2496? The?Journal?of?Clinical?Investigation? ? ? http://www.jci.org? ? ? Volume 118? ? ? Number 7? ? ? July 2008
Mutations in the nervous system–specific
HSN2 exon of WNK1 cause hereditary
sensory neuropathy type II
Masoud Shekarabi,1 Nathalie Girard,1 Jean-Baptiste Rivière,1 Patrick Dion,1 Martin Houle,2
André Toulouse,1 Ronald G. Lafrenière,1 Freya Vercauteren,1 Pascale Hince,1 Janet Laganiere,1
Daniel Rochefort,1 Laurence Faivre,3 Mark Samuels,1 and Guy A. Rouleau1
1Centre of Excellence in Neuromics, University of Montreal, Centre Hospitalier de l’Université de Montréal, Montreal, Quebec, Canada.
2McGill Cancer Centre, Montreal, Quebec, Canada. 3Centre de Génétique, Hôpital d’Enfants, Dijon, France.
Hereditary sensory neuropathies form part of the inherited periph-
eral neuropathies that are subdivided into 3 categories, depending
on the selective or predominant involvement of the motor or senso-
ry peripheral nervous system (PNS) (1). The most common of these
neuropathies affect both motor and sensory nerves. In the second
category, only the peripheral motor nervous system is affected, and
the neuropathy is classified as a distal hereditary motor neuropa-
thy. Finally, there are neuropathies in which sensory dysfunction
prevails, and these are referred to as hereditary sensory and auto-
nomic neuropathies (HSANs). Hereditary sensory and autonomic
neuropathy type II (HSANII; OMIM 201300) is an early-onset auto-
somal recessive disorder. It is characterized by loss of perception
to pain, touch, and heat attributable to a partial loss of peripheral
sensory nerves (2–4). In 2004, we reported mutations in the heredi-
tary sensory neuropathy type II (HSN2) gene, a single-exon ORF
identified in Quebec and Newfoundland families, as the cause of
HSANII (5). Subsequently, 3 independent groups also reported
causative HSN2 mutations in unrelated populations (6–10).
In 2001, large intronic deletions in the with-no-lysine(K)–1
(WNK1) gene were reported to cause Gordon hyperkalemia-hyper-
tension syndrome, also referred to as pseudohypoaldosteronism
type II (PHAII; OMIM 145260) (11). PHAII is a dominant disor-
der, the main feature of which is hypertension (12, 13). Members
of the WNK family contain a Ser/Thr catalytic domain similar to
that of other kinases. However, one of their unique characteristics
is that the well-conserved lysine residue of the active domain is
instead a cysteine (14). In the case of WNK1, this kinase domain
extends from the end of exon 1 to exon 4. WNK1 has an autoinhibi-
tory domain of its kinase activity (15). WNK1 has been shown to
interact with a number of cellular proteins (e.g., synaptotagmin-2,
MEKK2/3, protein kinase B) (16–18). Early experiments using
WNK1-specific antibodies demonstrated that WNK1 is not present
in all cells; rather, it was mostly localized to the polarized epithelia
of the liver and kidney (19). With the exception of a recent report
in which neural precursor cells were used (20) and of one in which
WNK1 expression was observed in the developing brain (21), very
little has been reported about WNK1 in the nervous system. Given
the symptoms of PHAII patients, WNK1 experiments conducted to
this day were done with kidney or liver tissues or in cellular models
derived from these. We now report that HSN2, which was initially
believed to lie within intron 8 of WNK1, is a nervous system–specific
exon of WNK1 that we will refer here as the WNK1/HSN2 isoform.
Compound heterozygous mutation in WNK1 and HSN2 cause HSANII.
A French family (Figure 1A) was referred to G.A. Rouleau’s group
in Montreal by L. Faivre for an HSN2 genetic analysis to confirm
the diagnosis of HSANII in an 18-year-old female. Clinically, she
had classic HSANII symptoms (2–4). Examination of the patient
revealed no dysautonomia or abnormal blood pressure. Vegetative
tests elicited by cutaneous stimulation revealed normal sympathet-
ic reflexes. Parasympathetic cardiac function was found to be nor-
mal during a forced breathing test. The only apparent autonomic
dysfunction observed in the patient was excessive hand sweating.
Nonstandard?abbreviations?used: DRG, dorsal root ganglia; HSANII, hereditary
sensory and autonomic neuropathy type II; HSN2, hereditary sensory neuropathy
type II; PHAII, pseudohypoaldosteronism type II; PNS, peripheral nervous system;
UTR, untranslated region; WNK1, with-no-lysine(K)–1.
Conflict?of?interest: The authors have declared that no conflict of interest exists.
