Human Reproduction Vol.21, No.12 pp. 3178–3184, 2006
doi:10.1093/humrep/del293
Advance Access publication August 24, 2006.
Mutations in the testis-specific NALP14 gene in men suffering
from spermatogenic failure
G.H.Westerveld1,4, C.M.Korver1, A.M.M.van Pelt2, N.J.Leschot3, F.van der Veen1,
S.Repping1 and M.P.Lombardi3
1
Department of Obstetrics and Gynaecology, Center for Reproductive Medicine, Amsterdam, 2Laboratory of Endocrinology,
Department of Biology, Faculty of Science, Utrecht University, Utrecht and 3Department of Clinical Genetics, Academic Medical Center,
Amsterdam, the Netherlands
4
To whom correspondence should be addressed at: Department of Obstetrics and Gynaecology, Center for Reproductive Medicine,
Academic Medical Center, Meibergdreef 9, H4-205, 1105 AZ Amsterdam, the Netherlands. E-mail: [email protected]
BACKGROUND: Because of the common use of ICSI and the potential genetic aetiology of spermatogenic failure,
concern has been raised about transmitting genetic disorders to ICSI offspring. However, to date, in only ~15% of all
cases of spermatogenic failure, an underlying genetic cause can be identified. We have previously established an association between spermatogenic failure and chromosomal region 11p15. In this study, we set out to explore whether
NALP14, a gene recently mapped to 11p15, has a function in spermatogenesis and whether mutations in NALP14 can
cause spermatogenic failure. METHODS: We applied two different multiple tissue northern (MTN) blots to determine tissue specificity of NALP14 and performed immunohistochemistry on human testis with anti-NALP14 antiserum. To determine imprinting status of NALP14, we tested the expression pattern of two single-nucleotide
polymorphisms (SNPs) in human testis. Finally, we performed a mutation screen of the NALP14 gene in 157 men with
azoospermia or severe oligozoospermia by direct sequencing; 158 normospermic men served as controls. RESULTS:
NALP14 was, as are the three other genes in 11p15, exclusively expressed in testis. Within the testis, the NALP14 protein was mainly expressed in A dark spermatogonia, mid and late spermatocytes and spermatids. The mutation
screen revealed five mutations that occurred only in the patient group. One of these unique mutations introduced an
early stop codon in the NALP14 sequence, predicted to result in a severely truncated protein. CONCLUSION: Our data
suggest that NALP14 has a function in spermatogenesis and that mutations in this gene might cause spermatogenic
failure.
Key words: chromosomal region 11p15/genetics/male infertility/NALP14/spermatogenesis
Introduction
Subfertility, that is the inability to conceive after 1 year of
unprotected intercourse, affects 10–15% of all couples. In
∼50% of cases, this subfertility is due to spermatogenic failure
(de Kretser and Baker, 1999). Since the introduction, in 1992,
of ICSI (Palermo et al., 1992), a technique in which a single
spermatozoon is injected into an oocyte, men with only a few
spermatozoa in their ejaculate are able to father offspring that
is genetically their own. The use of ICSI has increased tremendously over the years. In 2001, >110 000 ICSI cycles were
performed in Europe alone (Andersen et al., 2005).
Because of the common use of ICSI and the potential
genetic aetiology of spermatogenic failure, concern has been
raised about transmitting genetic disorders to ICSI offspring.
The search for genetic causes of spermatogenic failure has thus
far been relatively unrewarding. The only common and wellestablished genetic causes of human idiopathic spermatogenic
failure are numerical and structural chromosome abnormalities
and Y-chromosome deletions (Huynh et al., 2002; Silber and
Repping, 2002; Gianotten et al., 2004; Repping et al., 2004). In
addition, some infrequent mutations have been identified in the
USP9Y, SYCP3 and FSHR genes that cause spermatogenic failure (Tapanainen et al., 1997; Sun et al., 1999; Miyamoto et al.,
2003). Altogether, these genetic abnormalities explain only
∼15% of all cases of idiopathic spermatogenic failure (Gianotten
et al., 2004; Repping et al., 2004).
