequencing resource identifies 18 new candidate genes for autism spectrum disorder. Nat Neurosci. 2017;20(4):602–611. doi: 10.1038/nn.4524.
    1. Iossifov I, O'Roak BJ, Sanders SJ, Ronemus M, Krumm N, Levy D, et al. The contribution of de novo coding mutations to autism spectrum disorder. Nature. 2014;515(7526):216–221. doi: 10.1038/nature13908.
    1. O'Roak BJ, Stessman HA, Boyle EA, Witherspoon KT, Martin B, Lee C, et al. Recurrent de novo mutations implicate novel genes underlying simplex autism risk. Nat Commun. 2014;5:5595. doi: 10.1038/ncomms6595.
    1. Trost B, Engchuan W, Nguyen CM, Thiruvahindrapuram B, Dolzhenko E, Backstrom I, et al. Genome-wide detection of tandem DNA repeats that are expanded in autism. Nature. 2020;586(7827):80–86. doi: 10.1038/s41586-020-2579-z.
    1. Sanders SJ, Murtha MT, Gupta AR, Murdoch JD, Raubeson MJ, Willsey AJ, et al. De novo mutations revealed by whole-exome sequencing are strongly associated with autism. Nature. 2012;485(7397):237–241. doi: 10.1038/nature10945.
    1. Tammimies K, Marshall CR, Walker S, Kaur G, Thiruvahindrapuram B, Lionel AC, et al. Molecular diagnostic yield of chromosomal microarray analysis and whole-exome sequencing in children with autism spectrum disorder. JAMA. 2015;314(9):895–903. doi: 10.1001/jama.2015.10078.
    1. Schaaf CP, Betancur C, Yuen RKC, Parr JR, Skuse DH, Gallagher L, et al. A framework for an evidence-based gene list relevant to autism spectrum disorder. Nat Rev Genet. 2020;21(6):367–376. doi: 10.1038/s41576-020-0231-2.
    1. Hansen-Kiss E, Beinkampen S, Adler B, Frazier T, Prior T, Erdman S, et al. A retrospective chart review of the features of PTEN hamartoma tumour syndrome in children. J Med Genet. 2017;54(7):471–478. doi: 10.1136/jmedgenet-2016-104484.
    1. Ciaccio C, Saletti V, D'Arrigo S, Esposito S, Alfei E, Moroni I, et al. Clinical spectrum of PTEN mutation in pediatric patients. A bicenter experience. Eur J Med Genet. 2018;62:103596. doi: 10.1016/j.ejmg.2018.12.001.
    1. Amiri A, Cho W, Zhou J, Birnbaum SG, Sinton CM, McKay RM, et al. Pten deletion in adult hippocampal neural stem/progenitor cells causes cellular abnormalities and alters neurogenesis. J Neurosci. 2012;32(17):5880–5890. doi: 10.1523/JNEUROSCI.5462-11.2012.
    1. Fraser MM, Zhu X, Kwon CH, Uhlmann EJ, Gutmann DH, Baker SJ. Pten loss causes hypertrophy and increased proliferation of astrocytes in vivo. Cancer Res. 2004;64(21):7773–7779. doi: 10.1158/0008-5472.CAN-04-2487.
    1. Kwon CH, Luikart BW, Powell CM, Zhou J, Matheny SA, Zhang W, et al. Pten regulates neuronal arborization and social interaction in mice. Neuron. 2006;50:377–388. doi: 10.1016/j.neuron.2006.03.023.
    1. Kwon CH, Zhu X, Zhang J, Knoop LL, Tharp R, Smeyne RJ, et al. Pten regulates neuronal soma size: a mouse model of Lhermitte–Duclos disease. Nat Genet. 2001;29(4):404–411. doi: 10.1038/ng781.
    1. Page DT, Kuti OJ, Prestia C, Sur M. Haploinsufficiency for Pten and Serotonin transporter cooperatively influences brain size and social behavior. Proc Natl Acad Sci USA. 2009;106(6):1989–1994. doi: 10.1073/pnas.0804428106.
