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A simple and robust quantitative PCR assay to determine CYP21A2 gene dose in the diagnosis of 21-hydroxylase deficiency.

Congenital adrenal hyperplasia is an autosomal recessive disorder caused in 90%-95% of cases by a 21-hydroxylase deficiency (210HD) [3] (OMIM +201910), which leads to impaired synthesis of cortisol. 21OHD shows highly variable phenotypical expression, and cases are commonly classified as either classical or nonclassical forms. The absence of steroid 21-hydroxylase (210H; EC activity results in the classical form of 210HD, which manifests as a salt-wasting form, in which neither cortisol nor aldosterone can be synthesized, and by the simple virilizing form, in which residual activity of 210H allows aldosterone synthesis. The nonclassical form occurs when partial 210H activity is present. This form is late onset and may be asymptomatic or associated with signs of postnatal androgen excess (1).

The 210H enzyme is encoded by the CYP21A2 [4] (older nomenclature, CYP21, CYP21B) gene, which is 98% homologous to the CYP21A1P pseudogene (older nomenclature: CYP21P, CYP21A) in its coding sequence and 96% homologous in introns. Both genes lie in the human MHC at chromosome 6p21.3, a highly variable stretch of DNA. Most chromosomes bear 1 CYP21A1P pseudogene and 1 CYP21A2 gene, although deletions and duplications have been described (2-13). Intergenic recombinations are relatively frequent in this region (14), and are responsible for 95% of the mutations associated with 210HD; the remaining 5% of mutations are apparently not the result of gene conversion events (1-15). Among the intergenic recombinations, approximately 75% are deleterious or partially inactivating mutations, which are typically present in the CYP21A1P pseudogene and appear in the CYP21A2 gene as a result of gene conversion events (14). These mutations are easily detected by CYP21A2 gene sequencing, restriction digestion, or other methods (15). The remaining 20%-25% of mutations are CYP21A2 gene deletions or CYP21A1P/CYP21A2 chimeric genes formed by unequal meiotic crossover (1, 14,15). Such mutations lead to the absence of 210H activity and thus are very important for 21OHD diagnosis and carrier detection (2, 4-6, 10, 11, 13, 16). Other unequal meiotic crossover arrangements can produce duplicated CYP21A2 genes, which have been found in Dutch (12), Swedish (4, 8), Italian (6), and other populations, mainly with the presence of the severe G1n318X or I2G (c.293-13 A/C>G GenBank Ref. ID NM000500, according to the Human Genome Variation Society) mutations in 1 of the CYP21A2 genes (8,12). Consequently, to avoid false-positive results, assessment of the CYP21A2 gene copy number is very important when a mutation is present, especially the G1n318X or I2G mutation.

Until now such large rearrangements have been mainly detected by Southern blot analysis (17, 18), which is time-consuming, highly laborious, and requires large amounts of DNA. To avoid such limitations, PCR-based amplification analyses have been developed (19-25), but some of these have failed in CYP21A2 duplication detection (21, 23-25).

We report a real-time quantitative PCR (gPCR)-based method for the easy and rapid detection of these large rearrangements. The method can detect CYP21A2 deletions, CYP21A1P/CYP21A2 chimeric genes, and also CYP21A2 duplications.

Materials and Methods


DNA samples from 24 persons were used for the development of the quantitative method. Seven of these individuals were carriers of 2 copies of the CYP21A2 gene, 5 were carriers of only 1 copy of the gene, 2 were carriers of the G110de18nt deleterious mutation (c.331 338de18 Gen-Bank Ref. ID NM000500, according to the Human Genome Variation Society), 7 were carriers of 3 copies, 2 had no copies, and 1 was homozygous for the G110de18nt mutation. Once the method was developed and the criteria to assess gene copy numbers were established, we confirmed the method in an independent set of samples from 24 persons randomly selected from samples sent to our laboratory because 21OHD was suspected. In all cases, DNA was extracted from peripheral blood leukocytes by standard procedures. All study participants gave informed consent, and the procedures were approved by our institutions ethics committee.


Sequencing and Southern blot. The CYP21A2 gene sequencing and the Southern blot for the 24 samples used to develop the assay were carried out as previously described (26) and enabled the assessment of the CYP21A2 copy number. Thus, the presence of 2 copies of the CYP21A2 gene was assumed for individuals who were compound heterozygous for mutations or polymorphisms in the gene.

