Mutations in the nervous system--specific HSN2 exon of WNK1 cause hereditary sensory neuropathy type II

Abstract

Hereditary sensory and autonomic neuropathy type II (HSANII) is an early-onset autosomal recessive disorder characterized by loss of perception to pain, touch, and heat due to a loss of peripheral sensory nerves. Mutations in hereditary sensory neuropathy type II (HSN2), a single-exon ORF originally identified in affected families in Quebec and Newfoundland, Canada, were found to cause HSANII. We report here that HSN2 is a nervous system-specific exon of the with-no-lysine(K)-1 (WNK1) gene. WNK1 mutations have previously been reported to cause pseudohypoaldosteronism type II but have not been studied in the nervous system. Given the high degree of conservation of WNK1 between mice and humans, we characterized the structure and expression patterns of this isoform in mice. Immunodetections indicated that this Wnk1/Hsn2 isoform was expressed in sensory components of the peripheral nervous system and CNS associated with relaying sensory and nociceptive signals, including satellite cells, Schwann cells, and sensory neurons. We also demonstrate that the novel protein product of Wnk1/Hsn2 was more abundant in sensory neurons than motor neurons. The characteristics of WNK1/HSN2 point to a possible role for this gene in the peripheral sensory perception deficits characterizing HSANII.

Figure 1. Mutations in the WNK1/HSN2 gene.

(A) Segregation of the 2 mutations identified in the nuclear family. The single affected individual has compound heterozygous mutations. Sequencing traces show the 1-bp deletion (639delA, Arg214fsX215) identified in HSN2 (B) and the 2-bp deletion (1584_1585delAG, Asp531fsX547) identified in exon 6 of WNK1 (C), with sequencing traces from a normal control. mut, mutated sequencing trace; wt, control sequencing trace.

Figure 2. RNA expression analyses of Hsn2 messenger.

(A) Northern blotting of Hsn2 mouse tissues. The membrane was hybridized with Hsn2 probe (top) before it was stripped and rehybridized with a β-actin probe (bottom). (B) RT-PCR amplifications between putative exon 8B (primer 2) and Hsn2 (primer 3b). (C) Diagram of Wnk1 encompassing Hsn2. Below are the numbered primers (Supplemental Table 1). The positions of Wnk1 promoters are indicated by black arrows, and the sites detected by anti-HSN2 and anti-WNK1 (Alpha Diagnostic International) are indicated in red. Kin. D., WNK1 kinase domain. (D) RT-PCR amplifications from brain, spinal cord, DRG, sciatic nerve, kidney, testis, lung, and spleen cDNA. Bottom: Amplifications between exon 8 (primer 1) and Hsn2 (primer 3a). Top: Amplifications between Hsn2 (primer 4) and exon 10 (primer 5). Arrows 1, 2, and 3 are, respectively, at 250, 420, and 160 bp. (E) RT-PCR amplifications between Hsn2 and upstream and downstream region of Wnk1. Left: RT-PCR between a region upstream to the 5′UTR of P1 (primer 5′UTRP1) and Hsn2 (primer 3a) in DRG. The same panel shows the amplification between the same 5′UTR of the P1 region and exon 6 in the kidney. Middle: Amplifications between Hsn2 (primer 4) and exon 16 (primer 11). Arrows 1, 2, 3, and 4 are, respectively, at approximately 2.2 kb, 950 bp, 650 bp, and approximately 3.8 kb. Right: Amplifications between Hsn2 (primer 4) and exon 24 (primer not shown in B). (F) Comparison of Wnk1/Hsn2 isoforms with the most common isoform of Wnk1.

Figure 3. Flanking junction sites of the novel alternatively spliced WNK1/HSN2 isoforms.

(A) RACE revealed that exon 8B was specifically spliced in some transcripts from mouse brain and spinal cord and the amino acids encoded by this mouse putative exon 8B of the longer Wnk1/Hsn2 isoform are highly conserved across species. A comparison of the amino acids encoded by the exon 8B ORF in the mouse was made with different species/taxa (chicken, Xenopus, and zebrafish), and greater than 95% residues are fully or highly conserved (an asterisk below the residues indicates those that are fully conserved across the different taxa/species). (B) The amino acid sequences of the splice acceptor and splice donor sites that flank the HSN2 exon are highly conserved across species. The regions in blue represent the sequence flanking HSN2, whereas the regions indicated in black are spliced out during mRNA maturation. Splice acceptor and donor sites are in bold characters. The sequences presented were obtained from the UCSC Genome Bioinformatics Browser ( http://genome.ucsc.edu).

Figure 4. Separate Western immunodetections of WNK1 and WNK1/HSN2.

Various mouse tissues from adult or E13 animals were loaded and detected with anti-WNK1 (Alpha Diagnostic International) (top) or with IgG purified anti-HSN2 antibody (middle). Expression of WNK1 was observed in all the lysates, and the expression of WNK1/HSN2 was limited to lysates from neuronal tissues. The membrane detected in the top panel was stripped and then detected with the second antibody. An anti-actin antibody was used to confirm that the loading protein was equal in different lanes (bottom).

Figure 5. WNK1/HSN2 histological immunodetections.

(A) Immunohistochemistry detection of adult mouse DRG (from L5 sections) with anti-HSN2 antiserum (red). A clear immunoreactive signal is visible in the satellite cells (arrows) and in some of the neuronal somata (arrowheads). (B) Overlaid images of the detections with anti-HSN2 (red in A), a mix of axonal markers (SMI-31/32 mix; green), and nuclear staining (TOTO-3 iodide; blue). Colocalization of the signals (yellow overlay) shows that WNK1/HSN2 is expressed in some of the axonal fiber and satellite cells, which surround the neuronal somata (arrows). (C) Adult mouse sciatic nerve cross sections detected with the anti-HSN2 antiserum (red) show the presence of the protein in a mosaic distribution of axons. (D) Overlaid images of the detection with anti-HSN2 (red in C), the axonal markers (green), and nuclear staining (blue) show that not all axonal fibers express WNK1/HSN2 (yellow) and that some do not express WNK1/HSN2 (green). Cross sections of dorsal roots through which sensory axons pass (E) and of ventral roots through which motor axons transit (F) were detected with anti-HSN2 (red) and the axonal marker (green in E and F, insets). The majority of motor neuron axonal fibers showed weak or no WNK1/HSN2 signal. In contrast, the HSN2 signal was strong in most of the axonal fibers of the sensory neurons in the dorsal roots. Original magnification of insets, ×400.

Figure 6. WNK1/HSN2 protein expression in the adult mouse CNS.

(A) Low-magnification image of immunohistochemistry detections of adult mouse spinal cord cross section with anti-HSN2 antiserum. The anti-HSN2 antiserum gave a strong signal (red) in the superficial layers (LI and LII) of the dorsal horn, the dorsolateral funiculus (DLF), the lateral funiculus (LF), and the Lissauer tract (LT). (B) Overlaid images of the HSN2 signal (red in A), the signal from the anti–SMI-31/32 axonal marker (green), and the nuclear fluorescent labeling (blue). (C) Immunodetection of LI and LII with anti-HSN2 (red). (D) Overlaid images of the detection of HSN2 (red in C), NeuN (green), and the nuclear labeling (blue). Arrows indicate neurons where the colocalization is observed, confirming the presence of HSN2 protein in cells of this region.