Determining Gene Dosage

Ohio State University, 121 Hamilton Hall, 1645 Neil Ave., Columbus, OH 43210, Fax 614-292-7072, E-mail prior-1{at}medctr.osu.edu

Numerous investigations (both clinical and basic) in today's molecular biology laboratory require accurate quantification of DNA by the polymerase chain reaction (PCR). To name a few important examples, quantitative PCR has been used to quantify viral copy number, to perform gene expression studies, and to diagnose genetic diseases. In this issue of Clinical Chemistry, Poropat and Nicholson describe a quantitative PCR assay for the determination of gene dosage (i.e., the number of copies of a gene per somatic cell) of the PMP22 gene, a myelin gene involved in inherited neuropathies (1). The PMP22 gene is extremely sensitive to copy number; for example, it is duplicated in the autosomal dominant Charcot-Marie Tooth type I (CMT1A) disease and is deleted in the autosomal dominant hereditary neuropathy with liability to pressure palsies (HNPP). Because the expression of the disorders can be quite variable and CMT1A shows genetic heterogeneity, the DNA testing for these diseases establishes a secure diagnosis; it also permits genetic counseling and testing for high-risk family members. For molecular laboratories involved in genetic disorders, gene dosage determinations are clinically important and frequently performed. In addition to CMT1A and HNPP, there are many genetic disorders where the primary defect is due either to allelic deletions (e.g., Duchenne muscular dystrophy, spinal muscular atrophy, -thalassemia, growth hormone deficiency, and familial hypercholesterolemia) or to duplications (e.g., Klinefelter syndrome and Down syndrome). Furthermore, for the determination of the carrier state of disorders such as Duchenne muscular dystrophy and spinal muscular atrophy, the accurate determination of heterozygous deletions is essential.

Originally, gene dosage determinations were made from Southern blots. The protocol was laborious and required large amounts of DNA. Furthermore, optimal conditions were required; very high-quality blots were necessary, with consistent transfer and hybridization and low background. We have found that Southern autoradiographic bands >10 kb and <0.5 kb frequently produce weaker intensities that are not always adequate for dosage determinations. The difference between one and two copies may be relatively easy to detect, but differences between two and three copies, as in the case of CMT1A, can be more challenging and technically difficult.

The determination of gene dosage has been markedly improved by the use of PCR. Because the extension product of each primer serves as a template for the other primer, each cycle essentially doubles the amount of the DNA product produced in the previous PCR cycle. This produces an exponential accumulation of the specific fragment, up to several million-fold in a few hours. To obtain quantitative results, however, the PCR products must be measured during the exponential phase of the amplification process, for it is during the exponential phase that the amount of amplified products is proportional to the amount of starting DNA (2). This occurs when the primers, nucleotides, and the polymerase enzyme are in a large excess over that of the template concentration. It is critical that samples are assayed within the exponential phase of the PCR reaction, before the plateau phase when the amplification efficiency is decreased. Therefore, it is necessary to define the range of concentrations that give an exponential amplification over a defined range of cycle numbers. The simplest way to establish the exponential range is to remove small aliquots from a trial PCR reaction every few cycles during amplification, measure the band intensity, and determine the product linearity over several cycles. We have found that the conditions are relatively liberal; the PCR reaction can provide accurate quantification over a range of cycle numbers and starting DNA template concentrations (3). Poropat and Nicholson also found that the linearity for the PMP22 assay was well maintained over many cycles, and the authors selected 26 cycles for the quantitative analysis (1).

The selection of an internal control is of the utmost importance when developing a quantitative PCR assay. The internal control is co-amplified with the target of interest and serves as a control for several factors: differences in initial template concentrations between different samples, sample-to-sample variations in the PCR, the extent of any DNA degradation, and differences in the amounts of amplicon loaded onto the gel. The authors chose an endogenous internal control, the NF1 gene, which is present at a known dosage (disomic copy) and is then compared with the sequence of interest (PMP22 gene) (1). Thus, rather than directly comparing single bands, band ratios are calculated. Factors influencing the PCR should affect both products, and thus the ratio should be unaffected. It is important to choose an internal endogenous control that is not only well resolved from the target of interest but also is amplified at the same rate as the sequence of interest. Primers for both the target of interest and the control should therefore be designed to have similar Tm values. Ideally, the normal dosage control ratio should be about one. As shown, the NF1 sequence was an excellent control. The PMP22/NF1 ratios for CMT1A, unaffected, and HNPP samples were ~1.5, 1.0, and 0.5, respectively. Futhermore, the variances for the three ratios were small, and no overlap between the three populations was observed.