Southern blot analysis revealed that 5 individuals had only 1 copy of the CYP21A2 gene, due to gene deletion or to CYP21A2/CYP21A1P chimeric gene formation. Direct PCR product sequencing revealed the presence of the G110de18nt mutation in 2 heterozygous individuals. These 2 samples were further grouped with the samples with 1 gene copy because the primer (P3S) used for the gPCR annealed to the wild-type gene at the 110-codon stretch. It is important to point out that the Southern blot pattern of these 2 samples would be normal and different from CYP21A2 gene deletions and CYP21A1P/CYP21A2 chimeric genes.

The absence of CYP21A2 gene copies due to homozygous gene deletion was assessed by Southern blot analysis in 2 individuals. The presence of a homozygous G110de18nt mutation was determined in 1 individual by gene sequencing. Like the samples described above, this sample would show a normal Southern blot pattern, but it was included in the group of no CYP21A2 copies.

Finally, the presence of 3 copies of the gene was determined by direct sequencing in 2 individuals: 1 individual was a carrier of the 12G, IIe172Asn, and G1n318X mutations and possessed 3 different alleles at the -13 position in intron 2 (SNP rs6467 A/C plus the G mutation); in the 2nd individual the presence of a 3rd copy of the gene was demonstrated by the presence of heterozygosis along the 1st PCR product, despite the presence of Ile236Asn, Va1237G1u, and Met239Lys mutations, which prevented the antisense primer from binding on this allele. Results for both of these individuals were confirmed by Southern blot. A 3rd individual, identified from a prenatal diagnosis sample, had 3 copies of the CYP21A2 gene and was a compound heterozygous female with 2 severe mutations: G1n318X inherited from her mother and G1y291Ser inherited from her father. The duplication of the gene in 1 allele was suspected because of an unbalanced allele ratio in the sequencing electropherogram at the G1n318X mutation in the mother DNA as well as in the fetus. Gene duplication in 1 allele was later confirmed by the finding that 17-OH progesterone concentrations were within the reference interval in the amniotic fluid and by fetal echography showing ordinary female external genitalia. Four more individuals with 3 copies of the gene were characterized by gene sequencing. All of these individuals displayed the unbalanced G1n318X mutation in the electropherogram. This mutation was present in a particular haplotype, which was also present in the other 2 individuals with 3 copies; this haplotype presented 2 uncommon polymorphisms, a G>A change in intron 2 at position -79 and a C>T change at nucleotide 13 in the 3' UTR region of the gene.


PCR primers and probes. For the quantification of the CYP21A2 gene copy number, we amplified a fragment of 132 by in size that extended from exon 3 to intron 3. The forward primer used for this amplification was 5'-ACC TGT CCT TGG GAG ACT AC-3' (P3S), which allowed the specific amplification of the CYP21A2 gene, because it annealed to a segment of exon 3 when the G110de18nt mutation was not present. The reverse primer was 5'-TTA CCT CAC AGA ACT CCT GGG T-3' (BI3A). The MGB-TagMan [R] probe (Applied Biosystems) used for the quantification was designed 7 by away from the end of the P3S primer, and the sequence was 5'-TCT GGA AAG CCC ACA-3', with FAM dye at the 5' end as reporter. The quantification has been assessed as relative to the DSP gene in a duplex gPCR. To obtain greater similarity among both PCRS, we followed the same criteria for the DSP primers and probe design as for the CYP21A2 gene. Accordingly, we amplified a fragment of 132 by in size, extending along exon 2 of the DSP gene, by use of the forward primer 5'-TGC CTC CTT AGT CAA ACC GG-3' (DSP-S) and the reverse 5'-GGA ACA GGG AAA GCT TAC AGG-3' (DSP-A). The sequence of the MGB-TagMan probe (Applied Biosystems) for the DSP gene was 5'-TCC AGG CAC CAG AA-3' with VIC dye as reporter.


