Mutations in XPNPEP3 cause an NPHP-like kidney disease.
We performed a whole-genome search for linkage in 116 consanguineous kindred with NPHP and NPHP-like phenotypes. Linkage analysis in a 5-generation consanguineous family from northern Finland (A131), with 3 members affected with an NPHP-like disorder, yielded a significant logarithm of odds (LOD) score of LODmax
= 3.6, defining a new locus (NPHP
-like 1, NPHPL1
) on chromosome 22q13.2 (Figure A), within a 4.3-Mb interval flanked by markers SNP_A-1516630
(Figure B). The critical genetic region overlapped with a homozygous segment detected in a consanguineous kindred of Turkish descent (F543), having 2 members with an NPHP-like phenotype (Supplemental Figure 1; supplemental material available online with this article; doi:
). The NPHPL1
critical genetic interval contained 101 positional candidate genes (Figure B).
Positional cloning of the XPNPEP3 gene, as mutated in NPHPL1.
We performed mutation analysis by sequencing exons in 29 of the candidate genes, prioritizing proteins present in the Ciliary proteome database (http://ciliaproteome.org/; ref. 28
). Among them, we identified novel, likely pathogenic variants in only one gene, XPNPEP3
(Figure , b–f). Kindred A131 harbored a homozygous splice-site mutation (1357G>T) in an 80% conserved exonic position of the splice donor consensus (Figure , b–f). RT-PCR from lymphoblastoid cells showed that this mutation disrupts correct splicing by activating a cryptic splice site and introducing a frame shift (Supplemental Figure 2). The same mutation changed an amino acid residue (G453C) that is conserved throughout evolution, including E. coli
(protein ecAPP) (data not shown). In addition, the 2 affected individuals of kindred F543 had a homozygous 4-base deletion in exon 6 (c.931_934 delAACA), resulting in a premature stop codon (p.N311LfsX5) within the predicted prolidase domain, which is the catalytic domain (Figure , e and f). The mutation in family F543 segregated from both parents and was absent from 150 people of mixed European descent and 83 Turkish healthy control individuals. Likewise, the mutation of the northern Finnish family A131 segregated from both parents; it was absent from 118 Western European healthy control individuals (Table ) and was present once heterozygously in 100 Finnish control individuals, where it occurred on a shared haplotype of more than 2 Mb, suggesting a founder effect.
XPNPEP3 mutations in 2 consanguineous families with a NPHP-like kidney disease
We then examined 96 kindred with NPHP by direct sequencing of all exons and screened a multiethnic cohort of 376 additional kindred with NPHP using heteroduplex analysis (29
) and CELI
), followed by direct sequencing of all aberrant bands. We also examined 96 different kindred with Joubert syndrome and 95 different kindred with Senior-Loken syndrome using heteroduplex analysis (29
), with subsequent direct sequencing of all aberrant bands. In addition, we analyzed 100 DNA samples from patients with isolated respiratory chain complex I (RCCI) deficiency, using high-resolution melting point analysis. We detected no patients with 2 mutations in any of these additional 643 families with NPHP-like phenotype and in 100 index cases with isolated RCCI deficiency. Ten novel heterozygous mutations of unknown pathogenicity were identified in 10 patients. We did not find any kindred with 2 recessive mutations in any of 823 families with an NPHP-like phenotype (Table ).
We thus identified mutations in XPNPEP3
as a novel cause to our knowledge of an NPHP-like nephropathy (NPHPL1). XPNPEP3 is contained in the M24B subfamily of X-prolyl peptidases that comprises aminopeptidase P and the dipeptidase prolidase. XPNPEP3 has a predicted C-terminal catalytic domain and has greatest overall sequence identity (32%) to E. coli
aminopeptidase P. This high level of conservation, which includes the catalytic domain, suggests that XPNPEP3 is a functional aminopeptidase P and is 1 out of 3 X-prolyl aminopeptidases known to exist in mammals (31
) (Figure E). The E. coli
ortholog and the human XPNPEP3 contain 2 manganese ions that are required for peptidase activity, sites that are also conserved in XPNPEP3 (31
). Studies performed with the E. coli
) indicate that XPNPEP3 recognizes N-terminal peptides that have proline in their second position as substrate and cleaves the peptide bond between the first and second amino acid residues, releasing the N-terminal amino acid. In addition, MitoProt II analysis of XPNPEP3 detected an N-terminal mitochondrial localization signal with a cleavage site after amino acid 53, resulting in a 51-kDa protein of likely mitochondrial function (Figure E).