We have recently described an association of chromosomal
region 11p15 with spermatogenic failure (Gianotten et al.,
2003). Thus far, three genes, namely Zinc Finger 214 (ZNF214),
Zinc Finger 215 (ZNF215) and heterogeneous nuclear ribonucleoprotein G-T (HNRNP G-T), that are predominantly or exclusively expressed in testis have been identified from this region
(Alders et al., 2000; Elliott et al., 2000). In these genes, we have
previously identified several unique mutations which are likely
to be responsible for spermatogenic failure in at least some men
(Gianotten et al., 2003; Westerveld et al., 2004).
3178 © The Author 2006. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
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NALP14 mutations in men with spermatogenic failure
ZNF214
telomere
centromere
ZNF215
NALP14
HNRNP G-T
10 kB
Figure 1. Schematic representation of testis-specific genes in chromosomal region 11p15. Size and relative distance are drawn on scale. The
arrows indicate the orientation of the genes.
Recently, an additional gene (NALP14, also known as NOD5)
was mapped to chromosomal region 11p15 (NM_176822) in
between ZNF214 and HNRNP G-T (Figure 1). NALP14
belongs to the NALP protein family (also known as PANs or
PYPAFs). This family consists of 14 cytoplasmic proteins that
are characterized by a NACHT, leucine-rich repeat (LRR) and
PYD (also called PAAD or DAPIN) domain. The NALPs are a
subfamily of the larger CATERPILLER family, which is likely
to be a highly conserved gene family, because it is structurally
related to a similar family in plants (Tschopp et al., 2003; Ting
and Davis, 2005). As NALPs have been described only
recently, little is known about their function, but a role for
NALPs in apoptosis (by activation of caspases) and in proinflammation signalling processes has been suggested.
In this study, we set out to explore whether NALP14 has a
role in spermatogenesis and, subsequently, whether mutations
in the NALP14 gene can cause spermatogenic failure.
Materials and methods
Northern blot
Two human multiple tissue northern (MTN) blots (BD Biosciences,
San Jose, CA, USA) were used to test RNA expression of NALP14 in
various tissues. The MTN Blot Human I contained heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas RNA. The
MTN Blot Human II contained spleen, thymus, prostate, testis, ovary,
small intestine, colon and peripheral blood leukocyte RNA. Blots
were hybridized and washed according to manufacturer’s instructions.
The NALP14 radioactive-labelled PCR probe was a 0.76-kb fragment
containing part of exon 3 of NALP14 (forward primer, 5′-gggccatgaaagtattcagttc-3′; reverse primer 5′-aacatctcattgcctggcttat-3′). β-Actin
was used as an internal control probe.
Cellular expression
Peptide design, synthesis and purification of the peptide and the rabbit
antibody against NALP14 (NH2–NPNLRSLDLGNNDLQD–COOH)
was performed by making use of the double XP program of Eurogentec
(Eurogentec, Liege, Belgium).
Human testis tissues were fixed in formalin and embedded in paraffin.
Five-micrometre testis sections were dehydrated, and after a wash in
phosphate-buffered saline (PBS), endogenous peroxidase was blocked
with 0.3% H2O2 in PBS. The sections were incubated with 1% bovine
serum albumin (BSA) and 5% goat serum for 1 h at room temperature,
followed by incubation with the NALP14-antibody overnight at 4°C.
After washing, the sections were incubated with biotinylated anti-rabbit (Vector Laboratories Inc., Burlingame, CA, USA). Detection of
the antibody was performed with an ABC peroxidase staining kit
(Vector Laboratories Inc.) and DAB (Sigma, St Louis, MO, USA) as a
substrate (brown). Sections were counterstained with Mayers’s
haematoxylin (blue). As a negative control, the NALP14 specific antibody was blocked with the (NALP14) synthetic peptide for 2 h before
incubation with the testes sections.
