Genomic deoxyribonucleic acid (DNA) was extracted from ethylenediaminetetra-acetic (EDTA) blood samples obtained by standard procedures in all subjects. The methods applied for mutation screening differed somewhat between the Center for Medical Genetics Ghent (Belgian and Dutch patients) and the ROI (Italian patients). In the Dutch and Belgian probands, mutation screening of the
COL1A1 gene was initiated by polymerase chain reaction (PCR) amplification of the 52
COL1A1 exons, using forward and reverse primers. The PCR products were analyzed by gel electrophoresis using the Labchip GX Caliper, software version 2.1.322.0 (Caliper Life Sciences, Hopkinton, MA). All fragments were directly sequenced using the ABI PRISM 3730XL automated sequencer (Applied Biosystems, Foster city, CA). In the Italian probands, complete mutational screening of
COL1A1 coding regions and exon-intron junctions was performed on DNA by analyzing samples with denaturing high pressure liquid chromatography (DHPLC) followed by direct sequencing of samples with abnormal elution profile. The coding exons of
COL1A1, along with exon-intron junctions, were PCR-amplified. The results of amplification and the presence of well-sized PCR reaction products were analyzed by gel electrophoresis and visualized by ethidium bromide staining on 2% agarose gels. DHPLC analysis was carried out using the WAVE DNA Fragment Analysis System 3500HT (Transgenomic, Crewe, UK) equipped with a DNASep column (Transgenomic, Crewe, UK). Amplification products showing abnormal elution profiles were reamplified, purified with ExoSAP-IT (USB, Affymetrix, Santa Clara, CA, USA) and sequenced in both forward and reverse direction using BigDye Terminator chemistry version 3.1 and ABI PRISM 3100 automated DNA sequencer (Applied Biosystems, Foster City, CA, USA). Independently of the sequencing method applied, the obtained sequences for all
COL1A1 exons were compared to the wild-type sequences [GenBank:NG_007400.1] [
22]. If no causal mutation could be detected in any exon of the
COL1A1 gene, the appropiate procedure of those described above was repeated for the proband's 52
COL1A2 exons, using the wild-type reference sequences [GenBank:NG_007405.1][
22]. Presence of the mutation identified in a proband was subsequently analyzed in all participating relatives by PCR-amplification and sequencing of the corresponding mutated exon in the
COL1A1 or
COL1A2 gene.