Genechip or array technology can be applied for gene discovery, gene expression, gene mapping, and mutation detection. Microarrays typically consist of an ordered collection of microspots on a surface matrix, where each microspot contains a single defined sequence of nucleic acid (probes), which may either be large DNA fragments (cDNA’s or PCR amplicons) or allele-specific oligonu- cleotides, the latter being either pre-synthesised, or in some cases, synthesised in-situ (on-chip). The microarray technique is based on hybridisation of nucleic acids, whereby sequence comple- mentarities leads to the hybridisation between two single-stranded nucleic acid sequences, one of which (usually the allele-specific oligonucleotide) is immobilized on the surface matrix. In this sec- tion, the state-of-the-art with respect to microarrays technologies for the characterization of single nucleotide DNA variations (mutations) and deletions associated with the haemoglobinopathies will be presented [1].
7.1.1 MICROARRAY ANALYSIS FOR POINT MUTATIONS
Microarray analysis is potentially appropriate for characterizing point mutations within the globin genes associated with the disorders of haemoglobin synthesis, representing an approach for rapid genotyping, potentially interrogating a large number of samples for a large numbers of mutations simultaneously. The probes used for genotyping applications are usually allele-specific oligonucle-
otides. In some protocols the sample-probe hybridization step is followed by enzymatic mismatch discrimination to enhance allelic specificity. The detection of sample-probe hybridisation on the array requires that samples or probes are labelled, and this is usually achieved with a fluorescent moiety. However, the development of reliable microarrays or gene-chips for detecting disease-as- sociated mutations within a diagnostic setting has been hindered by difficulties in optimizing homo- geneous stringency conditions for a potentially wide and varied range of DNA sequences necessary for the simultaneous analysis of multiple mutations. This criteria is exacerbated by the requirement to discriminate alleles within samples which may differ by only a single nucleotide (eg homozygous wild-type or homozygous mutant versus heterozygote at a particular nucleotide position).
During the last decade there was considerable effort directed towards the standardization and vali- dation of genotyping microarrays. Amongst the more successful examples is an arrayed primer extension-based system which involves the extension of oligonucleotides probes that are designed to be a single nucleotide short relative to the base of the allele variant, called Arrayed Primer EX- tension (APEX) technology (APEX, Asper Biotech, Tartu, Estonia; http://www.asperbio.com) [2]. In this method the probes, designed to specifically interrogate known mutations and single-nucleo- tide polymorphisms in the gene of interest, are immobilized via their 5’end on a glass surface, while its 3’ end is free for enzymatic extension. Following an initial step based on the polymerase chain reaction (PCR), an amplicon from a DNA containing the region which harbours the mutation(s) or Single Nucleotide Polymorphisms (SNPs) under investigation is hybridized to the complementary probe sequence that is immobilized on the microarray chip. A single on-the-chip base extension of the probe is performed by incorporation of the appropriate dye-labeled dideoxynucleotide which is complementary to the variant base, followed by termination of the reaction. An automatic laser imaging system is used to read the APEX slide and detect which of the four fluorescent dideoxynu- cleotide labels (Fluorescein, Cy3, Cy5 and Texas Red) have been incorporated at each oligo-probe site on the microarray.
The APEX method has been adapted for large scale β-globin mutation and polymorphism detec- tion (amongst other disease-gene applications), [3, 4], The updated validated version, known as ThalassoChip is a β-thalassaemia genetic diagnostic tool based on Array Primer Extension (APEX) technology which has the ability to detect over 60 β-globin gene mutations and polymorphisms in a single step [5]. The optimized APEX reaction conditions are entirely reproducible, as long as prereq- uisites, such as good quality human genomic DNA at the appropriate concentration, a successful DNA fragmentation step and optimized PCR amplification conditions are fulfilled. A commercially available service for the diagnosis of β-thalassaemia mutations is available from Asper Biotech.
Although microarrays for point mutations provide diagnostic tool with potentially relatively low run- ning costs along with the possibility to determine a wide spectrum of mutations and polymorphisms in a single experiment, with a very few exceptions, many genotyping microarray technologies have not evolved beyond the prototype stage, or have proved to be economically unsustainable (eg the Nanogen Nanochip®microelectronic microarray system) [6].
is that the microarray chip is limited for detecting only known nucleotide changes for which the probes have been designed. Previously undetected polymorphisms and new mutations will be missed, and furthermore may even lead to false results through mismatched bases under the probes. Finally microarray platforms are technically quite demanding with respect to both sam- ple processing and data interpretation, and thus require highly trained operators. High-throughput sample preparation is also an advantage and microarray systems are generally not supported by integrated automated DNA extraction and PCR preparation systems. Finally, the costs of most plat- forms developed to date are higher than in-house technologies.
7.1.2 MICROARRAY ANALYSIS FOR DELETIONS/DUPLICATIONS OF CHROMOSOMAL REGIONS
The array comparative genomic hybridization (aCGH) technology is a method for copy number vari- ation across chromosomal regions and has become a valuable routine diagnostic tool in genetics. The high resolution, simplicity, high reproducibility and precise mapping of imbalances are the most significant advantages of aCGH over traditional cytogenetic methods.