Genomic imprinting
To determine imprinting status of NALP14, the allelic expression of
two known single-nucleotide polymorphisms (SNPs) in NALP14,
namely E808K (rs10839708) and L1010F (rs17280682), was determined in RNA extracted from testis tissue of a man who was heterozygous for these SNPs at DNA level. RNA was isolated with the use
of the RNeasy Mini Kit (Qiagen, Venlo, the Netherlands), according
to the manufacturer’s instructions. cDNA was synthesized from 1 μg
of RNA using SuperScript II reverse transcriptase (Invitrogen, Breda,
the Netherlands) and the internal primer NALP14_1R_RT
(GTAGCCGCCGTCATCATC) in a volume of 20 μl. Nested PCR
was performed using primers NALP14_1F_RT (TCCRGATTTTGGGCTGCTAT) and NALP14_1R_RT (GTAGCCGCCGTCATCATC). Amplification of this fragment was performed using the same
protocol as that described below in the Mutation analysis. Sequencing
was performed using the same primers as those used for PCR, on an
automated ABI Prism 3730 Genetic Analyser (Applied Biosystems,
Foster City, CA, USA).
Patients
As part of our ongoing research regarding the genetics of spermatogenic
failure, we consecutively included all men who attended our outpatient
clinic and gave informed consent from January 1998 until April 2004.
Patients with a history of orchitis, surgery of the vasa deferentia, bilateral orchidectomy, chemotherapy or radiotherapy, obstructive azoospermia, bilateral cryptorchidism, numerical or structural chromosome
abnormalities and Y-chromosome deletions were excluded. We performed a nested case-control study in a cohort consisting of 631 men
and included one control for every patient. The patient group comprised
157 men with idiopathic azoospermia or severe oligozoospermia,
defined as a total sperm count of <20 × 106 in two semen samples. The
control group comprised men with good semen parameters, defined as a
total sperm count of >240 × 106 in two semen samples. The Institutional
Review Board of the Academic Medical Center approved this study.
Mutation analysis
DNA was extracted from peripheral blood leukocytes according to
standard procedures. Whenever possible, parental DNA was used to
check for the inheritance pattern in case of a unique mutation.
We amplified all coding exons and intron/exon boundaries of the
NALP14 gene. Fifteen primer pairs (Table I) were designed with the
aid of Primer3 (Rozen and Skaletsky, 2000), using the NALP14
genomic sequence information from the Entrez Nucleotide database
of NCBI (www.ncbi.nlm.nih.gov/entrez) (NM_176822). PCR was
carried out in a total volume of 30 μl and contained 50 ng of DNA, 3
μl of 10 × PCR buffer (Roche, Woerden, the Netherlands), 0.2 mM
3179
G.H.Westerveld et al.
Table I. Primers
Exon
Primer sequence
Product size
1
F: TTATTCCATGTGCTTTTGGTTATTT
R: CCCTAAATCCCTCTTACAGAAACAT
F: CTAGCCATCATGAAAGCTCACTT
R: GAGTGTTTTGCTGATCCTTTTTG
F: TCCAAGTTCGGTATTCTTGACAT
R: CTTGGCTGGTACATGATTTCTTC
F: CAGTCTCTACCAGCAGAGGTTTAAG
R: AGCATCTCATTGCTTTTTAGTGAAC
F: AGCAGTTGTTGAAGAATCACCAT
R: GTTAACCCAAGCCTTCTGAGATT
F: TCTAGCTTGTTCACACCAGTAGATG
R: TTTTACTCGATCTTCATTCAAAGGC
F: AGTTATAAAGACCCCCATTTGACA
R: CCTCCTCCCTCTGACAAATATCTA
F: TGAGAGTTGGCTAGGCTGAAA
R: ATTGGCTTCCCCTAACGAGTA
F: AGATTGAAAAGAAAGGGATCCTG
R: TATGAATAGCAAATCCAGGCTGT
F: TTCAGGCAAGATTCCAAAGATTA
R: ACACAGAACCAAGCAAAATGACT
F: GAGAAGAGAAGCATGGGCTTT
R: GGGTAACAGGGAAAAAGCAAG
F: TGGCAAATAGATAAGGGATCAAAT
R: CTCAGCCTTTCTTTCAGTATCTCC
F: CTTATTTAACCTCACAATGGAAGGA
R: TACCTTCTAGCCCCATTTAGTTTTT
F: TGTGGGCTTTGTTGTGTCAT
R: GGTGGATTCTGAGGCCTGTA
F: TACAGAGAAAGTGGAGGGCTGTA
F: GTATCTGAAGCTAAGCACCCAAG
440
2
3A
3B
3C
3D
3E
4
5
6
7
8
9
10
11
295
is assigned a score based on the observed frequencies of such
occurrences in alignments of related proteins. Frequently observed
substitutions receive positive scores (highest score: 11), and seldom observed substitutions are given negative scores (lowest
score: –4). We used the ClustelW program (http://www.ebi.ac.uk/
clustalw/index.html) to determine the conservation throughout
species of the aligned amino acids of the NALP14 orthologues.
