Abstract
Approximately 90% of patients with osteogenesis imperfecta (OI) exhibit dominant COL1A1 or COL1A2 mutations; however, molecular analysis is difficult because these genes span 51 and 52 exons, respectively. We devised a PCR-denaturing high-performance liquid chromatography (DHPLC) procedure to analyze the COL1A1 or COL1A2 coding regions and validated it using 130 DNA samples from individuals without OI, 25 DNA samples from two cells to investigate the procedure's potential for preimplantation diagnosis, and DNA samples from 10 patients with OI. Three novel intronic variants in vitro were expressed using a minigene assay to assess their effects on splicing. The procedure is rapid, inexpensive, and reproducible. Analysis of samples from individuals without OI revealed six novel and some known polymorphisms useful for linkage diagnosis because of high heterozygosity. Analysis of two-cell samples confirmed the known genotype in 24 of 25 experiments; DNA failed to amplify in only one case. No incidence of allele dropout was recorded. DHPLC revealed six novel mutations, three of which were intronic, in all patients with OI, and these results were confirmed by means of COL1A1 and COL1A2 direct sequencing. Expression of intronic mutations demonstrated that variant 804 + 2_804 + 3delTG in intron 11 disrupts normal splicing, thereby leading to formation of two alternative products. Variants c.3046-4_3046-5dupCT (COL1A1) and c.891 + 77A>T (COL1A2) did not affect splicing. The described DHPLC protocol combined with the minigene assay may contribute to molecular diagnosis in OI. Moreover, this protocol will aid in counseling about prenatal and preimplantation diagnosis.
Osteogenesis imperfecta (OI) includes a group of inherited diseases that involve connective tissue and are characterized by bone fragility. The disorder is traditionally classified into types I to IV on the basis of phenotype and outcome,1 although other types have recently been described.2 Approximately 90% of patients with OI exhibit dominant mutations in the COL1A1 (MIM 120150) and COL1A2 (MIM 120160) genes, which encode α1 and α2 type I collagen chains, respectively,2 and some hundreds of pathogenic mutations have been described (OI Mutation Database on http://www.le.ac.uk/ge/collagen). Genotype-phenotype correlations have been reported.3,4 Type I OI demonstrates a milder clinical presentation and is frequently associated with impaired synthesis of structurally normal collagen5 owing to premature stop codons in the coding sequence of COL1A1,6 which are defined as class I mutations.2 The other types of OI, frequently due to class II mutations,2 involve glycine substitutions in the triple-helical domain.6 De novo mutations, observed in the proband but not in the parents, in particular in COL1A1, are often observed in patients with the most severe OI, ie, types II and III, whereas types I and IV are usually observed in familial clusters.7
OI is diagnosed on the basis of the typical phenotype, and is confirmed at radiography. Molecular diagnosis, although more specific, is limited by spreading of COL1A1 and COL1A2 mutations (51 and 52 codifying exons, respectively). In addition, causative mutations, often de novo, occur in all exons, and 20% to 30% of cases involve splice mutations within intron-exon boundaries.2 However, the increasing availability of procedures for scanning whole genes has dramatically increased the demand for the molecular diagnosis of OI. Indeed, molecular analysis can confirm the diagnosis, in particular in patients without well-defined phenotypes, thereby permitting early use of specific therapy.8 In addition, identification of the causative mutation enables prediction of the outcome of the disease and improves counseling of affected families about prenatal or preimplantation diagnosis.9,10
Automated direct gene sequencing, available in reference laboratories, is the criterion standard method for molecular diagnosis; however, it is expensive and time-consuming. Denaturing high-performance liquid chromatography (DHPLC) is a good alternative approach for gene scanning analysis because of its sensitivity and the possibility of automating the revelation phase.11,12
We generated a PCR-DHPLC procedure for analysis of the entire coding regions of COL1A1 and COL1A2 and validated the procedure on i) DNA samples from individuals without OI, also to define the most frequent polymorphisms of these genes; ii) DNA samples from two cells for a possible use in preimplantation diagnosis; and iii) DNA samples from patients with OI. We also expressed three novel intronic variants in vitro to assess their effect on the splicing process.
Materials and Methods
Subjects
To validate the DHPLC procedure and to define the frequency of the predicted polymorphisms within the COL1A1 and COL1A2 genes, we studied 130 DNA samples, from unrelated Italian subjects without OI, available in our bank of biological samples. Also analyzed were DNA samples from 10 patients with OI, supplied by the Cell Line and DNA Biobank from Patients Affected by Genetic Diseases. Written informed consent using a form approved by the local ethics committee was obtained for analysis and storage of each sample.
DNA Extraction from Blood Cells
Genomic DNA was extracted from peripheral blood leukocytes using the Nucleon BACC2 kit (Amersham Biosciences, GE Healthcare, Piscataway, NJ) and spectrophotometrically quantified at 260 and 280 nm. Thus, DNA samples were diluted at a working concentration of 60 ng/μL.
Single-Cell Preparation
Laser microdissection13 was used to obtain aliquots of two cells from the buccal mucosa of laboratory personnel. DNA was extracted according to the GenomiPhi DNA amplification kit V2 protocol (Amersham Biosciences), amplified using the same kit, and eluted in 10 mmol/L Tris-HCl (pH 8.5). Reamplification of whole genome from cells obtained via laser microdissection was checked using 1% agarose gel electrophoresis in 1× Tris-borate-EDTA buffer.