    1. Tilot AK, Bebek G, Niazi F, Altemus JB, Romigh T, Frazier TW, et al. Neural transcriptome of constitutional Pten dysfunction in mice and its relevance to human idiopathic autism spectrum disorder. Mol Psychiatry. 2016;21:118–125. doi: 10.1038/mp.2015.17.
    1. Tilot AK, Gaugler MK, Yu Q, Romigh T, Yu W, Miller RH, et al. Germline disruption of Pten localization causes enhanced sex-dependent social motivation and increased glial production. Hum Mol Genet. 2014;23(12):3212–3227. doi: 10.1093/hmg/ddu031.
    1. Frazier TW, Hardan AY. A meta-analysis of the corpus callosum in autism. Biol Psychiatry. 2009;66(10):935–941. doi: 10.1016/j.biopsych.2009.07.022.
    1. Just MA, Cherkassky VL, Keller TA, Kana RK, Minshew NJ. Functional and anatomical cortical underconnectivity in autism: evidence from an FMRI study of an executive function task and corpus callosum morphometry. Cereb Cortex. 2007;17(4):951–961. doi: 10.1093/cercor/bhl006.
    1. Fishman I, Keown CL, Lincoln AJ, Pineda JA, Muller RA. Atypical cross talk between mentalizing and mirror neuron networks in autism spectrum disorder. JAMA Psychiatry. 2014;71(7):751–760. doi: 10.1001/jamapsychiatry.2014.83.
    1. Frazier TW, Embacher R, Tilot AK, Koenig K, Mester J, Eng C. Molecular and phenotypic abnormalities in individuals with germline heterozygous PTEN mutations and autism. Mol Psychiatry. 2015;20(9):1132–1138. doi: 10.1038/mp.2014.125.
    1. Busch RM, Chapin JS, Mester J, Ferguson L, Haut JS, Frazier TW, et al. Cognitive characteristics of PTEN hamartoma tumor syndromes. Genet Med. 2013;15(7):548–553. doi: 10.1038/gim.2013.1.
    1. Busch RM, Srivastava S, Hogue O, Frazier TW, Klaas P, Hardan A, et al. Neurobehavioral phenotype of autism spectrum disorder associated with germline heterozygous mutations in PTEN. Transl Psychiatry. 2019;9(1):253. doi: 10.1038/s41398-019-0588-1.
    1. Muhle RA, Reed HE, Stratigos KA, Veenstra-VanderWeele J. The emerging clinical neuroscience of autism spectrum disorder: a review. JAMA Psychiatry. 2018;75(5):514–523. doi: 10.1001/jamapsychiatry.2017.4685.
    1. Hoang N, Cytrynbaum C, Scherer SW. Communicating complex genomic information: a counselling approach derived from research experience with autism spectrum disorder. Patient Educ Couns. 2018;101(2):352–361. doi: 10.1016/j.pec.2017.07.029.
    1. Tan MH, Mester J, Peterson C, Yang Y, Chen JL, Rybicki LA, et al. A clinical scoring system for selection of patients for PTEN mutation testing is proposed on the basis of a prospective study of 3042 probands. Am J Hum Genet. 2012;88(1):42–56. doi: 10.1016/j.ajhg.2010.11.013.
    1. Jaini R, Loya MG, King AT, Thacker S, Sarn NB, Yu Q, et al. Germline PTEN mutations are associated with a skewed peripheral immune repertoire in humans and mice. Hum Mol Genet. 2020;29(14):2353–2364. doi: 10.1093/hmg/ddaa118.
    1. Sarn N, Jaini R, Thacker S, Lee H, Dutta R, Eng C. Cytoplasmic-predominant Pten increases microglial activation and synaptic pruning in a murine model with autism-like phenotype. Mol Psychiatry. 2020 doi: 10.1038/s41380-020-0681-0.