Many studies have used fine-tiling oligonucleotide arrays for breakpoint analysis of deletions un- derlying several genetic diseases such as neurofibromatosis (7), Wilms tumor (8) or breast cancer (9). These studies not only confirm the power of the fine-tiling arrays to find breakpoint regions, but also underline the increasing importance of fine tiling array technology as a follow up after MLPA for the delineation of deletions and breakpoints in common and rare rearrangements. Similarly a custom fine-tiling array has been developed recently for high-resolution determination of deletion breakpoints in the α- and β-globin gene clusters (10). This array has been used to fine-map the po- sitions of breakpoint junctions supporting the design of gap-PCR primers for sequencing analysis in order to determine the exact deletion breakpoints. The design of gap-PCR assays for deletions characterized by aCGH, has an important diagnostic application, by providing a simple screening method in laboratories where MLPA is not available, especially appropriate if the specific deletions reach high frequencies in a local population.
Array design, experimental conditions and data analysis: Two custom fine tiling arrays covering the α- and β-globin gene clusters plus surrounding areas have been developed (Roche NimbleGen, Madison, WI, USA) (10). Design of the array was based on NCBI Build 36.1 (hg18). (NimbleGen Ar- rays User’s Guide: CGH Analysis v4.0). These customized fine-tiling arrays included 135,000 probes (12 x 135K format), with 12 identical sub-arrays per slide, allowing for simultaneous analysis of up to 12 different samples. The coverage of the probes on the Nimblegen fine-tiling array is shown in
Figure 7.1. The probes on the α-globin gene cluster array cover the 2 Mb telomeric region of chro- mosome 16p, including the α-globin gene cluster (position 1-2,000,000, according to UCSC Genome Browser, March 2006, hg18). The array for the β-globin gene cluster on chromosome 11p and sur- rounding region covers 0.6 Mb (position 4,900,000-5,500,000). The average spacing on each array is 20 bp, and the oligonucleotide probes have a length of 60-80 bp, involving an overlap between probes and approximately 3x coverage of the region of interest.
The design had to accommodate certain features of the sequences in the α- and β-globin gene clusters. For example both clusters contain non-unique sequences, such as the duplicated α- and γ-globin genes and the Alu- and LINE-repeat regions. The largest repeat unit in our region of inter- est is a ~7 kb LINE-repeat in the β-globin gene cluster. In order to increase specificity and to pre- vent false positive results, all the probes on the array were selected to be unique, which resulted in non-unique sequences of the globin gene clusters not being covered by the probes. Thus, in cases where the breakpoint is located within a repetitive sequence, the determination of the breakpoint position by the array may be inaccurate by up to a maximum of 7 kb from the true breakpoint.
The experimental conditions were based on the NimbleGen Arrays User’s Guide: CGH Analysis v4.0 and the data analysis was performed using the NimbleScan v2.5 and SignalMap v1.9 software (NimbleGen). Details are described in a recent publication by Phylipsen et al, 2011 (10). Application of the customized fine-tiling aCGH technology has demonstrated the capability to detect small and large rearrangements (from ~4 kb up to 2 Mb) in the α- and β-globin gene clusters with high reso- lution, as illustrated in Figure 7.2. Based on information provided by the array analysis, it has been possible to design primers to amplify relatively short products including the breakpoint sequence which can then be characterized easily by direct sequencing.
FIG. 7.1
Nimblegen custom fine-tiling array: schematic overview of the coverage of the probes. The vertical grey lines indicate where probes are located, uncovered areas are left in white. The array covers the telomeric 2 Mb of chro- mosome 16p including the α-globin gene cluster (a) and a 600 kb region including the β-globin gene cluster on chromosome 11p (b). The stippled bars below the figures represent the location of the globin gene clusters. Posi- tions are according to the UCSC Genome Browser (March 2006, hg18).
Concluding remarks: Currently, the high cost of aCGH technology limits its wider use in diagnostic applications, estimated at about 9x the cost of MLPA per sample (comparing reagents only, and excluding purchase of the required instruments. However, fine-tiling aCGH technology is a valu- able tool to support high resolution breakpoint characterization in α- and β-globin gene cluster rearrangements. In addition, the information provided can be used to design simple PCR-based tests to detect the variant alleles, useful to laboratories where specific deletions may reach a high frequency in the local population since gap-PCR protocols are easier and cheaper than MLPA and Southern blotting. Since it is not feasible that all laboratories can set-up and apply aCGH, an alter- native strategy for characterizing any rare new deletions might be to screen samples using MLPA and then refer any undefined samples to a centralized collaborating laboratory which has fine- tiling aCGH assays available, facilitating the design of gap-PCR assays if required.
FIG. 7.2
Detection of two α0-thalassaemia deletion mutations with the Nimblegen custom fine-tiling array: the aCGH re-
sults for 3 patients are shown below the schematic presentation of the 2Mb region of 16p containing the α- globin gene clusters covered by overlapping ‘tiled’ oligonucleotide probes. Patient A and B are carriers of the South African deletion (--SA) and patient C is a carrier of the --JB deletion.