Citation?for?this?article: J. Clin. Invest. 118:2496–2505 (2008). doi:10.1172/JCI34088.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 118 Number 7 July 2008
DNA was obtained from the daughter, brother, and both parents
(Figure 1A). Sequencing the HSN2 ORF identified a heterozygous
1-bp deletion (639delA, Arg214fsX215) in the affected daughter,
her asymptomatic brother, and the father (Figure 1B). Surprisingly,
no other mutation could be found in HSN2. Despite the multiple
studies (5–10) that authenticated the HSN2 ORF as causative of
HSANII and its apparent independence from WNK1, we decided to
screen the entire coding sequence of WNK1 and identified a second
mutation (1584_1585delAG, Asp531fsX547) in exon 6 of WNK1
(Figure 1C). This 2-bp deletion was inherited from the mother,
absent in the unaffected brother, and predicted to result in a trun-
cated protein at amino acid 547 of WNK1. The mother presented no
abnormal blood pressure or symptoms that could be connected to
HSANII. This should not be surprising, given that all WNK1 muta-
tions so far associated with hypertension were all large deletions
in the gene’s first intron that led to an overexpression of the gene
(11). Given that both mother and daughter presented no obvious
symptoms of abnormal blood pressure, it can be surmised that par-
tial loss of WNK1 function has no blood pressure phenotype. The
unexpected discovery of these compound heterozygous mutations
in the affected daughter — one carried in WNK1 and the other in
HSN2 — led us to speculate that HSN2 might be an alternative exon
of WNK1, rather than an independent gene.
Nervous tissues express an HSN2 mRNA with a size similar to that of
WNK1. Because the coding regions and regulatory elements of WNK1
and HSN2 are well conserved between mouse and human (86% iden-
tical), we used the mouse Wnk1 orthologous gene to test our hypoth-
esis. To detect the mRNA encoding the Hsn2 sequence, a Northern
blot of adult mouse tissues was probed with a 434-bp DNA fragment
that recognized the putative 3′ coding region of Hsn2. A single band,
with a size slightly greater than approximately 10 kb (Figure 2A, top),
appeared in nervous system tissues exclusively. This band was pre-
dominant in the spinal cord, but it was also detected in the brain and
dorsal root ganglia (DRG) of adult mice. At E13, the mouse embryo
appears to express this mRNA in its body rather than its distal limb
area and its nose area. The size of this mRNA was in accordance with
observations made by 3 independent groups that detected an approx-
imately 10.5-kb WNK1 mRNA in the brain (14, 22, 23). A smaller
kidney-specific isoform (~9.0 kb) in which exons 1–4 of WNK1 are
replaced by an alternative exon 4B has also been reported (22, 23).
Our result suggests that HSN2 is part of the WNK1 mRNA, making
it an unreported and novel nervous system–specific disease-causing
isoform of WNK1 (WNK1/HSN2). Past poly(A) Northern blot and RT-
PCR investigation of HSN2 failed to detect this species, because the
probes and primers used were designed to recognize what were the 2
putative untranslated region (UTR) of HSN2 (5), which now appear
to be spliced out of the WNK1/HSN2 mRNA. By comparison, Wnk1
mRNAs that do not contain Hsn2 sequences were detected using a
Wnk1-specific probe in a broad range of neuronal and non-neuronal
tissues (Supplemental Figure 4; supplemental material available
online with this article; doi:10.1172/JCI34088DS1). Expression of
Wnk1 in DRG was previously recorded in the course of a gene expres-
sion microarray study (24).
RT-PCR shows that Hsn2 has flanking exons that are those of Wnk1. To
further investigate whether Hsn2 is an alternatively spliced exon of
Wnk1, we performed RT-PCR reactions with primers flanking the
region between the coding region of Hsn2 and its neighboring Wnk1
exons (Figure 2C, primers 1 and 3a for exon 8 to Hsn2; primers 4 and 5
for Hsn2 to exon 10). When amplifications between Wnk1 exon 8 and
Hsn2 were performed, a 160-bp band was visible in all neuronal tissues
(Figure 2D, arrow 3) but absent from non-neuronal tissues. A very
weak 160-bp band is visible in the kidney, but in light of subsequent
immunodetections (see below), this may be due to contamination
of the kidney sample with adrenal glands; moreover, the Northern
Mutations in the WNK1/HSN2 gene. (A) Segregation of the 2 mutations identified in the nuclear family. The single affected individual has com-
pound 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.
2498? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 118 Number 7 July 2008
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 118 Number 7 July 2008
blot analysis (Figure 2A) established the absence of Hsn2-containing
mRNA in this organ. This 160-bp band corresponded to the predict-
ed size of a single poly(A) species that would contain both exon 8 of
Wnk1 and Hsn2; this was subsequently confirmed by the sequencing.
The same amplifications of the region between Wnk1 exon 8 and Hsn2
also yielded an additional band of approximately 420 bp in the brain
and spinal cord. Sequencing of this 420-bp fragment revealed the
existence of an mRNA that contained exon 8 of Wnk1, a novel exon
downstream of exon 8 (putative exon 8B in Figure 2, C and F) and the
Hsn2 exon. The splicing of this putative exon 8B in the brain Wnk1
mRNA was confirmed by RT-PCR reactions with primers in Hsn2
and the putative exon 8B (Figure 2C, primers 2 and 3b), which pro-
duced a band of 320 bp (Figure 2B). The putative exon 8B encodes an
ORF of 86 amino acids, and this peptide sequence was used to iden-
tify orthologous exon 8B sequences from birds, amphibians, and fish
(Figure 3A). When amplifications between Wnk1 exon 10 and Hsn2
were prepared (Figure 2C, primers 4 and 5), a single band of 250 bp
corresponding to the predicted size of a single poly(A) transcript
encoding Wnk1’s exons 10, 9, and Hsn2 (Figure 2D, top) was visible,
again only in neuronal tissues; this was confirmed by sequencing.
