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Technical Briefs |
1
Division of Genetic Disorders, Wadsworth Center, New York State Department of Health, Albany, NY 12201-0509
a author for correspondence: fax 518-402-4989,
Detection of hemoglobinopathies in newborns is critical for the identification of those infants in need of follow-up care [reviewed in Ref. (1)]. For example, infants homozygous for the sickle cell mutation are at greater risk for developing fatal pneumococcal infections and sepsis, which can be prevented by prophylactic antibiotic therapy. At present, the majority of newborn screening for hemoglobin (Hb) variants is done by electrophoresis, isoelectric focusing, or HPLC (2) using Hb extracted from dried blood spots. Nevertheless, detection of adult Hb variants often is complicated by the presence of fetal Hb in neonatal blood. Alternative approaches to these protein-based methodologies have been developed that directly detect the presence of hemoglobinopathy-associated mutations in newborn DNA (3), and some have been adapted to use blood spots (4)(5). Using the S and E mutations in the ß-globin gene as examples, we have developed a multiplexed, high-throughput methodology that uses an array of allele-specific fluorescent beads and the Luminex100 analyzer (Luminex Corporation, Austin, TX; www.luminexcorp.com). This methodology distinguishes between the S and E alleles and their wild-type counterparts, HbA and non-E, of the ß-globin gene in each specimen, making it possible to determine the genotype at each locus.
The methodology is based on the principle that fluorescent microspheres with unique fluorescent profiles, called classifications, can be cross-linked to different analyte-specific reagents and used to create a fluorescence-based array capable of simultaneously assaying multiple analytes in each sample (6). The bead classifications were obtained separately from the Luminex Corporation with surface carboxyl groups for chemical cross-linking to different analyte-specific reagents, which in our studies were 5'-amino-modified oligodeoxynucleotides. As indicated above, each bead classification has a unique spectral address based on its 658 nm/712 nm emission ratio when excited by the 635 nm laser in the Luminex100 instrument. The Luminex software uses this spectral profile to assign beads to their classifications, and each classification occupies a known position on a dot plot of 658 nm vs 712 nm fluorescence. Thus, multiple bead classifications can be combined in one sample, and the Luminex software processes the fluorescent signals to generate an array of bead classifications on the dot plot of 658 nm/712 nm fluorescence. Determination of the amounts of the different analytes bound to each bead classification is accomplished by coincident excitation of the beads with the 532 nm laser in the Luminex100 instrument. Thus, labeling bead-bound analytes with a fluorescent reporter molecule such as phycoerythrin, which emits at 575 nm when excited at 532 nm, produces a third fluorescent signal that allows the amounts of analytes bound to the beads to be quantified. Thus, in each sample, the amounts of multiple analytes can be determined from the emissions of a single fluorescent reporter molecule because the analyte specificity and position of each bead classification in the array is known.
The following oligodeoxyribonucleotides (ODNs) used in our studies were purchased from Oligos Etc, and the positions of the S and E mutations are indicated in bold: 5'-amino-modified 18mer ODNs for coupling to beads were HbA, GCAGACTTCTCCTCAGGA; HbS, GCAGACTTCTCCACAGGA; non-E, CAGGGCCTCACCACCAAC; HbE, CAGGGCCTTACCACCAAC; nonsense (NS), GCATACTCCGTCTCAAGG. 5'-Biotinylated ODNs complementary to HbA, HbS, non-E, HbE, and NS were also used to quality control coupling efficiency of 5'-amino ODNs to the beads.
The five 5'-amino-ODNs were coupled in separate reactions to five different bead classifications. More specifically, 2.5 x 106 beads were coupled to 0.5 nmol of ODN with freshly made 1-ethyl-3(3-dimethylaminopropyl)carbodiimide HCl, using the protocol provided by the Luminex Corporation.
PCR was performed after treatment of 1-mm blood-spot punches with methanol and boiling water as described (5). A 327-bp region of the ß-globin gene between nucleotides 62010 and 62336 of the GenBank sequence (Accession No. U01317.1) was amplified using Qiagen 10x PCR buffer and Taq polymerase with a 5'-biotinylated PCR primer (ACGGCTGTCATCACTTAG) and an unmodified reverse PCR primer (TCCCACATGCCCAGTTTC). Amplification conditions were 5 min at 95 °C and 35 cycles of 95 °C for 30 s, 57 °C for 30 s, and 72 °C for 30 s, followed by extension for 5 min at 72 °C. PCR amplification was verified by agarose gel electrophoresis using an aliquot of each reaction. Both the S and E loci are present in the region of the ß-globin gene that was amplified and are separated by 60 nucleotides.