488
477
Results
412
Tissue specificity
414
477
317
338
334
341
350
Hybridization of MTN Blots, containing human adult tissues,
with a NALP14 specific probe revealed that NALP14 is exclusively expressed in testis (lane 4, Figure 2A). Hybridization
with an internal control probe (β-actin) is shown in Figure 2B.
Cellular expression
Immunolabelling with anti-NALP14 showed clear brown DAB
staining in the cytoplasm of A dark spermatogonia, mid and late
pachytene spermatocytes and spermatids (Figure 3A). Also,
Sertoli cells showed some cytoplasmic staining for NALP14
(Figure 3A). Staining was absent in A pale spermatogonia
334
320
294
F, forward primer; R, reverse primer.
deoxyribonucleotide triphosphates (dNTPs), 12 pmol forward and
reverse primer, 2 mM MgCl2 and 0.5 U Taq polymerase. We used a
touchdown PCR program with a temperature range of 62–50°C with a
2°C decrement per cycle and one cycle increment per temperature step
and a final amplification for 20 cycles at 94°C for 30 s, 50°C for 30 s
and 72°C for 30 s with a final extension at 72°C for 5 min.
Mutation screening was performed by direct sequencing of both
sense and antisense strands, using the same primers as those used for
PCR, on an automated ABI Prism 3730 Genetic Analyzer (Applied
Biosystems). All sequences were analysed with the CodonCode
Aligner software (CodonCode Corporation, Dedham, MA, USA).
To predict the possible effect of silent exonic variants on splicing
activity, we used the ESE finder (Exonic Splicing Enhancer) program
(http://rulai.cshl.edu/tools/ESE/) (Cartegni et al., 2002). Similarly, to
predict the possible effect of intronic variants at the intron/exon boundaries and the branch site sequence on splicing activity, we used the
Alex Dong Li’s SpliceSiteFinder (http://www.genet.sickkids.on.ca/
∼ali/splicesitefinder.html).
For each missense mutation and for silent mutations and intronic
variants that were predicted to alter splice site activity, we evaluated
the frequency of these variants in controls using a restriction fragment
length polymorphism (RFLP) assay. If more than one SNP was
detected per PCR fragment or when no RFLP assay could be
designed, control samples were examined by direct sequencing.
Statistical analysis of genotype frequencies and distribution
between patients and controls was performed using a Mann–Whitney
U-test. A P value <0.05 was considered statistically significant.
We applied the BLOSUM62 Substitution Scoring Matrix to
describe the putative impact of identified amino acid changes
(Henikoff and Henikoff, 1992). The BLOSUM62 matrix is a
matrix in which every possible amino acid identity and substitution
3180
Figure 2. Northern blot analysis. (A) Human MTN Blot II (Clontech)
containing 2 mg of poly(A) + RNA per lane from each tissue, indicated at the top of the panel, hybridized to an exon 3 NALP14-specific
PCR probe. None of the tissues on the Human MTN Blot II (Clontech) showed a signal for NALP14 (data not shown). (B) Human
MTN Blot II (Clontech) hybridized with a β-actin probe.
