Exonic CLDN16 mutations associated with familial hypomagnesemia with hypercalciuria and nephrocalcinosis can induce deleterious mRNA alterations

Ana Perdomo-Ramirez,
Marian de Armas-Ortiz,
Elena Ramos-Trujillo,
Lorena Suarez-Artiles and
Felix Claverie-MartinEmail author View ORCID ID profile

BMC Medical Genetics201920:6

https://doi.org/10.1186/s12881-018-0713-7

Received: 26 July 2018
Accepted: 30 October 2018
Published: 8 January 2019

Open Peer Review reports

Abstract

Background

Familial hypomagnesaemia with hypercalciuria and nephrocalcinosis type 1 is an autosomal recessive disease characterized by excessive renal magnesium and calcium excretion, bilateral nephrocalcinosis, and progressive chronic renal failure. This rare disease is caused by mutations in CLDN16 that encodes claudin-16, a tight-junction protein involved in paracellular reabsorption of magnesium and calcium in the renal tubule. Most of these variants are located in exons and have been classified as missense mutations. The functional consequences of some of these claudin-16 mutant proteins have been analysed after heterologous expression showing indeed a significant loss of function compared to the wild-type claudin-16. We hypothesize that a number of CLDN16 exonic mutations can be responsible for the disease phenotype by disrupting the pre-mRNA splicing process.

Methods

We selected 12 previously described presumed CLDN16 missense mutations and analysed their potential effect on pre-mRNA splicing using a minigene assay.

Results

Our results indicate that five of these mutations induce significant splicing alterations. Mutations c.453G > T and c.446G > T seem to inactivate exonic splicing enhancers and promote the use of an internal cryptic acceptor splice site resulting in inclusion of a truncated exon 3 in the mature mRNA. Mutation c.571G > A affects an exonic splicing enhancer resulting in partial skipping of exon 3. Mutations c.593G > C and c.593G > A disturb the acceptor splice site of intron 3 and cause complete exon 4 skipping.

Conclusions

To our knowledge, this is the first report of CLDN16 exonic mutations producing alterations in splicing. We suggest that in the absence of patients RNA samples, splicing functional assays with minigenes could be valuable for evaluating the effect of exonic CLDN16 mutations on pre-mRNA splicing.