    1. Little RJ, Rubin DB. Statistical analysis with missing data. 2. New York: Wiley; 2002.
    1. Schafer JL, Graham JW. Missing data: our view of the state of the art. Psychol Methods. 2002;7(2):147–177. doi: 10.1037/1082-989X.7.2.147.
    1. Bhaskaran K, Smeeth L. What is the difference between missing completely at random and missing at random? Int J Epidemiol. 2014;43(4):1336–1339. doi: 10.1093/ije/dyu080.
    1. Muthén BO, Muthén LK, Asparouhov T. Regression and mediation analysis using MPlus. Los Angeles: Muthén & Muthén; 2016.
    1. Corp IBM. IBM SPSS Statistics for Windows. 260. Armonk: IBM Corp; 2018.
    1. Muthén LK, Muthén BO. Mplus User's Guide. 7. Los Angeles: Muthén & Muthén; 1998.
    1. Zablotsky B, Black LI, Maenner MJ, Schieve LA, Danielson ML, Bitsko RH, et al. Prevalence and trends of developmental disabilities among children in the united states: 2009–2017. Pediatrics. 2019;144(4):e20190811. doi: 10.1542/peds.2019-0811.
    1. Mighell TL, Evans-Dutson S, O'Roak BJ. A saturation mutagenesis approach to understanding PTEN lipid phosphatase activity and genotype-phenotype relationships. Am J Hum Genet. 2018;102(5):943–955. doi: 10.1016/j.ajhg.2018.03.018.
    1. Matreyek KA, Starita LM, Stephany JJ, Martin B, Chiasson MA, Gray VE, et al. Multiplex assessment of protein variant abundance by massively parallel sequencing. Nat Genet. 2018;50(6):874–882. doi: 10.1038/s41588-018-0122-z.
    1. Papa A, Wan L, Bonora M, Salmena L, Song MS, Hobbs RM, et al. Cancer-associated PTEN mutants act in a dominant-negative manner to suppress PTEN protein function. Cell. 2014;157(3):595–610. doi: 10.1016/j.cell.2014.03.027.
    1. Post KL, Belmadani M, Ganguly P, Meili F, Dingwall R, McDiarmid TA, et al. Multi-model functionalization of disease-associated PTEN missense mutations identifies multiple molecular mechanisms underlying protein dysfunction. Nat Commun. 2020;11(1):2073. doi: 10.1038/s41467-020-15943-0.
    1. Ganesan H, Balasubramanian V, Iyer M, Venugopal A, Subramaniam MD, Cho SG, et al. mTOR signalling pathway—a root cause for idiopathic autism? BMB Rep. 2019;52(7):424–433. doi: 10.5483/BMBRep.2019.52.7.137.
    1. Yehia L, Seyfi M, Niestroj LM, Padmanabhan R, Ni Y, Frazier TW, et al. Copy number variation and clinical outcomes in patients with germline PTEN mutations. JAMA Netw Open. 2020;3(1):e1920415. doi: 10.1001/jamanetworkopen.2019.20415.
    1. Yehia L, Ni Y, Feng F, Seyfi M, Sadler T, Frazier TW, et al. Distinct alterations in tricarboxylic acid cycle metabolites associate with cancer and autism phenotypes in cowden syndrome and Bannayan–Riley–Ruvalcaba syndrome. Am J Hum Genet. 2019;105(4):813–821. doi: 10.1016/j.ajhg.2019.09.004.
    1. Kern JK, Jones AM. Evidence of toxicity, oxidative stress, and neuronal insult in autism. J Toxicol Environ Health Part B. 2006;9(6):485–499. doi: 10.1080/10937400600882079.
    1. Abruzzo PM, Matte A, Bolotta A, Federti E, Ghezzo A, Guarnieri T, et al. Plasma peroxiredoxin changes and inflammatory cytokines support the involvement of neuro-inflammation and oxidative stress in Autism Spectrum Disorder. J Transl Med. 2019;17(1):332. doi: 10.1186/s12967-019-2076-z.
