Functionally distinct groups of inherited PTEN mutations in autism and tumour syndromes

Background Germline mutations in the phosphatase PTEN are associated with diverse human pathologies, including tumour susceptibility, developmental abnormalities and autism, but any genotype-phenotype relationships are poorly understood. Methods We have studied the functional consequences of seven PTEN mutations identified in patients diagnosed with autism and macrocephaly and five mutations from severe tumour bearing sufferers of PTEN hamartoma tumour syndrome (PHTS). Results All seven autism-associated PTEN mutants investigated retained the ability to suppress cellular AKT signalling, although five were highly unstable. Observed effects on AKT also correlated with the ability to suppress soma size and the length and density of dendritic spines in primary neurons. Conversely, all five PTEN mutations from severe cases of PHTS appeared to directly and strongly disrupt the ability to inhibit AKT signalling. Conclusions Our work implies that alleles causing incomplete loss of PTEN function are more commonly linked to autism than to severe PHTS cases.


Figure S1. PTEN mutants characterisation in bacteria and U87MG cells
Autism-related mutations proteins drastically reduce PTEN phosphatase activity when expressed in bacteria but they are able to regulate AKT when expressed in  Accordingly, for six of the seven mutants the measured released phosphate was significantly higher than detected with this inactive control (the exception being       Tables S2 and S3. Severity index scoring system. Based on the clinical description of the patients carrying PTEN germline mutations, the severity scores were created according to the number of sites affected as well as the presence of mental retardation/ development delay (MR/DD). The presence of macrocephaly was recorded but not included in the severity index score, as macrocephaly was seen in almost every subject, with the chance that it was not recorded in others. For each site affected by benign tumours or lesions it was assigned one point, two points for each site affected by malignant tumours, and one point for the presence of mental retardation. The severity index score is the sum of these numbers. Patients who scored one to three were classified as "mild", four to five as "moderate" and six or above as "severe".
The choice of mutations to analyse has been made based on the "index score average" resulting from the average of the scores of all patients carrying the same PTEN mutation. It should be noted that the characterised PTEN mutants associated with severe phenotypes (see figure 4) were selected at a mid-point of the progression of the project and that the data tabulated here contain very recently published information added since that time. Efforts have been made to minimise the chances of double counting but this cannot be excluded.

Expression vectors and recombinant PTEN protein expression
Plasmid and lentiviral expression vectors for untagged and glutathione S-transferase (GST)-tagged human PTEN have been previously described [58 59]. Antibody complexes were detected by 1 hour incubation at RT with HRP conjugated secondary antibodies (Vector Labs). Blots were developed with ECL plus (Millipore) and chemiluminescence imaged directly using an ImageQuant LAS4000 imaging system.

Lipid phosphatase assay
The preparation of 3- 33  Chemical) and radioactivity was counted using a Beckman scintillation counter.

Reverse Transcriptase quantitative PCR (qPCR)
Total cellular RNA was isolated from U87MG cells expressing PTEN WT and mutants by using TRIzol (Life Technologies -Invitrogen) and RNeasy Mini kit

Animal procedures
All animal procedures (breeding and sacrifice) were conducted in accordance with local ethical guidelines and approved animal care protocols (Berlin: Institutional Animal Care and Use Committee (IACUC) and the Landesamt für Gesundheit und Soziales (LAGeSO) -license T 0347/11.