Assessment of liquid microbead arrays for the screening of newborns for spinal muscular atrophy.
Not all disorders have an associated blood-born phenotypic marker. Spinal muscular atrophy (SMA) has an incidence of approximately 1 in 6000 births (4) and has recently been considered as one of the next generation of disorders to be included in NBS (5, 6). The disease course is characterized by degeneration of anterior horn cells leading to progressive, proximal muscular weakness (7). SMA is grouped into 3 clinical types on the basis of clinical course and the age of onset. SMA type I (MIM no. 25330) is characterized by muscle weakness and hypotonia at birth or within the 1st 3 months. Death from respiratory insufficiency usually occurs within the 1st 2 years of life. Individuals with SMA type II (MIM no. 253550) are able to sit, but they cannot stand or walk unaided. Type III (253400) patients are able to stand and walk, but in adulthood they often lose the ability to walk.
SMA is caused by mutations in the survival motor neuron 1 (SMN1)  gene (MIM no. 600354), with approximately 95% of affected individuals demonstrating a homozygous deletion of SMN1 exon 7 (8). The SMN1 gene is located in a region of chromosome 5 that contains 2 highly homologous copies: SMN 1, telomeric (SMN1), and SMN 2, centromeric (SMN2; MIM no. 601627) (5). SMN1 and SMN2 differ by 5 bases, including a single coding base in exon 7 (840 C>T) that does not alter the amino acid codon (6, 9, 10). Transcription of SMN1, however, results in full-length mRNA, in contrast to SMN2, which generates primarily a deleted species lacking exon 7 and a small amount of full-length transcript (11). SMN2 exon skipping is mediated by the exon 7 single-base change, which has been shown to alter an exon-splicing enhancer site (12).
Because of the alternative splicing, SMN2 does not compensate for the homozygous loss of SMN1. However, several recent studies have shown that the SMN2 copy number influences the severity of the disease (13). The small amount of full-length transcript generated by SMN2 produces a milder phenotype when the copy number of SMN2 is increased. These findings suggest a potential therapeutic strategy for S1VIA: shifting the transcription of SMN2 from the shortened form to the full-length species. However, recent reports suggest the therapeutic window for effective treatment may be short. Swoboda et al. (14) recently reported "severe and substantial postnatal progression of motor denervation ... with progression to generalized hypotonia and quadriparesis over a 1-2 week period." in 3 S1VIA type I infants immediately from birth. The success of the agents currently in clinical trials may depend on identifying individuals as early as possible to begin treatment before irreversible neuronal loss. This early identification could potentially be accomplished through the NBS for S1VIA.
The identification of S1VIA patients during the newborn period can be accomplished only by DNA testing because the disorder has no biochemical markers. Direct DNA testing is the next innovation in NBS (15,16), but currently in the US this testing is used primarily for reflex testing for 1st-tier-positive results. DNA testing would also not be influenced by clinical factors such as premature delivery, maternal nutrition, and illness (18). With its sizeable capacity for multiplexing, array technology has been touted as the application of choice for the 1st-tier analysis of DNA in NBS (17,18). Liquid bead arrays in particular (such as xMAP technology from Luminex) offer fast reaction times through their solution-phase kinetics, reproducibility, and relatively low per-sample costs.
We chose to examine the application of liquid microbead arrays for the NBS of SMA.
Materials and Methods
We used the Luminex array system, on the basis of the fluorescent detection of up to 100 unique polystyrene microspheres in a single reaction (19). Microbeads are dyed internally with a unique combination of red and infrared fluorphores and coupled with a specific oligonucleotide tag. The tag is complimentary to an antitag sequence that has been engineered in the 5' position of allele-specific PCR primers. After the allele-specific reaction and bead hybridization, detection is then carried out using a flow-cytometric system in which microsphere-DNA complexes are passed single file through 2 lasers, one that distinguishes the individual bead on the basis of its distinct spectral designation and another that registers the presence of the allele-specific PCR product associated with it.