NALP14 mutations in men with spermatogenic failure
Mutation analysis
In our 157 patients with idiopathic azoospermia or severe
oligozoospermia, we identified 25 sequence variants in total;
1 nonsense mutation, 14 missense mutations, 6 silent mutations and 4 intronic variants (Table II). Because silent and
intronic mutations can alter splice site activity, we applied the
ESEfinder and the Alex Dong Li’s SpliceSiteFinder to check if
any of these 10 mutations predicted alteration of splice site
activity. We found that one mutation, an A→G transition at the
third position of codon 751 (p.E751E, dbSNP rs1552726), predicted to remove the ESE consensus motifs of two SR (serine/
arginine rich) proteins, namely SRp40 and SRp55, thereby
potentially affecting correct splicing. The other nine silent and
intronic variants were not predicted to affect splice site activity.
We then screened our 158 normospermic controls for the
presence of the nonsense mutation, the 14 missense mutations
and the silent mutation that potentially alters splicing (Table II).
Of these 16 mutations, 11, namely 10 missense mutations and
the silent p.E751E mutation, were also found in controls.
Furthermore, the frequencies of these mutations did not differ
significantly between the patient and the control groups (Table II).
These were therefore classified as polymorphisms. Five of the
mutations, all present in heterozygous state, however, were
unique to the patient population. Clinical parameters of the five
patients with these unique mutations are listed in Table III.
Interestingly, one of these mutations was an early stop codon
mutation (p.K108X). This A→T change in exon 2 introduces a
Figure 3. Cellular expression. Photograph of cross section of human
testis tissue labelled with anti-NALP14 (A), and as a negative control,
anti-NALP14 blocked with the synthesized peptide (B). Arrowheads
indicate A dark spermatogonia; open arrows indicate A pale spermatogonia; asterisks indicate late spermatocytes and dark arrows indicate
Sertoli cells. The bar represents 20 μm.
and in B spermatogonia and early spermatocytes. Blocking
of the antibody with the synthesized peptide that served as a
negative control showed no staining in the seminiferous
tubules as expected (Figure 3B).
Genomic imprinting
Because chromosomal region 11p15 is known to be
imprinted, we determined the imprinting status of NALP14.
Sequence analysis of two known SNPs (E808K and L1010F)
showed that for each SNP both alleles are equally expressed
in the testis, indicating that NALP14 is not subject to
genomic imprinting (Figure 4).
A.
T
C T
C T
C C T
C T
G
C
T
T
T
C
T A T
C T
G C A A
C A A
A A G
A C T
G
A T A
1600
DNA
0 T
11
C
T
C
T
C
C
T
C
T
G
C
T
N
T
T
A
T
C
T
G
C
A
A
C
A
A
A
A
G
A
C
T
G
A
T
A
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
B.
T
C
T
C
C T
C T
G C
T
T
T
C
T A
T
C
T
G C A A C A
A
A
A G
A C
T
G
A T A
A
RNA
T
C
T
C
C
T
C
T
G
C
T
N
T
T
A
T
C
T
G
C
A
A
C
A
A
A
A
G
A
C
T
G
A
T
A
A
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
Figure 4. Imprinting status. Sequence analysis of the common L1010F polymorphism conducted on peripheral blood lymphocytes DNA (A) and
on RNA (B) extracted from testis tissue of a heterozygous carrier. Maternal and paternal alleles are equally expressed.
3181
G.H.Westerveld et al.