More RT-PCR reactions were then performed to investigate which
of the more distal exons of Wnk1 are also parts of the Wnk1/Hsn2
isoform. In order to investigate the exons upstream to Hsn2, sepa-
rate reactions were performed using a primer located in Hsn2 (Figure
2C, primer 3a) and a series of primers in Wnk1 exon 1 (Figure 2C,
primers 7–10). When analyzed on agarose gel, the reactions from
DRG, using primers 3a and 7, yielded a single band of approximately
2.2 kb (data not shown), and the amplifications between primers 3a
and 8, 9, or 10 yielded single bands as well (data not shown). WNK1
transcripts have been reported to be initiated from 2 distinct pro-
moters (P1 and P2) (25); to investigate which promoter was used for
the transcription of Wnk1/Hsn2, we used RT-PCR (Figure 2E, arrow
2). The existence of multiple Wnk1 promoters is an important point,
and multiple approaches (RT-PCRs and Western immunodetections)
were necessary to establish which promoter appears to be used for
WNK1/HSN2 (see Discussion). To observe the exons downstream to
Hsn2, reactions were performed using a primer in Hsn2 and a primer
in Wnk1 exon 16 (Figure 2C, primers 4 and 11). When loaded on gel,
these amplifications revealed an approximately 650-bp band from the
brain tissues and an approximately 950-bp band from the DRG (Fig-
ure 2E, arrows 3 and 4). The subsequent sequencing of these 2 frag-
ments showed that both the brain and DRG lacked exon 11 (462 bp)
and that the brain additionally lacked exon 12 (285 bp) (Figure 2F);
this alternative splicing confirmed previous reports in which exons
11 and 12 were shown to be skipped in some Wnk1 mRNAs expressed
in mouse tissues (13, 23). Further amplifications of the region
between the last exons of Wnk1 and Hsn2 were, however, more diffi-
cult because of the large distance between these primers; nonetheless,
a product corresponding to the expected mRNA size (3.8 kb) could
be amplified from Hsn2 to exon 24 of Wnk1 (Figure 2E, arrow 1).
A product was also amplified from Hsn2 to Wnk1 exon 25, but its
intensity was very weak (data not shown).
Rapid amplification of cDNA end reactions from Hsn2. To corroborate
the results of the various RT-PCR products, 5′ rapid amplification
of cDNA ends (5′RACE) reactions were also initiated to character-
ize Wnk1/Hsn2 isoforms (with or without putative exon 8B). DNA
sequencing of the region upstream to Hsn2 confirmed the presence
of a highly conserved splice junction (Figure 3B), and the mRNA
did contain Wnk1 exons 2–8 (with the inclusion of exon 8B in some
cases) and the Hsn2 exon. The distance between Wnk1 exon 1 and
Hsn2 was too large (~2.2 kb) for the 5′RACE reaction to proceed that
far from Hsn2. DNA sequencing of the region amplified by prim-
ers in Wnk1 exon 10 and Hsn2 also revealed a splice junction highly
conserved across species in the region 3′ to Hsn2 (Figure 3B). No
3′RACE reactions were done because of the size of Wnk1, but the
gene has been reported to have 2 alternative polyadenylation sites
(25). The distance between Hsn2 and these 2 polyadenylation sites
is too large (~4 kb) for direct amplifications to be possible, and this
portion of Wnk1 does not permit an easy distinction of Wnk1/Hsn2
and Wnk1 transcripts by PCR. It has, however, been reported that
the second site appears to be more abundantly used in tissues where
WNK1 expression is high, such as the brain (25).
Western blot detection of the Wnk1/Hsn2 isoform. In order to confirm
that Hsn2 is an exon of Wnk1, we prepared Western blots using whole
protein lysates from adult mice, and we separately detected them
with an antiserum specific to the C-terminal portion of HSN2 and
a purified commercial antibody that recognizes the N terminus of
WNK1 (Alpha Diagnostic International) (Figure 2C). Past reports
examining WNK1 expression in mouse showed that its MW was
slightly greater than 250 kDa (26, 27), and this was confirmed in
every tissue detected here with anti-WNK1 (Figure 4, top). By com-
parison, the anti-HSN2 antiserum detected a band only in tissues of
the nervous system, and the MW of this band was smaller (~230 kDa)
(Figure 4, middle). The anti-WNK1 antibody failed to reveal any
band at approximately 230 kDa in which WNK1/HSN2 was
detected with the anti-HSN2 antiserum. To confirm the specificity
of the signal detected with the anti-HSN2 antiserum, it was prein-
cubated with its antigenic peptide prior to its use for Western blot
and immunohistochemistry detections. This competition step was
found to prevent the detection of the approximately 230-kDa band
observed in the DRG (Supplemental Figure 1). WNK1 was previously
established to contain 2 distinct promoters (P1 and P2) (25) (Fig-
ure 2C), and 2 distinct bands could be observed (~230 and slightly
greater than ~250 kDa) when other anti-WNK1 antibodies were used
(R&D Systems and Kinasource; Supplemental Figure 2). Together,
the detections of Western blots with alternative anti-WNK1 antibod-
ies and the Western blot detection presented in Figure 4 suggest that
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 (bot-
tom). (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 posi-
tions of Wnk1 promoters are indicated by black arrows, and the sites
detected by anti-HSN2 and anti-WNK1 (Alpha Diagnostic Internation-
al) are indicated in red. Kin. D., WNK1 kinase domain. (D) RT-PCR
amplifications from brain, spinal cord, DRG, sciatic nerve, kidney, tes-
tis, 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 (prim-
er 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: Ampli-
fications 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.