    1. Rossignol DA, Frye RE. Mitochondrial dysfunction in autism spectrum disorders: a systematic review and meta-analysis. Mol Psychiatry. 2012;17(3):290–314. doi: 10.1038/mp.2010.136.
    1. Weissman JR, Kelley RI, Bauman ML, Cohen BH, Murray KF, Mitchell RL, et al. Mitochondrial disease in autism spectrum disorder patients: a cohort analysis. PLoS ONE. 2008;3(11):e3815. doi: 10.1371/journal.pone.0003815.
    1. Yenkoyan K, Harutyunyan H, Harutyunyan A. A certain role of SOD/CAT imbalance in pathogenesis of autism spectrum disorders. Free Radic Biol Med. 2018;123:85–95. doi: 10.1016/j.freeradbiomed.2018.05.070.
    1. Smith AM, Natowicz MR, Braas D, Ludwig MA, Ney DM, Donley ELR, et al. A metabolomics approach to screening for autism risk in the children's autism metabolome project. Autism Res. 2020;13(8):1270–1285. doi: 10.1002/aur.2330.
    1. Lustgarten MS, Jang YC, Liu Y, Qi W, Qin Y, Dahia PL, et al. MnSOD deficiency results in elevated oxidative stress and decreased mitochondrial function but does not lead to muscle atrophy during aging. Aging Cell. 2011;10(3):493–505. doi: 10.1111/j.1474-9726.2011.00695.x.
    1. Larson JC, Mostofsky SH. Motor deficits in autism. In: Tuchman R, Rapin I, editors. Autism: a neurological disorder of early brain development. London: Mac Keith Press; 2006.
    1. Dewey D, Cantell M, Crawford SG. Motor and gestural performance in children with autism spectrum disorders, developmental coordination disorder, and/or attention deficit hyperactivity disorder. J Int Neuropsychol Soc. 2007;13(2):246–256. doi: 10.1017/S1355617707070270.
    1. Licari MK, Alvares GA, Varcin K, Evans KL, Cleary D, Reid SL, et al. Prevalence of motor difficulties in autism spectrum disorder: analysis of a population-based cohort. Autism Res. 2020;13(2):298–306. doi: 10.1002/aur.2230.
    1. Kelleher RJ, 3rd, Bear MF. The autistic neuron: troubled translation? Cell. 2008;135(3):401–406. doi: 10.1016/j.cell.2008.10.017.
    1. Hooshmandi M, Wong C, Khoutorsky A. Dysregulation of translational control signaling in autism spectrum disorders. Cell Signal. 2020;75:109746. doi: 10.1016/j.cellsig.2020.109746.
    1. Hazlett HC, Gu H, Munsell BC, Kim SH, Styner M, Wolff JJ, et al. Early brain development in infants at high risk for autism spectrum disorder. Nature. 2017;542(7641):348–351. doi: 10.1038/nature21369.
    1. Piven J, Elison JT, Zylka MJ. Toward a conceptual framework for early brain and behavior development in autism. Mol Psychiatry. 2018;23(1):165. doi: 10.1038/mp.2017.212.
    1. Lainhart JE. Increased rate of head growth during infancy in autism. J Am Med Assoc. 2003;290:393–394. doi: 10.1001/jama.290.3.393.
    1. Lainhart JE, Bigler ED, Bocian M, Coon H, Dinh E, Dawson G, et al. Head circumference and height in autism: a study by the collaborative program of excellence in autism. Am J Med Genet A. 2006;140(21):2257–2274. doi: 10.1002/ajmg.a.31465.
    1. Winden KD, Ebrahimi-Fakhari D, Sahin M. Abnormal mTOR activation in autism. Annu Rev Neurosci. 2018;41:1–23. doi: 10.1146/annurev-neuro-080317-061747.
    1. Zhang J, Gao Z, Ye J. Phosphorylation and degradation of S6K1 (p70S6K1) in response to persistent JNK1 activation. Biochim Biophys Acta. 2013;1832(12):1980–1988. doi: 10.1016/j.bbadis.2013.06.013.

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