Objectives: A comparison of the individual genomes within a
species demonstrates that structural variation, including Copy Number Variation
(CNV), is a major contributor to phenotypic diversity and evolutionary adaptation.
CNVs lead to the deregulation of gene expression and could account for the
development of a number of genomic disorders. Thus, the development of
efficient, rapid and accurate CNV screening is of fundamental importance. We
report a method that enables the simultaneous determination of the copy numbers
of different genetic targets as well as the discrimination among highly
similar/almost identical DNA sequences that differ by only one single
Methodology: The PCR co-amplification and single-base extension technologies are used to identify the copy number of target sequences, the primary spinal muscular atrophy-determining gene, SMN1, and the disease modifier gene, SMN2, in a cohort of 160 subjects previously genotyped with MLPA-based and qPCR-based techniques. The copy numbers of SMN1/SMN2 were relative to a reference sequence of known genomic copy number (Albumin and Factor VIII genes).
Results: We developed an efficient and accurate quantification platform which can be adopted as an alternative to other technologies for CNV evaluation, like as MLPA-based or qPCR-based techniques. In addition, our method has proved effective in resolving a diagnostic event by questionable result in using the previously mentioned technologies.
Conclusions: The reliability, low-cost and potential for high-throughput make our method suitable for screening large populations as well as for use as a tool in clinical settings for genetic diagnosis/prognosis.
Keywords: Copy Number Variation; Gene Dosage; Hereditary Diseases; Multiplex-PCR; SNPs identification; Single Base Extension
2.4 Multiplex PCR and SBE
Following the primer extension, the reactions products
were purified by SAP (Amersham Biosciences), according to the manufacturer’s
instructions. The cleaned products were combined with 0.2 µl of GeneScan-120 LIZ Size Standard Mix and 9.8 µl of form amide and run on Applied Bio systems 3730
DNA Analyzer (Applied Bio systems). The peaks of dye intensities corresponding
to extensions of the SBE primers were determined by inspecting the output from
the ABI 3730 DNA Analyzer.The multiplex PCRs were
performed in a final volume of 25 µl. We
combined the template (30 ng of genomic DNA) with the KAPA2G Fast HS Ready Mix PCR
Kit Premix (Kappa Bio systems, Wilmington, MA, USA) and locus-specific primers
(10 µM each; Sigma Aldrich), as recommended by
the KapaTaq protocol. The thermo-cycling conditions consisted of an initial
denaturation step of 95°C for 2 min.,
followed by 26 cycles of 95°C for 15 s,
Base pair exchanges, C-to-T at position +6 in exon7 (c.840C>T) and G-to-A in the un translated region of exon8 (Burglen et al. 1996)  were used to distinguish between the two SMN genes (Figure 1A), while the regions of the ALB and F8 genes were used as internal references (Figure 1A and Materials and Methods). We unambiguously identified the SMN1 and SMN2 peaks as well as the reference peaks from the genotyping outputs (Figure 1B).