Clinical and biochemical phenotype of individuals with NPHPL1.
Clinical data of kindred A131 and F543 are presented in Table . Three affected individuals of kindred A131 had moderate renal insufficiency (glomerular filtration rate, 30%–40% of normal) at between 20 and 29 years of age (Table ). Two individuals of the northern Finnish family A131 had undergone renal biopsy, which yielded characteristic features of NPHP (Figure ). Renal ultrasound revealed features characteristic of NPHP (Table ), while extrarenal manifestations included essential tremor in all 3 affected individuals. Two affected individuals (A131 II-3 and A131 II-4) also had high frequency sensorineural hearing loss; A131 II-3 had arachnoid cysts on brain imaging, while A131 II-1 and A131 II-3 also had gout.
Renal histology of patient A131 II-4.
In the Turkish kindred (F543), the 2 affected individuals exhibited end-stage kidney disease by 8 and 9 years, respectively (Table ). Renal ultrasound and renal histology demonstrated NPHP (data not shown). In addition, both affected individuals manifested a mitochondriopathy, with an isolated complex I deficiency and decreased NADH-CoQ oxidoreductase activity. This complex I deficiency was not found in fibroblast cultures from family A131, with the splice-site mutation (Supplemental Tables 2 and 3), but in family F543, with a homozygous frame-shift mutation, it was detected in a muscle biopsy of F543-I (Supplemental Tables 4 and 5) and in a fibroblast culture of F543-II (Supplemental Tables 6 and 7). Additionally, both affected individuals had seizures and hypertrophic dilated cardiomyopathy. F543 II-1 also demonstrated chronic pancreatitis with pancreatic cysts, while F543 II-2 had a hepatopathy.
A signal peptide traffics XPNPEP3 to mitochondria.
To study the expression and subcellular localization of XPNPEP3, we characterized a rabbit polyclonal anti-human XPNPEP3 antibody (HPA000527, Sigma-Aldrich), hereafter called α-XPNPEP3. Upon immunoblotting, α-XPNPEP3 specifically recognized transiently transfected V5-tagged human XPNPEP3 (XPNPEP3-V5) as well as endogenous XPNPEP3 in lysate from murine inner medullary collecting duct (IMCD3) cells (Supplemental Figure 3B). It also specifically detected overexpressed XPNPEP3-V5 in mitochondria, where it colocalized with the anti-V5 antibody (Supplemental Figure 3, A and C–E) as determined by immunofluorescent staining. Upon immunoblotting of cell lines from human, monkey, or dog, the α-XPNPEP3 cross-reacted with single bands at 51 kDa, representing the expected size for full-length XPNPEP3, following cleavage of the mitochondrial signal peptide (data not shown). Immunoblotting of multiple tissues from adult mouse detected single bands in cell lysates of kidney, heart, liver, skeletal muscle, brain, and testis (Supplemental Figure 4).
Because of the predicted targeting of XPNPEP3 to mitochondria, we fractionated whole kidney homogenates from mice into mitochondrial and cytosolic fractions. Immunoblotting with α-XPNPEP3 yielded a single band in the mitochondrial fraction at approximately 51 kDa, consistent with the product predicted to result from cleavage of the mitochondrial signal peptide following mitochondrial import (Figure A). The cytosolic fraction showed a doublet at approximately 57 kDa, compatible with unprocessed XPNPEP3. Immunofluorescent microscopy in IMCD3 cells stably expressing human full-length XPNPEP3-GFP, which contains the mitochondrial leader sequence, were localized to mitochondria (Figure B). In contrast, in IMCD3 cells that stably express a cDNA, which lacks the mitochondrial leader sequence (ΔN-XPNPEP3-GFP), XPNPEP3 was found diffusely in the cytoplasm (Figure C). Expression of XPNPEP3 in mitochondria was also confirmed by transmission electron microscopy in ultrathin rat kidney sections, using immunogold-labeled α-XPNPEP3 (Figure D).
