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*Nephro-Urology and Clinical and Molecular Genetics Units, Institute of Child Health, University College London, England; University Department of Medical Genetics, St. Mary’s Hospital, Manchester, England; Department of Clinical Genetics, Western General Hospital, Edinburgh, Scotland; and ¶Telethon Institute of Genetics and Medicine, Naples, Italy.
Correspondence to Dr. Leila Romio, Clinical and Molecular Genetics Unit, Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK. Phone: 44-20-7905-2263; Fax: 44-20-7916-0011;
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-tubulin, suggesting that OFD1 is associated with the centrosome. First, it is concluded that OFD1 mutations would generally be predicted to result in unstable transcripts or nonfunctional proteins. Second, OFD1 is expressed in human organogenesis; on the basis of the metanephric expression pattern, the results suggest that OFD1 plays a role in differentiation of metanephric precursor cells. E-mail: lromio@ich.ucl.ac.uk |
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Feather et al. (13) mapped OFD1 to Xp22.2 to 22.3, and Ferrante et al. (14) identified Cxorf5 (renamed OFD1; accession number Y15164) as the mutated gene in seven kindreds. OFD1 has an open reading frame of 3033 bp over 23 exons (15). Due to the use of alternative 3' splice sites in intron 9, two mRNA species, here designated OFD1a and OFD1b, are generated. Although the OFD1b transcript is the longer form, OFD1a has a longer translated region. The two putative OFD1 proteins have different C-termini; the larger form (here designated ODF1a) is predicted to have 1011 amino acids (aa), whereas OFD1b has 367 aa. Although many OFD1 anomalies are developmental aberrations, no published data exists on OFD1 expression in human development. We here describe further OFD1 mutations in six kindreds, including two families with several members affected by PKD and report, for the first time, an overview of OFD1 expression in human organogenesis and subcellular localization in human embryonic renal cell lines.
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Patient Identification and Collection of Samples
Clinical details of two families with multiple members affected by OFD1 and associated PKD have been described (7,10). For mutation analyses, we used these families and four other kindreds who are described in Results; all were assessed at Clinical Genetics centers and patients were studied with renal ultrasonography (US). Fifteen terminated normal human fetuses were collected under research ethical committee permission and were staged by external morphology (16) as between 7 and 14 wk of gestation, spanning early organogenesis. Sections of mature kidney tissue were also examined from areas that appeared histologically normal adjacent to three Wilms tumors, removed at 28 to 72 mo of age. The metanephros forms in the sixth week from the ureteric bud, which branches to form collecting ducts, and the mesenchyme, which differentiates into S-shaped bodies and glomeruli, the first of which form by eight weeks; this process reiterates until 34 wk. New nephrons are not formed after birth.
Mutation Detection
DNA was isolated from native or transformed leukocytes by standard techniques, after obtaining written consent. PCR amplified the entire OFD1a coding region (exons 1 to 23) including splice junctions (14). Exon 10a, which confers the additional genetic information in the OFD1b transcript, was not analyzed for mutations. PCR reactions contained 30 ng genomic DNA, 2 mM MgCl2, 200 µM dTTP, dGTP, dCTP, dATP, 25 pmol of each primer, 1 x PCR buffer, 1 x Q solution, and 0.1 U of HotStartTaq DNA polymerase (Qiagen, Crawley, UK) in 25 µl final volume. Cycling parameters were: 94°C for 12 min, 35 cycles of 94°C for 15 s, 55°C for 15 s, and 72°C for 25 s. PCR products from one proband and, where available, one unaffected married-in member of each pedigree, were screened for sequence changes by denaturing HPLC (dHPLC), using the WAVE DNA fragment analysis system (Transgenomic Inc, Crewe, UK). Column temperatures and acetonitrile gradients were determined using WaveMaker v. 3.4.4 (Transgenomic Inc). Proband PCR products showing altered elution properties to controls were sequenced on both strands using ABI Big Dye Terminator Sequencing Kit and an ABI 377 Sequencer (Perkin Elmer, Forster City, CA). Sequences were analyzed using Sequence Analysis v. 3.4.1 (Perkin Elmer). Sequence changes in probands were then assessed to determine co-segregation with clinical disease.