2500? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 118 Number 7 July 2008
it is most likely the second ATG (+640) that is used in the translation
of WNK1/HSN2. The 2 alternative polyclonal anti-WNK1 antibod-
ies (Kinasource and R&D Systems) recognize portions of the pro-
tein that are downstream to the second ATG, while the anti-WNK1
antibody from Alpha Diagnostic International recognizes a portion
(TSKDRPVSQPSLVGSKE) of the protein (14) that is located between
the first and the second ATG of the promoters P1 and P2.
Immunohistological investigation of WNK1/HSN2 distribution. While
WNK1/HSN2 was observed in both the CNS and PNS, the DRG
and sciatic nerves are the tissues with the highest expression (Fig-
ure 4). We therefore chose to perform immunohistochemistry to
investigate which cells inside the DRG and sciatic nerve expressed
WNK1/HSN2. In DRG, the signal was predominantly in the satel-
lite cells that envelop sensory neurons (Figure 5, A and B, arrows),
but low expression was also observed in the cell bodies of neurons
(Figure 5, A and B, arrowheads). The DRG anti-HSN2 detections
were overlaid with parallel detections made using a cocktail of
anti–SMI-31 and anti–SMI-32 (neuronal markers). The identity
of satellite cells was established with an antibody specific to the
glutamine synthetase (28) (data not shown). In cross sections of
the sciatic nerve, a strong WNK1/HSN2 signal was visible in the
Schwann cells that surround axons (Figure 5D) and in a mosaic
distribution of axons (Figure 5, C and D). The same neuron-spe-
cific antibodies were used to make parallel detection and overlay
images. Given the distribution of WNK1/HSN2-positive axons in
the sciatic nerve, which includes fibers from sensory and motor
neurons, separate cross sections from both dorsal (Figure 5E) and
ventral roots were prepared (Figure 5F). WNK1/HSN2 expression
in the 2 roots revealed a striking difference, as the signal was sub-
stantially stronger in dorsal roots (almost exclusively containing
sensory axons) than in ventral roots (almost exclusively containing
motor axons). Given the weak expression of WNK1/HSN2 in the
cell body of the DRG neurons, it generally appears that neurons
express more WNK1/HSN2 in the axon than the cell body in vivo.
To further investigate this, we prepared primary cultures of sen-
sory neurons from DRG and detected these with anti-HSN2 and
anti-WNK1 (Alpha Diagnostic International) antibodies. These
detections were subsequently overlaid with detections made with
the antibodies recognizing the neuronal markers mentioned above
(Supplemental Figure 3, A–D). The results showed that in these
primary neurons, WNK1/HSN2 is expressed in both the cell body
and axons, while WNK1 is only expressed in the cell body and not
in the axons. Moreover, immunohistochemistry detection of both
DRG and sciatic nerve using anti-WNK1 showed that the protein
was ubiquitously expressed in the neuronal somata of the DRG
neurons but absent from axonal fibers of sciatic nerve (Supple-
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).
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 118 Number 7 July 2008
mental Figure 3, E–H). The Schwann cells surrounding the fibers
of the sciatic nerve express WNK1 (Supplemental Figure 3H,
arrow) in addition to WNK1/HSN2 (Figure 5D).
The expression of WNK1/HSN2 in components of the CNS was
also investigated by immunohistochemistry (Figure 6). Cross sec-
tions of adult mouse spinal cord were prepared and detected with
the anti-HSN2 antiserum. In the spinal cord, we observed a strong
signal in superficial layers (LI and LII) (Figure 6, A and B), which
receive the neuronal projections that carry a variety of sensory infor-
mation (including most nociceptive information from the PNS).
The expression of WNK1/HSN2 also appeared in the fibers of the
Lissauer tract (Figure 6B). The neuronal nature of fibers express-
ing WNK1/HSN2 in LI and LII was confirmed using the neuronal
marker SMI (Figure 6B) and a detection using an anti-NeuN anti-
body (Figure 6D). The axon fibers of dorsolateral funiculus (DLF)
and lateral funiculus (LF), which contain ascending sensory fibers,
also expressed WNK1/HSN2 (Figure 6, A and B).
At the time of the publication of the identification of HSN2, the
most remarkable feature of this gene was its single-exon structure.
