Three rare diseases in one Sib pair: RAI1, PCK1, GRIN2B mutations associated with Smith-Magenis Syndrome, cytosolic PEPCK deficiency and NMDA receptor glutamate insensitivity

Abstract

The National Institutes of Health Undiagnosed Diseases Program evaluates patients for whom no diagnosis has been discovered despite a comprehensive diagnostic workup. Failure to diagnose a condition may arise from the mutation of genes previously unassociated with disease. However, we hypothesized that this could also co-occur with multiple genetic disorders. Demonstrating a complex syndrome caused by multiple disorders, we report two siblings manifesting both similar and disparate signs and symptoms. They shared a history of episodes of hypoglycemia and lactic acidosis, but had differing exam findings and developmental courses. Clinical acumen and exome sequencing combined with biochemical and functional studies identified three genetic conditions. One sibling had Smith-Magenis Syndrome and a nonsense mutation in the RAI1 gene. The second sibling had a de novo mutation in GRIN2B, which resulted in markedly reduced glutamate potency of the encoded receptor. Both siblings had a protein-destabilizing homozygous mutation in PCK1, which encodes the cytosolic isoform of phosphoenolpyruvate carboxykinase (PEPCK-C). In summary, we present the first clinically-characterized mutation of PCK1 and demonstrate that complex medical disorders can represent the co-occurrence of multiple diseases.

Keywords: Developmental delay; Dysmorphism; Hypoglycemia; Lactic acidemia; Multiple genetic disorders; Protein structure-function.

Fig. 1

Photographs of propositae. Patient 1 (A–C) was obese and had a broad square-shaped face, midfacial hypoplasia, mildly upslanting palpebral fissures, a broad nasal bridge, a full-tipped nose, short tapering fingers and mild 5^th finger clindodactyly. In contrast, patient 2 (D–F) was slight and had no dysmorphic features.

Fig. 2

Identification and characterization of RAI1 and PCK1 mutations. (A) Family pedigree. (B) Sanger sequence traces showing a heterozygous RAI1 mutation (NM_030665:c.2273G>A, NP_109590.3:p.W758X) only in patient 1 (II-1). (C) Quantification of RAI1 steady state mRNA levels in cultured skin fibroblasts of patient 1 and an unaffected control. The nearly 50% decrease in RAI1 steady state mRNA levels in patient lymphoblastoid cells compared to unaffected control lymphoblastoid cells suggests that the p.W758X causes nonsense mediated mRNA decay. β-actin mRNA levels were used for normalization. (D) Sanger sequence traces showing that both patients (II-1 and II-2) had a homozygous PCK1 mutation (NM_002591.3:c.134T>C, NP_002582.3:p.I45T), whereas the parents (I-1 and I-2) were heterozygous for the mutation. (E) NP_002582.3:p.I45T alters an isoleucine (arrowhead) conserved across species to Drosophila melanogaster (see also Figure S-1 for more extensive species comparison, indicating this hydrophobic residue is conserved in the GTP utilizing enzymes all the way through Ascaris suum).

Fig. 3

Structural and Functional Properties of the Mutant PEPCK-C. (A) The overall structure of crystallized recombinant I45T PEPCK-C indicating the location of the I45T mutation with respect to the overall enzyme structure and the active site (indicated by the molecules of βSP and GTP rendered as ball and stick models colored by atom type). The N- and C-termini are also labeled. (B) The comparison of WT (light blue, PDB 3DT7) and I45T PEPCK-C (grey, PDB 4OX2, this work) in the vicinity of I45 illustrates that the local structure of the hydrophobic pocket is not perturbed upon the threonine for isoleucine substitution. 2Fo-Fc density corresponding to the side-chain of T45 is illustrated as a blue mesh rendered at 1.2 σ. (C) Kinetic characterization of recombinant I45T PEPCK-C compared with the wild type (WT) enzyme, showing very similar kinetic parameters (K_M for both OAA and GTP and k_cat), and heat stability (t_1/2) at two temperatures (*WT data is from [20]). (D) Western blots of PEPCK-C and PEPCK-M in frozen liver from patient 1 and two controls. Figures above show western blots specific for PEPCK-C and PEPCK-M and the reference proteins β-actin or GAPDH, with the amount of micrograms of protein homogenate beneath, for patient 1 and human controls A or B. Graphs below these respective blots comparing the normalized signal intensity (corrected for the ratio to their reference proteins) of ratios of PEPCK-C (blue) and PEPCK-M (red) in the patient vs controls A and B. (E) Half lives of mutant I45T and wt PEPCK-C in T-Rex 293 cultured stable inducible cell lines. Figure above shows Western blots at indicated time intervals before and after tetracycline induction of PEPCK-C I45T (upper row), PEPCK-C wt (lower row), and GAPDH as a reference protein. Graph below shows relative amounts of PEPCK-C I45T/GAPDH (solid circles) and PEPCK-C/GAPDH wt (solid squares) over 24 h after induction, to establish the mutant and wild type protein half-lives.

Fig. 4

Identification and characterization of a de novo GRIN2B (GluN2B) mutation in patient 2. (A) Sanger sequence traces showing a heterozygous GRIN2B mutation (NM_000834.3:c.1238A>G, NP_000825.2:p.E413G) only in patient 2 (II-2). (B) Predicted quaternary structure of tetrameric human GluN1/GluN2B, based on the GluA2 AMPA receptor structure (left; 3KG2) [52]. The bi-lobed ligand binding domain (yellow) of GluN2B and related glutamate receptors adopts a clamshell-like structure, which binds glutamate (blue) within the cleft. Ligand-protein interactions between L-glutamate (spheres) and wild-type GluN2B (middle) or GluN2B-E413G (right) ligand binding domains, are modeled from GluN1/GluN2A crystallographic data (2A5T) [15]. Nearby residues and crystallographically conserved waters are shown (sticks). Hydrogen bonds are depicted by the black dashed lines. (C) The composite concentration-response curves and fitted EC₅₀ values are shown for human GluN1/GluN2B (wild type) or GluN1/GluN2B-E413G (E413G) current responses (100 μM glycine in all solutions, V_HOLD −40 mV). (D) The composite glycine concentration-response curves and fitted EC₅₀ values at human GluN1/GluN2B (wild type) or GluN1/GluN2B-E413G (E413G) receptors (1 mM glutamate present in all solutions). EC₅₀ values were obtained by fitting the curves in (C) and (D) with Response (%) = 100 / (1 + (EC₅₀ / [agonist])^N). N is the Hill slope, which ranged between 1.1 – 1.4; n = 11–20 oocytes).