Ire1 is an ER resident transmembrane protein that functions as a transducer of the unfolded protein response (UPR). Its luminal domain senses unfolded proteins, triggering the activation of the cytosolic domain for the transcriptional up regulation of chaperones and other protein folding enzymes to help restore ER homeostasis. Ire1 is unprecedented in nature with two functional domains, a kinase and an endoribonuclease. Upon activation, Ire1 autophosphorylates, and cleaves an intron from HAC1 mRNA. In the absence of autophosphorylation or lack of nuclease activity, a UPR response cannot be mounted. While the roles of Ire1's kinase and nuclease domains during the activation of UPR are understood to some extent, little is known about the mechanism of attenuation of the response. In this study we used a mutagenesis-based approach, both in vivo and in vitro, to characterize the importance of conserved residues in Ire1 during the attenuation of UPR. We present here that in wild type cells, spliced HAC1 mRNA disappears some time after UPR induction, upon recovery of the ER protein folding capacity. In contrast, cells with a mutation in the conserved DFG kinase motif, Ire1-D828A, show sustained HAC1 splicing, even after recovery of ER protein folding capacity. Mutating the DFG kinase motif inactivated the Ire1 kinase, although ATP binding and RNase activation were normal. Limited proteolysis and further biochemical studies showed that the DFG mutation altered Ire1 conformation such that it was no longer responsive to ATP binding. Taken together, it suggests that Ire1-D828A is rendered conformationally unresponsive to the attenuating signals from the ER luminal domain, causing the sustained HAC1 splicing. In addition, we also observed sustained HAC1 splicing in the Ire1 phosphomimetic activation loop mutant, hinting at the importance of loop dephosphorylation in the attenuation process. Thus, we show that the Ire1 kinase domain in addition to its role in activation of UPR also plays an equally important role in responding to the attenuating signal. In addition, we established that the timely down regulation of UPR is as important an event as activation, because sustained UPR activation was toxic to cellular growth

Congenital sodium diarrhea is a rare and life-threatening disorder characterized by a severe, secretory diarrhea containing high concentrations of sodium, leading to hyponatremia and metabolic acidosis. It may occur in isolation or in association with systemic features such as facial dysmorphism, choanal atresia, imperforate anus, and corneal erosions. Mutations in the serine protease inhibitor, Kunitz-Type 2 (SPINT2) gene have been associated with congenital sodium diarrhea and additional syndromic features. We present a child with congenital sodium diarrhea, cleft lip and palate, corneal erosions, optic nerve coloboma, and intermittent exotropia who was found to have biallelic mutations in SPINT2. One mutation, c.488A > G, predicting p.(Tyr163Cys), has been previously associated with a syndromic form of congenital sodium diarrhea. The other mutation, c.166_167dupTA, predicting p.(Asn57Thrfs*24) has not previously been reported and is likely a novel pathogenic variant for this disorder. We found only one other report of an optic nerve coloboma associated with SPINT2 mutations and this occurred in a patient with congenital tufting enteropathy. Our patient confirms an association of ocular coloboma with presumed loss of SPINT2 function.

Purpose

Haploinsufficiency of USP7, located at chromosome 16p13.2, has recently been reported in seven individuals with neurodevelopmental phenotypes, including developmental delay/intellectual disability (DD/ID), autism spectrum disorder (ASD), seizures, and hypogonadism. Further, USP7 was identified to critically incorporate into the MAGEL2-USP7-TRIM27 (MUST), such that pathogenic variants in USP7 lead to altered endosomal F-actin polymerization and dysregulated protein recycling.

Methods

We report 16 newly identified individuals with heterozygous USP7 variants, identified by genome or exome sequencing or by chromosome microarray analysis. Clinical features were evaluated by review of medical records. Additional clinical information was obtained on the seven previously reported individuals to fully elucidate the phenotypic expression associated with USP7 haploinsufficiency.

Results

The clinical manifestations of these 23 individuals suggest a syndrome characterized by DD/ID, hypotonia, eye anomalies,feeding difficulties, GERD, behavioral anomalies, and ASD, and more specific phenotypes of speech delays including a nonverbal phenotype and abnormal brain magnetic resonance image findings including white matter changes based on neuroradiologic examination.

Conclusion

The consistency of clinical features among all individuals presented regardless of de novo USP7 variant type supports haploinsufficiency as a mechanism for pathogenesis and refines the clinical impact faced by affected individuals and caregivers.