The exon mapped within the intron on the same strand and in the
same orientation as another gene, WNK1 (5). While the character-
ization of the HSN2 ORF was hampered the existence of an incom-
plete EST sequence, the ORF was nonetheless found to have three
methionines in 5′ and an AATAAA poly(A) addition signal in 3′.
While the HSN2 ORF was novel, WNK1 had been extensively stud-
ied in the context of tissues that are affected in PHAII patients. No
symptom overlap was apparent between PHAII and HSANII.
We have now established that HSN2 is as an alternatively spliced
exon of WNK1 and that this selectively occurs in nervous tissues.
The WNK1/HSN2 nervous system isoforms appear to include
either HSN2 alone or HSN2 along with a novel exon (putative exon
8B). EST clones encompassing both Wnk1 and Hsn2 were previous-
ly observed in a mouse E10.5 cDNA library (I.M.A.G.E. 30862659
and 30862741). However, while sequencing these ESTs would have
validated our observations, they are no longer available. Prior to
this report, only 2 reports examined WNK1 expression in the ner-
vous system (20, 21). In one of them, by Sun et al., neural precursor
cells and the anti-WNK1 antibody from Alpha Diagnostic Inter-
Separate Western immunodetections of WNK1 and WNK1/
HSN2. Various mouse tissues from adult or E13 animals
were loaded and detected with anti-WNK1 (Alpha Diagnos-
tic International) (top) or with IgG purified anti-HSN2 anti-
body (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).
WNK1/HSN2 histological immunodetections. (A) Immunohistochemistry
detection of adult mouse DRG (from L5 sections) with anti-HSN2 anti-
serum (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.
2502? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 118 Number 7 July 2008
national were used, and a protein said to have a MW of approxi-
mately 230 kDa instead of approximately 250 kDa as shown here
was observed. It is possible that when Sun et al. identified WNK1,
they referred to the antibody specification sheet (catalog WNK11;
Alpha Diagnostic International), which indicates that the MW
of WNK1 is 230 kDa. However, a number of independent groups
reported the MW of WNK1 to be approximately 250 kDa (17, 26,
27, 29). Even though the neuron-specific isoforms (HSN2 alone
or HSN2 with exon 8B) of WNK1 incorporate additional amino
acids, their MW is nonetheless lower than that of the previously
reported WNK1 long isoform, even considering the skipping of
exon 11 in the DRG and of exons 11 and 12 in the brain. This lower
MW of WNK1/HSN2 is likely attributable to the use of the second
promoter (P2) of WNK1 (25). When the P1 promoter is used, trans-
lation is initiated at the ATG (+1), and when the P2 promoter is
used, translation is initiated at the more downstream ATG (+640).
In both instances the kinase domain of WNK1 is retained, but the
use of the second ATG makes the mRNA 642 bp shorter and the
protein 214 amino acids smaller. An in vitro study has previously
shown that both promoters (P1 and P2) are active and that the
second ATG of the P2 promoter is actually sufficiently used to
make this form the dominant one in the kidney (25). No exami-
nations have thus far been made in CNS or PNS tissues, but past
investigation of the region immediately upstream of the P2 pro-
moter with DNA analysis software (TESS; http://www.cbil.upenn.
edu/cgi-bin/tess/tess) identified transcription binding sites that
are recognized by neuronal transcription factors (e.g., NF-ATp,
HES-1) (21). Unfortunately our 5′RACE reactions did not proceed
far enough to provide information about which of the two pro-
moters was used for WNK1/HSN2 transcription. RT-PCR using
a primer upstream of the 5′UTR of P1 and a primer in Hsn2 were
prepared with RNA from DRG; the same 5′UTR of P1 primer was
used in separate reactions with a primer located in exon 6 of Wnk1
to amplify kidney cDNA (Figure 2E, arrow 2). The first amplifica-
tion gave no product (even though long-range PCR conditions had
allowed us to amplify larger fragment such as the one between the
Hsn2 and Wnk1 exon 24). The second amplification, however, pro-
duced a fragment of the expected size (~2.5 kb). This suggests that
the transcription of Wnk1/Hsn2 occurs through the second pro-
moter. The comparison of different anti-WNK1 antibodies (Figure
4 and Supplemental Figure 2) also suggested that it is the second
ATG that is used to initiate the translation of WNK1/HSN2.
The recessive mutations described in HSANII, here and in pre-
vious reports (5–10), all lead to truncations of the WNK1/HSN2
nervous system–specific protein. The disease-causing mutations
in WNK1 identified to date were large, heterozygous intronic
deletions that increase the gene expression (11). This impact on
the expression level in PHAII patients may explain the absence of
hypertension in individuals affected with HSANII, as the expres-
sion of the WNK1 isoform (in which the HSN2 exon is not incorpo-
rated) should not be affected. It is hard to speculate whether some
HSANII cases may later be found to have mutations in WNK1 and
not in the HSN2 portion of WNK1/HSN2. However, genetic data
presented here suggest that one mutation in the HSN2 exon is
sufficient to cause the HSANII phenotype when combined with
a mutation in WNK1 on the other allele. Moreover, homozygous
mutations disrupting WNK1 isoforms (without HSN2) may be
lethal, which would explain why all loss-of-function mutations
reported to date were located in the HSN2 exon. A 2003 report
supports this possibility, as homozygous mutations of the mouse
WNK1 were shown to be embryonically lethal (30).
