| 1. Detection of copy number variants (CNVs) in the α-globin gene clusterCopy number variants (CNVs) in the human genome are increasingly recognized as being highly prevalent and contributing to human diseases because they affect expression levels of genes. Allelic copy number variation (CNV) may alter the functional effects of an affected gene of the targeted locus. It is well known that for α-thalassemia, the most common form of the genetic disorder worldwide, there exists a typical relationship between clinical phenotypes and genotypes involving the copy number changes in the human α-globin gene cluster. A normal person possesses four copes of α-globin gene due to existing two copes of each chromosome. The different types of a-thalassemia mainly result from deletions at16p13.3. One,2,3,4of the a-globin genes may deleted, thus resulting in the following four different clinical phenotypes by turnes:"silent carrier" with no detectable phenotypes,"a-thalassemia trait" with a typical microcytosis and hypochromia of red blood cells, HbH disease with having mild to severe anemia, and the lethal hemoglobin (Hb) Bart’s hydrops fetalis syndrome. On the other hand, the duplications of α-globin genes, such as the triplicated a-globin genes (αααanti3.7or αααanti42) or the quadruplicated a-globin genes (аααα), have been verified as an important modulator of clinical severity of β-thalassemia.Recently, many genome-wide, array-based techniques such as array comparative genomic hybridization (array CGH), SNP genotyping arrays and deep sequencing-based approaches, have been developed to identify the CNVs. These assays are high throughput with high resolutions from several kilobases (kb) to several megabases (Mb) within the human genome. But they are not appropriate to detect particular disease-causing copy-number changes, which involving only one or two locus. Some targeted approaches have been used to address this issue, such as MAPH (Multiplex Amplifiable Probe Hybridization), MLPA (multiplex ligation-dependent probe amplification), QMPSF (Quantitative multiplex PCR of short fluorescent fragments), and real-time quantitative PCR (qPCR). They are mainly based on PCR, usually allow exploration of limited locus involving disease-related CNVs. For these technologies of typing CNVs in the targeted region, each has its advantages and limitations. For instance, the disadvantage of MLPA is the more laborious and costly design of a good probe set. Furthermore, an operator has to take very long time to perform an overnight hybridization step (about16h) for running the MLPA test.Real-time quantitative PCR is particularly attractive in clinical practice for its short turnaround time, easy automation, and avoidance of carryover contamination. It has been usually used for confirmative detection of known CNVs in the targeted regions and has been widely applied to quantify gene copy numbers of many different loci having relevance to genetic disorders. The multiple fluorescent channels of the instrument allow multiplex PCR in a single reaction. But it is generally believed that multiplex PCR is limited to the amplification of a few amplicons, due to spurious interactions of the primers in the reaction mixture and the target with higher amplification efficiency in the reaction inhibiting the lower ones, thus leading to inaccurate copy number results.Here we use a-globin gene as a model system, developing a nested multiplex real-time qPCR, a TaqMan probe-based approach that can directly measure the allelic ratio in copy number variation (CNV) regions of a genome. We used three pairs of tailed-oligonucleotide primers to capture the sequence of specific locus, three hydrolysis probes and one pair of universal primers to detect the signals of different locus. The tailed-oligonucleotide primers included a forward primer (F) and reverse one(R). F was relatively long, containing a target-specific sequence at the3’end, a common sequence used for universal PCR amplification at the5’end, and a barcode sequence for hybridizing hydrolysis probes in the middle. While R was a shorter oligonucleotide, containing a target-specific sequence at the3’end and a common sequence at the5’end. The copy number status of al/a2can be determined by profiling typical amplification plots of the3genes (al, a2and β-actin) for a sample tested. The copy number determination of α1/a2in the sample with different a-globin genotypes can be performed by calculation of the amplicon dose through compairing α1and/or a2with β-actin genes according to the2-△△Cq formula.To test the performance of different primers and probes in one tube, we used human genomic DNA templates by5-fold serially diluted, each replicating3samples. The data were obtained from the standard curve generated by the Stratagene software. The efficiency of three gene amplification plots was in the range of99.1%-100.3%, so that meant all the targets in the tube reacting well. When the template concentration reached as low as3.25ng, the Cq value was still in accord with the genotype. So the sensitivity of the method was at least at3.25ng.The reproducibility of the approaches was determined with11different genotype.In case of al, we used DNA from-α37carrier with1copy, control person with2copies and αααqnti3.7carrier with3copies. For a2, we used DNA from persons with0to3a2copies. The numbers of α1or a2copies in these individuals were also determined by Gap-PCR or MLPA. From the results, we could see a clear differentiation between1to3al or a2copies, without any region of overlap.We analyzed95clinical samples, among which16different genotypes containing various copies of α1and a2. We determined the genotype by the measured value, just in complete accordance with the actual samples. All the16genotypes were identified correctly, so our method attained100%specificity. Compared with other existing methods, this nested multiplex qPCR assay described in this study appears to be an accurate, reliable, quick and easily operated method for the determination of known CNVs in the targeted region. First, as shown here, we chose short fragments no longer than50bp to hybridize to the genomic templates. This, in one side, facilitates us searching suitable sequences in related genomic regions. In the other side, we could acquire them through generally synthesis, thus avoid using the specially prepared probes which needed in MLPA. Second, we use universal primer