Polymerase Chain Reaction
Primers suitable for amplification of each DNA region to be analyzed were selected using Primer 3 software (http://frodo.wi.mit.edu). The sequence of selected primers and the size of the amplified genomic regions are given in Tables 1 and 2. The final volume of PCR mix was 50 μL. The mix contained 10× PCR buffer II, 25 mmol/L MgCl2, 2.5 mmol/L deoxyribonucleotide triphosphate, 20 mmol/L forward oligo, 20 mmol/L reverse oligo, 5 U/mL AmpliTaq DNA polymerase (Applied Biosystems, Inc., Foster City, CA), and 60 ng/mL genomic DNA. Further details of the PCR mix and assembly and the PCR conditions are available on request. The amplification was checked using 2% agarose gel electrophoresis with a 50-bp stepladder as size marker.
Table 1.
Primers and Length of Various Amplicons of COL1A1⁎
| Exon | Forward primer | Reverse primer | Amplicon length (bp) |
|---|---|---|---|
| 1 | 5′-ACCCCTACCACAGCACCTC-3′ | 5′-GTGAGCTCCCTCCTGTCTCA-3′ | 636 |
| 2 | 5′-ATCCAAGTGTGCCTCTTAGA-3′ | 5′-GTTTGCTAATGCTGCTCCC-3′ | 414 |
| 3-5 | 5′-GGAGCAGCATTAGCAAACCT-3′ | 5′-CACAAACTGTGAAGGGTATGT-3′ | 567 |
| 6 | 5′-CACCAGGAAGTGCATGATGT-3′ | 5′-CTTCTGTCATCCATGCTCCC-3′ | 202 |
| 7 | 5′-ACAGAGGGATCACCATGACC-3′ | 5′-GGCATATGAAGACGTCCTGG-3′ | 115 |
| 8-9 | 5′-ATGAGGGCAGGAGAGATGCT-3′ | 5′-GTTCCCAAATGTGGTGGAGT-3′ | 454 |
| 10-11 | 5′-AACCTGACCTGCAACAATCC-3′ | 5′-GGACTTGGGGAGCTTAAATG-3′ | 368 |
| 12-14 | 5′-ACCTCCCAAGGCTCTTTCTC-3′ | 5′-GGCCAGTCCCTAGAGTTCCT-3′ | 521 |
| 15 | 5′-GATCCCTGAGCTCTGGAA-3′ | 5′-GAGGCCTACAGGCCACACTC-3′ | 157 |
| 16 | 5′-TCTTCCTTCTCGCTGACATC-3′ | 5′-TGAGGGTCATGCTTAGAGGAG-3′ | 126 |
| 17 | 5′-ACCTTTGTCCTGGGTTCTCC-3′ | 5′-AACAGGCAAGGACTCTGAGG-3′ | 303 |
| 18-19 | 5′-GTCCCCGACTCAGTGTCC-3′ | 5′-CCTGCTCCCCAGATGAGAG-3′ | 400 |
| 20-21 | 5′-ATCTGGGGAGCAGGAAGAC-3′ | 5′-AGTGAACTCCGCGACACAC-3′ | 528 |
| 22-23 | 5′-CTCATTGCCTGGCTGGTG-3′ | 5′-CTCATCCCAGACCCTACAC-3′ | 405 |
| 24 | 5′-CACCTCCATCATGCTTCTCC-3′ | 5′-ACAGGACAATGGCAGGGG-3′ | 147 |
| 25 | 5′-CTGCTTTCGTGCCTCCC-3′ | 5′-GTCCCTGGCTCTTCATGG-3′ | 161 |
| 26 | 5′-TAACAGGGAAAAGGCAGAGG-3′ | 5′-AGGGGAACTCAGGGTTAGGA-3' | 397 |
| 27-29 | 5′-CTGCAGGAGGGGTGCTAGAG-3' | 5′-CCGGCTGCTCCCTCTTAC-3′ | 477 |
| 30 | 5′-CGTGCTTTCCAGCAGAGTG-3′ | 5′-GCACCTTGACGGATGCAG-3′ | 150 |
| 31 | 5′-GCCCTGTCCTTCCCTTC-3′ | 5′-CCTTCCACGCTGCCCTC-3′ | 138 |
| 32 | 5′-ACAAGCCTGGGAGATACCAA-3′ | 5′-GGGACAGATCCCAGAGAGAA-3′ | 296 |
| 33-34 | 5′-GAGGGCCTCTCAGGAAACC-3′ | 5′-ACAGCTCAGTTTGGCAGGAC-3′ | 311 |
| 35-36 | 5′-GCTCTCCTGGGGTCATCTACT-3′ | 5′-CTGACGCCTTTGTCCTCATT-3′ | 425 |
| 37-38 | 5′-TGCCTCCATTACTGCTCCTC-3′ | 5′-GAGAACAGCCAACTCATCCG-3′ | 425 |
| 39 | 5′-GAGTATCACCCGCCTCTCTG-3′ | 5′-TGAGGATAGGAGGGGCTGTC-3′ | 313 |
| 40 | 5′-TGACTGAGGACCCAATGATG-3′ | 5′-GCAAACAGGGGTGAGGTG-3′ | 320 |
| 41-42 | 5′-CCCTATCTCTGGCCTGACTC-3′ | 5′-GGAAGAGGGCTTAGGCAAG-3′ | 213 |
| 43-44 | 5′-CATGCCAGTACCCTCAGCAT-3′ | 5′-GTGAAGTCCGACACCCATC-3′ | 445 |
| 45-46 | 5′-GCCTGACAGTTTGTCCCTTTC-3′ | 5′-GCTTGGGGCTCAGGAAG-3′ | 571 |
| 47 | 5′-AGAATAATGAAGGGGCATGG-3′ | 5′-CTTCTCCAGAGAGGCAAAGG-3′ | 266 |
| 48 | 5′-CCGGTAATCCCCACTCTCTT-3′ | 5′-TAGGAGGGAGGGAGAGGCTA-3′ | 351 |
| 49 | 5′-CATATGGTGCTGGCTGCTC-3′ | 5′-ATGTCCCTTCTGAGCACTGG-3′ | 312 |
| 50 | 5′-CTCCCTCCCTCCTACTCCTG-3′ | 5′-TGTCCATCACCCTTAGCAGA-3′ | 406 |
| 51 | 5′-CTCTCTGAGGACCCTGGACA-3′ | 5′-GTGGGAGTGATGGAGAGAGG-3′ | 421 |
| 52 | 5′-CCTCTCTCCATCACTCCCAC-3′ | 5′-TCAGTTTGGGTTGCTTGTCTG-3′ | 287 |
| poly(A) | 5′-TGCATTCATCTCTCAAACTTAG-3′ | 5′-CATTGTTTCCTGTGTCTTCTG-3′ | 294 |
| poly(A) | 5′-AGAGACAACTTCCCAAAGCAC-3′ | 5′-GCCCCTTCCCCATGTCTAC-3′ | 217 |
NCBI reference sequence: NG_007400.1.