Human XPNPEP3 is targeted to mitochondria by an amino-terminal mitochondrial signal sequence.
No evidence for XPNPEP3 in primary cilia, basal bodies, and centrosomes in vitro.
NPHP is considered a ciliopathy, because 10 different causative genes (NPHP1–NPHP9
) share the feature of subcellular function of their respective gene products at primary cilia, basal bodies, or centrosomes (1
). We therefore examined XPNPEP3 for organellar targeting to the cilia/basal body/centrosome complex. In IMCD3 cells that stably express the human full-length XPNPEP3-GFP, which contains the mitochondrial leader sequence, XPNPEP3-GFP was not detected in primary cilia, basal bodies, or centrosomes (Figure ). We conclude that loss of XPNPEP3 leads to an NPHP-like phenotype (NPHPL1), without any apparent ciliary localization, departing from the paradigm of all other genes mutated in NPHP.
Full-length human XPNPEP3-GFP transiently expressed in IMCD3 cells localizes to mitochondria and not to the cilium/basal body/centrosome complex in IMCD3 cells.
XPNPEP3 exhibits cell type–specific expression in kidney.
As the most prominent phenotype in individuals with suppressed XPNPEP3 function was manifested in the kidney, we performed immunofluorescent microscopy of rat kidney sections to study cell type–specific expression. We found XPNPEP3 to be expressed specifically in distal convoluted tubule and cortical collecting duct cells (Supplemental Figure 5). Of note, XPNPEP3 expression was detected not in principal cells but in intercalated cells, which are known to lack primary cilia (Supplemental Figure 5).
xpnpep3 suppression phenocopies ciliary and basal body zebrafish morphants.
Depletion of several cystogenic proteins, including NPHP2, CEP290/NPHP6, and numerous BBS proteins, have been shown to perturb convergence and extension movements in zebrafish (18
), a noncanonical Wnt phenotype, which in the mammalian nephron manifests as failure of orientation of the axis of cell division and has been causally linked to tubular dilatation (20
). We therefore wondered whether suppression of xpnpep3
might phenocopy this defect or whether loss of this gene/protein might affect hitherto unknown pathways. We therefore designed a translation-blocking morpholino (MO) against the sole Danio rerio
ortholog (by reciprocal BLAST), a transcript that is expressed as early as 12 hours after fertilization, as determined by RT-PCR (data not shown). At the mid-somite stage, embryos injected with xpnpep3
= 50–100 embryos/injection; scored blind) displayed quantifiable gastrulation defects reminiscent of ciliary gene morphants (22
); these included a shortened body axis, small anterior structures, broadening and kinking of the notochord, and elongated somites (Supplemental Figure 6). These phenotypes were unlikely to be caused by nonspecific action of the MO: not only did we observe an increase in affected embryos correlated with dose, but rescue with capped full-length human XPNPEP3
mRNA resulted in significant amelioration of the phenotype (χ2
= 34.45, P
< 0.0001). These phenotypes are likely independent of the XPNPEP3 mitochondrial activity: coinjection of xpnpep3
MO with a human rescue construct devoid of the mitochondrial localization signal (ΔN-XPNPEP3
) rescued the phenotype in a manner indistinguishable from that of the WT human construct (χ2
= 3.47, P
= 0.17). Therefore, targeting of XPNPEP3 to the mitochondrion is not required to rescue the convergence and extension phenotypes, suggesting that this protein might also have mitochondrial-independent activity.
Cleavage of known cystic disease proteins by XPNPEP3.
