FREM1 Mutations Cause Bifid Nose, Renal Agenesis, and Anorectal Malformations Syndrome
2009; Elsevier BV; Volume: 85; Issue: 3 Linguagem: Inglês
10.1016/j.ajhg.2009.08.010
ISSN1537-6605
AutoresAnas M. Alazami, Ranad Shaheen, Fatema Alzahrani, Katie Snape, Anand Saggar, Bernd Brinkmann, Prashant Bavi, L.I. Al-Gazali, Fowzan S. Alkuraya,
Tópico(s)Global Maritime and Colonial Histories
ResumoAn autosomal-recessive syndrome of bifid nose and anorectal and renal anomalies (BNAR) was previously reported in a consanguineous Egyptian sibship. Here, we report the results of linkage analysis, on this family and on two other families with a similar phenotype, which identified a shared region of homozygosity on chromosome 9p22.2-p23. Candidate-gene analysis revealed homozygous frameshift and missense mutations in FREM1, which encodes an extracellular matrix component of basement membranes. In situ hybridization experiments demonstrated gene expression of Frem1 in the midline of E11.5 mouse embryos, in agreement with the observed cleft nose phenotype of our patients. FREM1 is part of a ternary complex that includes FRAS1 and FREM2, and mutations of the latter two genes have been reported to cause Fraser syndrome in mice and humans. The phenotypic variability previously reported for different Frem1 mouse mutants suggests that the apparently distinct phenotype of BNAR in humans may represent a previously unrecognized variant of Fraser syndrome. An autosomal-recessive syndrome of bifid nose and anorectal and renal anomalies (BNAR) was previously reported in a consanguineous Egyptian sibship. Here, we report the results of linkage analysis, on this family and on two other families with a similar phenotype, which identified a shared region of homozygosity on chromosome 9p22.2-p23. Candidate-gene analysis revealed homozygous frameshift and missense mutations in FREM1, which encodes an extracellular matrix component of basement membranes. In situ hybridization experiments demonstrated gene expression of Frem1 in the midline of E11.5 mouse embryos, in agreement with the observed cleft nose phenotype of our patients. FREM1 is part of a ternary complex that includes FRAS1 and FREM2, and mutations of the latter two genes have been reported to cause Fraser syndrome in mice and humans. The phenotypic variability previously reported for different Frem1 mouse mutants suggests that the apparently distinct phenotype of BNAR in humans may represent a previously unrecognized variant of Fraser syndrome. In 2002, Al-Gazali et al. reported a second-cousin consanguineous Egyptian family in which four siblings had bifid nose associated with anorectal and renal abnormalities (BNAR [MIM 608980]).1Al-Gazali L.I. Bakir M. Hamud O.A. Gerami S. An autosomal recessive syndrome of nasal anomalies associated with renal and anorectal malformations.Clin. Dysmorphol. 2002; 11: 33-38Crossref PubMed Scopus (17) Google Scholar One of the children was born with bilateral renal agenesis and died within the first hour of life. The surviving three presented with unilateral renal agenesis, low-pitched crying, short and thick oral frenula, incurved fifth toe, anteriorly placed anus, and stenosis of the anal opening. Importantly, the bifid nose with bulbous nasal tip was not associated with hypertelorism. This therefore represented an apparently distinct autosomal-recessive phenotype. We have also observed this rare disorder in two consanguineous families of Afghani and Pakistani origin (Figure 1A), in which the phenotype is essentially similar although kidney involvement is more variable (Table 1).Table 1Comparison of Frequency of Key Clinical Features between BNAR and Fraser SyndromeBNARFraser Syndromec.2721delGc.1945C>Tc.4318G>AOverallOverallCryptophthalmos0/40/30/20%85–88%Syndactyly0/30/30/30%62–95%Abnormal genitalia0/30/30/30%40–66%Bifid nose4/43/32/2100%15%Ear malformation0/30/30/30%59–75%Airway malformation0/30/32/222%31–58%Anorectal malformation2/40/30/222%16–32%Renal agenesis4/41/31/266%45–77%Fraser syndrome data are based on Slavotinek and Tifft10Slavotinek A.M. Tifft C.J. Fraser syndrome and cryptophthalmos: review of the diagnostic criteria and evidence for phenotypic modules in complex malformation syndromes.J. Med. Genet. 