Table 2.
Primers and Length of Various Amplicons of COL1A2⁎
| Exon | Forward | Reverse | Amplicon length (bp) |
|---|---|---|---|
| 1 | 5′-ATTGGTGGAGGCCCTTTT-3′ | 5′-GAGTCTGCCCTCCAAGTGTA-3′ | 408 |
| 2 | 5′-GATCCCTGCCATACTTTTGA-3′ | 5′-TATTCAGTAGCCCCGCCTAT-3′ | 402 |
| 3 | 5′-GCTCATGAGTTGAATTTGAGG-3′ | 5′-TGCCTTCCATCTCCAGAATA-3′ | 547 |
| 4 | 5′-GCTGAAGACTATAGCAGCTTCC-3′ | 5′-CTTCTGCAGTGCATTACCTG-3′ | 407 |
| 5 | 5′-TTTCCACCCTACTTGCACAT-3′ | 5′-CAGTGCACACAAAGACCAGT-3′ | 416 |
| 6 | 5′-AGAGGAAGGGCTCAAAAAGT-3′ | 5′-AAATGGCGTGGTAAAATGTG-3′ | 393 |
| 7-9 | 5′-AATGGCACTGCTAAGTTGGT-3′ | 5′-CTGTCAGGCATATTCAGCTTT-3′ | 539 |
| 10 | 5′-CAAAAGCTGAATATGCCTGAC-3′ | 5′-AGATGCCTTCGATTCATGTT-3′ | 398 |
| 11 | 5′-CAGCAAGCATACCATAAGCA-3′ | 5′-CTCCAGGGATTTGAAAGTGA-3′ | 385 |
| 12 | 5′-CCAAGAGAGATTAATGGCAAA-3′ | 5′-AGGAAAGGAAATGGAACAAGA-3′ | 412 |
| 13 | 5′-CAAAAACTCAATCCTTCTCC-3′ | 5′-GCTTTGTTCAGTTTCCAACC-3′ | 296 |
| 14-15 | 5′-GGTTGGAAACTGAACAAAGC-3′ | 5′-GCCTTCAAAAGACCTCACAG-3′ | 415 |
| 16 | 5′-AATATCCCCACCCTGGATAC-3′ | 5′-GTCCATTTTGAAGGGAAGG-3′ | 383 |
| 17-18 | 5′-GTAGCCAAGATGGCAGAATC-3′ | 5′-GCTGTAGAGGCAGCACACAT-3′ | 548 |
| 19 | 5′-CTCTACAGCCCATCACCTCC-3′ | 5′-GGCAGAGGTGGTATTTCAGG-3′ | 200 |
| 20-21 | 5′-TCTCTTTACCTTGACCCACAA-3′ | 5′-GCCTCTGGTGTGTTAGGGTG-3′ | 425 |
| 22-23 | 5′-TTTTGCAGGATGCTCATCTA-3′ | 5′-TAATGCCAGGTGTGATTTGC-3′ | 477 |
| 24 | 5′-ACCCTAGGCAACAAACAAAA-3′ | 5′-GTCACCTGCTTCAATTTTCC-3′ | 400 |
| 25 | 5′-GAGAAGGGAATGAGGGAAAT-3′ | 5′-CCCTGAGACTGGACTGATTC-3′ | 420 |
| 26 | 5′-CCTGCTAAAAATCCATCTCCT-3′ | 5′-TCTGTACACAAGTTAGCACCT-3′ | 305 |
| 27-28 | 5′-CCTGAGGCTTTGAGACATCTT-3′ | 5′-TTGTGGTGGAGAAGAGAGGT-3′ | 418 |
| 29 | 5′-ACCTCTCTTCTCCACCACAA-3′ | 5′-TCCATCAGCACCAAAGTATG-3′ | 412 |
| 30 | 5′-TACCAAGTTCTGGGATGGATA-3′ | 5′-GCATCAGAGACTTGTTGCAG-3′ | 370 |
| 31 | 5′-CTCGGAAGCTACACAAATGTAA-3′ | 5′-CCCCACAGTTTGTTGCTATT-3′ | 393 |
| 32 | 5′-CCCTCATCTCTTCAGTCACC-3′ | 5′-ATTGTGAAAACTTGGGCATC-3′ | 390 |
| 33 | 5′-TGGAAATGGCCTTGAATTAT-3′ | 5′-ATAGACCCAGGAGAGAAAGG-3′ | 399 |
| 34 | 5′-CACTTTTGATGATACGGGGTG-3′ | 5′-TTTCCTGCTGCTCTATCACA-3′ | 256 |
| 35-36 | 5′-GTTCTCTCTCCCTCCCAGTT-3′ | 5′-TTACAGCTCTGGTATTCCGA-3′ | 365 |
| 37 | 5′-TTAAATCCCTTCTCCCACCT-3′ | 5′-ATCCTTCCGTTATTTTCCATC-3′ | 409 |
| 38 | 5′-GGGAATGATCCACTTGAAGA-3′ | 5′-AAAGAAAAGCGGCGAGAGTC-3′ | 171 |
| 39 | 5′-CCCAAATTCTTGGAGTTGATG-3′ | 5′-AGCTGACTTCAGACCAGGAG-3′ | 244 |
| 40 | 5′-GCATCACATTGTTTGCATCT-3′ | 5′-GACAGATTTCCTGGCTCAAC-3′ | 404 |
| 41 | 5′-CAATCTTCAAGCCAACCTGT-3′ | 5′-CCAGCATTTCATATTGCAGA-3′ | 471 |
| 42 | 5′-TTGTGTGACCCATTCACATT-3′ | 5′-TTGGCCTAAGCAATTTTCAT-3′ | 406 |
| 43-44 | 5′-TGAGCACTGGAAGTGATGAA-3′ | 5′-CAAGTGTGGGGAAGTCAGA-3′ | 416 |
| 45 | 5′-TGGGCTTCACTTCTGACTTC-3′ | 5′-CCTCTGCTGGTAATCTGCTT-3′ | 398 |
| 46 | 5′-AAGCAGATTACCAGCAGAGG-3′ | 5′-CAGTTTATGTGCGAGATGGC-3′ | 289 |
| 47-48 | 5′-CAATCCGGAGTCCATTTAAC-3′ | 5′-GGGCTAACTTTAATGGGTTGT-3′ | 499 |
| 49 | 5′-CGTGTGAAGCTCAACTGAAA-3′ | 5′-GCTCCATTAATTGGGTGTGA-3′ | 486 |
| 50 | 5′-TGGGGTAGACAATCAAAAATG-3′ | 5′-AAGCCCATTTTTGTCAGAGA-3′ | 400 |
| 51 | 5′-TTTCCTAAGCTTGGATCTGAGT-3′ | 5′-CCTTGGGGGCAGTCTAAGT-3′ | 446 |
| 52 | 5′-CAGAATGACACATGCCAAAC-3′ | 5′-TTTTCTCTTTTGCCCACAAT-3′ | 397 |