Cell Culture
Human embryonic kidney (HEK) 293 cells were transfected to express full-length OFD1. This line was generated by adenoviral transformation of primary cultures, but it is uncertain what type of metanephric cell, if any, it faithfully represents. Indeed, Shaw et al. (17) reported that they express neuronal proteins as well as cytokeratins. Cells were cultured in Dulbecco Modified Eagle Medium (DMEM, Invitrogen) with 10% fetal calf serum (FCS), 1000 U/L penicillin G, and 1 g/L streptomycin. The day before transfection, cells were plated at subconfluent density. They were incubated for 6 h in serum-free medium containing 30 µl of Lipofectamine reagent (Invitrogen) and 10 µg of DNA per 10-cm-diameter plate. DNA used were: (1) myc tag/enhanced green fluorescence protein (EGFP)/OFD1 in pcDNA3 vector (Invitrogen), with OFD1 cDNA corresponding to the OFD1a open reading frame; (2) vector with the myc-EGFP construct only. Cells were analyzed at 36 h after transfection. To produce renal mesenchymal lines, metanephroi were dissected from normal human embryos (two at 10, one at 11, and one at 12 wk gestation), diced into five pieces, placed onto plastic tissue culture plates, and fed DMEM/Hams F12 medium (Invitrogen) supplemented with 5% FCS, epidermal growth factor (10 µg/L), hydrocortisone (36 µg/L), 3,3',5-triiodo-L-thyronine sodium salt (4 ng/L), insulin/transferrin/selenium Sigma media supplement, and antibiotics. Outgrowths were passaged, and monolayers were characterized at passages four-eight (data not shown and reference 18).
RNA Analyses
For RT-PCR, RNA (500 ng), extracted from isolated organs with Tri-reagent, was treated with deoxyribonuclease I and subjected to reverse transcription with Superscript II (Life Technologies BRL). cDNA was diluted to a volume of 100 µl, and 2 µl was used as a template with the following primers sets synthesized by MWG-Biotech AG:
All PCR reactions were performed in a volume of 50 µl, with 0.25 units of HotStartTaq polymerase, 200 µM dNTPs, 2 mM MgCl2, and 3 pmol of primers. PCR conditions were: 10 min at 94°C followed by 30 cycles of amplification (30 s at 94°C, 30 s at 58°C, and 1 min at 72°C) with final elongation of 5 min at 72°C. PCR products were electrophoresed through 2% agarose gels containing ethidium bromide and sequences of the PCR products confirmed using the ABI Prism Big Dye Terminator Cycle Sequencing kit and an ABI 377 automated sequencer. OFD1 primer set 1 amplifies cDNA derived from both OFD1 cDNA isoforms to generate a 225-bp OFD1a band, and an 850-bp OFD1b band. In preliminary experiments, the former band was preferentially amplified in the PCR conditions used, while the product from OFD1b was barely visible. Therefore, we designed a second set of OFD1 primers (primer set 2) on the region of 630 bp in exon 10a unique to OFD1b cDNA. For Northern blot, 25 µg RNA was run on a formamide agarose gel and transferred to a Nylon membrane (Hybond N+; Amersham Pharmacia Biotech, Buckinghamshire, UK). The membrane was hybridized with either the OFD1/GFP cDNA, described above, or a 18S rRNA probe labeled with 32PdCTP by random priming, using the Megaprime DNA labeling system (Amersham Pharmacia Biotech). Hybridization was performed with Quikhybsolution (Stratagene, La Jolla, CA) at 68°C. X-ray films were exposed to filters for up to 7 d at -70°C.
Protein Analyses
Whole OFD1 proteins were not available, so we selected an oligopeptide, CDVQKQIEEQKEEEKIREQQVKER (867 to 891 aa in OFD1a), to generate antibodies in rabbits. Oligopeptide synthesis and production of antisera were undertaken by Sigma-Genosys (Cambridge, UK). Bleeds from two animals were tested by ELISA against the immunizing peptide to ensure that antisera had high titer and avidity. Both antisera gave similar results, and for simplicity in Results, we have not distinguished between results derived from either. For Western blot, tissues were lysed in RIPA buffer with protease inhibitors (19). Protein concentration was quantified (BCA kit; Pierce, Rockford, IL), and 50 µg was mixed with an equal volume of sample buffer (1 ml glycerol, 0.5 ml mercaptoethanol, 3 ml 10% sodium dodecyl sulfate, 1.25 ml Tris, pH 6.7, and 1 mg of bromophenol blue), heated at 95°C for 5 min, and loaded onto SDS-polyacrylamide gels. After electrophoresis and transfer by electroblotting, membranes were stained with Ponceau S to confirm equality of total protein transfer (data not shown). Blots were washed with deionized water, placed in blocking solution (5% nonfat milk, 0.1% Tween-20 in PBS) for 1 h, then incubated overnight at 4°C with primary antibodies directed against the following proteins: OFD1a antiserum (1:500); OFD1a antiserum (1:500) preincubated for 2 h at room temperature with immunogenic peptide as a negative control; preimmune serum (1:500) as another negative control; WT1 (1:250; Santa Cruz); PAX2 (1:500; Zymed); hepatocyte growth factor (HGF; 1:200; R&D Systems); glial cell line-derived neurotrophic factor (GDNF; 1:200; R&D Systems). Appropriate horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse secondary antibodies (1:5000, DAKO, High Wycombe, UK) were applied, and bound second antibody was detected with ECL reagent (Amersham Life Science). Some blots were stripped and reprobed with either mouse anti-human 
-actin (1:10.000), a housekeeping protein, or anti-myc (1:5000; Invitrogen).
