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Original Article |

Support for the N -Methyl-D-Aspartate Receptor Hypofunction Hypothesis of Schizophrenia From Exome Sequencing in Multiplex Families

Andrew E. Timms, PhD; Michael O. Dorschner, PhD; Jeremy Wechsler, MS; Kyu Yeong Choi, PhD; Robert Kirkwood, BA; Santhosh Girirajan, MBBS, PhD; Carl Baker, BS; Evan E. Eichler, PhD; Olena Korvatska, PhD; Katherine W. Roche, PhD; Marshall S. Horwitz, MD, PhD; Debby W. Tsuang, MD, MSc
JAMA Psychiatry. 2013;70(6):582-590. doi:10.1001/jamapsychiatry.2013.1195.
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Importance Schizophrenia is a complex genetic disorder demonstrating considerable heritability. Genetic studies have implicated many different genes and pathways, but much of the genetic liability remains unaccounted for. Investigation of genetic forms of schizophrenia will lead to a better understanding of the underlying molecular pathways, which will then enable targeted approaches for disease prevention and treatment.

Objective To identify new genetic factors strongly predisposing to schizophrenia in families with multiple affected individuals with schizophrenia.

Design We performed genome-wide array comparative genomic hybridization, linkage analysis, and exome sequencing in multiplex families with schizophrenia.

Setting Probands and their family members were recruited from academic medical centers.

Participants We intended to identify rare disease-causing mutations in 5 large families where schizophrenia transmission appears consistent with single-gene inheritance.

Intervention Array comparative genomic hybridization was used to identify copy number variants, while exome sequencing was used to identify variants shared in all affected individuals and linkage analysis was used to further filter shared variants of interest. Analysis of select variants was performed in cultured cells to assess their functional consequences.

Main Outcome Measures Rare inherited disease-related genetic mutations.

Results No segregating rare copy number variants were detected by array comparative genomic hybridization. However, in all 5 families, exome sequencing detected rare protein-altering variants in 1 of 3 genes associated with the N -methyl-D-aspartate (NMDA) receptor. One pedigree shared a missense and frameshift substitution of GRM5, encoding the metabotropic glutamate receptor subtype 5 (mGluR5), which is coupled to the NMDA receptor and potentiates its signaling; the frameshift disrupts binding to the scaffolding protein tamalin and increases mGluR5 internalization. Another pedigree transmitted a missense substitution in PPEF2, encoding a calmodulin-binding protein phosphatase, which we show influences mGluR5 levels. Three pedigrees demonstrated different missense substitutions within LRP1B, encoding a low-density lipoprotein receptor–related protein tied to both the NMDA receptor and located in a chromosome 2q22 region previously strongly linked to schizophrenia.

Conclusions and Relevance Exome sequencing of multiplex pedigrees uncovers new genes associated with risk for developing schizophrenia and suggests potential novel therapeutic targets.

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Figures

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Figure 1. Schizophrenia pedigrees studied by array comparative genomic hybridization and whole-exome sequencing. Filled-in symbols indicate affected individuals.

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Figure 2. Functional studies of metabotropic glutamate receptor subtype 5 (mGluR5). A, Schematic of mGluR5, showing location of variants. TM indicates transmembrane. B, Coimmunoprecipitation of cotransfected FLAG-tagged mGluR5 with HA-tagged tamalin in HEK293 cells. Upper panel documents equal expression levels and need for FLAG-tagged construct, given that the frameshift deletes the C-terminal epitope recognized by the native antibody. Lower panel represents cotransfection and coimmunoprecipitation with HA-tagged tamalin. (Images are cut from same film, same exposure to delete irrelevant lanes.) IB indicates immunoblot; IP, immunoprecipitation; TCL, total cell lysates; and WT, wild type.

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Figure 3. Effects of GRM5 variants on metabotropic glutamate receptor subtype 5 (mGluR5) trafficking. Agonist-induced internalization assays of mGluR5 variants on transfection of GRM5 of primary rat hippocampal neurons with 3,5 dihydroxyphenylglycine treatment. Int indicates internalized; norm, normalized ratio to WT; Sur, surface; and WT, wild type. *Significant value.

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Figure 4. Immunoblot showing influence of PPEF2 on FLAG-tagged metabotropic glutamate receptor subtype 5 (mGluR5) on cotransfection in the presence of the 3,5 dihydroxyphenylglycine agonist in HEK293 cells.

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