2002; 39: 623-633Crossref PubMed Scopus (168) Google Scholar and van Haelst et al.21van Haelst M.M. Scambler P.J. Hennekam R.C. Fraser syndrome: a clinical study of 59 cases and evaluation of diagnostic criteria.Am. J. Med. Genet. 2007; 143A: 3194-3203Crossref PubMed Scopus (77) Google Scholar Open table in a new tab Fraser syndrome data are based on Slavotinek and Tifft10Slavotinek A.M. Tifft C.J. Fraser syndrome and cryptophthalmos: review of the diagnostic criteria and evidence for phenotypic modules in complex malformation syndromes.J. Med. Genet. 2002; 39: 623-633Crossref PubMed Scopus (168) Google Scholar and van Haelst et al.21van Haelst M.M. Scambler P.J. Hennekam R.C. Fraser syndrome: a clinical study of 59 cases and evaluation of diagnostic criteria.Am. J. Med. Genet. 2007; 143A: 3194-3203Crossref PubMed Scopus (77) Google Scholar The apparent autosomal-recessive mode of inheritance suggested that autozygosity mapping would determine the disease locus in these consanguineous families. After obtaining written and informed consent from all patients or their legal guardians (in accordance with a protocol approved by the King Faisal Specialist Hospital institutional review board, protocol no. 2080006), we implemented genome-wide multipoint parametric linkage analysis, using the Affymetrix 250K StyI GeneChip platform (Affymetrix, Santa Clara, CA, USA). Resulting genotyping data were analyzed with the EasyLinkage software package, assuming fully penetrant autosomal-recessive inheritance and a disease allele frequency of 0.0001, with a consanguinity loop utilized for each family. Analysis of all surviving affected individuals yielded a maximum LOD score of 6.62 between SNP markers rs10124106 and rs10963391, a 4.4 Mb interval on 9p22.2–p23 encompassing a total of 28 annotated genes (Figure S1, available online). This was in agreement with data obtained with the use of microsatellite markers and signified that a single locus, but on different haplotypes, was the likely cause of the syndrome in all three families. Candidate-gene selection was prioritized according to expression levels in the developing kidney and assessment of the mouse knockout phenotype, if known. The most promising candidate gene that met our criteria was FRAS1-related extracellular matrix protein 1 (FREM1 [MIM 608944]), which encodes a basement membrane protein. Deficiency of the orthologous Frem1 in mouse results in a phenotype that includes renal agenesis.2Kiyozumi D. Sugimoto N. Sekiguchi K. Breakdown of the reciprocal stabilization of QBRICK/Frem1, Fras1, and Frem2 at the basement membrane provokes Fraser syndrome-like defects.Proc. Natl. Acad. Sci. USA. 2006; 103: 11981-11986Crossref PubMed Scopus (87) Google Scholar, 3Smyth I. Du X. Taylor M.S. Justice M.J. Beutler B. Jackson I.J. The extracellular matrix gene Frem1 is essential for the normal adhesion of the embryonic epidermis.Proc. Natl. Acad. Sci. USA. 2004; 101: 13560-13565Crossref PubMed Scopus (104) Google Scholar Primers were therefore designed to flank all known FREM1 exons, as identified on the UCSC and Ensembl websites, and were directly sequenced with the dideoxy chain-termination method (Amersham ET Dye Termination Sequencing Kit). Samples were processed on a MegaBACE 1000 (Molecular Dynamics, Sunnyvale, CA, USA). Sequence analysis with SeqMan II (DNASTAR, Madison, WI, USA) revealed a single basepair (bp) deletion in exon 17 of FREM1 in the Egyptian family (c.2721delG; NM_144966), predicting a frameshift at amino acid 908 and a premature truncation 17 residues downstream (p.V908SfsX17; Figure 1B). In the Afghani sibship, we identified a transition in exon 12, predicted to cause an Arg-to-Trp change (c.1945C>T [p.R649W]), whereas