The 2 fragments described above were simultaneously amplified in the same well in a 25-[micro]L reaction containing 1 X TagMan Universal PCR Master Mix, No AmpErase [R] UNG (Applied Biosystems), 900 nmol/L of each CYP21A2 primer, 1080 nmol/L of each DSP primer, 250 nmol/L of each probe, and 50-200 ng of DNA. The reaction was subjected to 95 [degrees]C for 10 min, followed by 40 cycles of 95 [degrees]C for 15 s and 60 [degrees]C for 1 min in an ABI7300 Real-Time PCR System (Applied Biosystems). Data were analyzed with the 7300 System SDS Software (Version


The efficiencies of the CYP21A2 and DSP PCRS were assessed with the dilution series method (27), in which threshold cycle (Ct) values were plotted against the logarithm of the input amount of DNA. The duplex PCBs previously described were carried out in triplicate with 8 serial dilutions, from 1:1 to 1:500, starting with 250 ng of template DNA. Then we performed calibration curves by plotting the Ct values against the logarithms of input DNA. Slope values from -3.90 to -3.20 were considered adequate because they correlated with 80%-105% PCR efficiencies. The efficiency was calculated for templates with 3, 2, and 1 CYP21A2 copy.


To assess the CYP21A2 copy number, the difference in Ct ([Delta]Ct) for CYP21A2 and DSP genes was calculated and plotted against the logarithm of the CYP21A2:DSP ratios (%) (ratio of CYP21A2 and DSP copy number). The resulting curves obtained through linear regression analysis were used to calculate the CYP21A2 copy number.


The SD and the CV (%) of [Delta]Ct values and the CYP21A2: DSP ratios obtained were used to determine the variability of the method. For intraassay variation, the 24 samples used for the method development were run in duplicate in the same plate; and for the intraassay variation, the plates were run on 3 different days.

The variation due to the amount of DNA used in the assay was assessed from [Delta]Cts and CYP21A2:DSP ratios obtained with 50 ng, 100 ng, and 200 ng of input DNA for the 3 groups of samples: 1, 2, and 3 gene copies.


The linear regression, the ANOVA test, and data plots were performed with the SPSS version 12.0 for Windows and Instant version 3.33 programs.



The efficiency rates for the CYP21A2 gene PCR were 81%, 84%, and 99% for templates with 3, 2, and 1 gene copy, respectively, and for the DSP gene PCR were 84% for templates with 3 and 2 CYP21A2 gene copies and 95% for templates with 1 copy (Fig. 1). These results were obtained with dye fluorescence threshold values of 0.055 and 0.05 for CYP21A2 and DSP genes, respectively, and an automatic baseline. Regression linear analysis for [Delta]Ct values and the logarithm of the input amount of DNA showed slopes <0.1 for all the templates, indicating that the efficiencies for both genes were very similar.


The mean (SD) [Delta]Ct obtained, taking into account the results of all the plates with 100 ng of input DNA, were -1.91 (0.20) for samples with 3 CYP21A2 gene copies, -0.86 (0.17) for samples with 2 copies, and +0.80 (0.29) for samples with 1 copy. Similar results were obtained with 50 ng and 200 ng of DNA (Table 1). The ANOVA tests showed no significant differences in [Delta]Ct values between samples with the same number of copies; these differences became significant, however, when we compared [Delta]Ct values for samples with a different number of copies (P <0.001) for 100 ng of template DNA, as well as for 50 ng and 200 ng (Fig. 2).


Based on the results of the duplex PCR described in Materials and Methods, we considered samples with amplification of the DSP gene but without amplification of the CYP21A2 gene to be deficient in functional CYP21A2 genes. This result was found in the 2 samples homozygous for the gene deletion and in the sample homozygous for the G110de18nt mutation. The samples with 1 or more CYP21A2 copies showed amplification for both genes, and we inferred the number of copies, as described in Materials and Methods (see Fig. 1 in the Data Supplement that accompanies the online version of this article at The mean (SD) CYP21A2:DSP ratios, taking into account the results of all the plates with 100 ng of sample DNA, were 1.45 (0.11) for samples with 3 CYP21A2 copies, 1.05 (0.07) for samples with 2 copies, and 0.52 (0.06) for samples with 1 copy (Table 1). The ANOVA test, which we applied to the slopes of the curves so that the reproducibility of the method could be assessed, revealed no significant differences. Similar results were obtained with 50 ng and 200 ng of sample DNA (Tables 1 and 2).



