Haplotype analysis of CLDN19 single nucleotide polymorphisms in Spanish patients with familial hypomagnesemia with hypercalciuria and nephrocalcinosis

Methods

Samples selected

Our study included 27 FHHNC patients (17 females and 10 males) from 26 families in different regions of Spain and 13 controls. In this series there were 24 patients from a Spanish cohort reported previously (19 homozygous for the p.G20D mutation and 5 heterozygous)^[11] and 3 new patients who were subsequently identified [P551 (Family 28), P479 (Family 29) and P590 (Family 30) (Table 1)]. The controls included two patients homozygous for a different CLDN19 mutation, p.G122R and p.Q57E, respectively. Diagnosis was based on the established clinical criteria and confirmed by the identification of a CLDN19 mutation.^[1,5,11] DNA was isolated from peripheral blood using the QIAamp DNA Blood Mini kit (Qiagen, Hilden, Germany). Ethics authorization for DNA analysis was granted from the Ethics Committee of Clinical Research, Hospital Nuestra Señora de Candelaria (Santa Cruz de Tenerife, Spain). All patients or their parents and healthy controls signed an informed consent for the genetic analysis before inclusion.

Haplotype analysis

Four SNPs, rs10890211, rs11210708, rs7548008 and rs12141833, located in CLDN19 introns 3 and 4 (Fig. 1), were selected based on their allelic frequencies calculated from the GenBank database (National Center for Biotechnology Information). Genotyping was performed by polymerase chain reaction (PCR) amplification from genomic DNA and sequencing. Primers pairs used were: C19Int3F (5'-ACACTGATGGCTCTGTCTGC-3') and C19Int3R (5'-TTGGGCTCATTTCCTGATCT-3'), and C19Int4F (5'-CCCAGGCATACACACAGATG-3') and C19Int4R (5'-AGGGTGAAGGTTTCTGCTCA-3'). PCR reactions were carried out using the following conditions: 94ºC for 5 minutes, 30 cycles of 94ºC for 30 seconds, 54ºC for 45 seconds, and 72ºC for 45 seconds, and a final extension at 72ºC for 5 minutes. PCR products were sequenced with the BigDye terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA). Sequencing reactions were purified with Performa DTR Gel Filtration Cartridges (EdgeBio, Gaithersburg, MD, USA) and analyzed on a 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Haplotype analysis in new patients using microsatellite markers D1S463, D1S193 and D1S447, flanking the CLDN19 locus, was performed as described previously.^[10,11]

Mutation screening

CLDN19 exons and flanking intronic regions of the three new patients were amplified by PCR using primers and conditions described previously.^[11] PCR products were sequenced as described above.

Results

The main clinical and biochemical characteristics of the 3 new diagnosed patients are as follows. Patient P551 was a two-year-old boy was referred to the hospital at two months of age because of the ultrasound finding of hyperechogenicity of renal pyramids and cortex. He was diagnosed with nephrocalcinosis, hypercalciuria (2.98 mmol/d, normal range <2.5 mmol/d), hypomagnesemia (0.57 mmol/L, normal range 0.65-1.05 mmol/L) and severe myopia. His glomerular filtration rate (GFR) was 74 mL/min per 1.73 m² at 2 months of age. Patient P479 was a 14-year-old girl who was referred to the hospital at age of 7 months because she had two UTIs. Radiological examination revealed bilateral nephrocalcinosis. The patient presented with hypomagnesemia (0.49 mmol/L, normal range 0.65-1.05 mmol/L) and hypercalciuria (5.35 mmol/d, normal range <2.5 mmol/d). At age of 13 months, her GFR was 95 mL/min per 1.73 m². Ophthalmologic examination revealed myopia and nystagmus. Patient P590 was a 33-year-old woman who was diagnosed with nephrocalcinosis at 6 years of age. She had recurrent UTIs including pyelonephritis. Eye examination showed horizontal nystagmus and at age of 14 years, she had surgery for a left divergent strabismus. Laboratory data obtained at age of 24 years when the patient was admitted to the hospital for acute pyelonephritis showed hypomagnesemia (0.49 mmol/L, normal range 0.65­-1.05 mmol/L) and hypercalciuria (6.4 mmol/d; normal range <6.2 mmol/d). At age of 30 years, her GFR was 30 mL/min/per 1.73 m², and at age of 33 years she started hemodialysis and a kidney transplant. We identified homozygous mutation p.G20D in the CLDN19 gene of these three patients. Their parents were heterozygous carriers. The results of microsatellite analysis demonstrated the presence of a mutation-carrying haplotype (Fig. 2) as had been previously found for the rest of the patients.^[11]