the Pakistani family harbored a different transition in exon 25, which predicted a Gly-to-Ser alteration (c.4318G>A [p.G1440S]) (Figure 1B). All mutations segregated with the disease state and were confirmed via bidirectional sequencing, and all patients were homozygous for their respective mutations. To validate the two missense mutations, we examined 121 Afghani and 97 Indian subcontinental normal control individuals, respectively. No trace of either mutation was found, indicating that these sequence variants are not common polymorphisms within the two populations. Furthermore, protein sequence alignment of FREM1 orthologs demonstrated that the affected residues are highly conserved across species, from human to fugu and for the G1440S alteration, even down to the Florida lancelet and the Ciona intestinalis sea squirt (Figure 1C), lending further credence to the pathogenicity of these amino acid substitutions. Frem1 is part of a ternary complex that includes two other extracellular matrix proteins, Fras1 (MIM 607830) and Frem2 (MIM 608945).2Kiyozumi D. Sugimoto N. Sekiguchi K. Breakdown of the reciprocal stabilization of QBRICK/Frem1, Fras1, and Frem2 at the basement membrane provokes Fraser syndrome-like defects.Proc. Natl. Acad. Sci. USA. 2006; 103: 11981-11986Crossref PubMed Scopus (87) Google Scholar To a large extent, these three proteins are functionally interdependent. They exhibit strict colocalization in the epithelial basement membrane of several organs within the developing mouse,2Kiyozumi D. Sugimoto N. Sekiguchi K. Breakdown of the reciprocal stabilization of QBRICK/Frem1, Fras1, and Frem2 at the basement membrane provokes Fraser syndrome-like defects.Proc. Natl. Acad. Sci. USA. 2006; 103: 11981-11986Crossref PubMed Scopus (87) Google Scholar, 4Chiotaki R. Petrou P. Giakoumaki E. Pavlakis E. Sitaru C. Chalepakis G. Spatiotemporal distribution of Fras1/Frem proteins during mouse embryonic development.Gene Expr. Patterns. 2007; 7: 381-388Crossref PubMed Scopus (28) Google Scholar, 5Petrou P. Chiotaki R. Dalezios Y. Chalepakis G. Overlapping and divergent localization of Frem1 and Fras1 and its functional implications during mouse embryonic development.Exp. Cell Res. 2007; 313: 910-920Crossref PubMed Scopus (36) Google Scholar and although one Frem1 mutant did display normal Fras1 localization,3Smyth I. Du X. Taylor M.S. Justice M.J. Beutler B. Jackson I.J. The extracellular matrix gene Frem1 is essential for the normal adhesion of the embryonic epidermis.Proc. Natl. Acad. Sci. USA. 2004; 101: 13560-13565Crossref PubMed Scopus (104) Google Scholar compromising the expression of any one of these three proteins generally leads to reduced deposition of the other two.2Kiyozumi D. Sugimoto N. Sekiguchi K. Breakdown of the reciprocal stabilization of QBRICK/Frem1, Fras1, and Frem2 at the basement membrane provokes Fraser syndrome-like defects.Proc. Natl. Acad. Sci. USA. 2006; 103: 11981-11986Crossref PubMed Scopus (87) Google Scholar, 5Petrou P. Chiotaki R. Dalezios Y. Chalepakis G. Overlapping and divergent localization of Frem1 and Fras1 and its functional implications during mouse embryonic development.Exp. Cell Res. 2007; 313: 910-920Crossref PubMed Scopus (36) Google Scholar, 6Petrou P. Pavlakis E. Dalezios Y. Chalepakis G. Basement membrane localization of Frem3 is independent of the Fras1/Frem1/Frem2 protein complex within the sublamina densa.Matrix Biol. 2007; 26: 652-658Crossref PubMed Scopus (24) Google Scholar Indeed, mutations of either FRAS1 or FREM2 lead to Fraser syndrome (FS [MIM 219000]),7Jadeja S. Smyth I. Pitera J.E. Taylor M.S. van Haelst M. Bentley E. McGregor L. Hopkins J. Chalepakis G. Philip N. et al.Identification of a new gene mutated in Fraser syndrome and mouse myelencephalic blebs.Nat. Genet. 2005; 37: 520-525Crossref PubMed Scopus (135) Google Scholar, 8McGregor L. Makela V. Darling S.M. Vrontou S. Chalepakis G. Roberts C. Smart N. Rutland P. Prescott N. Hopkins J. et al.Fraser syndrome and mouse blebbed phenotype caused by mutations in FRAS1/Fras1 encoding a putative extracellular matrix protein.Nat. Genet. 