Intraassay variation. The [Delta]Ct variability values [mean (SD)] when the method was performed with 100 ng of DNA were 13% [-1.93 (0.25)], 20% [-0.77 (0.16)], and 47% [+0.79 (0.37)], corresponding to 3, 2, and 1 CYP21A2 copy, respectively. These results are similar to those observed for 50 ng of DNA and slightly lower than those observed for 200 ng (data not shown). The 99% Cls were -2.14 to -1.72, -0.90 to -0.64, and +0.48 to +1.10 for 3, 2, and 1 CYP21A2 copy, respectively; these Cls as well as the [Delta]Ct intervals obtained from mean and SD values did not overlap.


The intrassay variation for the CYP21A2:DSP ratios [mean (SD)] were 10% [1.51 (0.15)], 6% [0.97 (0.06)], and 15% [0.54 (0.08)] for samples with 3, 2, and 1 CYP21A2 copy, respectively. The reported data and the 99% Cls, (1.37-1.65), (0.92-1.02), and (0.48-0.60), for the 3 groups were clearly separated as they were when 50 ng and 200 ng of input DNA were used (data not shown).

Interassay variation. The interassay variation obtained from [Delta]Ct values was 10% for samples with 3 CYP21A2 copies, 20% for carriers of 2 copies, and 36% for samples with 1 copy. The mean (SD) [Delta]Ct and the 99% CIs calculated for the different CYP21A2 copy numbers did not overlap. Similar results were found for 50 ng of DNA, and results were slightly higher when 200 ng of DNA was added to the reaction (Table 1).

The interassay variation for the CYP21A2:DSP ratios was 8% for 3, 7% for 2, and 11% for 1 gene copy. Again, the mean (SD) ratios and the 99% CIs calculated in each case did not overlap, as was the case when 50 ng and 200 ng of input DNA were used (Table 1).

Variation induced by the amount of DNA. The variation induced in the [Delta]Ct values by the amount of DNA was 15% for samples with 3 gene copies, 21% for samples with 2 copies, and 39% for samples with 1 gene copy. The variability rates induced in the CYP21A2:DSP ratios were 9%, 7%, and 13% for samples with 3, 2, and 1 copy, respectively (Table 3).


The reported data allow the establishment of the criterion for the assessment of the CYP21A2 copy number. Thus, a sample is diagnosed as having 2 CYP21A2 copies when [Delta]Ct values are between -1.35 and -0.25. A heterozygous chimeric CYP21A1P/CYP21A2 gene or CYP21A2 deletion is determined by [Delta]Ct values between +0.20 and +2.00, and heterozygous duplication is diagnosed by [Delta]Ct values between -2.50 and -1.50. A homozygous chimeric CYP21A1P/CYP21A2 gene or CYP21A2 deletion is assigned by the absence of CYP21A2 gene amplification in the presence of DSP gene amplification. In any case, DSP Ct values have to be <27 for 100 ng and <29 for 50 ng of input DNA.


These 24 additional samples were analyzed in duplicate and showed the following [Delta]Ct values: -1.30 to -0.33 in 21 samples, -2.45 to -2.19 in 2 samples, and +0.77 to +0.85 in 1 sample.


Southern blot analysis has long been the reference method for the detection of large deletions and of CYP21A1P/ CYP21A2 chimeric genes associated with 21OHD. Nevertheless, this method requires large quantities of DNA and is too laborious and time-consuming to be routinely used for 21OHD molecular diagnosis (15-28).

To facilitate diagnosis of gross deletions and chimeric genes, other strategies have been developed. Most of these methods are based on PCR (19-24) or real-time gPCR (25), but most fail to detect CYP21A2 gene duplications (21, 23, 25), a shortcoming that can lead to misidentification of a nonpathological allele with 2 CYP21A2 genes, 1 mutated plus 1 wild type, as a pathological 21OHD allele (12).

Taking into account all the problems and limitations of current methods for 21OHD diagnosis, we developed a new method based on the relative quantification of the CYP21A2 gene using the DSP gene as a reference. The DSP gene was chosen as the reference gene because it is located on chromosome 6, as is CYP21A2, and no variations in its copy number have been described in the Database of Genomic Variation ( variation/). In both cases, primers and probes annealed to a region of the genome where no polymorphisms have been described to date. Any such variation in DSP primers and/or probe DNA sample binding sites that impaired amplification would produce an increase in Ct values of this gene, and the sample would not be considered for analysis; a possible CYP21A2 allele dropout due to the presence of variations under the PCR primers and/or probe would be detected by sequencing. On the other hand, the high specificity of the CYP21A2 gPCR was demonstrated by the absence of CYP21A1P amplification in samples with no CYP21A2 gene copies.

