Variants in PRKAR1B cause a neurodevelopmental disorder with autism spectrum disorder, apraxia, and insensitivity to pain

Abstract

Purpose: We characterize the clinical and molecular phenotypes of six unrelated individuals with intellectual disability and autism spectrum disorder who carry heterozygous missense variants of the PRKAR1B gene, which encodes the R1β subunit of the cyclic AMP-dependent protein kinase A (PKA).

Methods: Variants of PRKAR1B were identified by single- or trio-exome analysis. We contacted the families and physicians of the six individuals to collect phenotypic information, performed in vitro analyses of the identified PRKAR1B-variants, and investigated PRKAR1B expression during embryonic development.

Results: Recent studies of large patient cohorts with neurodevelopmental disorders found significant enrichment of de novo missense variants in PRKAR1B. In our cohort, de novo origin of the PRKAR1B variants could be confirmed in five of six individuals, and four carried the same heterozygous de novo variant c.1003C>T (p.Arg335Trp; NM_001164760). Global developmental delay, autism spectrum disorder, and apraxia/dyspraxia have been reported in all six, and reduced pain sensitivity was found in three individuals carrying the c.1003C>T variant. PRKAR1B expression in the brain was demonstrated during human embryonal development. Additionally, in vitro analyses revealed altered basal PKA activity in cells transfected with variant-harboring PRKAR1B expression constructs.

Conclusion: Our study provides strong evidence for a PRKAR1B-related neurodevelopmental disorder.

Fig. 1. Facial phenotypes and distribution of observed PRKAR1B variants.

(a) Top left: individual 1 at the age of 3 years. Top right: individual 4 the age of 7 years. Bottom left: individual 5 the age of 7 years. Bottom right: individual 6 at the age of 3 years. (b) Distribution of the observed variants within the PRKAR1B gene. Exons are shown as boxes, introns as a blue line (introns are not to scale). Light blue color indicates protein-coding sequence. (c) Mutated amino acid (AA) positions within the R1β protein and number of affected individuals. A color shift to red indicates a higher degree of intolerance towards AA variation throughout evolution; according to the respective position’s evolutionary trace (ET) score (see Supplemental Methods for further details).

Fig. 2. Functional consequences of the observed variants on R1β protein function.

(a) PKA enzymatic activity assay: basal and total PKA enzymatic activity in lysates of HEK293 cells transfected with PRKAR1B expression constructs (wild type [WT], p.Q167L, p.E196K and p.R335W [p.Gln167Leu, p.Glu196Lys and p.Arg335Trp]). One-way analysis of variance (ANOVA) was performed for both basal and total PKA activity data sets; a Bonferroni multiple comparison test was used for basal activity data that produced a significant ANOVA statistic. (b) Fluorescence resonance energy transfer (FRET) in HEK293 cells transfected with R1β-Venus (WT, p.Q167L, p.E196K and p.R335W) and Cα-Cerulean vectors. A Mann–Whitney U-test was used to check for statistical significance as data were not normally distributed.

Fig. 3. Expresssion of PRKAR1B during human development.

(a) Normalized expression levels of different PRKAR genes and a selection of reporter genes in embryonic stem cells (ESCs), neural progenitor cells (NPCs), and neural crest cells (NCCs), based on RNA-Seq data from different sources.^– For each individual set of expression data (ESC, NPC, and NCC), 0% reflects the gene with the lowest, and 100% the gene with the highest level of expression. The median expression level of each data set is 50% (dashed line). Genes scoring higher than 50% can be considered to be more highly expressed than the majority of genes in their respective data set. (b) Upper row: sagittal section of a human embryo at Carnegie stage 22 and corresponding 3D model of the embryonic brain (yellow: mesencephalon; green: subpallium; light blue: diencephalon; purple: hypothalamus; pink: rhombencephalon). A RNAscope PRKAR1β probe has been used to hybridize PRKAR1B messenger RNA (mRNA) (red). The section has been counterstained with hematoxylin (blue). A corresponding positive and negative control is shown in Fig. S2. Lower row: magnified sections show PRKAR1B expression in the pituitary, diencephalon, mesencephalon, and hypothalamus.