2003; 34: 203-208Crossref PubMed Scopus (195) Google Scholar, 9Vrontou S. Petrou P. Meyer B.I. Galanopoulos V.K. Imai K. Yanagi M. Chowdhury K. Scambler P.J. Chalepakis G. Fras1 deficiency results in cryptophthalmos, renal agenesis and blebbed phenotype in mice.Nat. Genet. 2003; 34: 209-214Crossref PubMed Scopus (102) Google Scholar an autosomal-recessive condition characterized most notably by cryptophthalmos (due to failure of palpebral fissure development) in ∼90% of human cases, syndactyly in ∼60% of cases, as well as renal agenesis and abnormal or ambiguous genitalia (Table 1).10Slavotinek A.M. Tifft C.J. Fraser syndrome and cryptophthalmos: review of the diagnostic criteria and evidence for phenotypic modules in complex malformation syndromes.J. Med. Genet. 2002; 39: 623-633Crossref PubMed Scopus (168) Google Scholar A set of four spontaneous mouse mutants, known as bleb mutants, each mapping to a different chromosome, have long been considered to represent mouse models of FS.11Smyth I. Scambler P. The genetics of Fraser syndrome and the blebs mouse mutants.Hum Mol Genet. 2005; 14 (Spec. No. 2): R269-R274Crossref PubMed Scopus (71) Google Scholar The hallmark of these bleb mutants is the formation of subepidermal blisters, appearing at midgestation, which then become hemorrhagic, possibly as a result of in utero friction.3Smyth I. Du X. Taylor M.S. Justice M.J. Beutler B. Jackson I.J. The extracellular matrix gene Frem1 is essential for the normal adhesion of the embryonic epidermis.Proc. Natl. Acad. Sci. USA. 2004; 101: 13560-13565Crossref PubMed Scopus (104) Google Scholar, 9Vrontou S. Petrou P. Meyer B.I. Galanopoulos V.K. Imai K. Yanagi M. Chowdhury K. Scambler P.J. Chalepakis G. Fras1 deficiency results in cryptophthalmos, renal agenesis and blebbed phenotype in mice.Nat. Genet. 2003; 34: 209-214Crossref PubMed Scopus (102) Google Scholar, 12Petrou P. Makrygiannis A.K. Chalepakis G. The Fras1/Frem family of extracellular matrix proteins: structure, function, and association with Fraser syndrome and the mouse bleb phenotype.Connect. Tissue Res. 2008; 49: 277-282Crossref PubMed Scopus (32) Google Scholar Although this often leads to embryonic lethality, mice that are born at term do not display further blistering, an indication that this defect is temporally restricted.3Smyth I. Du X. Taylor M.S. Justice M.J. Beutler B. Jackson I.J. The extracellular matrix gene Frem1 is essential for the normal adhesion of the embryonic epidermis.Proc. Natl. Acad. Sci. USA. 2004; 101: 13560-13565Crossref PubMed Scopus (104) Google Scholar, 7Jadeja S. Smyth I. Pitera J.E. Taylor M.S. van Haelst M. Bentley E. McGregor L. Hopkins J. Chalepakis G. Philip N. et al.Identification of a new gene mutated in Fraser syndrome and mouse myelencephalic blebs.Nat. Genet. 2005; 37: 520-525Crossref PubMed Scopus (135) Google Scholar, 8McGregor L. Makela V. Darling S.M. Vrontou S. Chalepakis G. Roberts C. Smart N. Rutland P. Prescott N. Hopkins J. et al.Fraser syndrome and mouse blebbed phenotype caused by mutations in FRAS1/Fras1 encoding a putative extracellular matrix protein.Nat. Genet. 2003; 34: 203-208Crossref PubMed Scopus (195) Google Scholar In addition, most bleb mice also exhibit cryptophthalmos, syndactyly, and renal malformation, traits that link them to human FS.11Smyth I. Scambler P. The genetics of Fraser syndrome and the blebs mouse mutants.Hum Mol Genet. 2005; 14 (Spec. No. 2): R269-R274Crossref PubMed Scopus (71) Google Scholar The classic bleb mutants blebbed, myelencephalic blebs, and eye blebs are now known to harbor mutations in, respectively, Fras1, Frem2, and Grip1 (which encodes a scaffolding protein required for basolateral targeting of Fras1 and Frem2) (MIM 604597).7Jadeja S. Smyth I. Pitera J.E. Taylor M.S. van Haelst M. Bentley E. McGregor L. Hopkins J. Chalepakis G. Philip N. et al.Identification of a new gene mutated in Fraser syndrome and mouse myelencephalic blebs.Nat. Genet. 