For immunohistochemistry, tissues were fixed in 4% paraformaldehyde overnight, dehydrated, wax-embedded, and sectioned at 5 µm. Sections were dewaxed in Histoclear (National Diagnostics, Atlanta, GA) and rehydrated through graded ethanols. Antigens were unmasked by digestion in 1 mg/ml trypsin for 10 min and then treated with 3% hydrogen peroxide in PBS (pH 7.4) for 30 min to quench endogenous peroxidase activity. Nonspecific binding was blocked by incubation with 10% FCS in PBS for 30 min before overnight incubation at 4°C with either OFD1a anti-serum or preimmune serum (1:350) or antiserum preincubated with immunizing peptide. Washed slides were exposed to a streptavidin-biotin peroxidase system followed by diaminobenzidine (Dako, ABC Kit) and counterstained with hematoxylin.
For immunocytochemistry, mesenchymal cells were plated on four-well Lab-Tek chamber slides (Nunc, Naperville, IL) at a density of 50,000 per well, cultured overnight, then fixed for 10 min in methanol at -20°C. After blocking for 1 h with 10% FCS in PBS, they were incubated overnight at 4°C with OFD1 antiserum (1:100), preimmune serum or anti-serum preincubated with immunizing peptide. In some experiments, for double labeling, the cells were additionally incubated with mouse monoclonal -tubulin antibody (1:50). The cells were washed three times in PBS and 0.1% Tween-20, and incubated for 1 h with donkey anti-rabbit FITC (1:100) or donkey anti-mouse Cy5 (1:500) secondary antibodies; the emission spectra for these fluorochromes is non-overlapping. After immunocytochemistry, some slides were also incubated for 5 min with the nuclear marker propidium iodide (4 mg/L). Fluorescence was visualized on a confocal laser scanning microscope (Leica Aristoplan, Heidleberg, Germany).
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We then stained normal metanephroi of 7, 10, 13, and 14 wk of gestation and the histologically grossly normal areas adjacent to three postnatal Wilms tumors. In the four normal fetal developing metanephroi, a consistent expression pattern was observed: immunoreactive OFD1a protein was detected in the outer nephrogenic zone of the cortex in the mesenchyme (Figure 4A). From these wax-embedded sections, it was difficult to discern the exact subcellular localization of immunoreactivity, although positive signal was not detected in renal cell nuclei. Minimal positive signal was detected in later nephron precursors, including vesicles, comma, and S-shaped bodies. Consistent staining was also not observed in developing glomeruli (data not shown). Immunoreactive OFD1a protein was not detected in normal appearing areas of postnatal kidney sections (data not shown). Cranial OFD1 expression was also observed in an 8-wk embryo: positive signals were detected in nasal (Figure 4B) and developing skull (not shown) cartilage, oral mucosa (Figure 4C), skin (not shown), brain (not shown), and tongue myocytes (Figure 4D). Adjacent metanephric sections probed with antiserum preincubated with immunizing peptide (Figure 4E) showed no mesenchymal signal. Other tissues probed with preimmune serum (Figure 4, F through H) showed no specific signals.
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-chain. Furthermore, the three lines also expressed WT1 (52 kD) and PAX2 (doublet at 46 and 48 kD). In contrast, in the fourth line examined, HGF, WT1, and PAX2 were barely detectable using similar methodology (N84; Figure 5A). On the basis of these results, we speculate that lines N75, N73, and N70 represent "induced" renal mesenchyme, while N84 appears more like "uninduced" mesenchyme or stroma. Next, we examined OFD1a expression in the mesenchymal lines using Western blot (Figure 5B): OFD1a protein was not detected in line N84, whereas bands were observed at 120 kD in the other three lines (Figure 5B).
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-tubulin (Figure 6E), a well-characterized centrosomal marker (24); immunoreactive OFD1a and -tubulin co-localized exactly (Figure 6F). Although these cell lines are not clonal, this centrosomal localization was observed in over 98% of cells in cultures of the N75 and N70 lines when the whole thickness of the cell was examined by focusing up and down.
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OFD1 Mutations are Highly Penetrant for Dysmorphic Features
Tracking mutations through two pedigrees showed that the mutation is highly penetrant; females with mutations had classic dysmorphic features, but three other females without such features did not have the mutant allele. This observation, also made by Ferrante et al. (14), is important for genetic counseling regarding prenatal diagnosis. It is also pertinent if a renal transplant from a living female relative is proposed for an OFD1 individual with renal failure.