Assays were created to identify the approximately 95% of affected individuals with homozygous deletions in exon 7 of the SMN1 gene. Two different chemistries for the Luminex system were evaluated; the Tag-it[TM] detection system and F1ex1VIAP assays from TM Bioscience, and the MultiCode[R]-PLx System from Eragen Biosciences.
All samples were sent to the Molecular Pathology Laboratory at the Ohio State University for S1VIA mutation analysis or carrier detection, and informed consent was obtained for the use of all samples for SMN analysis. All approved procedures for the handling of study participants were performed in compliance with the ethical standards of the Ohio State University.
To compare the sensitivity, specificity, and repeat rate for each of these technologies in an NBS setting, we created a series (20) of 367 blood spots including blood samples from 164 affected individuals, 46 known carriers, and 157 unaffected individuals. In all affected and carrier samples, SMN1 and SMN2 gene copy numbers were independently determined using a previously published quantitative PCR (21). SMN2 copy numbers ranged from 1 to 5 in affected samples and from 1 to 3 in known carriers (Table 1). Homozygous SMN1 exon 7 deletions were also excluded from unaffected samples using the same method.
We spotted 50 [micro]L of whole blood onto blood spot cards, which were then dried overnight at room temperature. DNA was extracted using the Gentra Capture Card Kit (Gentra Systems) into a volume of 50 [micro]L according to the manufacturer's protocol using a Biomek NX Laboratory Automation Workstation (Beckman Coulter). Blood spot cards were then stored at room temperature in the dark. Samples for either protocol were analyzed in 96-well plates containing 92 samples, 2 controls positive for SMN1 exon 7 homozygous deletions, and 2 template blanks. Independent extractions were performed on each sample for both assays investigated.
LIQUID BEAD ARRAYS: TAG-IT PROTOCOL
Multiplex assays were designed based on protocols for previously published reactions from TM Bioscience (22). The initial PCR used previously described primer sequences for SMN exon 7 and cystic fibrosis transmembrane conductance regulator (ATP-binding cassette subfamily C, member 7; CFTR) exon 4 as amplification controls, and reaction conditions with primer concentrations adjusted to 25 nmol/L for SMN and 30 nmol/L for CFTR (21). The reaction program consisted of 95 [degrees]C for 2 min, 45 cycles of 95 [degrees]C for 10 s, 55 [degrees]C for 30 s, 72 [degrees]C for 30 s, and a final extension of 72 [degrees]C for 3 min. PCR products were treated with 2 units shrimp alkaline phosphatase (Roche Diagnostics) and 5 units exonuclease I (USB) at 37 [degrees]C for 30 min followed by 99 [degrees]C for 15 min to inactivate the enzymes. Thermocycling was performed in DNA Engine Tetrad 2 Peltier Thermal Cycler (Bio-Rad Laboratories).
ALLELE-SPECIFIC PRIMER EXTENSION (ASPE)
The multiplex allele-specific primer extensions were carried out using a primer designed to specifically recognize the single base in exon 7, which distinguishes SMN1 from SMN2 and a second primer to an invariant base in exon 4 of the CFTR gene as a control of amplification. The antitag sequence (bold) corresponding to microbead 37 was placed 5' to the allele-specific primer for SMN1 exon 7 (5' CTT TTC ATC TTT TCA TCT TTC AAT GCT ATT TTT TTT AAC TTC CTT TAT TTT CCT TAC AGG GTT TC 3'). The antitag sequence (bold) for microbead 12 was also placed in the same position for the allele-specific primer for CFTR exon 4 (5' TAC ACT TTC TTT CTT TCT TTC TTT GGC CTG TGC AAG GAA GTA TTA CCT TC 3'). Both primers were ordered PAGE purified (Invitrogen).