2005; 37: 520-525Crossref PubMed Scopus (135) Google Scholar, 8McGregor L. Makela V. Darling S.M. Vrontou S. Chalepakis G. Roberts C. Smart N. Rutland P. Prescott N. Hopkins J. et al.Fraser syndrome and mouse blebbed phenotype caused by mutations in FRAS1/Fras1 encoding a putative extracellular matrix protein.Nat. Genet. 2003; 34: 203-208Crossref PubMed Scopus (195) Google Scholar, 9Vrontou S. Petrou P. Meyer B.I. Galanopoulos V.K. Imai K. Yanagi M. Chowdhury K. Scambler P.J. Chalepakis G. Fras1 deficiency results in cryptophthalmos, renal agenesis and blebbed phenotype in mice.Nat. Genet. 2003; 34: 209-214Crossref PubMed Scopus (102) Google Scholar, 13Takamiya K. Kostourou V. Adams S. Jadeja S. Chalepakis G. Scambler P.J. Huganir R.L. Adams R.H. A direct functional link between the multi-PDZ domain protein GRIP1 and the Fraser syndrome protein Fras1.Nat. Genet. 2004; 36: 172-177Crossref PubMed Scopus (120) Google Scholar The fourth classic mutant, head blebs (heb), though it was similarly found to contain a truncating mutation in Frem1,3Smyth I. Du X. Taylor M.S. Justice M.J. Beutler B. Jackson I.J. The extracellular matrix gene Frem1 is essential for the normal adhesion of the embryonic epidermis.Proc. Natl. Acad. Sci. USA. 2004; 101: 13560-13565Crossref PubMed Scopus (104) Google Scholar bears a significantly milder phenotype. None of the homozygous heb embryos or adults in the original report was found to exhibit cryptophthalmos, a characteristic finding in the other bleb mutants. Syndactyly also was not present, although polydactyly was.14Varnum D.S. Fox S.C. Head blebs: a new mutation on chromosome 4 of the mouse.J. Hered. 1981; 72: 293PubMed Google Scholar In fact, 97% of E17 embryos demonstrated an “open eyelids” phenotype that is in marked contrast to Fras1−/− mice, for example, in which adults were reported to show 20% bilateral and 75% unilateral cryptophthalmos.9Vrontou S. Petrou P. Meyer B.I. Galanopoulos V.K. Imai K. Yanagi M. Chowdhury K. Scambler P.J. Chalepakis G. Fras1 deficiency results in cryptophthalmos, renal agenesis and blebbed phenotype in mice.Nat. Genet. 2003; 34: 209-214Crossref PubMed Scopus (102) Google Scholar In the course of normal mammalian development, the eyelids expand across the corneal surface, temporarily fusing then subsequently reopening. In mice, this temporary closure transpires between E15.5 and E16.5 and remains in place until roughly two weeks after birth, when complete separation occurs.15Findlater G.S. McDougall R.D. Kaufman M.H. Eyelid development, fusion and subsequent reopening in the mouse.J. Anat. 1993; 183: 121-129PubMed Google Scholar Due to the failure of eyelid fusion in heb, the eyes suffer from in utero mechanical damage and reduced eyeball growth. As adults, these mice keep their eyelids closed as a means of coping with their atrophic eyes.14Varnum D.S. Fox S.C. Head blebs: a new mutation on chromosome 4 of the mouse.J. Hered. 1981; 72: 293PubMed Google Scholar The temporary epithelial fusion of both halves of the eyelid involves participation of cells of the periderm layer.16Maconnachie E. A study of digit fusion in the mouse embryo.J. Embryol. Exp. Morphol. 1979; 49: 259-276PubMed Google Scholar Interestingly, whereas Fras1 remains restricted to the basement membrane of the advancing eyelid, Frem1 is also found in the rounded periderm cells that lie along the plane of eyelid fusion, which may help explain the unique “open eyes at birth” phenotype of the heb mutants.5Petrou P. Chiotaki R. Dalezios Y. Chalepakis G. Overlapping and divergent localization of Frem1 and Fras1 and its functional implications during mouse embryonic development.Exp. Cell Res. 2007; 313: 910-920Crossref PubMed Scopus (36) Google Scholar Two further Frem1 mutants have also been described. The first, labeled bat, also displays an “open eyes at birth” phenotype, though cryptophthalmos was also observed.3Smyth I. Du X. Taylor M.S. Justice M.J. Beutler B. Jackson I.J. The extracellular matrix gene Frem1 is essential for the normal adhesion of the embryonic epidermis.Proc. Natl. Acad. Sci. USA. 2004; 101: 13560-13565Crossref PubMed Scopus (104) Google Scholar In addition, 20% of homozygous bat mutants revealed unilateral renal agenesis, whereas the original heb mutants were described as having normal kidneys.14Varnum D.S. Fox S.C. Head blebs: a new mutation on chromosome 4 of the mouse.J. Hered. 