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Recently, we reported that mutations in the R-spondin 4 (RSPO4) gene underlie inherited anonychia/hyponychia. Here, we studied five consanguineous Pakistani families with recessive inheritance of a combination of anonychia and hyponychia. Homozygous mutations were identified in the RSPO4 gene in all five families. Three families had a splice site mutation at the exon 2–intron 2 boundary. One family had a 26 bp deletion encompassing the start codon, and the final family had a missense mutation changing the initiating methionine to isoleucine. We demonstrated by in situ hybridization that Rspo4 is exclusively expressed in the mesenchyme underlying the digit tip epithelium in the mouse at embryonic day 14.5 (e14.5). These findings expand our understanding of the role of RSPO4 in nail development and disease.
Congenital absence of the nails in humans is referred to as anonychia/hyponychia congenita (OMIM 206800), a rare autosomal-recessive condition in which the only phenotype is the absence or severe hypoplasia of all fingernails and toenails. Using homozygosity mapping, we recently identified a region of linkage on chromosome 20p13 and identified a spectrum of mutations in the R-spondin 4 (RSPO4) gene in several affected families from India, Pakistan, Finland, and the United Kingdom (
To investigate further the molecular basis of anonychia, here we studied five consanguineous families from Pakistan (N1–N5) that show recessive inheritance of a combination of anonychia and hyponychia (Figure 1a–e). The five families come from different geographic regions of Pakistan. All patients exhibited either complete absence of the nail plate and matrix, with only the nail bed present, or hyponychia, with some remnants of rudimentary, fragile nail plates (Figure 1f and g). No evidence for associated anomalies of ectodermal appendages, including hair, teeth, and sweat glands was noted in any of the affected individuals.
We obtained DNA from 55 members of the five families, including 25 affected and 30 unaffected individuals. The Institutional Review Board of Columbia University approved all described studies. The study was conducted according to Declaration of Helsinki Principles and participants gave their written informed consent. Genomic DNA was isolated from peripheral blood collected in EDTA-containing tubes using the PUREGENE DNA isolation kit (Gentra System, Minneapolis, MN). All samples were collected after informed consent had been obtained and in accordance with the Institutional Review Board.
To confirm that each family was linked to chromosome 20, genotyping was first performed using the markers D20S117, D20S199, and D20S906, which are closely mapped to the RSPO4 gene. All anonychia families were found to be linked for each of the three markers.
To screen for a mutation in the human RSPO4 gene, exons 1–5 and flanking splice junctions were PCR amplified from genomic DNA. The primers used for the PCR were described previously (
). After purification in Performa DTR gel filtration cartridges (Edge Biosystems, Gaithersburg, MD), PCR fragments were directly sequenced by an ABI PRISM 310 automated sequencer (Applied Biosystems, Foster City, CA) with a Big Dye terminator cycle sequencing kit (Applied Biosystems) and the primers. Homozygous mutations were identified in RSPO4 in all five Pakistani families. Families N1, N2, and N4 have a novel splice site mutation at the exon 2–intron 2 boundary (IVS-1G>A), predicted to result in aberrant splicing of RSPO4 (Figure 2a). Family N3 has a 26 bp deletion, which includes the start codon in exon 1 and is predicted to lead to expression of a protein lacking the first 16 amino acid residues (Figure 2b). Family N5 has a missense mutation changing the initiating methionine of RSPO4 to isoleucine (M1I) (Figure 2c). Each of these mutations is predicted to impair severely the synthesis of a functional RSPO4 protein. Two of these mutations are novel, whereas the third (N3: -9-+17del26) was previously identified in our earlier studies (
, family P2). No correlation has thus far been observed between the various mutations detected and specific phenotypic alterations in the small number of patients studied here and in previous studies. Figure 2d summarizes all previously reported RSPO4 mutations from our group and others, as well as those identified in this study.
Recently, a detailed analysis of the expression patterns of the four Rspo family members during mouse embryogenesis was reported (
). Interestingly, Rspo4 expression was detected from e7 to e17 by reverse transcription-PCR on cDNA derived from mouse embryos. Whole mount in situ hybridization during embryogenesis revealed Rspo4 expression in the groove of the neural fold at e8.5, in the forebrain at e9.5, and in the developing heart and limbs from e9.5 to e10.5. From e15.5 to e17.5, Rspo4 expression was observed in a number of tissues, with the highest level of expression in the developing tooth and various elements of the skeleton (
). As inferred from the anonychia phenotype, this broad pattern of expression suggests that in all body sites except the digit tip, the function of Rspo4 may be compensated for by the presence of another family member.
We have previously shown Rspo4 expression in the tip of the digits, arising between e14.5 and e15.5 (
). To localize the expression of Rspo4 and Rspo3 to a particular compartment in early nail development, we performed whole mount in situ hybridization on e14.5 mouse embryos. Digoxigenin-labeled antisense riboprobes specific to the mouse Rspo3 and Rspo4 genes were synthesized and in situ hybridization was performed on the limbs of e14.5 embryos as described (
). The stained embryos were post-fixed in 4% paraformaldehyde in phosphate buffered saline, and cryosectioned after embedding in Tissue-Tek® OCT compound (Fisher Scientific, Hampton, NH). The images of sections were obtained using an HRC Axiocam fitted onto an Axioskop2 plus microscope (Carl Zeiss, Thornwood, NY). We demonstrate that Rspo4 is exclusively expressed in the mesenchyme underlying the digit tip epithelium (Figure 3b). Moreover, we show that in comparison to Rspo3, which is expressed more intensely and in a broader region of the digit tip (Figure 3a), Rspo4 expression is weaker and more restricted.
To extend these findings further, we next examined the expression of Rspo4 by reverse transcription-PCR in e14.5 mouse dermis and epidermis from dorsal skin. Dissected skin was enzymatically digested, allowing for a separation of the epidermis from the dermis. RNA was subsequently extracted from each dissected tissue using an RNeasy Mini Kit (Qiagen, Valenica, CA). RNA was also extracted from adult mouse dorsal whole skin. Reverse transcription was carried out using oligo (dT) primer and SuperScript™ III (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The primers mRspo4 F: 5′-CAGCAGAGGCTCTTCCTCTTCATC-3′ and mRspo4 R: 5′-GAGCCACAGGTCTTCCCATTGTGT-3′ were used to amplify a 326 bp product. Consistent with our in situ hybridization results, the expression of Rspo4 was restricted to the dermis and was not present in the epidermis (Figure 3c).
We conclude that although Rspo3 is expressed at the same place and time as Rspo4 in the mouse digit tip, RSPO3 is apparently not able to compensate for RSPO4 in this region in humans, because the phenotype arises in the absence of RSPO4 despite the presence of RSPO3. Given that RSPO3 is located on human chromosome 6, it is not a candidate in this form of human anonychia. In mice, Rspo3 knockouts die at e10 due to abnormal placental development (
). To date, we have found no evidence for locus heterogeneity, and all anonychia families studied thus far are both linked to and have mutations in RSPO4 on chromosome 20.
Conflict of Interest
The authors state no conflict of interest.
We appreciate the participation of family members in this study. This work was supported in part by NSPHS NIH Grant R01AR44924 from NIAMS (to A.M.C.), Association for International Cancer Research (to D.P.K.), and a grant from The Ministry of Education, Culture, Sports, Science, and Technology in Japan (to Y.I.).
R-spondin3 is required for mouse placental development.