Two independent references were used simultaneously to avoid biases due to possible individual variations in the copy number of one reference sequence. Because ALB on chromosome 4 is present in two copies per diploid genome and F8 on chromosome X is present in two copies in females and in a single copy in males, we examined the reliability of our system by looking at the relative ratios of the two reference signals across males and females. The ALB/F8 ratios found in males (1.85 ± 0.10) were consistently doubled with respect to those found in females (0.98 ± 0.12), underlining the reliability and the quantitative nature of the assay. In addition, the fixed relative ratios between the two reference signals across individuals indicated stability in their copy numbers, confirming them as suitable references in all of the samples. It is important to note that the reference sequence can be any genomic region with stable copy numbers. Control samples with known SMN1/SMN2 copy numbers (1/1, 1/2, 1/3, 2/2, 2/1 and 3/1) were run alongside the test samples. First, the controls were used to determine the proportionality between SMN1 and SMN2 relative signal intensities, separately for exon 7 and exon 8, showing the linearity of the assay in response to the varying copy numbers of the two SMN genes (exon7 R2=0.9915 and exon8 R2=0.9653) and further confirming the quantitative nature of the assay. Second, the target-to-reference ratios in the control subjects allowed for the assessment of the SMN1 and SMN2 copy numbers in the test samples with an unknown SMN dosage. The copy numbers of the SMN1 and SMN2 genes in the Test samples (T) compared to those of the Control samples (C) were calculated separately for males and females by the following equations:
Peak heights SMN1 (T)/[ALB+F8(T)] and Peak heights SMN2(T)/[ALB+F8(T)]
Peak heights SMN1(C)/ [ALB+F8(C)] Peak heights SMN2(C)/ [ALB+F8(C)]
This comparison resulted in a dosage quotient that indicated the copy number of each SMN gene in the test sample. For each individual, we quantified the SMN1 and SMN2 dosage independently for exon7 and exon 8, enabling the identification of the gene-conversion events that result in the creation of a hybrid SMN gene. The gene conversion of SMN1 to SMN2 in exon 7 is one cause of SMA. The +6 C-to-T substitution in SMN2 exon 7 decreases the activity of an exotic splice enhancer and alters the splicing pattern so that the SMN2 mRNA excludes the exon 7 sequences. Consequently, SMN2 produces insufficient amounts of the full-length SMN transcript and protein to rescue the SMA phenotype [26,28]. However, the conversion of SMN2 to SMN1 in exon 7 could be used as a therapeutic approach for SMA . By identifying individuals with unequal SMN1/SMN2 ratios in the two exons we were able to distinguish between the hybrids genes derived from conversion events. Individual P86 had an SMN1/SMN2 ratio of 1:3 for exon7 and 2:2 for exon8, while individual P80 had a ratio of 0:3 in exon 7 and 1:2 in exon 8 (Figure 1B), indicating a conversion from SMN1 to SMN2 in exon7. Due to this conversion, individuals P86 and P80 became an SMA carrier and an SMA-affected individual, respectively.
The evaluation of both the variation in gene expression and the variation in allelic expression within and among individuals/populations is of fundamental value in biomedical research, where it may lead to the discovery of the causative genes of common hereditary diseases and their mechanism of action . Coupling genotyping to gene expression studies could reveal the existence of direct and/or indirect mechanisms that require further dissection to appropriately elucidate a particular phenotype. In conclusion, we report a rapid and simple quantitative assay that allows for the identification and allele-specific characterisation of CNVs. The assay has the potential for high-throughput, as 96 samples can be analysed in only a few hours, and it is a cost-effective tool. In addition, the assay we describe here could be easily employed in gene expression studies, allowing for the quantitative and allele-specific expression screening of a large number of genes, providing insight into questions of crucial importance to our understanding of the genomics of gene regulation.
Table 1: PCR and SBE primer sequences.
Table 2: A cohort of 160 individuals with different SMN1 and SMN2 copy numbers.
Figure 1A: SBE genotyping performed for target SMN genes and the reference Albumin and Factor 8 genes. For target SMN genes genotyped residues were C/T at position +6 in exon 7 and G/A at position +236 in exon 8. For reference Albumin (ALB) and Factor8 (F8) genes, genotyped residues were A at position +102 in exon 12 and T at position +52 in exon 8, respectively. Arrows represent SBE primers.
Figure 1B: SBE genotyping of multiplex PCR analysis from healthy controls, SMA patients and carriers with different SMN1/SMN2 ratios. SMN1 and SMN2 in exons 7 and 8 as well as ALB and F8 are indicated in colour code under respective peaks in sequencing outputs. Sample name, gender and SMN1/SMN2 copy numbers, separately reported for exon 7 and exon 8, and are indicated on the left side of each output. F = female; M = male; Individuals P80 (affected subject) and P86 (healthy carrier) experienced gene conversion of SMN1 to SMN2 in exon 7. Males have only one copy of the F8 gene since it is on chromosome X.
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