We validated the duplex PCR, confirming by serial dilution method that PCR efficiency was optimal (81%99%) and similar for CYP21A2 and DSP genes for the 3 templates tested (27) (Fig. 1). These results were also confirmed by the independence of [Delta]Ct values of the amount of DNA analyzed; nevertheless, variability was higher with 200 ng than with smaller amounts of sample DNA. Therefore, 50-100 ng of input DNA for the analysis is recommended. After method validation, we used linear regression studies to assess CYP21A2 copy number. The CYP21A2 copy number obtained for each sample tested, expressed as the ratio of the CYP21A2 and DSP gene copies, was in accord with the results obtained by Southern blot and direct sequencing.

Although we initially assessed CYP21A2 copy number through linear regression studies, we later found that estimations of [Delta]Ct values could be used to assess copy number, because the ranges of [Delta]Ct values were clearly separated into 3 groups according to the CYP21A2 gene copy number (Fig. 2). Moreover, intra- and interassay variation analyses showed that these 3 defined groups did not overlap. Therefore, we used [Delta]Ct values directly for the CYP21A2 copy number assessment, avoiding the CYP21A2:DSP ratio calculation and thus facilitating the analysis. In all of the samples, the results obtained in this way were concordant with the results obtained by Southern blot combined with sequencing. The results of the 3 samples without CYP21A2 gene copies were also concordant with Southern blot and sequencing results. The [Delta]Ct values obtained with the 24 additional samples fulfilled the criterion previously established. Therefore we found that the samples could be ascribed to 1 of the 3 defined groups: 21 samples had 2 CYP21A2 gene copies, 2 samples had 3 copies, and 1 sample had a heterozygous gene deletion or chimeric gene. These samples were further analyzed by sequencing and Southern blot when necessary, and the results were concordant.

The main limitation of this method, with respect to the Southern blot, is lack of characterization of the arrangement of CYP21A1P and CYP21A2 genes; however, for 21OHD diagnostic purposes, carrier detection, genetic counseling, and prenatal diagnosis, it is essential to know the exact functional gene copy number that an individual has, whereas detailed knowledge of this arrangement is less important.

In summary, the present method offers an accurate alternative to Southern blot and other methods described previously for 21OHD diagnosis and has the following advantages: (a) it requires only a small amount of DNA (50-100 ng); (b) it is neither time-consuming (2 h) nor laborious, and can analyze many samples simultaneously; and (c) it is effective in the detection of CYP21A2 gene duplication, which is one of its most important advantages (21, 23, 25).

In conclusion, the proposed method is easy to use in a molecular diagnosis laboratory and together with the CYP21A2 gene sequencing might be a definitive way to detect almost all, common as well as rare, 21OHD alleles.

Grant/funding support: L.L. has support from Fondo Invetigaciones Sanitarios del Instituto de Salud Carlos III, and F.D. is supported by grants from RTICCS from Instituto de Salud Carlos III and FEDER.

Financial disclosures: None declared.

Received February 13, 2007; accepted June 19, 2007.

Previously published online at DOI: 10.1373/clinchem.2007.087361

Acknowledgments: We thank Dr. F. Barros, Dr. M. De la Fuente, Dr. A. Ferndndez-Marmiesse, and M. Pastoriza for technical support and Peter Rees for the English language correction.


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[3] Nonstandard abbreviations: 210HD, 21-hydroxylase deficiency; 210H, steroid 21-hydroxylase; gPCR, quantitative PCR; Ct, threshold cycle; [Delta]Ct, difference in Cts between CYP21A2 and DSP genes.

[4] Human genes: CYP21A2, gene encoding steroid 21-hydroxylase enzyme (cytochrome P450, family 21, subfamily A, polypeptide 2). Older nomenclature CYP21, CYP21B; CYP21AIP pseudogene (cytochrome P450, family 21, subfamily A, polypeptide 1 pseudogene). Older nomenclature CYP21P, CYP21A; DSP, gene that encodes desmoplakin protein.


[1] Fundacion Pdblica Gallega de Medicina Genomica (Unidad de Medicina Molecular), Santiago de Compostela, Spain.

