Claudin-19 mutations and clinical phenotype in Spanish patients with familial hypomagnesemia with hypercalciuria and nephrocalcinosis

Abstract

Familial hypomagnesemia with hypercalciuria and nephrocalcinosis is an autosomal recessive tubular disorder characterized by excessive renal magnesium and calcium excretion and chronic kidney failure. This rare disease is caused by mutations in the CLDN16 and CLDN19 genes. These genes encode the tight junction proteins claudin-16 and claudin-19, respectively, which regulate the paracellular ion reabsorption in the kidney. Patients with mutations in the CLDN19 gene also present severe visual impairment. Our goals in this study were to examine the clinical characteristics of a large cohort of Spanish patients with this disorder and to identify the disease causing mutations. We included a total of 31 patients belonging to 27 unrelated families and studied renal and ocular manifestations. We then analyzed by direct DNA sequencing the coding regions of CLDN16 and CLDN19 genes in these patients. Bioinformatic tools were used to predict the consequences of mutations. Clinical evaluation showed ocular defects in 87% of patients, including mainly myopia, nystagmus and macular colobomata. Twenty two percent of patients underwent renal transplantation and impaired renal function was observed in another 61% of patients. Results of the genetic analysis revealed CLDN19 mutations in all patients confirming the clinical diagnosis. The majority of patients exhibited the previously described p.G20D mutation. Haplotype analysis using three microsatellite markers showed a founder effect for this recurrent mutation in our cohort. We also identified four new pathogenic mutations in CLDN19, p.G122R, p.I41T, p.G75C and p.G75S. A strategy based on microsequencing was designed to facilitate the genetic diagnosis of this disease. Our data indicate that patients with CLDN19 mutations have a high risk of progression to chronic renal disease.

Figure 1. CLDN19 sequence analysis, pedigrees of three families and multiple sequence alignments.

(A) Electropherograms showing homozygous p.G122R mutation in patient P243, mutations p.I41T, p.G75C, p.G75S in heterozygous state in patients P406, P203 and P206, respectively, and controls (bottom panels). The arrows indicate the nucleotide positions with the changes. (B) Pedigrees of families 3, 4 and 18 with genotypes showing 4 compound heterozygous patients, heterozygous parents and two heterozygous siblings. (C) The amino acid sequence alignment of claudin-19 in six species (Homo sapiens, Mus musculus, Monodelphis domestica, Xenopus laevis, Danio rerio and Takifugu rubripes) for the new variants identified. p.I41T and p.G122R are in completely conserve positions in claudin-19 orthologs to Fugu, while p.G75C and p.G75S are in a moderately conserved position.

Figure 2. Schematic representation of the claudin-19 protein and localization of missense CLDN19 mutations.

Novel mutations identified in this study are in boldface.

Figure 3. Analysis of three polymorphic microsatellite markers flanking the CLDN19 locus reveal a founder effect for the common p.G20D mutation.

(A) The chromosomal region containing the CLDN19 locus is shown. Arrows indicate the distances of microsatellite markers from the location of the CLDN19 mutation. (B) Pedigrees and haplotypes of FHHNC Spanish families 1 to 9 and 11 to 16. Haplotypes for markers D1S463, D1S193 and D1S447 (top to bottom) are represented by vertical bars. The disease-associated alleles are shown in black and the others in gray. Numbers indicate the allele size in nucleotides. Black circles and squares represent affected subjects; black circles and squares within open frames represent heterozygous individuals; gray symbols represent individuals without mutation; and unfilled symbols represent family members who were not analyzed. Squares represent males and circles females. The p.G20D mutation was heterozygous in patients of families 3, 4 and 12, and homozygous in the rest of the patients.

Figure 4. Analysis of three polymorphic microsatellite markers flanking the CLDN19 locus reveal a founder effect for the common p.G20D mutation.

Pedigrees and haplotypes of FHHNC Spanish families 17 to 20 and 22 to 27. Haplotypes for markers D1S463, D1S193 and D1S447 (top to bottom) are represented by vertical bars. The disease-associated alleles are shown in black and the others in gray. Numbers indicate the allele size in nucleotides. Black circles and squares represent affected subjects; black circles and squares within open frames represent heterozygous individuals; gray symbols represent individuals without mutation; and unfilled symbols represent family members who were not analyzed. Squares represent males and circles females. The p.G20D mutation was heterozygous in the patient of family 18, and homozygous in the rest of the patients. Three unrelated individuals, P243, P429 and P565, were included as controls; P243 and P429 are homozygous for mutations G122R and Q57E, respectively, and P565 is a healthy control. The p.G20D-associated haplotype was absent in these controls.