GLRB is the third major gene of effect in hyperekplexia

Glycinergic neurotransmission is a major inhibitory influence in the CNS and its disruption triggers a paediatric and adult startle disorder, hyperekplexia. The postsynaptic α-subunit (GLRA1) of the inhibitory glycine receptor (GlyR) and the cognate presynaptic glycine transporter (SLC6A5/GlyT2) are well-established genes of effect in hyperekplexia. Nevertheless, 52% of cases (117 from 232) remain gene negative and unexplained. Ligand-gated heteropentameric GlyRs form chloride ion channels that contain the a1 and b-subunits (GLRB) in a 2α:3β configuration and they form the predominant population of GlyRs in the postnatal and adult human brain, brainstem and spinal cord. We screened GLRB through 117 GLRA1- and SLC6A5-negative hyperekplexia patients using a multiplex-polymerase chain reaction and Sanger sequencing approach. The screening identified recessive and dominant GLRB variants in 12 unrelated hyperekplexia probands. This primarily yielded homozygous null mutations, with nonsense (n=3), small indel (n=1), a large 95 kb deletion (n=1), frameshifts (n 5 1) and one recurrent splicing variant found in four cases. A further three cases were found with two homozygous and one dominant GLRB missense mutations. We provide strong evidence for the pathogenicity of GLRB mutations using splicing assays, deletion mapping, cell-surface biotinylation, expression studies and molecular modelling. This study describes the definitive assignment of GLRB as the third major gene for hyperekplexia and impacts on the genetic stratification and biological causation of this neonatal/paediatric disorder. Driven principally by consanguineous homozygosity of GLRB mutations, the study reveals long-term additive phenotypic outcomes for affected cases such as severe apnoea attacks, learning difficulties and developmental delay

Aberrant phase separation and nucleolar dysfunction in rare genetic diseases.

Thousands of genetic variants in protein-coding genes have been linked to disease. However, the functional impact of most variants is unknown as they occur within intrinsically disordered protein regions that have poorly defined functions(1-3). Intrinsically disordered regions can mediate phase separation and the formation of biomolecular condensates, such as the nucleolus(4,5). This suggests that mutations in disordered proteins may alter condensate properties and function(6-8). Here we show that a subset of disease-associated variants in disordered regions alter phase separation, cause mispartitioning into the nucleolus and disrupt nucleolar function. We discover de novo frameshift variants in HMGB1 that cause brachyphalangy, polydactyly and tibial aplasia syndrome, a rare complex malformation syndrome. The frameshifts replace the intrinsically disordered acidic tail of HMGB1 with an arginine-rich basic tail. The mutant tail alters HMGB1 phase separation, enhances its partitioning into the nucleolus and causes nucleolar dysfunction. We built a catalogue of more than 200,000 variants in disordered carboxy-terminal tails and identified more than 600 frameshifts that create arginine-rich basic tails in transcription factors and other proteins. For 12 out of the 13 disease-associated variants tested, the mutation enhanced partitioning into the nucleolus, and several variants altered rRNA biogenesis. These data identify the cause of a rare complex syndrome and suggest that a large number of genetic variants may dysregulate nucleoli and other biomolecular condensates in humans