Table II. Variants
DNA
Protein
Patients (n = 157)
+/+ n (%)
Controls (n = 158)
+/–n (%)
–/– n (%)
Nonsense mutation
aag-taga
p.K108X
156
Missense mutations
act-aat
cgg-cag
gat-gtta
aaa-aga
tcg-ttg
gct-acta
aca-ata
gtg-atg
gac-ggca
gag-aag
tct-act
ttg-tcg
ctt-ttt
atg-ataa
p.T48N (rs12801277)
p.R55E
p.D86V
p.K92R (rs16921697)
p.S98L
p.A375T
p.T397I
p.V441M
p.D522Q
p.E808K (rs10839708)
p.S951T
p.L954S
p.L1010F (rs17280682)
p.M1019I
152 (97)
95 (61)
156
145 (92)
146 (93)
156
156
156
156
31 (20)
145 (92)
145 (92)
95 (61)
156
5 (3)
57 (36)
1
11 (7)
11 (7)
1
1
1
1
79 (50)
12 (8)
12 (8)
53 (34)
1
Silent mutations
ttt-ttc
tat-tac
ggt-ggg
ttt-ttc
cag-caa
gaa-gag
p.F166F (rs7123944)
p.Y338Y
p.G386G
p.F400F
p.Q484Q
p.E751E (rs1552726)
154
155
156
143 (91)
155
151 (97)
3
2
1
14 (9)
2
5 (3)
0
0
0
0
0
1
Intronic variants
Intron 2 g-a
Intron 2 t-g
Intron 5 t-c
Intron 5 t-g
IVS2+4A
IVS2+47G
IVS5+40C
IVS6–26G
156
136 (87)
156
147 (94)
1
21 (13)
1
10 (6)
0
0
0
0
1
P value
0
+/+ n (%)
158
0
5 (3)
0
1 (1)
0
0
0
0
0
47 (30)
0
0
9 (6)
0
0.47
0.86
0.43
0.24
0.57
0.10
0.12
0.62
0.57
+/– n (%)
–/– n (%)
0
0
155 (98)
94 (59)
158
144 (91)
150 (95)
158
157
157
158
24 (15)
151 (96)
149 (94)
91 (58)
158
3 (2)
57 (36)
0
14 (9)
7 (4)
0
1
1
0
78 (49)
6 (4)
7 (4)
58 (37)
0
0
7 (4)
0
0
1 (1)
0
0
0
0
56 (35)
1 (1)
2 (1)
9 (6)
0
ND
ND
ND
ND
ND
152 (96)
ND
ND
ND
ND
ND
6 (4)
ND
ND
ND
ND
ND
0
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
+/–, heterozygous; –/–, homozygous mutant; +/+, homozygous wild type; ND, not determined.
a
Unique mutation in patient population.
Table III. Patient characteristics
Patient ID
AMC0038
AMC0157
AMC0221
AMC0420
AMC0522
Mutation
p.D522Q
p.M1019I
p.K108X
p.A375T
p.D86V
Age
(years)
30
27
48
47
32
Semen analysis
Testis volume (ml)
Volume (ml)
Concentration
(106/mla)
Progressive
motile sperm (%)
Normoform
spermatozoa (%)
Left
Righta
3.8
5.5
3.4
2.0
3.4
0.1
2.8
1.5
2.7
2.0
3
26
10
1
9
NA
23
0
17
13
NA
13
11
11
10
NA
6
11
14
8
FSH (IU/l)
Testosterone
(nmol/l)
NA
6.0
4.3
3.6
4.9
NA
15.5
15.7
10.5
13.7
FSH, follicle-stimulating hormone; NA, not available.
a
Volume as determined by scrotal ultrasound.
stop codon at amino acid position 108, resulting in a shortened
protein of only 107 instead of the normal 1093 amino acids.
This truncated protein lacks the functional NACHT and LRR
domains (Figure 5A and B).
The four other unique mutations were all missense mutations. The first missense mutation was an A→T transition in
exon 1 that changes an aspartic acid at codon 86 into a valine
(p.D86V) (Figure 5A and B). This amino acid change gives a
–3 score in the BLOSUM62 matrix. The p.D86V mutation is
located in the PYD domain, known to be involved in protein
binding (Staub et al., 2001). The second mutation was a G→A
transition in exon 3 that changes an alanine into a threonine at
codon 375 (p.A375T) (Figure 5A and B). This amino acid
3182
change gives a –1 score in the BLOSUM62 matrix. The
p.A375T mutation is located just proximal to the NACHT
domain. The third missense mutation was an A→G transition
that changes a glutamine into an aspartic acid at codon 522
(p.D522Q) (Figure 5A and B). This amino acid change gives a
0 score in the BLOSUM62 matrix. The p.D522Q mutation is
located just proximal to the LRR region. The last unique
mutation was a G→A transition that changes a methionine
into an isoleucine at codon 1019 (p.M1019I) (Figure 5A and B).