| poly(A) | 5′-TCCTTCCATTTCTTCTGCAC-3′ | 5′-GCACCATCAACATTCTTCAA-3′ | 436 |
| poly(A) | 5′-TCCACATTGTTAGGTGCTGA-3′ | 5′-TTTGGAAAACCAAACATGC-3′ | 423 |
NCBI reference sequence: NG_007405.1.
Denaturing High-Performance Liquid Chromatography
DHPLC analysis was performed using the WAVE System 3500 (Transgenomic, Inc., Omaha, NE), working at partially denaturing conditions. To differentiate unequivocally the peaks from the background, the DHPLC procedure was set up to obtain chromatograms with peaks greater than 3 mV. For each amplified segment, we first used the temperature(s) indicated by Wavemaker Utility software (Transgenomic, Inc.). After mutated samples were identified, the temperature was reset by increasing or decreasing it at intervals of 1°C to experimentally define the optimal temperature for identifying all variants. The optimal temperatures for each DNA segment for COL1A1 and COL1A2, respectively, are given in Supplemental Tables S1 and S2 (available at http://jmd.amjpathol.org). Before injection (10 mL per sample) on the reverse-phase chromatographic column (DNASep, 4.5 × 50 mm; Transgenomic, Inc.), all DNA samples were denatured at 95°C for 6 minutes, then hybridized by slowly cooling at room temperature to enable the heteroduplex to form. Direct sequencing was used to confirm and identify the mutation in samples in which DHPLC analysis demonstrated abnormal elution profiles.
Direct Sequencing
Before sequencing, PCR products were purified using the QIAquick PCR Purification kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions. Sequencing analysis was performed using Sanger's method on an ABI-3730 automated analyzer (Applied Biosystems, Inc.). We referred to the wild-type sequence available at National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov), accession number NG_007400.1 for COL1A1 and NG_007405.1 for COL1A2. For the nomenclature of sequence variants, we referred to Human Genome Variation Society nomenclature guidelines (http://www.hgvs.org). Technical details of sequencing analysis are available on request.
Splice-Site Prediction Analysis
Genomic sequence fragments with and without the genetic variant under study were submitted for splice-site prediction to the servers Berkeley Drosophila Genome Project (http://www.fruitfly.org/seq_tools/splice.html) and NetGene2 (http://www.cbs.dtu.dk/services/NetGene2).
Generation of Minigene Construct
A DNA fragment approximately 1 kb long was directly amplified from genomic DNA from patients with OI heterozygous for the mutation to be expressed, and cloned into the enhanced version of green fluorescent protein plasmid (pEGFP)–C3 vector (Clontech, Saint-Germain-en-Laye, France). Primers used to amplify the DNA fragment for minigene construction for each gene variant to be studied are given in Table 3. The PCR products were obtained using HotStartTaq DNA polymerase (Qiagen GmbH). Each PCR product was digested using an appropriate restriction enzyme and cloned into XhoI-BamHI previously digested pEGFP-C3 vector. All clones were sequenced, and a wild-type and a mutated form of each variant were retained for expression experiments.
Table 3.