1981; 72: 293PubMed Google Scholar Occasional syndactyly was also reported in bat. The second, Qbrick/Frem1−/−, exhibits cryptophthalmos, syndactyly, and renal agenesis.2Kiyozumi D. Sugimoto N. Sekiguchi K. Breakdown of the reciprocal stabilization of QBRICK/Frem1, Fras1, and Frem2 at the basement membrane provokes Fraser syndrome-like defects.Proc. Natl. Acad. Sci. USA. 2006; 103: 11981-11986Crossref PubMed Scopus (87) Google Scholar This phenotypic variability may be due to differences in genetic background (which in the case of bat, for example, was shown to significantly modulate phenotype penetrance),3Smyth I. Du X. Taylor M.S. Justice M.J. Beutler B. Jackson I.J. The extracellular matrix gene Frem1 is essential for the normal adhesion of the embryonic epidermis.Proc. Natl. Acad. Sci. USA. 2004; 101: 13560-13565Crossref PubMed Scopus (104) Google Scholar or they may be caused by allelic differences between the three mutants. This latter possibility is further supported by the fact that although both bat and Qbrick/Frem1−/− utilize C57BL/6 mice, the deposition of Fras1 in the embryonic epidermis is unaffected in bat, whereas in Qbrick/Frem1−/− the deposition is substantially reduced. Both human FREM1 and its mouse ortholog are composed of a putative signal sequence, 12 chondroitin sulfate proteoglycan (CSPG) repeats,17Staub E. Hinzmann B. Rosenthal A. A novel repeat in the melanoma-associated chondroitin sulfate proteoglycan defines a new protein family.FEBS Lett. 2002; 527: 114-118Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar a Calx-β domain, and a C-terminal type C lectin-like domain (Figure 1D). On the basis of previous delineation of the CSPG repeats,18Kiyozumi D. Osada A. Sugimoto N. Weber C.N. Ono Y. Imai T. Okada A. Sekiguchi K. Identification of a novel cell-adhesive protein spatiotemporally expressed in the basement membrane of mouse developing hair follicle.Exp. Cell Res. 2005; 306: 9-23Crossref PubMed Scopus (38) Google Scholar the Afghani missense mutation lies in the fourth CSPG repeat, the truncating frameshift mutation in the sixth, and the Pakistani missense mutation in the tenth repeat. The original mouse heb mutant incorporates a LINE1 insertion in the seventh CSPG repeat, and bat contains a donor splice site mutation leading to frameshift in the twelfth repeat.3Smyth I. Du X. Taylor M.S. Justice M.J. Beutler B. Jackson I.J. The extracellular matrix gene Frem1 is essential for the normal adhesion of the embryonic epidermis.Proc. Natl. Acad. Sci. USA. 2004; 101: 13560-13565Crossref PubMed Scopus (104) Google ScholarQbrick/Frem1−/− knocks out the entire gene by replacing the initiation codon.2Kiyozumi D. Sugimoto N. Sekiguchi K. Breakdown of the reciprocal stabilization of QBRICK/Frem1, Fras1, and Frem2 at the basement membrane provokes Fraser syndrome-like defects.Proc. Natl. Acad. Sci. USA. 2006; 103: 11981-11986Crossref PubMed Scopus (87) Google Scholar Correlating our patient phenotypes to the mouse models on the basis of the site of mutation is not straightforward. BNAR appears to be a mixture of the heb's lack of cryptophthalmos and the bat and Qbrick/Frem1−/− mutants' presence of renal agenesis. Temporal and spatial expression data have been published on Frem1 previously.3Smyth I. Du X. Taylor M.S. Justice M.J. Beutler B. Jackson I.J. The extracellular matrix gene Frem1 is essential for the normal adhesion of the embryonic epidermis.Proc. Natl. Acad. Sci. USA. 2004; 101: 13560-13565Crossref PubMed