Our data led us to hypothesize that, in addition to its mitochondrial functions (which potentially explain the RCCI defects observed in family F543), XPNPEP3 might affect the function of ciliary proteins biochemically, most likely through its N-terminal proline cleavage activity. To identify such candidate substrates, we parsed the ciliary proteome (28
) for proteins with a proline in the second position (after N-terminal methionine cleavage). To reduce incidence of false-positives, we used a stringent version of the proteome that was restricted to reciprocal orthologs (minimal E
value of 10–30
) identified in at least 2 independent ciliary studies, thereby parsing 426 likely ciliary proteins for substrate candidacy. We identified 51 proteins with sequences that fulfilled our search criteria (Supplemental Table 8). Interestingly, 3 candidate substrates (centrosomal protein 290 kDa/NPHP6 [CEP290/NPHP6], Alstrom syndrome 1 [ALMS1], and leucine rich repeat containing 50 [LRRC50]) are known to cause cystic renal disease (9
To verify our computational predictions, we next sought to test whether XPNPEP3 can cleave these 3 candidate substrates. Expression of recombinant human XPNPEP3 in bacteria was not possible due to the protein being insoluble under numerous experimental conditions. We therefore focused on the E. coli ortholog of XPNPEP3, ecAPP, which we were able to express and purify to more than 99% homogeneity as detected by Coomassie staining and Western blot (Figure A). Three 9–amino acid peptides identical to the N-termini of each of CEP290/NPHP6, ALMS1, and LRRC50 (without the start methionine that is cleaved after translation) were synthesized (Figure B) and incubated with purified ecAPP, followed by mass spectrometry. We found that CEP290/NPHP6, ALMS1, and LRRC50 are each cleaved efficiently by ecAPP (Figure C). By contrast, peptides containing residues other than proline in the second position could not be processed by ecAPP (data not shown). Importantly, the cleavage activity of the enzyme is not promiscuous, since we found that dynein, another ciliary protein that also contains a proline in the second residue but is not involved in cystic kidney diseases, is poorly digested by ecAPP (Figure C). These data suggested that XPNPEP3 cleavage of other cystogenic proteins might be relevant to their biological function(s).
Peptide cleavage by the XPNPEP3 ortholog, ecAPP.
Requirement of XPNPEP3 N-terminal cleavage for early gastrulation in zebrafish.
To probe the importance of the likely XPNPEP3 cleavage sites in vivo, we asked whether cleavage resistance was deleterious to protein function. Focusing on LRRC50 (each of CEP290/NPHP6 and ALMS1 encode mRNA more than 7 kb, rendering them difficult to transcribe in vitro at sufficient purity), we investigated whether a cleavage-resistant human capped mRNA can rescue the gastrulation phenotypes caused by MO-driven suppression of endogenous lrrc50. Injection of 4.5 ng of a translation-blocking MO induced early gastrulation phenotypes that include all the hallmarks of cilia-related convergent extension defects and which could be rescued by coinjection of 150 pg of WT human capped LRRC50 mRNA (Figure ). However, coinjection of MO with mRNA encoding an N-terminal Pro-Val point mutation, followed by blind scoring of embryos, showed complete failure of rescue (WT vs. Pro-Val, χ2 = 39.20, P < 0.0001; n = 80–110 embryos/injection). This experiment could not exclude the possibility that the observed loss of function could be due to the presence of the Val at this position. However, repetition of this assay with Asp or Arg yielded similar data (Figure ). Still, the possibility remained that the observed failure to rescue might still be driven by the loss of the proline for reasons other than cleavage. To assess this possibility, we searched the zebrafish genome for other N-terminal aminopeptidases and identified an alanyl aminopeptidase (ZFIN ID, ZDB-Gene-030131-1253), which predicts that Ala at the position of the N-terminal proline should render lrrc50 subject to cleavage. Consistent with the notion that it is the cleavage that functionalizes lrrc50, injection of MO with an Ala-encoding human mRNA rescued the phenotypes in a manner indistinguishable from WT (Figure ; χ2 = 0.26, P < 0.87).
N-terminal cleavage of LRRC50 is required to rescue lrrc50 morphant phenotypes in zebrafish.