The Fibulin-1 Gene (FBLN1) Is Disrupted in a T(12;22) Associated
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98 ORIGINAL ARTICLE J Med Genet: first published as 10.1136/jmg.39.2.98 on 1 February 2002. Downloaded from The fibulin-1 gene (FBLN1) is disrupted in a t(12;22) associated with a complex type of synpolydactyly P Debeer,EFPMSchoenmakers*, W O Twal, W S Argraves, L De Smet, J-P Fryns, W J M Van de Ven ............................................................................................................................. See end of article for authors’ affiliations J Med Genet 2002;39:98–104 ....................... Correspondence to: Dr P Debeer, Centre for Molecular analysis of the reciprocal chromosomal translocation t(12;22)(p11.2;q13.3) cosegregating Human Genetics, with a complex type of synpolydactyly showed involvement of an alternatively spliced exon of the Herestraat 49, B-3000 fibulin-1 gene (FBLN1 located in 22q13.3) and the C12orf2 (HoJ-1) gene on the short arm of chromo- Leuven, Belgium; some 12. Investigation of the possible functional involvement of the fibulin-1 protein (FBLN1) in the philippe.debeer@med. observed phenotype showed that FBLN1 is expressed in the extracellular matrix (ECM) in association kuleuven.ac.be with the digits in the developing limb. Furthermore, fibroblasts derived from patients with the complex *Present address: type of synpolydactyly displayed alterations in the level of FBLN1-D splice variant incorporated into the Department of Human ECM and secreted into the conditioned culture medium. By contrast, the expression of the FBLN1-C Genetics, University Medical Centre Nijmegen, splice variant was not perturbed in the patient fibroblasts. Based on these findings, we propose that the Nijmegen, The Netherlands t(12;22) results in haploinsufficiency of the FBLN1-D variant, which could lead to the observed limb ....................... malformations. he formation of digits is a complex, tightly regulated on sequence data obtained during random sequence sam- process which involves apoptosis and in which several pling, and mapping within the rearranged 2.6 kb BamHI frag- Tpathways and signalling molecules are involved. The pres- ment on chromosome 12p. Part of the DNA produced in this entation of a small family with a very specific congenital hand first round was then reamplified using the same universal malformation associated with an apparently balanced auto- vectorette primer and the following (nested) chromosome 12 somal reciprocal translocation provided us with the oppor- specific primer (5′-GGTAATGCATGCTACATGGTGGCTTCA tunity to identify novel genes involved in limb development. GGG-3′). PCR products were analysed on, and recovered from, Previously, we described the clinical phenotype of three standard 1% agarose gels and subcloned using the pGEM-T patients from one family sharing a complex type of Easy system (Promega). synpolydactyly (fig 1) associated with a constitutional http://jmg.bmj.com/ balanced t(12;22)(p11.2;q13.3) and the exclusion of synpoly- Sequencing of the wild type FBLN1-D and construction dactyly associated HOXD13 mutations in these patients.1–3 All of FBLN1 probes affected subjects carry the constitutional balanced transloca- The sequence of the presumed wild type FBLN1-D was deter- tion, whereas unaffected family members have a normal mined as follows: first, the wild type FBLN1-D cDNA was iso- karyotype. The cosegregation of the phenotype with this lated with the FBLN1-D specific cDNA synthesis primer ′ ′ translocation suggested that a synpolydactyly associated gene (5 -CCACCTATAAGAAAAGAT-3 ). This primer is located might be localised at one or both of the chromosomal downstream of the translocation breakpoint, so only the pre- breakpoints involved in this translocation. As a first step sumed wild type transcript, that is, originating from the non- on September 26, 2021 by guest. Protected copyright. towards the identification of the genes involved in the disrupted gene, was isolated. Subsequently, overlapping frag- translocation, we were able to pinpoint the breakpoint region ments covering the whole FBLN1-D cDNA were amplified by on chromosome 12 to a 2.6 kb BamHI fragment.4 PCR and subcloned in the pGEM-T Easy vector. The following primer sets were used to amplify the overlap- Here, we describe the cloning of the translocation break- ′ point and the identification of two genes directly targeted by ping cDNA fragments: RT1-up: 5 -CTCATTTTTTAATGCG AAGGCTAAG-3′; RT1-low: 5′-AAGAAA AGATGGATGATG this translocation. One of these genes, FBLN1, turned out to be CAGAGTG-3′; RT2-up: 5′-TGCGGGACTCTTTTGACATCATC- 3′; the ideal positional and functional candidate for the observed RT2-low: 5′-TCTTTCTTTCCTTGGCCCTTAGC-3′; RT3-up: 5′- phenotype. We therefore examined the consequences of the CGC AACTGCCAAGACATTGATGA-3′; RT3-low: 