Primers Used To Amplify Various Fragments for Minigene Construction and Reverse Primers Used for RT-PCR Analysis
| Gene variant | Primer name | Primer sequence |
|---|---|---|
| Minigene construction | ||
| c.804 + 2_804 + 3delTG | ColA1SaL-10Exo, forward | 5′-GCATAACGTCGACAAGCTGGAAAACCTGG-3′ |
| Col1A1Bam-12Exo, reverse | 5′-CGTATTGGGATCCAGCAGGACCAGCATC-3′ | |
| c.3046-4_3046-5dupCT | Col1A1Xho-42Exo, forward | 5′-CGTATTGCTCGAGGCAAACAAGGTC-3′ |
| Col1A1Bam-44Exo, reverse | 5′-CGTATTGGGATCCACGATCACCACTC-3′ | |
| c.891 + 77A>T | Col1A2Xho-16Exo, forward | 5′-CGTATTGCTCGAGTCCCATTGGGTCTGC-3′ |
| Col1A2Bam-19Exo, reverse | 5′-CGTATTGGGATCCAACAAGTCCTCTGGCAC-3′ | |
| RT-PCR analysis c.804 + 2_804 + 3delTG | Col1A1-12Exo, reverse | 5′-GACAGCCTCTTACCTTAGGAC-3′ |
| c.3046-4_3046-5dupCT | Col1A1-44Exo, reverse | 5′-GTCTCACCACGATCACCACTC-3′ |
| c.891 + 77A>T | Col1A2-19Exo, reverse | 5′-CAAGTCCTCTGGCACCAGTAGC-3′ |
Cell Culture, Minigene Expression, and Analysis of Transcripts
HeLa cells were grown in a 5% CO2 incubator at 37°C in Dulbecco's modified Eagle's medium (Invitrogen Corp., Carlsbad, CA) supplemented with 10% fetal bovine serum (HyClone Laboratories, Inc., Logan, UT). HeLa cells, seeded at 6 × 105 per well in 60-mm dishes 16 hours before transfection, were transfected with 5 mg pEGFP-C3 minigene constructs using the calcium phosphate precipitation method. Twenty-four hours after transfection, cells were collected, and total RNA was extracted using the MasterPure Complete DNA and RNA Purification Kit (Epicenter Biotechnologies, Madison, WI). Then 1 mg total RNA was reverse transcribed using M-MLV Reverse Transcriptase (Invitrogen Corp.) according to the manufacturer's instructions. After total RNA retro-transcription, 100 ng cDNA was amplified using the forward primer RT-GFP-FOR 5′-CACATGGTCCTGCTGGAGTTC-3′ coupled with each of the reverse primers corresponding to the variants given in Table 3. This set of primers does not amplify eventual endogenously expressed COL1A1 transcripts. PCR products were separated on a 2% agarose gel, and individual bands were excised and sequenced.
Results
DHPLC Procedure
To assess the reproducibility of the DHPLC procedure, we analyzed DNA samples bearing known gene variants. In particular, 10 samples, each carrying several known polymorphisms for the COL1A1 and COL1A2 genes, were singly tested 10 times using the novel procedure (PCR, DHPLC, and sequencing). The results obtained after the 10 independent re-analyses were unequivocally concordant. The results also concurred when the same experiments were independently performed by three different operators (M.I., A.E., R.I.) on 10 other DNA samples, 8 bearing gene variants and 2 with no gene variants. Figure 1 shows an example of DHPLC analysis on a DNA fragment corresponding to a COL1A1 exon affected by a mutation.
Figure 1.
DHPLC profiles obtained in a patient with OI bearing a mutation in COL1A1 (profile A) and in a control individual bearing a variant in COL1A1 when testing DNA from two cells of buccal mucosa (profile B).
Allele Frequency of Single-Nucleotide Polymorphisms
To determine the occurrence and frequency of COL1A1 and COL1A2 gene variants, we analyzed DNA samples from 130 unrelated individuals without OI. Characteristics and allele frequencies of the 34 variants identified are given in Tables 4 (COL1A1) and 5 (COL1A2). Twenty variants including four novel variants were identified in COL1A1 (Table 4). In the present series, the allele frequency of 10 of these variants was low, barely 5.0%. In contrast, the three novel variants (c.4006-33_4006-31del, c.4006-13C>T, and c.4006-6delC) identified in intron 50 were in complete linkage disequilibrium with each other and demonstrated a frequency of 41.2%. The remaining six COL1A1 variants occurred in coding regions. Four of these latter variants were synonymous, including the novel c.1005T>A (p. G335G), and two were not synonymous. Fourteen variants including the two novel variants c.594 + 18delT and c.1350 + 11A>T were identified in COL1A2 (Table 2). Ten variants occurred in noncoding regions; two were observed in the 3′UTR; and two affected coding regions, one of which is an amino acid change.
Table 4.
Characteristics of Putatively Neutral SNPs Identified in COL1A1
| Position | dsSNP rs no. | Nucleotide substitution⁎ (Amino acid change)† | Minor allele Frequency‡ |
|---|---|---|---|
| Intron 8 | rs16948767 | c.642 + 18 A>C | 0.05 |
| Intron 8 | rs73987448 | c.642 + 25 T>C | 0.05 |
| Intron 8 | rs67207840 | c.643-36delT | 0.05 |
| Intron 9 | rs11079898 | c.697-30 A>G | 0.05 |
| Intron 12 | rs17639446 | c.859-14 T>G | 0.10 |
| Intron 19 | rs41317361 | c.1300-8 C>T | 0.10 |
| Intron 22 | rs41316653 | c.1515 + 30 C>G | 0.05 |
| Intron 28 | rs2696247 | c.1930-14 T>C | 0.12 |
| Intron 37 | rs2075559 | c.2560-18 G>C | 0.04 |
| Intron 49 | rs2734275 | c.3814 + 4 A>T | 0.35 |
| Intron 50 | NA | c.4006-33_4006-31del | 0.41 |
| Intron 50 | NA | c.4006-13 C>T | 0.41 |
| Intron 50 | NA | c.4006-6delC | 0.41 |
| Intron 51 | rs2249492 | c.4249-12 G>A | 0.10 |
| Exon 16 | NA | c.1005 T>A (p.G335G) | 0.04 |
| Exon 33/34 | rs2734272 | c.2298 T>C (p.T766T) | 0.13 |
| Exon 45 | rs1800215 | c.3223 G>A (p.A1075T) | 0.03 |
| Exon 48 | rs1800218 | c.3459 T>C (p.D1153D) | 0.05 |
| Exon 51 | rs2586486 | c.4171 C>A (p.Q1391K) | 0.23 |
| Exon 51 | rs1800219 | c.4179 C>T (p.S1393S) | 0.03 |
dsSNP, Single nucleotide polymorphism database (available on http://www.ncbi.nlm.nih.gov/projects/SNP); NA, not available; p, synonymous mutation.