Scopus (104) Google Scholar However, the expression pattern of this gene in midline is generally lacking. Therefore, and given the highly penetrant bifid nose component of BNAR, we decided to assay Frem1 expression in the developing nose of E11.5 whole-mount embryos. Two Frem1 probes were utilized, one spanning c.4446–48523Smyth I. Du X. Taylor M.S. Justice M.J. Beutler B. Jackson I.J. The extracellular matrix gene Frem1 is essential for the normal adhesion of the embryonic epidermis.Proc. Natl. Acad. Sci. USA. 2004; 101: 13560-13565Crossref PubMed Scopus (104) Google Scholar and the other spanning c.2070–2854 (NM_144966). SP6- and T7-tagged primers were used for generating, respectively, sense and antisense digoxygenin-labeled RNA probes with the MaxiScript Kit (Ambion, Austin, TX, USA) and Roche's DIG RNA Labeling Mix (Indianapolis, IN, USA). Embryos were permeabilized with proteinase K (10 μg/ml) at 37°C for 4 min, and in situ hybridization was performed with the InsituPro VSi (Intavis AG, Koeln, Germany) in accordance with a manufacturer-recommended protocol. Both antisense probes demonstrated strong and specific staining in the snout, as well as in the midline, where the two medial nasal processes fuse (Figures 2A and 2B). After in situ hybridization, examination of 5 μm sections of paraffin-embedded embryos revealed that Frem1 expression in the developing nose was mainly in the epithelial-mesenchymal transitional region at the midline (Figures 2C and 2D). Localization of Frem1 in this area, coupled with the consistent bifid nose phenotype of our patients, strongly argues for an important role by Frem1 in the fusion of the medial nasal processes during gestation. Among the Fras1/Frem proteins, Frem1 is unique in several aspects. It is expressed predominantly from the underlying mesenchyme, although to a lesser extent also from the epithelia,3Smyth I. Du X. Taylor M.S. Justice M.J. Beutler B. Jackson I.J. The extracellular matrix gene Frem1 is essential for the normal adhesion of the embryonic epidermis.Proc. Natl. Acad. Sci. USA. 2004; 101: 13560-13565Crossref PubMed Scopus (104) Google Scholar, 5Petrou P. Chiotaki R. Dalezios Y. Chalepakis G. Overlapping and divergent localization of Frem1 and Fras1 and its functional implications during mouse embryonic development.Exp. Cell Res. 2007; 313: 910-920Crossref PubMed Scopus (36) Google Scholar and is targeted with the use of a Grip1-independent pathway. Fras1 and Frem2 are strictly secreted from the epiderm and require Grip1 for correct basolateral targeting.2Kiyozumi D. Sugimoto N. Sekiguchi K. Breakdown of the reciprocal stabilization of QBRICK/Frem1, Fras1, and Frem2 at the basement membrane provokes Fraser syndrome-like defects.Proc. Natl. Acad. Sci. USA. 2006; 103: 11981-11986Crossref PubMed Scopus (87) Google Scholar, 13Takamiya K. Kostourou V. Adams S. Jadeja S. Chalepakis G. Scambler P.J. Huganir R.L. Adams R.H. A direct functional link between the multi-PDZ domain protein GRIP1 and the Fraser syndrome protein Fras1.Nat. Genet. 2004; 36: 172-177Crossref PubMed Scopus (120) Google Scholar Although all three overlap in the basement membrane, Frem1 expression in the ocular region is slightly delayed in comparison to the others.4Chiotaki R. Petrou P. Giakoumaki E. Pavlakis E. Sitaru C. Chalepakis G. Spatiotemporal distribution of Fras1/Frem proteins during mouse embryonic development.Gene Expr. Patterns. 