An SNP haplotype analysis was performed to check whether our 27 FHHNC patients had a common p.G20D (c.59G>A) linked haplotype. The results revealed that almost every patient shared the same allele sequence in the vicinity of the mutation, A-A-G-C-G, where the bold A is the c.59A mutant allele (Table 1). To assess whether this strong association of a specific haplotype was indeed the result of a founder effect, fifteen unrelated Spanish individuals not carrying the p.G20D mutation were genotyped for the four SNPs. The most common alleles in these individuals for the SNPs rs10890211, rs11210708 and rs7548008 were the C (19/30: 63.3%), A (20/30: 66.7%), and T (23/30: 76.7%) alleles, respectively, which were never associated with c.59G>A among the disease chromosomes (Table 1). While the most common allele for SNP rs12141833 (18/12: 60%) in the controls was linked with the mutation in only a few cases (Table 1). A comparison of the allele frequency of each SNP between our controls, global population and Iberian population in Spain, and the FHHNC patients is shown in Table 2. The overall distribution of this haplotype was signifi cantly different between the patients and controls (P<0.0001).

Discussion

In 2006, Konrad et al^[7] reported the first CLDN19 mutations in patients with FHHNC. They identified the same homozygous mutation, p.G20D, in seven unrelated Spanish/Hispanic patients and suggested a founder effect. Several studies^[7,10,11] showed that most Spanish patients present mutation in the CLDN19 gene; as far as we know only three patients have mutations in CLDN16. ^[8,15] The p.G20D mutation in a homozygous state results in absence of claudin-19 protein in the cell membrane and a complete loss of function.^[7,12] Subsequently, two studies using microsatellite markers with FHHNC families from Spain and from the South of France further supported the founder effect hypothesis.^[10,11] It should be noted that a large immigration from Spain to France occurred after the Spanish civil war in 1936-1939.

The results of the present study showed that the SNP haplotype A-A-G-C-G was specifically linked to p.G20D, and are therefore consistent with the existence of a founding ancestor in our cohort. Founder effects have been also identified for FHHNC in patients from three North African families harboring the same CLDN16 p.A139V mutation^[10] and in 14 families who originated from Germany or eastern European countries carrying CLDN16 p.L151F mutation.^[5] Mutations that persist following their occurrence in a single founder may refl ect selection-favoring heterozygotes, perhaps by providing the carriers with a survival advantage. However, there is no apparent reason why the p.G20D carriers should have a benefit.

In conclusion, the previously described founder effect for p.G20D supported by the conservation of a specific mutation-linked SNP haplotype shown in this study suggests that the c.59A allele arose from one independent ancestral allele. This effect explains the high frequency of the mutation in the Spanish FHHNC families. Our finding is fundamental for the implementation of a protocol for genetic diagnosis, genetic counseling of affected families, and search of new therapies.

We are grateful to the patients and their parents for their cooperation in the study. Cordoba-Lanus E, Gonzalez-Acosta H and Claverie-Martin F are members of the Renaltube Group (www.renaltube.org).

Funding: This work was supported by a research grant (PI11/00342) from Fondo de Investigacion Sanitaria (co-funded by the European Regional Development Fund, "A way to build Europe") to F.C.M. This funding was granted as coordinated project Renaltube.

Ethical approval: This study was approved by the Ethics Committee of Clinical Research, Hospital Nuestra Señora de Candelaria, Santa Cruz de Tenerife, Spain.

Competing interest: None of the authors have a fi nancial relationship with a commercial entity that has an interest in the subject of this study. Contributors: Martin-Nuñez E, Cordoba-Lanus E and Gonzalez-Acosta H performed the experiments, analyzed the data and helped to draft the manuscript. Oliet A and Izquierdo E performed the clinical evaluation and analyzed the clinical data. Claverie-Martín F participated in the design of the experiments, analysis of data and coordination, and wrote the manuscript. All authors contributed to the intellectual content and approved the final version of the manuscript.

References

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Received January 9, 2014  Accepted after revision March 6, 2014