The other type of control that is used in quantitative PCRs is an exogenous DNA sequence synthesized in vitro and used in a competitive PCR format (4). In the competitive PCR method, a known number of copies of a synthetic internal control are directly introduced with the patient sample into the PCR mixture, and the dosage ratios are measured. The major advantage of this technique is that the internal control is amplified with the same set of primers that amplify the target sequence. The internal standard is designed, using a PCR mutagenesis protocol, such that it contains the same sequences for the forward and reverse primers to anneal (5). However, the amplicons of the internal standard can be distinguished from those of the target by a difference in size or sequence. The efficiencies of the amplification of the patient DNA and the internal control are very similar, thus allowing one to make highly quantitative dosage determinations. This is a more complex PCR method because it requires the additional step of generating the internal control and it is often used when one needs to make more highly quantitative measurements, such as quantifying RNA in gene expression studies or determining the viral copy number. We have recently developed a competitive PCR assay for the determination of spinal muscular atrophy (SMA) carrier status (6). SMA is a motor neuron disease characterized by the degeneration of spinal cord horn cells and muscular atrophy. SMA is an autosomal recessively inherited disorder with an incidence of about 1 in 10 000 live births and a carrier frequency of 1 in 40 to 1 in 60. In ~95% of SMA patients, exon 7 of the spinal motor neuron gene (SMN) is deleted (7). Carrier detection for the heterozygous state proved to be more technically challenging because the SMA region is characterized by the presence of many repeated elements. The SMN gene itself is present in two almost identical copies, a telomeric and a centromeric copy on chromosome 5; it is the telomeric copy that is deleted in the SMA. However, the centomeric copy number fluctuates: Approximately 5% of healthy individuals lack the centromeric copy, whereas many of the more mildly affected SMA patients have three copies. Thus, a straightforward dosage assay using the centromeric gene as the internal control would not be reliable. We therefore generated an exogenous in vitro internal SMN control and have used this for dosage determinations and identification of SMA carriers (6).

In my opinion, dosage ratios should always be quantified using an automated detection system, thus removing the subjectivity of qualitative visual analysis. Furthermore, for quality control purposes, the ratio values should be statistically analyzed both between and within runs. The authors used a laser-based detection system that proved to be highly sensitive and eliminated the need for radioisotopes.

In summary, gene dosage determination by quantitative PCR is an accurate, rapid, and sensitive method for identifying deletions or duplications. The assay does not require major equipment purchases and can be performed in a diagnostic laboratory. The dosage test is based simply on amplifying not only the target of interest but also an internal control, which is then used in calculating the dosage ratio. Dosage ratios are used as a means of correcting any errors due to differences in initial template concentrations and efficiencies of PCR. In the article by Poropat and Nicholson (1), the internal control was an endogenous target amplified simultaneously with the gene of interest, and the dosage ratio was calculated. As long as one is careful to choose an internal control that is not a member of a homologous gene family or a repetitive DNA sequence, this is a straightforward technique and can often be used for dosage determinations. For more complex loci, the more technically demanding competitive PCR may be necessary. This technique requires the synthesis of an exogenous internal control that is then added to the PCR reaction. Because the genomic DNA and the internal control utilize the same primers, efficiencies of amplification should be equivalent, thus allowing for extremely sensitive dosage measurements. This technique is not only applicable for complex gene loci but is often used to quantify RNA when one may be interested in relatively small differences in gene expression (8).

References

1. Poropat RA, Nicholson GA. Determination of gene dosage at the PMP22 and androgen receptor loci by quantitative PCR. Clin Chem 1998;44:724-730. [Abstract/Free Full Text]
2. Lubin M, Elashoff JD, Wang S, Rotter J, Toyoda H. Precise gene dosage determination by polymerase chain reaction: theory, methodology, and statistical approach. Mol Cell Probe 1991;5:307-317. [ISI][Medline] [Order article via Infotrieve]
3. Prior TW, Papp AC, Snyder PJ, Highsmith WE, Friedman KJ, Perry TR, et al. Determination of carrier status in Duchenne and Becker muscular dystrophies by quantitative polymerase chain reaction and allele-specific oligonucleotides. Clin Chem 1990;36:2113-2117. [Abstract/Free Full Text]
4. Gilliland G, Perrin S, Bunn HF. Competitive PCR for quantitation of mRNA. Innis MA Gelfand DH Sninsky JJ White TJ eds. PCR protocols: a guide to methods and applications 1990:60-69 Academic Press San Diego. .
5. Celi FS, Zenilman ME, Shuldiner AR. A rapid and versatile method to synthesize internal standards for competitive PCR. Nucleic Acids Res 1993;21:1047.[Free Full Text]
6. McAndrew PE, Parsons DW, Simard LR, Rochette C, Ray PN, Mendell JR, et al. Identification of proximal spinal muscular atrophy carriers and patients by analysis of SMNT and SMNC gene copy number. Am J Hum Genet 1997;60:1411-1422. [ISI][Medline] [Order article via Infotrieve]
7. Lefebvre S, Burglen L, Reboullet S, Clermont O, Burlet P, Viollet L, et al. Identification and characterization of a spinal muscular atrophy-determining gene. Cell 1995;80:155-165. [ISI][Medline] [Order article via Infotrieve]
8. Foley KP, Leonard MW, Engel JD. Quantitation of RNA using the polymerase chain reaction. Trend Genet 1993;9:380-384. [ISI][Medline] [Order article via Infotrieve]