[2] Universidad de Santiago de Compostela, Departamento de Fisiologia, Santiago de Compostela, Spain.

* Address correspondence to this author at: Unidad de Medicina Molecular, Fundacion Pdbhca Gallega de Medicina Genomica, Hospital Clinico Universitario, 15706 Santiago de Compostela, Spain. Fax 34-981951473; e-mail
Table 1. OCt values and CYP21A2:DSP ratios obtained from
data of plate replicates. (a)

 [Delta] Ct *
Amount of DNA copy number Mean SD CI (99%)

50 ng 3 -1.85 0.23 (-1.94)-(-1.76)
 2 -0.98 0.14 (-1.04)-(-0.92)
 1 +0.81 0.31 (+0.63)-(+0.93)
100 ng 3 -1.91 0.20 (-1.99)-(-1.83)
 2 -0.86 0.17 (-0.93)-(-0.79)
 1 +0.80 0.29 (+0.67)-(+0.93)
200 ng 3 -2.00 0.35 (-2.14)-(-1.86)
 2 -0.92 0.21 (-1.00)-(-0.84)
 1 +1.08 0.59 (+0.79)-(+1.25)

 CYP21A2:DSP ratio (b)
Amount of DNA copy number Mean SD CI (99%)

50 ng 3 1.45 0.11 1.40-1.50
 2 1.05 0.07 1.02-1.08
 1 0.52 0.06 0.50-0.55
100 ng 3 1.50 0.12 1.45-1.55
 2 0.99 0.06 0.97-1.02
 1 0.52 0.06 0.49-0.55
200 ng 3 1.46 0.14 1.40-1.52
 2 1.00 0.07 0.97-1.03
 1 0.52 0.08 0.49-0.55

(a) Interassay variation is also shown, defined by SD, for ACt values
and CYP21A2:DSP ratios according to the amount of DNA added to the
reaction and to the CYP21A2 copy number.

(b) Ratio of CYP21A2 and DSP gene copy number.

* [Delta] Ct is the difference in threshold cycles between CYP21A2
and DSP genes.

Table 2. Slopes from linear regression studies for plate replicates
using 50, 100, and 200 ng of DNA. (a)

of DNA Replicate B [SD.sub.b]

50 ng 1st 1.857 0.008
 2nd 1.866 0.008
 3rd 1.829 0.009
100 ng 1st 1.842 0.008
 2nd 1.843 0.006
 3rd 1.855 0.008
200 ng 1st 1.813 0.009
 2nd 1.910 0.009
 3rd 1.879 0.008

of DNA Replicate S [SD.sub.s]

50 ng 1st -0.162 0.006
 2nd -0.171 0.006
 3rd -0.167 0.006
100 ng 1st -0.173 0.006
 2nd -0.176 0.004
 3rd -0.167 0.005
200 ng 1st -0.159 0.006
 2nd -0.131 0.006
 3rd -0.149 0.006

of DNA Replicate [r.sup.2] [SD.sub.res]

50 ng 1st 0.960 0.0367
 2nd 0.961 0.0344
 3rd 0.953 0.0399
100 ng 1st 0.958 0.0377
 2nd 0.979 0.0268
 3rd 0.952 0.0351
200 ng 1st 0.944 0.0466
 2nd 0.925 0.0551
 3rd 0.947 0.0460

(a) Intercepts (B), slopes (S), correlation coefficients (r.sup.2)
as well as their SD ([SD.sub.B], [SD.sub.s], and [SD.sub.res],
respectively) were calculated for each amount of DNA.

Table 3. Variability in CYP21A2 copy number related to the amount
of DNA added to the reaction.

 [Delta] Ct *
copy number Mean SD CI (99%)

3 -1.91 0.28 (-1.98)-(-1.84)
2 -0.92 0.20 (-0.97)-(-0.87)
1 -0.92 0.36 (+0.83)-(+1.01)

 CYP21A2:DSP ratio (b)
copy number Mean SD CI (99%)

3 1.47 0.13 1.44-1.50
2 1.01 0.07 0.99-1.03
1 0.52 0.07 0.50-0.54
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Title Annotation:Molecular Diagnostics and Genetics
Author:Parajes, Silvia; Quinterio, Celsa; Dominguez, Fernando; Loidi, Lourdes
Publication:Clinical Chemistry
Date:Sep 1, 2007
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