Functional AssessmentIn Vivoof the Mouse Homolog of the Human Ala-9-Ser NHE6 Variant

Abstract

Christianson syndrome (CS) is an X-linked neurogenetic disorder resulting from loss-of-function (LoF) mutations in SLC9A6, which encodes the endosomal Na+/H+ exchanger 6 (NHE6). NHE6 regulates proton efflux from endosomes and, thus, participates in regulating cargo processing and trafficking. LoF mutations in NHE6 cause aberrant acidification of endosomes. While CS arises in males generally due to clear LoF mutations, other potentially hypomorphic variants have emerged, yet most of these variants have not been evaluated for functional effects, particularly in vivo. Here we characterize an SLC9A6 variant that has been previously reported in patients, yet now also appears in exome datasets of largely control individuals—c.25G>T, p.A9S. By heterologous expression in cell lines, we show that human NHE6A9S is expressed and localizes in a manner comparable to control NHE6. By genome editing, we generated the equivalent NHE6 mutation in mouse—p.A11S—and determined that male NHE6A11S mice have normal brain size at 6 months of age and do not show cerebellar degeneration or defective neuronal arborization. Neurons from male NHE6A11S mice also did not demonstrate an abnormality in intraendosomal pH compared with controls. These findings are in contrast to findings in NHE6-null mice previously reported and indicate that the NHE6A11S variant functions at a level equivalent to control NHE6 for many of the assays performed. These data stand in support of the population genetic data, which are also evaluated here, indicating that the A9S variant is unlikely to confer disease susceptibility with high penetrance.

Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

“…Although this experimental cell system has proven informative in deciphering NHE6 function, we cannot exclude the possibility that any deleterious consequences of the A9S and R568Q variants may be more subtle and manifested only in certain neuronal cell-types. However, during the preparation of this manuscript, Ouyang and co-workers (88) reported that mice genetically manipulated by CRISPR/Cas9 technology to harbor the homologous human NHE6 A9S variant (i.e., mouse A11S) appeared normal in terms of brain morphology, NHE6 subcellular localization and intra-endosomal pH of hippocampal neurons. Though a behavioural assessment of these mice was not described, we envision that human A9S and possibly the R568Q variants are likely benign and possibly misattributed as the cause of the patients' neurological symptoms.…”

Generation of PathogenicTPP1Mutations in Human Stem Cells as a Model for CLN2 Disease

“…Samples containing 20 µg of protein were then boiled in sample buffer at 95 °C for 5 min before loading onto a 4-12% SDS-PAGE gel (Novex #NP0321Box). Following separation of proteins by electrophoresis, the gel was transferred to a nitrocellulose membrane (Novex #LC2000), and Western blot was performed using standard procedures (Ouyang et al, 2019; Xu et al, 2017). The Western blot was analyzed with the Li-CoR Odyssey Imaging System.…”

Correction of a pathogenic mutation in iPSCs derived from a patient with Christianson syndrome using CRISPR/Cas9 genome editing

“…Following separation of proteins by electrophoresis, gels were transferred to nitrocellulose membranes (Novex #LC2000). Western blots were performed using standard procedures (Lizarraga et al, 2021; Ouyang et al, 2019; Xu et al, 2017) and were analyzed with the Li-CoR Odyssey Imaging System. Proteins were detected using rabbit anti-NHE6 antibody (1:1,000 working dilution; Covance #048) (Ouyang et al, 2013) and mouse anti-α-Tubulin (1:5,000 working dilution; Sigma #T6074).…”