Does Occurrence of PKD Correlate with Genotype in OFD1?
Two OFD1 proteins are predicted to be produced from two alternatively spliced mRNAs (14); the shorter one terminates at the beginning of exon 11. Four of the previously published mutations occur beyond this point (Figure 1); in these cases, the sequence of OFD1b, the shorter protein, would be predicted to be unchanged. Two of these patients did not have PKD on US (14), while the other two were not assessed by US (20). Conversely, most patients with mutations before this point have PKD (Figure 1). One interpretation is that the shorter protein is responsible for normal kidney growth, and the longer protein prevents dysmorphic features. Against this, it can be argued that premature stop codons in the later part of the molecule will lead to nonsense-mediated decay of the OFD1b mRNA. In future, exactly which mRNA alleles exist could be established if suitable mutant cells expressing OFD1 were available. A further argument against a simple renal phenotype/genotype correlation is that the severity of PKD (i.e., cyst numbers and severity of renal failure at certain ages) can vary markedly in one pedigree, all of whom will carry the same mutation, as described previously by Feather et al. (10). In addition, we are aware of one individual with OFD1 dysmorphology who developed cysts on US over several years (SAF, personal observation); hence, the lack of cysts does not preclude their evolution.
OFD1 Is Expressed in Developing Human Organs
This study reports for the first time that OFD1 is expressed in human organogenesis. De Conciliis et al. (15) demonstrated by Northern blot that OFD1 was expressed in adult human organs. Transcripts corresponding to OFD1a RNA were found in kidney, pancreas, skeletal muscle, liver, lung, brain, heart, and placenta, while a larger species, postulated to be the OFD1b transcript, was noted in some organs, including kidney and brain. In-situ hybridization studies in mice (14) demonstrated expression at embryonic day 12 in genital ridges, with transcripts detected later in gestation in craniofacial structures, including oral and nasal cavity epithelia, the nervous system, and metanephros. In the current study, using the highly sensitive technique of RT-PCR, OFD1a and OFD1b transcripts were found in a wide variety of developing human organs, including heart and lung. PCR is so sensitive that we may be detecting low levels of transcripts. In contrast, however, using less sensitive Northern blot analysis, significant signals were only observed in brain, metanephros, tongue, and limb, which is similar to previous in-situ results in mice (14). We generated an antiserum to the carboxyterminal of the putative OFD1a protein and validated it in HEK293 cells transfected with OFD1a cDNA. In normal human embryos, OFD1a immunolocalized to oral mucosa, nasal cavities, cranial cartilage, and brain. Hence, during development, several cell types and lineages express the gene. OFD1a expression was observed in the mesenchyme of the outer metanephric cortex. These cells are highly proliferative (21), correlating with epithelial transformation. In fetal kidneys, OFD1a protein was apparently downregulated as the mesenchyme transformed into nephron epithelia and consistent glomerular staining was not observed. Moreover, OFD1a immunoreactivity was absent in sections of postnatal kidneys, consistent with the hypothesis that it may have a role in renal differentiation.
OFD1 Is an Intracellular Protein
In normal human embryonic renal mesenchymal lines, OFD1 protein immunolocalized to perinuclear "dots;" furthermore, -tubulin immunoreactivity overlapped with OFD1. These findings are consistent with OFD1 being a centrosome-associated protein. The centrosome is a small nonmembranous organelle, often associated with the nuclear membrane, composed of two centrioles surrounded by electron-dense pericentriolar matrix. Interestingly, the centrosomal matrix is the site of microtubule nucleation and contains a high proportion of coiled coil proteins (24). Further experiments, incorporating immunoelectron microscopy and biochemical studies to assess protein-protein interactions, will be necessary to determine the sub-centrosomal localization of OFD1. Although, the precise function of centrioles is unclear, they are known to be essential for the generation of the primary cilium (24). Expression of several proteins implicated in both human and murine PKD, including polycystin-1 and -2, polaris, and cystin, has recently been described in the primary cilium complex (28,29). Morover, OFD1 contains a large number of coiled-coil domains and heptad repeats (15,27) and polycystins interact via coiled-coil domains (30). This raises the possibility that these molecules are involved in the same biologic pathway. Functional cell biology experiments are clearly required to assess potential roles of OFD1a during both nephrogenesis and in cystic kidney disease. In addition further studies are required to establish whether renal mesenchymal cells have primary cilia. Furthermore, while all developing organs that we studied expressed both OFD1a and OFD1b isoforms on RT-PCR, it remains possible that the two OFD1 proteins are not expressed synchronously, and the pattern of OFD1b protein remains to be determined.
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