GenBank-EMBL Accession No. NM_000088.3.
GenBank-EMBL Accession No. NP_000079.2.
Minor allele frequencies were calculated from 260 Italian control alleles (novel polymorphisms in bold).
Table 5.
Characteristics of Putatively Neutral SNPs Identified in COL1A2
| Position | dsSNP rs No. | Nucleotide substitution⁎ (Amino acid change)† | Minor allele Frequency‡ |
|---|---|---|---|
| Intron 6 | rs406226 | c.280-68A>G | 0.03 |
| Intron 12 | NA | c.594 + 18delT | 0.09 |
| Intron 15 | rs2293739 | c.738 + 86T>A | 0.03 |
| Intron 15 | rs2293740 | c.738 + 98C>A | 0.03 |
| Intron 18 | rs42518 | c.936 + 14C>T | 0.08 |
| Intron 18 | rs28754326 | c.936 + 46G>A | 0.04 |
| Intron 18 | rs42519 | c.937-3C>T | 0.25 |
| Intron 23 | NA | c.1350 + 11A>T | 0.04 |
| Intron 28 | rs421587 | c.1665 + 15A>G | 0.19 |
| Intron 28 | rs2301643 | c.1666-41G>A | 0.09 |
| 3′UTR | rs1060399 | c.⁎194C>T | 0.32 |
| 3′UTR | rs3917 | c.⁎654_⁎655ins7 | 0.17 |
| Exon 25 | rs412777 | c.1446A>C (p.P482P) | 0.05 |
| Exon 28 | rs42524 | c.1645C>G (p.P548A) | 0.13 |
dsSNP, Single nucleotide polymorphism database (available on http://www.ncbi.nlm.nih.gov/projects/SNP); NA, not available; p, synonymous mutation.
GenBank-EMBL Accession No. NM_000089.3.
GenBank-EMBL Accession No. NP_000080.2.
Minor allele frequencies were calculated from 260 Italian control alleles (novel polymorphisms in bold).
Two-Cell Analysis
The novel DHPLC procedure was used to analyze 25 DNA samples from different donors, extracted from two-cell samples obtained by means of laser capture. For all 25 samples, we previously performed the DHPLC analysis of COL1A1 and COL1A2 on DNA from leukocytes. In particular, 20 of 25 samples had variants in COL1A1, and 5 of 25 samples had variants in COL1A2. Of the 25 samples, 16 had a single variant; 8 had two variants in two different exons of the same gene; and 1 had three variants, two in COL1A1 and one in COL1A2. In 24 of 25 samples (96.0%), analysis of the DNA from buccal cells provided the expected result; ie, it correctly identified the variant(s) already identified in DNA from leukocytes.14 In 1 of 25 samples (a sample carrying two variants in COL1A1), the result was negative because both tracts containing the variants failed to amplify at PCR. No instances of allele dropout were recorded in the 24 samples correctly analyzed. An example of DHPLC analysis performed on DNA from buccal cells is shown in Figure 1.
Analysis of Patients with OI
The primary characteristics of the mutations identified in the 10 patients with OI are given in Table 6. In the first five cases, the analysis revealed a known pathogenic variant, ie, four glycine substitutions, two of which were identified in a single patient (patient 1), and a frameshift and a nonsense mutation. Patients 6 to 10 demonstrated novel variants. In particular, patient 6 exhibited a frameshift mutation in COL1A1; patient 7 demonstrated a threonine-isoleucine substitution in exon 52 of COL1A1; patient 8 exhibited a 2-bp deletion in intron 11 of COL1A1 that caused defective splicing of intron 11 (Figure 2B); patient 9 demonstrated a proline substitution in exon 17 of COL1A2 and a CT duplication in intron 42 of COL1A1 that does not affect splicing (Figure 2C); and patient 10 exhibited a nucleotide substitution at residue +77 in intron 17 of COL1A2 that does not affect splicing (Figure 2D). No other mutations were identified in COL1A1 or COL1A2 in patient 10 at DHPLC analysis. Direct gene sequencing of all coding regions and exon-intron boundaries of COL1A1 and COL1A2 was also performed; however, no other potentially pathogenic variants were identified. In the other nine patients, direct sequencing invariably confirmed the results obtained at DHPLC.
Table 6.