substantially reduces the amplification bias induced by direct amplification of the target. The quantification accuracy of target amplification strongly depends on whether amplification efficiency varies with the primers. In contrast, the method involves a nested reaction step that generates probes with a universal primer-binding sequence, which is used as the target for the following amplifications with a common primer pair. Consequently, the PCR amplification efficiencies among differently target products can be equalized easily. Moreover, our assay could exactly differentiate3vs.2copies of template DNA and accurately diagnose the clinical samples. At last, the protocol included only two steps, not needing long time for hybridization and ligation, so the entire process is in less than2hours. Furthermore, the assay was based on the real time qPCR which is widely applied in the clinical laboratories.In conclusion, we developed a new approach for genotyping common deletions and triplications in a-globin gene cluster through analyzing CN values of al and a2. We directly obtained the results of genetic diagnosis for clinical samples, skipping the steps of testing blood cells as in the past. So this assay could be used as an alternative approach applied for molecular screening of alpha-thalassemia. Moreover, for the assay had the property of universal, high-throughput performance and precise resolution, it has the potential to be developed as a general application to detect CNV in a given DNA region, not only confirmation of unknown CNV in genetic analysis but also molecular screening for known CNV related to the genomic disorders. 2. Molecular diagnosis of common deletions with a-thalassemia in ChineseIn general, there are2a-globin genes in a-globin gene cluster. When deletion occurs, it will result in abnormally clinical phenotypes. The deletion of a-globin gene is the mainly molecular genesis of a thalassemia, Up to95%of recognized a-thalassemia involves deletion of one or both a-globin genes in chromosome16pl3.3. The--SEA deletion type,0copy of a-globin gene, is the most common mutation, accounting for48.54%in the China population. While the-a3.7and-α42deletion types, one copy of a-globin gene, with the rate of47.49%.During meiosis, misalignment and unequal crossover between the homologous X-, Y-, and Z-box segments at the a-globin gene cluster generates a single gene deletion and reciprocal gene triplication. If the crossover occurs between the homologous Z2and Zl boxes, also referred to as a "rightward crossover", this produces α-a3.7single-gene deletion allele and the reciprocal aaaanti3.7triplicated allele. However, if the crossover occurs between the X2and XI boxes (a "leftward crossover"), α-α42single-gene deletion allele and the reciprocal αααant4.2triplicated allele are generated.The existed methods are mainly used for detecting the deletions, such as Southern blotting and Gap-PCR. Southern blotting is known with highly sensitive and specific, but as labor-intensive and time-consuming, thus not an ideal screening tool. The gap-PCR analysis is currently applied to diagnose seven a-globin gene deletions in a single reaction, and also developed to detect αααanti37and αααanti4.2triplications. Both based on multiplex amplification of junction fragments of the a-thalassemia determinants. But the technique requires post-PCR processing steps, thus vulnerable to carry over contamination. MLPA (multiplex ligation-dependent probe amplification) is a new, high resolution method to detect copy number variation in genomic sequences, permitting multiple targets to be amplified with only a single primer pair. But the specially prepared hybridization probes in the method hinder its popularization and application.Real-time quantitative PCR is particularly attractive in clinical practice because of its short turnaround time, easy automation, and avoidance of carryover contamination. It has been widely used to relative quantify of gene expression. And the multiple fluorescent channels of the instrument allow multiplex PCR in a single reaction. Our method is based on the real time qPCR, and incorporated universal primers through first step of ligation dependent reaction, so that simultaneously detecting the common deletions and triplications in single tube with similar efficiency.In our method, the efficiencies of four targets were approximately equal according to the results, so the relative quantity of starting copy number was authentic. We attributed it to two factors. The first factor is that our method reduced the amplification bias by avoiding direct amplification of the targets, but involved a ligation step that generates probes with a universal primer-binding sequence, which was used as the target for the following amplifications with a common primer pair, thus efficiencies among differently ligated products can be equalized easily. The second factor is the use of exonuclease I (Exo) after ligation step. Exo I could degenerate unligated single-stranded probes to diminish non-specific background, so the bias of next amplification step could be reduced as far as possible.The protocol contained two steps of reaction; only taking5hours in whole process. Other ligation dependent methods such as MLPA wasted too long time in hybridization step (16hours). For MLPA used low amounts (fmol level) of probe oligonucleotides to prevent most of PCR primers being consumed by linear amplification. As was known, the concentration of probe is low; the time it takes to anneal a significant number of probe molecules to a target band is increased. But long time with the reaction would greatly obstruct its application in the clinical screening. In our method, we used high concentration of probes which over excess amounts to DNA template, this promoting the probe anneal to the templates in a short time. After several cycling, ligation of probes to their target sequences had to be complete. Then exonuclease I was used to degenerate the excess probes, which were avoided to affect the next step of reaction.In conclusion, using the protocol presented here, we could quickly diagnose common deletions in a-thalassemia in less than3hours. The high throughput ability allowed the analysis of hundreds of samples per day without the need for complex and time consuming optimization procedures for each primer combination. |
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