While our results indicate that WNK1/HSN2 is expressed in com-
ponents of both the CNS and PNS, the HSANII symptoms indicate
a defect in peripheral sensory perception. This is consistent with
the disease phenotype in which expression of neuronal WNK1/
HSN2 appears to be stronger in the sensory neurons than in motor
neurons. The primary event of this disorder is liable to occur in
sensory fibers, as a very high number of unmyelinated sensory
fibers are observed in HSANII. In conjunction with the absence of
motor deficit, this constitutes a distinctive pathological hallmark
of the disease (31). The expression of WNK1/HSN2 in the outer
edge of the DLF, which is where the Lissauer tract travels, may be
very relevant for the development of HSANII symptoms, as cells
from the Lissauer tract has been reported to be missing in familial,
congenital, and universal insensitivity to pain (32, 33). These obser-
vations suggest that a more detailed investigation of WNK1/HSN2
expression in components of the posterior horn (e.g., the substan-
tia gelatinosa, where the pain-sensing unmyelinated C fibers are
located) (34) at different levels of the spinal cord is needed. Even
though no strong evidence supports the hypothesis that myelina-
ting Schwann cells are involved in HSANII, it may eventually be
interesting to test whether Schwann cells normally proliferate in
WNK1/HSN2 protein expression in the adult mouse CNS. (A) Low-mag-
nification 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 funicu-
lus (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) Immunodetec-
tion of LI and LII with anti-HSN2 (red). (D) Overlaid images of the detec-
tion of HSN2 (red in C), NeuN (green), and the nuclear labeling (blue).
Arrows indicate neurons where the colocalization is observed, confirm-
ing the presence of HSN2 protein in cells of this region.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 118 Number 7 July 2008
the event of neuronal damage or degeneration. Biopsies of the sural
nerve, which contains only sensory fibers, from an HSANII famil-
ial case study where multiple affected individuals were diagnosed
showed not only a complete absence of myelination but also no
evidence of Schwann cell proliferation (35). Based on our observa-
tion of WNK1/HSN2 expression in satellite and Schwann cells, it
can be hypothesized that the symptoms worsen when these cells
respond (28, 36) to the damage sensory neurons suffer because
of mutant WNK1/HSN2 expression, although this is speculative.
WNK1/HSN2 could act as molecular switch that initiates the pro-
liferation of these cells. In other tissues, WNK1 was shown to acti-
vate ERK5 through a MEKK2/3-dependent mechanism, a signal-
ing pathway involved in cell growth and proliferation (18). Though
WNK1/HSN2 appears to be expressed predominantly in the PNS,
it is also expressed in the CNS. The primary afferents of DRG that
transmit sensory stimuli (among them nociceptive) signals are
structures that enter the spinal cord through the dorsal root entry
zone (DEZ). Once inside the spinal cord, these primary afferents
make synapses with second-order neurons of laminae I, II (external;
Figure 6A), V, and VI, and from there these fibers take different
pathways to transmit HSANII-relevant sensory and nociceptive
impulses to structures of the brain stem and diencephalons (37).
The gray matter of the spinal cord appeared stained when we used
anti-HSN2 and overlaid images of the anti-HSN2 detection and
detection of the axonal markers SMI-31/32 (Figure 6), suggesting
that any specific HSN2 staining in the gray matter (with the excep-
tion of the DEZ) comes mainly from axons, and not cell bodies. The
expression of WNK1/HSN2 in the brain was not investigated, but
a comprehensive analysis of this will eventually be informative and
may help to better understand the disease. Given the strong expres-
sion of WNK1/HSN2 in the dorsal roots, it appears probable that
the axons of the sciatic nerve expressing this protein are sensory.
The difference in the level of WNK1/HSN2 expression observed
in the neuronal somata of DRG (Figure 5A) and primary culture
prepared from the same DRG (Supplemental Figure 3A) may be
the consequence of the activation the neuronal pathway typical of
cultured primary neurons. The mechanical, chemical, and new in
vitro environment stresses these neurons undergo when the prima-
ry culture is established may also activate the expression of WNK1/
HSN2. Together, the histological observations suggest that WNK1/
HSN2 may have a critical role in the development of the pain-sen-
sory pathways in both the CNS and PNS. WNK1 expression in the
PNS during development has not yet been well investigated. Only a
few observations made in the CNS were reported, and these showed
that WNK1 expression could be seen in the granular layer and cer-
ebellar Purkinje cells, and only weak staining could be observed in
the molecular layer and white matter (21). The observation that
WNK1 is primarily present in the cell body while WNK1/HSN2 is
in the axon may suggest a role in sensory axon maintenance, which
is compatible with the neuropathy seen in HSANII.