Mutations Observed in 10 Patients with Osteogenesis Imperfecta
| Patient | Gene | Location | Nucleotide Substitution⁎ | Amino acid change⁎ | Effect |
|---|---|---|---|---|---|
| 1 | COL1A1 | Exon 37 | c.2461G>A | p.G821S (Gly-Ser) | Glycine substitution |
| COL1A1 | Exon 1 | c.77G>A | p.G26D (Gly-Asp) | Glycine substitution | |
| 2 | COL1A1 | Exon 11 | c.769G>A | p.G257R (Gly-Arg) | Glycine substitution |
| 3 | COL1A1 | Exon 25 | c.1696G>A | p.G566R (Gly-Arg) | Glycine substitution |
| 4 | COL1A1 | Exon 7 | c.579delT | p.G194V fsX71 | Frame shift |
| 5 | COL1A1 | Exon 39 | c.2644 C>T | p.R882X (Arg-Stop) | Nonsense |
| 6 | COL1A1 | Exon 44 | c.3114delG | p.T1039PfsX69 | Frame shift |
| 7 | COL1A1 | Exon 52 | c.4292C>T | p.T1432I (Thr-Ile) | Missense |
| 8 | COL1A1 | Intron 11 | c.804 + 2_804 + 3delTG | Defective splicing | |
| 9 | COL1A2 | Exon 17 | c.2428C>T | p.P810S (Pro-Ser) | Proline substitution |
| COL1A1 | Intron 42 | c.3046-4_3046-5dupCT | No defective splicing | ||
| 10 | COL1A2 | Intron 17 | c.891 + 77A>T | No defective splicing |
Novel mutations in bold.
Gene banks for nomenclature are reported in Materials and Methods.
Figure 2.
A: Schematic structure of the pEGFP-C3-Col1A(1-2) minigene constructs and position of the unclassified variants. Green fluorescent protein and COL1A1 or COL1A2 exons are shown as large boxes, and the lines between exons represent the corresponding introns. The thin box indicates the CMV promoter (pCMV), and the black arrows represent the location of the primers used for the splicing pattern analysis. Also shown are alternative predicted splicing donor (d) and acceptor (a) sites, as well as classic splicing donor (D) and acceptor (A) sites. B–D: Splicing pattern analysis of the pEGFP-C3-Col1A(1-2) minigene constructs carrying different genetic variants (M) are compared with their relative wild-type (W) form. The structures of the PCR products are schematically shown to the right of each gel. B: The c.804 + 2_804 + 3delTG variant causes complete failure of the normal splicing pattern, which is replaced by a new splicing pattern with the presence of a predominant form that retains intron 11 and a less representative form in which the presence of an alternative donor (d) site into exon 10 causes the alternative splicing of exon 11 and part of exon 10. C and D: The variants c.3046-4_3046-5dupCT in COL1A1 and c.891 + 77A>T in COL1A2 do not affect splicing.
In Vitro Analysis of Putative Splice-Site Mutations
Three novel variants identified in patients 8, 9, and 10 (Table 6) involved intronic nucleotides. In silico analysis predicted that the c.804 + 2_804 + 3delTG mutation in intron 11 of COL1A1 would abolish the canonical 5′ splice donor site of intron 11 and create an alternative splice donor site in exon 10. In contrast, no changes in the splicing process were predicted in the presence of the intronic c.3046-4_3046-5dupCT microduplication (COL1A1) and the intronic c.891 + 77A>T substitution (COL1A2).
The minigene system was used to verify the results of in silico analysis. The exon of interest was cloned together with at least one exon downstream and one exon upstream, depending on the length of the introns, thereby conserving the genomic context insofar as possible (Figure 2A). The splicing pattern analysis revealed that loss of the intron 11 splice donor site caused by the c.804 + 2_804 + 3delTG mutation completely obliterated the normal splicing pattern (Figure 2B) and led to transcription of two alternative splicing products: a major product with retention of intron 11, resulting in the premature formation of a stop codon in the mRNA with consequent formation of a truncated form of the protein (p.Gly269GlufsX12); and a minor product in which an alternative donor site in exon 10 caused the exclusion of exon 11 and part of the 3′ end of exon 10, again resulting in premature formation of a stop codon in the mRNA, with consequent formation of a truncated form of the protein (pGlu243PhefsX16) (Figure 2B). In contrast, the splicing pattern analysis of the COL1A1 exon 42-44 and COL1A2 exon 16-19 genomic fragments revealed that the two variants, c.3046-4_3046-5dupCT in COL1A1 (Figure 2C) and c.891 + 77A>T in COL1A2 (Figure 2D), did not affect splicing.
Discussion
The novel DHPLC-based procedure described herein has proved to be a simple, efficient, and reproducible method for analysis of the COL1A1 and COL1A2 genes. In addition, it is inexpensive; the cost of reagents and supplies is approximately €20 (approximately $30 US) to test both genes in each patient. Thus, inasmuch as more than 90% of OI cases are due to mutations in COL1A1 and COL1A2, the procedure can be used to support the diagnosis, in particular in clinically controversial OI cases. Indeed, molecular analysis of COL1A1 and COL1A2 should be based on scanning of the entire coding regions and the exon-intron boundaries of the two genes because of the absence of hotspot mutation regions and the relevant rate of splicing mutations (20% to 30%) that affect noncoding regions.2 In addition, because a few OI cases are due to mutations in the LEPRE1,15,16 CRTAP,16,17 PPIB,18 FKPB65,19 and SERPINH120 genes, it is necessary to exclude with high predictivity mutations in COL1A1 and COL1A2 before analyzing a series of potentially causative genes. The whole COL1A1 and COL1A2 genes must be sequenced to differentiate patients with OI due to de novo mutations (5% to 7% of cases) in which the parents have no symptoms and are negative at mutational analysis21 from patients without symptoms who exhibit a recessive inheritance pattern and, thus, a 1:4 risk of transmitting the disease to sons. Molecular analysis is the only tool that enables couples carrying OI mutations to plan reproductive strategies by offering them the option of either prenatal diagnosis, at 10 to 12 weeks by testing DNA samples from chorionic villi, or preimplantation diagnosis.22 Indeed, the procedure described herein has a high analytical sensitivity (96.0%) for testing DNA extracted from two cells and, thus, can be used for both prenatal and preimplantation diagnosis. Of note, in 25 cases, we recorded only 1 PCR failure in analysis of DNA from two-cell samples and no cases of allele dropout, which usually limit preimplantation diagnosis.