2007; 7: 381-388Crossref PubMed Scopus (28) Google Scholar Ablation of Fras1 or Frem2 causes complete loss of the ternary complex, but in the bat mutant of Frem1, deposition of Fras1 is unaffected,3Smyth I. Du X. Taylor M.S. Justice M.J. Beutler B. Jackson I.J. The extracellular matrix gene Frem1 is essential for the normal adhesion of the embryonic epidermis.Proc. Natl. Acad. Sci. USA. 2004; 101: 13560-13565Crossref PubMed Scopus (104) Google Scholar whereas in Qbrick/Frem1−/−, only residual levels of Fras1 and Frem2 are detectable in the skin basement membrane.2Kiyozumi D. Sugimoto N. Sekiguchi K. Breakdown of the reciprocal stabilization of QBRICK/Frem1, Fras1, and Frem2 at the basement membrane provokes Fraser syndrome-like defects.Proc. Natl. Acad. Sci. USA. 2006; 103: 11981-11986Crossref PubMed Scopus (87) Google Scholar Even more intriguing is the supplementary role of Frem1 in periderm cells, a role that is first apparent at E16.5 and becomes almost exclusive by E18.5. Notably, in the adult mouse, Frem1 is not required for complex stabilization, because, unlike Fras1 and Frem2, no appreciable levels of Frem1 are present in the P30 kidney, gut, testes, or esophagus.19Pavlakis E. Makrygiannis A.K. Chiotaki R. Chalepakis G. Differential localization profile of Fras1/Frem proteins in epithelial basement membranes of newborn and adult mice.Histochem. Cell Biol. 2008; 130: 785-793Crossref PubMed Scopus (11) Google Scholar Finally, unlike the phenotypic consistency observed in mice bearing Fras1, Frem2, and Grip1 mutations, the range of phenotype is considerably wider with Frem1 mutants. Reassessment of the literature demonstrates that at least some of these mutants fall outside the scope of FS. These observations may explain why our patient phenotype differs from that of classic FS, given that we have shown in this study that nonsense and missense mutations in FREM1 cause BNAR, a disorder with features that are both overlapping and distinct from FS. Previous studies have failed to identify pathogenic FREM1 mutations in patients with classic FS,20Short K. Wiradjaja F. Smyth I. Let's stick together: the role of the Fras1 and Frem proteins in epidermal adhesion.IUBMB Life. 2007; 59: 427-435Crossref PubMed Scopus (45) Google Scholar and this suggests that FREM1 mutations are exceedingly rare in this context or that they lead to BNAR or to other phenotypes that are not recognized clinically as FS. On the other hand, the remarkable phenotypic variability observed in murine Frem1 mutants argues for the classification of BNAR as an atypical FS subtype. Future studies on the genetics of FS should include “atypical” cases for examination of the contribution of FREM1 to the genetics of this syndrome. In summary, this report further underscores the differences between Frem1 and Fras1/Frem2 and establishes, for the first time to our knowledge, a role for FREM1 in human craniofacial and renal development. We are grateful to the family members for their enthusiastic and generous participation. We thank Raafat El-Sayed and Catalino Santos for invaluable support with the mouse work, Valerie Atizado and Valorie Balde for their critical help with mouse sections, Mohammad Rajab and Shamsa Al-Enazi for rapid DNA sequencing, Hassan Omirah for whole-mount embryo photography, Brigitte Loddenkötter and Carsten Hohoff for DNA extraction of control samples, Haya Al-Saud for whole-genome amplification of various controls, and Salma Wakil, Batoul Baz, Khushnooda Ramzan, Rana Al-Omar, and Abeer Al-Mostafa for their tremendous help in processing all Affy chips. We are particularly grateful to Irfan Saadi for his critical review of the manuscript. This study was approved and funded by the King Faisal Specialist Hospital & Research Centre (RAC no. 2080006). Download .pdf (.03 MB) Help with pdf files Document S1. One Figure The URLs for data presented herein are as follows:Ensembl Genome Browser, http://www.ensembl.org/index.htmlHuman Genome Browser Gateway (UCSC), http://genome.ucsc.edu/cgi-bin/hgGatewayMultalin (multiple sequence alignment), http://www-archbac.u-psud.fr/genomics/multalin.htmlOnline Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/omim/UCSC Genome Browser, http://genome.ucsc.edu/ FREM1 Mutations Cause Bifid Nose, Renal Agenesis, and Anorectal Malformations SyndromeAlazami et al.The American Journal of Human GeneticsNovember 13, 2009In Brief(The American Journal of Human Genetics 85, 414–418; September 11, 2009) Full-Text PDF Open Archive
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