The Role of Extended Phenotyping and Functional Testing in Variant Interpretation of a Complex Patient with Overlapping Traits

Abstract

Purpose : Biallelic pathogenic variants in G protein subunit beta 5 (GNB5) gene causes developmental delay, seizures, hypotonia, sinus node dysfunction and retinal defects featuring bradyopsia and rod ON-bipolar dysfunction. This study investigated the pathogenicity of a homozygous variant of uncertain significance in GNB5 annotated as GNB5L: c.920T>G (p.L307R); GNB5S: c.794T>G (p.L265R) identified in a patient through Whole Exome Sequencing who was newborn screening positive for 3-methylcrotonyl-CoA carboxylase (3-MCC) deficiency with clinical features unexplained by 3-MCC.

Methods : Various clinical signs were ascertained by chart review. Detailed eye exams and extended electroretinogram (ERG) were performed. Fibroblast cell line was derived from patient’s skin biopsy for gene expression and protein localization using digital PCR, Western Blot and Immunohistochemistry (IHC). Bioluminescence Resonance Energy Transfer (BRET) – based signaling assay and measuring stability of Regulator G-protein signaling (RGS) complexes in expression assays were used to investigate the impact of the variant on the function of Gβ5-s.

Results : The ocular features of the 9 year-old proband included high myopia, normal fundus and a subtle cone photo-transduction recovery deficit. Her extra-ocular features included severe intellectual disability (ID) and repeated cardiac arrests. The 3-MCC deficiency was confirmed with molecular finding of homozygous MCCC1 (c.1394C>T p.T465I) variant and decreased enzyme activity. The severity of her ID was atypical for 3-MCC deficiency and more in keeping with GNB5-disorder. Cardiac arrest is only known to GNB5-related disorder. In vitro study showed severe loss of function of abolishing the ability of Gβ5-s containing RGS complexes to deactivate signaling via D2 dopamine receptor due to destabilization of RGS complexes. Fibroblast culture demonstrated minimal change in GNB5 expression and translation by digital PCR and Western blots. Differences in subcellular co-localization of Gβ5 and its binding partners were seen with IHC.

Conclusions : Proband’s ERG phenotype is consistent with defective function of GNB5L whereas her cardiac and neurodevelopmental phenotype together with the in vitro functional analysis indicates defective GNB5S function. This study highlights the importance of detailed phenotyping and need of functional assays to aid variant classification.

This is a 2021 ARVO Annual Meeting abstract.