Other methods that have been described for scanning of COL1A1 and COL1A2 include denaturing gradient gel electrophoresis10 and single-strand conformation polymorphism11; however, both methods are less sensitive than DHPLC. Direct sequencing is efficient, but is expensive and cumbersome because of the high number of amplicons to be tested. Direct sequencing of the COL1A1 and COL1A2 coding regions may be performed also using cDNA obtained after retrotranscription of RNA extracted from fibroblasts.23 This is an excellent procedure in principle because it reduces the length of tracts to be analyzed; however, it is invasive because of fibroblast sampling and is associated with a low detection rate2,24 because large rearrangements might cause degradation of anomalous transcription that would, therefore, be missed at analysis. Next-generation sequencing25 is a promising tool for analysis of large genes, including noncoding regions, but is used in only a few laboratories.
With use of our DHPLC procedure, we identified 20 polymorphisms in the COL1A1 gene and 14 in the COL1A2 gene in 130 Italian control individuals. Most of these polymorphisms have already been classified, and the allelic frequency obtained in the present study was comparable to that obtained in other populations.6 However, four polymorphisms in COL1A1 and two in COL1A2 are novel. Three novel polymorphisms, ie, c.4006-33_4006-31del, c.4006-13C>T, and c.4006-6delC in intron 50 of COL1A1, are in linkage disequilibrium with each other and exhibit an allelic frequency of 41.2%. Two COL1A1 polymorphisms have a high allelic frequency: 35.0% for c.3814 + 4A>T and 23.4% for c.4171 C>A. Similarly, two COL1A2 polymorphisms have a high allelic frequency: 25.0% for c.937-3C>T, and 31.8% for c.*654_*655ins7. Because of high heterozygosity, analysis of these markers may be used for prenatal diagnosis in families in which the causative mutation is not known.9,26 Some polymorphisms identified in the present and other studies lie within exon-intron boundaries, in regions where splicing mutations responsible for up to 30% of OI cases2 have been identified. Furthermore, some of them involve coding regions. Thus, it is important to have a record of innocuous gene variants observed in unaffected individuals with the same genetic background as the patients with OI undergoing molecular testing. Indeed, the large study by Chan et al26 reported that COL1A1 and COL1A2 evolved differently among ethnic groups, and consequently collagen gene variants must be evaluated in the context of the genetic background of each patient.27
In the patients with OI analyzed in the present study, six COL1A1 mutations were identified, some already classified and some novel. Among the novel mutations, patient 7 demonstrated a threonine-isoleucine substitution that we consider pathogenic because i) a polar amino acid (threonine) is replaced by a neutral amino acid (isoleucine), and OI substitutions of charged amino acids are often associated with a lethal outcome2; ii) no other potentially pathogenic variants were observed in COL1A1 and COL1A2; iii) substitution of the adjacent threonine (ie, c.4291) is a known pathogenic mutation2; and iv) the mutation was not identified in 260 alleles from control individuals. In addition, three novel intronic variants were identified. Patient 8 exhibited a novel mutation, c.804 + 2_804 + 3delT, in COL1A1; patient 9 demonstrated two novel mutations, the p.P810S proline substitution in COL1A2, which is a typical pathogenic change in OI, and the intronic c.3064-4_3046-5dupCT variant in COL1A1; and patient 10 exhibited the novel COL1A2 intronic c.891 + 77A>T variant. Inasmuch as 20% to 30% of mutations causative of OI lie in intron-exon boundaries, ie, at the end or start of intronic sequences that act as the acceptor or donor site in the splicing process, and can also contain a series of nonpathogenic variants, it is crucial to establish the effect of a novel intronic variant on splicing, in particular in patients with OI in whom molecular analysis does not enable identification of other variants, as in patient 10. In silico prediction may contribute to this evaluation; however, functional analysis using the minigene system is the most specific means by which to assess the effect of the variant28–30 because it overcomes the need to obtain patient RNA for transcription analysis because genetic tests are routinely performed on genomic DNA. By using the minigene system, we demonstrated that the novel mutation c.804 + 2_804 + 3delTG in intron 11 of COL1A1 greatly altered the splicing process, whereas the splicing pattern was not affected by either the c.3046-4_3046-5dupCT COL1A1 variant, identified in a patient with OI who had another potentially pathogenic variant, or the COL1A2 c.891 + 77A>T gene variant observed in a patient with OI without any other mutations in COL1A1 or COL1A2 and, thus, presumed affected by a mutation in another gene.
In conclusion, the novel DHPLC procedure reported herein is an effective approach to scanning analysis of the COL1A1 and COL1A2 genes in patients with OI and may also be used for prenatal and preimplantation diagnosis. We suggest that functional analysis be used to characterize novel mutations that potentially might affect the splicing process.
Acknowledgments
Samples from patients with OI were obtained from the Cell Line and DNA Biobank from Patients Affected by Genetic Diseases (G. Gaslini Institute)–Telethon Genetic Biobank Network, Project No. GTB07001A.
Footnotes
Supported by grant DGRC 1901/2009 from Regione Campania and grant PS 35-126/Ind from Ministero dell'Istruzione, dell”Universita della Ricerca.
A.F. and M.I. contributed equally to this work.
CME Disclosure: None of the authors disclosed any relevant financial relationships.
Supplemental material for this article can be found on http://jmd.amjpathol.org at doi 10.1016/j.jmoldx.2011.06.006.
Supplementary Data
References
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