HNF1B mutations associate with hypomagnesemia and renal magnesium wasting

Abstract

Mutations in hepatocyte nuclear factor 1B (HNF1B), which is a transcription factor expressed in tissues including renal epithelia, associate with abnormal renal development. While studying renal phenotypes of children with HNF1B mutations, we identified a teenager who presented with tetany and hypomagnesemia. We retrospectively reviewed radiographic and laboratory data for all patients from a single center who had been screened for an HNF1B mutation. We found heterozygous mutations in 21 (23%) of 91 cases of renal malformation. All mutation carriers had abnormal fetal renal ultrasonography. Plasma magnesium levels were available for 66 patients with chronic kidney disease (stages 1 to 3). Striking, 44% (eight of 18) of mutation carriers had hypomagnesemia (<1.58 mg/dl) compared with 2% (one of 48) of those without mutations (P < 0.0001). The median plasma magnesium was significantly lower among mutation carriers than those without mutations (1.68 versus 2.02 mg/dl; P < 0.0001). Because hypermagnesuria and hypocalciuria accompanied the hypomagnesemia, we analyzed genes associated with hypermagnesuria and detected highly conserved HNF1 recognition sites in FXYD2, a gene that can cause autosomal dominant hypomagnesemia and hypocalciuria when mutated. Using a luciferase reporter assay, we demonstrated HNF1B-mediated transactivation of FXYD2. These results extend the phenotype of HNF1B mutations to include hypomagnesemia. HNF1B regulates transcription of FXYD2, which participates in the tubular handling of Mg(2+), thus describing a role for HNF1B not only in nephrogenesis but also in the maintenance of tubular function.

Figure 1.

Renal length on latest ultrasound in those with HNF1B mutations. Crosses, renal length of the four solitary functioning kidneys after regression of contralateral MCDKs. Circles, all other (26 of 30) kidneys from the HNF1B mutation cohort for which lengths were accurately measured. Top, middle, and bottom lines indicate the 95th, 50th, and fifth centiles for age as based on data from our hospital's radiology department (see the Concise Methods section). Note absence of compensatory hypertrophy in single remaining kidneys (crosses).

Figure 2.

Plasma total magnesium, calcium, and GFR values in mut+ and mut− patients assessed at stages 1 through 3 CKD. (A) Median plasma magnesium value for each patient is shown, together with mut− and mut+ group medians (short horizontal bars). The lower limit of normal is indicted by the dotted horizontal line. Note the lower magnesium values in the mut+ cohort. Eight of 18 HNF1B mutation carriers had hypomagnesemia versus only one of 48 in the mut− group. (B and C) Plasma calcium and GFR values (time matched for the plasma magnesium points shown in A). P values refer to comparisons of the median (ranges) between the two groups.

Figure 3.

HNF1 recognition sites in the FXYD2 promoter. (A) Localization of HNF1 recognition sites at the FXYD2 locus. The FXYD2 gene has different splice variants, starting with either exon γ-b (FXYD2b; NM021603) or exon γ-a (FXYD2a; NM001680). Two identical pairs of HNF1-binding sites (pair A and pair B) were identified. Pair A is 62 nucleotides (nt) upstream of the transcription initiation site of exon γ-a, whereas pair B is 2128 nt further upstream. In each pair, the HNF1-binding sites are in opposite orientation and separated by 6 nt. Note the high mammalian conservation at both sites (derived from UCSC human genome browser). No HNF1 binding sites were found upstream of exon γ-b. (B) Sequence of established HNF1 recognition sites. The size of each letter is in approximate proportion to the frequency of finding the denoted nt at this position in the recognition site. (C) Alignment of the putative HNF1 recognition sites with the canonical sequence. Pair A and pair B contain sites 1/2 and sites 3/4, respectively. Sites 1 and 3 are in the upper strand (+), whereas sites 2 and 4 are in the lower strand (−). R denotes puRine (A or G).

Figure 4.

RT-PCR analyses in a DCT cell line. Hnf1a, Hnf1b, and Fxyd2b transcripts are expressed in mouse DCT cells. From left to right, lanes show consistent results from three independent cDNA preparations from DCT cells, mouse whole kidney (kidney), and reaction buffer only (neg). Note that Fxyd2a transcripts were not detected in DCT cells but were present in the whole kidney. Hprt was included as a housekeeping gene.