The sequences of HSN2 and of the putative exon 8B have no
known motif that suggests a particular function, and so how their
insertion in WNK1 affects this protein’s function remains to be
elucidated. The role of WNK1 in PHAII is partly thought to be
attributable to its interaction with WNK4, with ion channels, and
with cotransporters through which sodium absorption and potas-
sium wasting is modulated (13). Under normal conditions, WNK1
participates in the regulation of a number of K+ channels such as
ROMK1 (38) and Na+, K+, 2Cl–, and Na+Cl– cotransporters such as
NKCCs and NCCs (39). These interactions indicate that WNK1
plays an important role in the regulation of ionic transport across
the plasma membrane. In the context of HSANII, it is important
to note that the activity of a very relevant ion transporter, TRPV4,
is regulated by WNK1 and WNK4 (40). TRPV4 is a vanilloid recep-
tor involved in thermal and mechanical nociception (40). TRPV4–/–
mice were found to exhibit hypoalgesic responses to pressures of
the tail and acid applications and they have a delayed response to
escape from hot temperature (41, 42). How the alternative splic-
ing of HSN2 and/or of the putative exon 8B to form a nervous
system–specific WNK1/HSN2 isoform affects the normal activity
of WNK1 or its affinity for WNK4, TRPV4, or other pain receptors
activity is unknown. At this point, it may only be postulated that
WNK1/HSN2 is involved in the proper localization and/or ionic
regulation of TRPV channels in the nervous system.
Our study of Wnk1/Hsn2 stresses the importance of tissue-specific
alternative splicing, as it shows that mutations in different splice
variants may affect protein function differently and consequently
lead to very different tissue-specific pathologies. The 9 laminopathies
(including lipodystrophies, muscular dystrophies, and progeroid
syndromes) that are caused by mutations of the LMNA/C gene are
good examples of such a phenomenon (43, 44). Unlike the situation
with WNK1, there is no association between the position or the exon
where the mutation occurs and the tissue or system affected.
Mutation detection. After written informed consent was received from the fam-
ily, blood samples were collected and DNA was extracted from peripheral
blood lymphocytes using a standard protocol. All 28 exons of WNK1 and the
single predicted exon of HSN2 were amplified by PCR using flanking intron-
ic primers before they were sequenced with an ABI 3700 sequencer, according
to the manufacturer’s recommended protocol (Applied Biosystems).
RNA isolation and mRNA purification. All animal experiments were approved
by the Institutional Committee for Animal Protection (CIPA) of the Cen-
tre Hospitalier de l’Université de Montréal. Total RNA was prepared from
the different tissues of C57BL/6 adults and E13 embryos according to the
method described by Chomczynski and Sacchi (45), and poly(A) RNA was
subsequently purified using an Oligotex mRNA Midi Kit (QIAGEN).
Northern blot analyses. Purified poly(A) RNA (2 μg) from the different tis-
sues (C57BL/6 adult and E13 embryo) was electrophoresed on a denatur-
ing formamide/formaldehyde gel and transferred onto a Hybond nylon
membrane (GE Healthcare). The membrane was hybridized with a PCR-
amplified murine Hsn2 fragment of 435 bp that recognized the 3′ region of
the gene. This 435-bp probe was generated using the primers 5′-CATGCT-
CAAACACCAAGTTCTT-3′ and 5′-TGAAGCAGATAAGACCTGCTGA-3′
that cover the region found between position 768 and 1,203 bp past the 5′
Hsn2 spliced-in sequence (Figure 3B). The membrane was also probed with
β-actin DNA fragments. Probes were radiolabeled with [α-32P]dCTP–labeled
Random prime PCR fragment (Rediprime II Random Prime Labelling Sys-
tem; GE Biosciences). Before use, the fragments were sequenced to confirm
their identity. Hybridization was performed according to the procedure
previously described by Houle et al. (46). Wnk1 Northern blotting was per-
formed with an amplified DNA template using 5′-TGACATCGAAATCG-
GCAGAGGCT-3′ and 5′-GGGTACGGGTAGAATTAGCAGAAG-3′ primers.
The probe spans from the end of exon 1 to exon 6 (850 bp).
Analysis of Wnk1 splicing events by RT-PCR. cDNA synthesis and PCR analy-
sis have been described previously by Herblot et al. (47). Total RNA (1 μg)
was used as template for first-strand DNA synthesis. Long-range PCR was
performed using Long PCR Mix (Fermentas). For each amplification of a
specific cDNA, 18S, was coamplified as internal control to correct for varia-
tions in loading. Oligonucleotide sequences are listed in Supplemental
2504? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 118 Number 7 July 2008
Table 1. cDNAs were amplified for 35 cycles, and PCR conditions were: initial
denaturation of 94°C for 3 minutes, then 35 cycles of 93°C for 10 seconds,
55°C for 30 seconds, and 68°C for 2 minutes and 45 seconds and then 1
cycle of 68°C for 10 minutes. The amplified fragments were analyzed by
agarose gel electrophoresis, and when the sequence of specific bands was
investigated, the band was then purified with the QIAEX kit (QIAGEN) and
cloned in TOPO PCRII (Invitrogen). Sequence analysis was performed on
the ABI 3700 sequencer at the Génome Québec Innovation Centre.
5′RACE assay. RNA was used in conjunction with the GeneRacer kit
(Invitrogen) for the 5′RACE and 3′RACE assays. PCR products from the RACE
reaction were analyzed on agarose gel and subcloned into TOPO-4 Vector
(Invitrogen). Ninety-six clones were chosen, and 23 were sent for sequencing.