Hybridization of the biotinylated PCR products to the five different ODN-coupled bead classifications was performed in tetramethylammonium chloride (TMAC) buffer (1x is 3 mol/L TMAC, 1 g/L sodium dodecyl sulfate, 50 mmol/L Tris-HCl, pH 8.0, 4 mmol/L EDTA, pH 8.0) as follows. Duplicate samples containing 5 µL of each PCR were diluted to 17 µL with water and denatured at 98 °C for 10 min. To each sample, 33 µL of 1.5x TMAC containing 5000 of each of the five ODN-coupled bead classifications was added while at 98 °C. The samples were mixed and immediately transferred to a 45 °C heating block. The samples were hybridized at 45 °C for 30 min and diluted to 150 µL with 100 µL of 1x TMAC (45 °C). The beads were pelleted at 8000g for 1 min, and the supernatant was removed. The beads were labeled for 7 min with 50 µL of freshly made 0.5 mg/L streptavidin-phycoerythrin (at 45 °C; Molecular Probes) in 1x TMAC. The samples were again diluted with 100 µL of 1x TMAC (at 45 °C), the beads were pelleted, and the supernatants were removed. Each sample was resuspended in 100 µL of 1x TMAC at 45 °C, and the samples were analyzed using the Luminex100 instrument set in Multiplex Analysis mode to exclude multimers of beads and collect median fluorescent intensities (MFIs) on 100 events per bead classification. In each sample, the background MFI obtained from the NS ODN-coupled beads was subtracted from the MFIs of the four ß-globin allele-specific MFIs, and the mean background-subtracted MFIs were calculated for duplicate samples. The intersample variation of duplicate background values (as MFIs) was 70–130.
To test this genotyping methodology, blood spots from 12 individuals
with known ß-globin genotypes were screened. Representative data from
these experiments are shown in Fig. 1
. From these results, it appears that mean background-subtracted
MFIs <50 can be considered negative. However, further experimentation
is required to confirm this observation. The data show that individuals
1, 2, and 3 were homozygous for both HbA and non-E; individuals 4, 5,
and 6 were HbA/S heterozygotes and homozygous for non-E; individuals 7,
8, and 9 were homozygous for both HbS and non-E; and individuals 10,
11, and 12 were HbA homozygotes and non-E/HbE heterozygotes. These data
also demonstrate an additional advantage of this multiplexed
fluorescent bead array-based approach. Because it is very rare for an
individual to be negative for all four of the ß-globin alleles being
assessed in this study, the majority of samples will yield positive
background-subtracted MFIs for at least one ß-globin allele, which
will also serve as an internal positive control. Those samples negative
for all of the alleles being assessed would most likely represent PCR
failures and require retesting.
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The interexperimental variation for this genotyping methodology was
also assessed. As shown in Table 1
, the standard deviations calculated from the
background-subtracted MFIs from the three experiments did not interfere
with the ability to correctly designate the ß-globin genotype of
these individuals. These data suggest that the results from each
experiment are correct and that the retesting rate should be low.
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In conclusion, we have shown that this multiplexed fluorescent bead array-based genotyping methodology can be used to accurately determine the allelic composition at two ß-globin loci, using dried blood spots from newborns. The array of fluorescent beads is flexible and could be expanded to include additional ß-globin mutations or other clinically important genetic loci. This method would also be adaptable to automation to meet the high throughput requirements of a large newborn screening program.
Acknowledgments
This research was also supported by the Wadsworth Center Genomics Core Facility. We thank Dr. Michelle Caggana (Division of Genetic Disorders, Wadsworth Center) for providing laboratory workspace and access to equipment and supplies.
References
The following articles in journals at HighWire Press have cited this article:
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  Y.-C. Lin, W.-H. Sheng, S.-C. Chang, J.-T. Wang, Y.-C. Chen, R.-J. Wu, K.-C. Hsia, and S.-Y. Li Application of a Microsphere-Based Array for Rapid Identification of Acinetobacter spp. with Distinct Antimicrobial Susceptibilities J. Clin. Microbiol., February 1, 2008; 46(2): 612 - 617. [Abstract] [Full Text] [PDF]
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 B.-K. Ha, R. S. Hussey, and H. R. Boerma Development of SNP Assays for Marker-Assisted Selection of Two Southern Root-Knot Nematode Resistance QTL in Soybean Crop Sci., July 16, 2007; 47(S2): S-73 - S-82. [Abstract] [Full Text] [PDF]
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 S. C. Johnson, D. J. Marshall, G. Harms, C. M. Miller, C. B. Sherrill, E. L. Beaty, S. A. Lederer, E. B. Roesch, G. Madsen, G. L. Hoffman, et al. Multiplexed Genetic Analysis Using an Expanded Genetic Alphabet Clin. Chem., November 1, 2004; 50(11): 2019 - 2027. [Abstract] [Full Text] [PDF]
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S. Faucher, A. Martel, A. Sherring, T. Ding, L. Malloch, J. E. Kim, M. Bergeron, P. Sandstrom, and F. F. Mandy Protein Bead Array for the Detection of HIV-1 Antibodies from Fresh Plasma and Dried-Blood-Spot Specimens Clin. Chem., July 1, 2004; 50(7): 1250 - 1253. [Full Text] [PDF]
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R. Bellisario, R. J. Colinas, and K. A. Pass Simultaneous Measurement of Thyroxine and Thyrotropin from Newborn Dried Blood-Spot Specimens Using a Multiplexed Fluorescent Microsphere Immunoassay Clin. Chem., September 1, 2000; 46(9): 1422 - 1424. [Full Text] [PDF]
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, HbA; 
, HbS; 
, non-E;
, HbE. The dashed line indicates the MFI value below
which samples were considered negative.