5′-CAGCTT translocation induced disruption on the expression of FBLN1- CAGGACGGCATGAAATG-3′; RT4-up: 5′-AGAACACGCTGGG D, one of the four variants of FBLN1, in the possible genesis of CTCCTACCTC-3′; RT4-low: 5′-CGGACCGTGTCTGTCTTCT the limb malformations observed in our patients. CCTG-3′; RT5-up: 5′-GCCCTATTGGGCATACATGCATC-3′; RT5- low: 5′-CTCGCCGGCAGTAACACTGGTAG-3′; RT6-up: 5′- MATERIALS AND METHODS GACGAGGTGGTCTGCTCCTGCTT-3′; RT6-low: 5′-CGCACT ′ ′ Vectorette PCR CGTCCACATCAACACAG-3 ; RT7-up: 5 -TCAAGAGCC ′ ′ Vectorette PCR experiments were performed essentially as AGGAGACCGGAGA-3 ; RT7-low: 5 -CTGGTAGCCCACGAA ′ ′ ′ described by Schoenmakers et al.5 Genomic DNA was digested GCAGGAG-3 ; RT8-up: 5 -CCCGCCGCCCATGGAGCGCGC-3 ; ′ ′ with BamHI and a vectorette linker with BamHI compatible RT8-low: 5 -CGGCACTGCTGCTTGCAGGGCCCGC-3 . cohesive ends was ligated to the digested DNA. This ligated DNA was used in two consecutive rounds of (hemi-nested) ............................................................. PCR amplification. In a first round a universal vectorette ′ ′ Abbreviations: Approved gene symbols were obtained from the HUGO primer (5 -CGAATCGTAACCGTTCGTACGAGAATCGCT-3 )was Nomenclature Committee: FBLN1 = human fibulin-1 gene; FBLN1 = used in combination with a specific primer (5′- human fibulin-1 protein; fbln1 = mouse fibulin-1 gene; fbln1 = mouse CTTGAAGGAGAGAGGCAGGGAAACTGAAGTGG-3′), based fibulin-1 protein; ECM, extracellular matrix www.jmedgenet.com FBLN1 disruption in synpolydactyly 99 J Med Genet: first published as 10.1136/jmg.39.2.98 on 1 February 2002. Downloaded from Figure 1 Clinical and radiological appearance of the hand malformations associated with the t(12;22). Annealing temperatures of 60°C were used for all primer Detection of fibulin-1 in conditioned culture medium http://jmg.bmj.com/ combinations. DMSO (10% final concentration) was added and extracellular matrix of cultured fibroblasts when using sets RT5, RT7, and RT8. Sequencing was Age matched, normal foreskin fibroblasts (ATCC CRL 2056, performed using standard protocols and T7 and SP6 primers. Rockville, MD) and synpolydactyly fibroblasts isolated from FBLN1 probes for northern blot analysis were constructed skin biopsy (obtained during surgery after informed consent) by PCR. The FBLN1-D specific probe was amplified using were grown in 100 mm tissue culture plates (Corning, Corning, primers 5′-ACCATCTCCCACA-3′ (position 1793-1805 in NY) in Dulbecco’s Minimal Essential medium (DMEM) supple- FBLN1-D mRNA) and 5′-CAGCAATGATTTG-3′ (position 2162- mented with 10% iron supplemented bovine calf serum 2174). Similarly, a FBLN1-C specific probe was constructed (Hyclone, Logan, UT) until they reached 90% confluency. The on September 26, 2021 by guest. Protected copyright. using primers 5′-CCAAGCTGCCTCTGAGAATA-3′ (position medium was then aspirated and replaced with DMEM supple- 1749-1768 in FBLN1-C mRNA) and 5′- mented with insulin, transferrin, and selinious acid (5 µg/ml, 5 GCTCTGCAGACACAAAGATG-3′ (position 2038-2057). For the µg/ml, and 5 ng/ml, respectively, Becton Dickinson Labware probe residing within the part common to all FBLN1 variants, Bedford, MA). After an additional three days in culture, the primers RT5-up and RT5-low were used. conditioned culture medium (CCM) was collected and the cell layer washed with phosphate buffered saline (pH 7.4). The cell 3′ RACE analysis layer was then extracted with 10 mmol/l EDTA for five minutes 3′ RACE was performed according to the method described by with gentle agitation. The extraction buffer was collected and Schoenmakers et al6 with minor modifications. RNA used for centrifuged for 10 minutes at 2000 × g. A 400 µl aliquot of the the 3′ RACE analysis was derived from skin fibroblasts. First supernatant was concentrated with 10 µl of Strataclean resin strand cDNA synthesis was performed using the poly (A) spe- (Stratagene, La Jolla, CA) by vortexing for one minute and cen- ′ ′ × cific primer AP2 (5 -AAGGATCCGTCGACATC(T)17-3 )ora trifugation at 13 000 g for 10 seconds. Similarly, a 500 µl aliq- FBLN1-D specific primer (5′-CCACCTATAAGAAAAGAT-3′). uot of CCM was absorbed with Strataclean resin and 10 µlof2 Specific 3′ RACE primers were designed on the published × Laemmli sample buffer containing β-mercaptoethanol was FBLN1-D sequences. In the first PCR round, a specific FBLN1-D added to each sample and extracts were run on SDS-PAGE primer (5′-CCAACGATGTCACATGCGTGT-3′, position 1760- using 4-20% gels (Invitrogen/Novex, Carlsbad, CA). After SDS- 1780 in FBLN1-D mRNA), located in the first FBLN1-D specific PAGE, proteins were transferred to PVDF membrane and exon (exon 18) was used in combination with the AP2 specific probed with antibodies against FBLN1 (1 µg/ml) in Tris buffered primer UAP2 (5′-CUACUACUACUAAAGGATCCGTCGACATC- saline (pH 7.4) containing 0.1% Tween 20 and 5%