As mentioned above, MLPA is a very useful technique in hemoglobinopathy diagnostics after point mutations and known deletions have been excluded by applying sequence and gap- PCR analysis. However, the exact breakpoints of deletions defined by MPLA will remain unknown because the distance between the MLPA probes varies from a few hundred base pairs up to >10 kb. Although not essential for demonstrating the presence of a deletion defect, knowledge of the exact breakpoint is important from a scientific point of view to unravel the mechanisms leading to these deletions. In addition, characterizing the breakpoints allows the design of a gap-PCR method specific for the new mutation. This can be of use for molecular diagnostics as an alternative to MLPA in populations where specific deletions occur at a significant frequency or for specific mutation detection in families.
The standard method to characterize such rearrangements is to randomly design primers in the breakpoint region and perform PCR across the breakpoint. The breakpoint region is determined by the most proximal and most distal MLPA probe still present and the first probe involved in the deletion. The distance between MLPA probes generally range between 3-10 kb which requires the use of long-range PCR and primer walking to sequence the breakpoint completely (117). Since this procedure is breakpoint specific and rather time consuming, other methods are needed to improve the resolution of the breakpoint position and to develop primers more closely located toward the breakpoint. In addition, it is necessary to develop techniques for quick and precise detection of deletions for diagnostic purposes.
The array comparative genomic hybridization (aCGH) technology (Figure 11) is a promising method in characterizing copy number variations. The development of aCGH in the past few years has facilitated the identification of the molecular basis of many genetic diseases (118-124). Originally, aCGH was developed as a research tool for the investigation of genomic imbalances in cancer (125) and has become an essential and routine diagnostic tool in genetics to search for copy number variation. The high resolution, simplicity, high reproducibility and precise mapping of imbalances are the most significant advantages of aCGH over traditional cytogenetic methods.
A fine-tiling oligonucleotide array was first used for breakpoint analysis of deletions causing neuroblastoma at sub-kilobase resolution on four different chromosomes (126). The same type of array was used to delineate the deletion breakpoints in chromosome 1p for patients with Wilms tumors (127). More recently, fine-tiling arrays have been used to unravel complex rearrangements involving the BRCA1 gene (128), the MECP2 gene (129), the GJB2 and GJB6 genes (130), the STK11 gene (131) and to characterize different balanced and unbalanced rearrangements in a group of 12 patients with phenotypic abnormalities (132). These studies not only confirmed the power of fine-tiling arrays to find breakpoint regions, but also underline the increasing importance of array technology as a follow up after MLPA for the characterization of deletions and breakpoints in common and rare rearrangements. Therefore, the aCGH technique was also used in the current study to improve diagnostics for hemoglobinopathies.
Chapter 2.4 describes the design and validation of a custom fine-tiling array for the detection of rearrangements in the globin gene clusters. Herewith we have shown that aCGH technology is suitable for high-resolution mapping of breakpoints and that it is a valuable tool for the design of simple gap-PCR assays. The latter might be useful as a quick screening method for the more local occurring deletions or in laboratories where aCGH or MLPA is not available.
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Figure 10 Outline of the MLPA technique. After hybridization to their target sequence in the sample DNA, the probe oligonucleotides are enzymatically ligated. One cosmid probe oligonucleotide contains a non-hybridizing stuffer sequence of variable length. In case of synthetic probes, the unique length of the product is determined by the length of the target sequence. Ligation products can be amplified using PCR primer sequences X and Y. Amplification products are separated by electrophoresis. Relative amounts of probe amplification products, as compared to a reference DNA sample, reflect the relative copy number of target sequences (Adapted from MRC Holland, http://www.mlpa.com).
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Figure 11 Outline of the array CGH technique. Test DNA and reference DNA samples are prepared separately. One sample is labeled with cyanine-5 fluorescent dye (red), and the other with cyanine-3 (green). Samples are then mixed and hybridized onto the array slide. By scanning the slide after ~72 hours of hybridization, the intensity of both fluorescent signals is measured. Differences in signal intensities, indicating copy number variations, between the two samples are visualized in a data plot (Adapted from Roche NimbleGen, http://www.nimblegen.com).
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C. Prenatal diagnosis of hemoglobinopathies
As mentioned above, it is estimated that about 7% of the world population is a healthy carrier of a hemoglobinopathy. This results in approximately 350,000 severely affected children being born each year. Hemoglobinopathies occur mainly in areas where malaria is or has been endemic, because of the protective effect of the hemoglobinopathy traits against the lethal complications of malaria infection. However, the number of patients in north European countries is increasing due to recent migration (7;9;10;133).
In many endemic countries, prenatal diagnosis has been offered for several decades as part of a national primary prevention program by applying different techniques, including