Genetic Cause of Antenatal Bartter Syndrome

Large Deletion of the 5th End of the ROMK1 Gene Causes Antenatal Bartter Syndrome

Abstract. Mutations in exon 5 of the ROMK1 gene ( KCNJ1) have recently been observed in antenatal Bartter syndrome patients. This study describes a homozygous deletion removing KCNJ1 exons 1 and 2 observed in a consanguineous family with antenatal Bartter syndrome.

Absence of the untranslated exon 1 led to the deletion of transcription elements located in this exon that may cause the disease. Deletion of exon 1 transcription elements should lead to the absence of hROMK2-K5 transcripts, whereas hROMK1 transcripts should normally be transcripted. Consequently, probably only hROMK2-K5 transcripts are expressed in the medullary thick ascending limb of Henle.

Antenatal Bartter syndrome (ABS) is an inherited tubular disorder beginning in utero and resulting in polyhydramnios and premature delivery [1] . Affected neonates have severe salt loss, moderate metabolic alkalosis and hypokalemia, renin and aldosterone hypersecretion, and severe hypercalciuria with early nephrocalcinosis. A disorder of the medullary thick ascending limb of Henle has been demonstrated. Recent molecular studies have shown that mutations in the genes encoding the Na-K-2Cl cotransporter [2] [3] , the chloride channel CLC-NKB [4] , and the potassium channel ROMK have been detected in ABS patients [5] [6] .

The ROMK gene (locus KCNJ1) encodes a K+ channel that belongs to the family of the inwardly rectifying K+ channel (Kir family) [7] . Kir family channels comprise two predicted transmembrane domains flanking an H5-like region which forms part of the channel pore. At least five distinct hROMK transcripts have been identified in the human kidney [8] [9] . They encode three peptides that differ in their N-terminal extensions (hROM-K1, -K2, -K3). Exon 5 of KCNJ1 is common to all of the transcripts and encodes most of the channel protein. The mutations described previously in ABS patients have been located in exon 5 [5] [6] . We report a large deletion of exons 1 and 2 within KCNJ1 in a consanguineous ABS family. In this family, previous linkage analysis with microsatellite markers in 11q24-25 and 15q15-21 suggested a linkage between KCNJ1 and ABS and allowed exclusion of gene NKCC2 encoding the Na-K-2Cl cotransporter [6] .

Materials and Methods


Clinical analysis of three ABS patients from the same family was made from the records of Armand Trousseau and Felix -Guyon hospitals.

Mutation Analysis

DNA was extracted from whole blood lymphocytes of all family members IV and V, except IV-4, V-2, and V-7. The five exons and the exon-intron boundaries were amplified using the sets of primers described previously [5] [6] . Amplification products were separated in Nusieve 3:1 agarose (FMC BioProducts, Rockland, ME) and studied by single-strand conformational polymorphism (SSCP) analysis. SSCP was carried out on a GenePhor Electrophoresis Unit (Pharmacia Biotech) using the GeneGel Clean 15/24 according to the manufacturer’s instructions (Pharmacia Biotech).

Southern Blotting

Genomic DNA was digested with BamHI. The digested products were analyzed by electrophoresis on 0.8% agarose gels (17 h at 44 V). Alkaline transfer to nylon membrane Genescreen+ (New England Nuclear) was performed over 4 h. The membrane was then hybridized with P32 -labeled PCR KCNJ1 probes and human Cot-1 DNA (Life Technologies).


Clinical Phenotype

A consanguineous family of Indian extraction in which three neonates presented with ABS is reported Patient V-1 was born at 31 wk after a pregnancy marked by polyhydramnios from the 28th week. Polyuria and sodium loss necessitated parenteral infusion to maintain normal hydration during the first 6 wk of life. At 1 yr of age, he suffered from failure to thrive (-3 SD), polyuria (230 ml/kg per d), and repeated unexplained episodes of fever. Nephrocalcinosis and renal lithiasis at 2.5 yr of age were associated with hypercalciuria (12 mg/kg per d). Mild hypokalemia (3.4 mmol/L), metabolic alkalosis (27 mmol/L), isosthenuria (270 mosmol/L), and renin hypersecretion also occurred at the same age. Indomethacin was given until 5 yr of age resulting in growth catch-up and decrease in polyuria. Patient V-7 died on the first day of life. The pregnancy was marked by polyhydramnios from the 20th week and premature delivery at 24 wk. Patient V-10 was born prematurely at 32 wk after a pregnancy marked by polyhydramnios. Moderate hypokalemia (3.5 mmol/L), renin hypersecretion (50 pg/ml), aldosteronism (1900 pg/ml), and hypercalciuria (urinary calcium/creatinine = 2.44 mmol/mmol) were identified at this time.

Mutation Analysis

Amplification of exons 1, 2, and 3 is presented in . Exons 1 and 2 could not be amplified in the two affected patients. Amplification of exons 1 and 2 of all of the other family members gave a product of the expected size. Amplification of exons 3, 4, and 5 gave a product of the expected size for all of the samples studied. Amplification products of exons 3, 4, and 5 were analyzed by SSCP. Normal SSCP patterns were observed for all of the family samples studied. Amplification of exons 1 and 2 in 50 unaffected subjects gave a product of the expected size.

Southern Blot Analysis

KCNJ1 sequence (GenBank accession no. U65406) revealed seven restriction sites for BamHI . The 12.6-kb fragment contains exons 4 and 5, and the 3.6-kb fragment contains exons 1 and 2. In the two patients, the 3.6-kb fragment was absent and no additional fragments were observed, thus confirming the homozygous deletion of exons 1 and 2 .

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