Ortholog identification. Orthologous sequences were identified by perform-
ing a tBLASTn search of GenBank genome sequences using the peptide
sequence encoded by exon 8B of the mouse gene.
Antibody production and immunodetection. DNAstar software version 5.02
was used to examine protein sequence homology, and freely available soft-
ware (ANTIGENIC; http://immunax.dfci.harvard.edu/Tools/antigenic.
html) was used to explore the peptide sequence of HSN2 with good anti-
genicity. Following these analyses, the best candidate protein sequences
were selected for the synthesis of peptides (Sheldon Biotechnology Centre,
McGill University) to be used to prepare rabbit polyclonal antibodies at our
facilities. An LSPQSVGLHCHLQPVT peptide corresponding to a sequence
in the 3′ end of the HSN2 exon was the one that allowed the preparation
of the best antibody in terms of antigenicity and specificity. The peptides
were injected with complete and subsequently with incomplete Freund’s
adjuvant in rabbit to produce an antiserum specific to HSN2 protein
(McGill animal facility). Either crude serum (1:1,000) or IgG purified anti-
body (1:5,000; Montage; Millipore) was used for the Western blot (7.5%)
and histological immunodetections. C57BL/6 adult mouse tissues were
prepared in SUB lysis buffer (8 M urea, 0.5% SDS, 200 mM β-mercaptoeth-
anol) and resolved by SDS-PAGE. Anti-WNK1 antibody (Alpha Diagnostic
International) was used at 1:1,000. The anti-WNK1 made to recognize the
kinase domain (Kinasource) was used at 1 μg/ml, and the anti-WNK1 from
R&D Systems was used at 0.5 μg/ml. TOTO-3 iodide (Molecular Probes;
Invitrogen) was used to generate nuclear staining.
Immunohistochemistry was performed as described previously (48). A
cocktail of anti–SMI-31 and anti–SMI-32 (SMI Monoclonals; Covance)
was used at 1:1,000 as an axonal marker. Neuronal marker NeuN anti-
body (Upstate) was used at 1:150. Alexa Fluor 555 secondary anti-rabbit
and Alexa Fluor 488 secondary anti-mouse antibodies (Molecular Probes;
Invitrogen), respectively, were used (1:1,000) to visualize rabbit and mouse
primary antibodies. Our observations were carried out using a Leica TCS
SP5 broadband confocal microscope. The system was equipped with the
AOBS (acousto-optical beam splitter) for optimal beam splitting. Control
immunodetections were made using the preimmunized serum obtained
from rabbits, which subsequently yielded the anti-HSN2 antiserum, after
their exposure to the HSN2 antigenic peptide, and these did not show a
specific signal in either immunohistochemistry or Western blots.
For competition experiments, anti-HSN2 antiserum was incubated with
5-fold excess of its antigenic peptide overnight at 4°C. The same amounts
of protein lysates from adult mouse DRG were loaded on a SDS-PAGE
and transferred on PVDF membrane. One strip was incubated with anti-
HSN2 antiserum alone, and a second strip incubated with the anti-HSN2/
peptide mix overnight at 4°C.
Primary cultures of DRG sensory neurons. Adult mouse sensory neuronal cul-
tures were established essentially as described by Seilheimer et al. (49) with
some modifications. DRG were dissected from adult mouse C57BL/6 and
incubated with 10 mg/ml of collagenase D (Roche) for 45 minutes at 37°C.
Trypsin (0.25%) was added at the end of the collagenase D treatment for
30 minutes at 37°C. The tissues were washed once with cold Neurobasal
medium (Invitrogen) plus 2% inactivated goat serum and then triturated in
warm Neurobasal medium plus serum using fire-polished Pasteur pipettes.
One milliliter of the cell suspension was overlaid on 1 ml of 35% Percoll in
saline (Pharmacia; Amersham Biosciences) and centrifuged at 10°C at 285 g
for 15 minutes. The cell pellet, which includes sensory neurons, was washed
in 5 ml of fresh medium and resuspended in fresh warm medium with
50 ng/ml of nerve growth factor and plated on pol-d-lysine/laminin–coated
coverslips. They were cultured for 48 hours prior to immunocytochemistry.
Immunostaining was performed essentially as described previously (50).
The authors would like to thank all family members for their cooper-
ation. J.-B. Rivière is the recipient of a Canadian Institutes of Health
Research (CIHR) Doctoral Research Award. The CIHR supported
G.A. Rouleau with a research grant (IG1-78904) for this project.
Received for publication September 28, 2007, and accepted in
revised form April 16, 2008.
Address correspondence to: Guy A. Rouleau, Centre of Excel-
lence in Neuromics, University of Montreal, Centre Hospitalier
de l’Université de Montréal, 1560 Sherbrooke East, Room Y-3633,
Montreal, Quebec H2L 4M1, Canada. Phone: (514) 890-8000 ext.
24594; Fax: (514) 412-7602; E-mail: firstname.lastname@example.org.
Masoud Shekarabi and Nathalie Girard contributed equally to
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