This amino acid change gives a +1 score in the BLOSUM62
matrix. The p.M1019I mutation is located in the LRR region.
Analysis of the aligned orthologues of NALP14 showed that
the p.D86V, p.D522Q and p.M1010I missense mutations are
NALP14 mutations in men with spermatogenic failure
PYD
52
2Q
D
A3
75
T
M
10
19
I
NALP14
D
86
K1 V
08
X
A.
NACHT
LRR
B.
Mutations
1
MADSSSSSFF PDFGLLLYLE ELNKEELNTF KLFLKETMEP EHGLTPWTEV KKARREDLAN
61
LMKKYYPGEK AWSVSLKIFG KMNLKDLCER AKEEINWSAQ TIGPDDAKAG ETQEDQEAVL
121
GDGTEYRNRI KEKFCITWDK KSLAGKPEDF HHGIAEKDRK LLEHLFDVDV KTGAQPQIVV
181
LQGAAGVGKT TLVRKAMLDW AEGSLYQQRF KYVFYLNGRE INQLKERSFA QLISKDWPST
241
EGPIEEIMYQ PSSLLFIIDS FDELNFAFEE PEFALCEDWT QEHPVSFLMS SLLRKVMLPE
301
ASLLVTTRLT TSKRLKQLLK NHHYVELLGM SEDAREEYIY QFFEDKRWAM KVFSSLKSNE
361
MLFSMCQVPL VCWAACTCLK QQMEKGGDVT LTCQTTTALF TCYISSLFTP VDGGSPSLPN
421
QAQLRRLCQV AAKGIWTMTY VFYRENLRRL GLTQSDVSSF MDSNIIQKDA EYENCYVFTH
481
LHVQEFFAAM FYMLKGSWEA GNPSCQPFED LKSLLQSTSY KDPHLTQMKC FLFGLLNEDR
541
VKQLERTFNC KMSLKIKSKL LQCMEVLGNS DYSPSQLGFL ELFHCLYETQ DKAFISQAMR
601
CFPKVAINIC EKIHLLVSSF CLKHCRCLRT IRLSVTVVFE KKILKTSLPT NTWDGDRITH
661
CWQDLCSVLH TNEHLRELDL YHSNLDKSAM NILHHELRHP NCKLQKLLLK FITFPDGCQD
721
ISTSLIHNKN LMHLDLKGSD IGDNGVKSLC EALKHPECKL QTLRLESCNL TVFCCLNISN
781
ALIRSQSLIF LNLSTNNLLD DGVQLLCEAL RHPKCYLERL SLESCGLTEA GCEYLSLALI
841
SNKRLTHLCL ADNVLGDGGV KLMSDALQHA QCTLKSLVLR RCHFTSLSSE YLSTSLLHNK
901
SLTHLDLGSN WLQDNGVKLL CDVFRHPSCN LQDLELMGCV LTNACCLDLA SVILNNPNLR
961
SLDLGNNDLQ DDGVKILCDA LRYPNCNIQR LGLEYCGLTS LCCQDLSSAL ICNKRLIKMN
1021
LTQNTLGYEG IVKLYKVLKS PKCKLQVLGL CKEAFDEEAQ KLLEAVGVSN PHLIIKPDCN
1081
YHNEEDVSWW WCF
Figure 5. Functional domains and protein sequence of NALP14. (A) The encoded 1093 amino acid protein contains two functional domains
(PYD and NACHT) and a leucine-rich repeat region. The position of the five unique mutations is indicated by an arrow. (B) Amino acid sequence
and location of the unique mutations identified. The nonsense mutation is marked in red and the missense mutations are marked in grey. The functional domains are highlighted in the same colours as in panel A.
located in conserved amino acids of the gene, whereas the
p.A375T mutation is located in a non-conserved region of
NALP14 (data not shown).
