A biochemical difference between HAI-2 variants causing syndromic and non-syndromic Congenital Sodium Diarrhea

Variants in the SPINT2 gene, encoding HAI-2, cause the syndromic form of autosomal recessive Congenital Sodium Diarrhea (CSD), a life-threatening disease with early-onset persistent diarrhea and stools containing elevated levels of sodium. A newly discovered variant, HAI-2 Y68C, causes a milder non-syndromic CSD-like condition in an isolated case. The present investigation compares biochemical features of the HAI-2 Y68C with two newly discovered syndromic CSD-causing variants, HAI-2 Y129C and R148H, and two previously characterized syndromic CSD-causing variants, HAI-2 F161V and G168S. HAI-2 contains two Kunitz domains that inhibit a range of serine proteases, including matriptase and prostasin. HAI-2 is co-located with target proteases mainly in the ER and the early secretory pathway but also on the apical plasma membrane of polarized cells. All investigated variants of HAI-2 were expressed with a similar steady-state protein level and weak pericellular staining as wild-type HAI-2. However, none of the CSD-causing variants were capable of inhibiting prostasin as efficiently as wild-type HAI-2. In addition, the HAI-2 Y68C variant was unable to inhibit matriptase, constituting a clear biochemical difference between the single variant causing non-syndromic CSD and the variants causing syndromic CSD. The HAI-2 Y68C variant may affect the phenotype by altering matriptase catalyzed activation of prostasin in the early secretory pathway. We hypothesize that CSD, whether syndromic or non-syndromic, is caused by a lack of inhibitory activity of HAI-2 in the early secretory pathway and/or on the apical plasma membrane, resulting in uncontrolled proteolytic activity leading to proteolytic modification of sodium transporters/channels like NHE3 and ENaC. NHE3 and ENaC are normally located on the apical plasma membrane and are mainly responsible for the uptake of sodium from the gut lumen. Oral administration of a protease inhibitor mimicking HAI-2 wild-type could potentially help to reduce the degradation of proteins important for sodium uptake across the apical plasma membrane.