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

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.

Keywords: Bioinformatics analysis; Claudin-16; Exonic splicing enhancer; Minigene; Missense mutation; Pre-mRNA splicing; Splicing defects.

Fig. 1

Diagrammatic representations of claudin-16. a The protein contains four TMDs (1–4) and two ECSs (1–2). ECS1 contains the ion selectivity filter while ECS2 is involved in claudin–claudin interactions. The location of the mutations included in this study is indicated. b Hypothetical protein structures of mutant claudin-16 proteins translated from the aberrant transcripts detected in Fig. 3a. Truncated exon 3 induced by mutations c.446G > T and c.453G > T would result in loss of the terminal part of ECS1 and part of TMD2. Skipping of exon 3 produced by mutation c.571G > A would cause loss of the terminal part of ECS1, TMD2, the cytoplasmic region and part of TMD3. Skipping of exon 4 induced mutations c.593G > A and c593G > C would result in loss of part of TMD3, ECS2, TMD4, and part of the cytoplasmic C terminus

Fig. 2

Location of presume missense mutations analysed in this study and schematic representation of CLDN16 minigenes. a Boxes represent the five coding exons and black lines in between indicate intron sequences. Coding and non-coding regions appears in blue and grey, respectively. Exons and introns sizes are not at scale. Small numbers indicate NNSplice scores of donor and acceptor splice sites. b Structure of CLDN16 minigenes used in the splicing reporter assay. Blue and black boxes represent CLDN16 exons and 5′ and 3′ exons of the shuttle vector pET01, respectively. Black lines indicate intron sequences. The minigenes were constructed by introducing a CLDN16 genomic fragment containing exons 2, 3 or 4 and flanking intronic sequences into the vector as described in Materials and Methods. The position of each mutation and the restriction sites used for cloning are indicated. Arrows above and below the vector exons represent primers used in the RT-PCR assays. LTR, long terminal repeat promoter of the Rous sarcoma virus; Poly A, polyadenylation site. The minigenes will express a poly-A+ RNA containing the spliced exons

Fig. 3

Minigene assay of CLDN16 exonic mutations revealed altered pre-mRNA splicing. a RT-PCR fragments produced by minigenes and separated by agarose gel electrophoresis. Wild-type and mutant pET01ex2, pET01ex3 and pET01ex4 constructs were transfected into COS7 cells, and the mRNAs were analysed as described in Material and Methods. All assays were performed in triplicate. The identities of the RT-PCR products are illustrated schematically on the left of each panel. b Electropherograms of anomalous RT-PCR fragments produced by minigenes containing the mutations indicated. c-f Schematic representation of minigene splicing in the presence and absence of mutations. The location of mutations is indicated. Mutations c.453G > T and c.446G > T activate a cryptic acceptor splice site internal to exon 3 that induces inclusion of a truncated exon 3. Mutation c.571G > A produced a major band corresponding to the normally spliced transcript together with a minor band that correspond to skipping of exon 3. Both c.593G > C and c.593G > A inactivate the splice site and result in exon 4 skipping