We were able to determine the inheritance pattern in one man
with a unique mutation (p.D522Q) in NALP14. The carrier of
this p.D522Q mutation inherited the mutation from his mother.
Unfortunately, no family members from the other four patients
with unique NALP14 mutations were available for the study.
Discussion
Our data demonstrate that NALP14 is exclusively expressed in
human testis and mainly in A dark spermatogonia, mid and late
spermatocytes and spermatids, but not in mitotically active A
pale and B spermatogonia. We found that NALP14 is not subject
to genomic imprinting although it is located in chromosomal
region 11p15, which is an imprinted region. By performing a
mutation screen, we discovered five unique mutations, including
3183
G.H.Westerveld et al.
an early stop codon mutation in our patient population of men
with spermatogenetic failure.
Little is known about the function of NALP14 in spermatogenesis, but a general role for NALPs in apoptosis by activation
of caspases and in pro-inflammation signalling processes has
been suggested (Inohara and Nunez, 2003; Tschopp et al.,
2003). Apoptosis in particular seems to be required for normal
spermatogenesis by matching the number of germ cells with
the supportive capacity of Sertoli cells through controlled cell
death (Said et al., 2004). Caspases have a central role in this
controlled cell death and are under the influence of diverse
regulators including NALP protein family members (Inohara
and Nunez, 2003; Said et al., 2004). NALPs have a PYD
domain, with functional similarity to the death domain (DD),
that is known to be involved in protein–protein interaction and
participation in stress signalling pathways, leading either to
NF-κB activation or to apoptosis (Tschopp et al., 2003). The
expression of NALP14 in mid and late pachytene spermatocytes
indicates that NALP14 could be involved in the apoptotic
processes that occur often during the meiotic divisions.
Our finding that NALP14 is exclusively expressed in human
testis seems to be in contrast with a recent article that studied
expression of NALP14 in mice and demonstrated expression
not only in testis but also in ovary (Horikawa et al., 2005).
However, in this study, expression of NALP14 in mouse testis
was four times higher than expression of NALP14 in mouse
ovary. Although we did not observe any signal for NALP14 in
human ovary in our northern blot analysis, we cannot exclude
low-level expression of NALP14 in human ovary.
Although the five unique mutations were all in heterozygous
state and were each found in only one patient with spermatogenic failure, these mutations provide, in our opinion, an
indication that NALP14 mutations can lead to spermatogenic
failure. Among the five unique mutations, the most striking is
the p.K108X nonsense mutation. This mutation leads to one
inactive allele and likely to a reduction in NALP14 expression.
The other four variants are all missense mutations, and their
effect could lie in altered secondary and tertiary structure of the
NALP14 protein.
It is tempting to speculate that these mutations could have a
dominant negative effect as recently been demonstrated for
SYCP3 (Miyamoto et al., 2003). Unfortunately, no functional
assays exist to determine whether our identified heterozygous
mutations cause the phenotype, and segregation analysis in
families is hampered as most patients deny permission to contact family members.
In summary, NALP14 seems to be important for spermatogenesis, because it is distinctly expressed in germ cells, and
mutations in this gene occur in patients with spermatogenic
failure. We suggest that these unique mutations in NALP14
cause spermatogenic failure by disturbance of the controlled
cell death in the tubules of the testis. More research, however,
is required to investigate the role of NALP14 in spermatogenesis in general and specifically the effect of the unique mutations found on normal spermatogenesis. As a mouse
homologue of the human NALP14 has been identified, a
NALP14 knockout mouse could provide further insight into
the precise function of NALP14.
3184
Acknowledgements
We thank Dr Axel Dietrich and Mrs HL Roepers-Gajadien for their
technical assistance.
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Submitted on September 15, 2005; resubmitted on May 8, 2006, June 16, 2006;
accepted on June 26, 2006