Allele-specific multiplex reactions consisted of 5 [micro]L of enzyme-treated product, 1x PCR buffer, 25 nmol/L for the SMN1 exon 7 and CFTR allele-specific primers, 1.25 mmol/L Mg[CL.sub.2], 5 [micro]mol/L of dATP, dGTP, dTTP, and biotin-l4-dCTP (Invitrogen), 1 x reaction buffer, and 1.7 units Amplitaq polymerase (Applied Biosystems). The reaction program consisted of 96 [degrees]C for 2 min followed by 40 cycles of 94 [degrees]C for 30 s, 54 [degrees]C for 30 s, and 74 [degrees]C for 30 s.
Bead hybridizations were carried out using 2 [micro]L of ASPE product and approximately 112 beads/[micro]L (2240 beads total) of F1eXMAP microspheres 12 and 37 in standard Luminex sheath fluid (Luminex,). Bead-DNA mixtures were incubated for 99 [degrees]C for 2 min and 30 min at 37 [degrees]C. A reporter solution consisting of 2 [micro]g of Streptavidin R-phycoerythrin conjugate diluted in 1 x sheath fluid was then added to each well and incubated at room temperature for 20 min. Detection was then carried out on a Luminex 200 cytometer running IS software version 2.3 with parameters set to collect a minimum of 100 events for each bead. Median fluorescent intensity (MFI) was recorded for each allele-specific primer.
LIQUID BEAD ARRAYS: MULTICODE-PLX PROTOCOL
An assay to amplify both for SMN1 exon 7 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was developed in conjunction with EraGen Biosciences and reactions were based on standard protocols (23). Multiplex reactions for SMN1 exon 7 and the GAPDH promoter included 2 [micro]L of template, 1 x MC PCR 555 solution (Eragen Biosciences), 50 nmol/L PCR primers (SMN1 exon 7 forward: (isoC)AGC TAT TTT TTT TAA CTT CCT), SMN1 exon 7 reverse: (iso C) TCA TAA TGC TGG CAG ACT TA / GAPDH forward: (isoC) TTT CAT CCA AGC GTG TAA, GAPDH reverse: TGA GAT TGG CCC GAT), and 1 x TITANNM[TM] Taq Polymerase (Clontech) in a final volume of 10 [micro]L. Samples were cycled for 95 [degrees]C for 2 min followed by 30 cycles of 95 [degrees]C 10 s, 55 [degrees]C for 30 s, and 72 [degrees]C for 30 s. Allele-specific amplification was carried out by the addition of 5 [micro]L total to the previous reaction volume containing 1 x MC TSE 555 solution (Eragen Biosciences) and 10 nN1 each primer for SMN1 exon 7 reverse (TAG3-c3-CAC CTT CCT TCT TTT TGA TTT TGT CTG) (Eragen Biosciences), and GAPDH (TAG05-(C3spacer)-GAC ACT AGG GAG TCA AGG A) (Eragen Biosciences). Samples were cycled for 95 [degrees]C for 30 s followed by 5 cycles of 95 [degrees]C for 10 s, and 65 [degrees]C for 2 min, and a final extension of 65 [degrees]C for 5 min. Thermocycling was performed in DNA Engine Tetrad 2 Peltier Thermal Cycler (Bio-Rad Laboratories).
For the bead hybridization, 1 [micro]L of the Fast-Shot 30-bead array (Eragen Biosciences) containing a mixture of microbeads 1-30 diluted in 34 [micro]L of MC Hybridization solution (Eragen Biosciences) were added to each sample and incubated for 10-min in the dark at room temperature. Finally, 2 [micro]g of streptavidin R-phycoerythrin conjugate (Prozyme) in 34 [micro]L of standard Luminex sheath fluid (Luminex) was added to the bead-DNA extension product mixture and incubated in the dark at room temperature for 15 min. Fluorescent signals were then recorded by use of a Luminex 200 cytometer with IS software version 2.3 collecting a minimum of 30 events for each bead, according to the manufacturer's instructions. MFI was again recorded.
Liquid microbead applications for DNA detection on the Luminex system follow the same basic procedure as presented in Fig. 1. The corresponding protocols for each assay were both based on this general strategy but modified in concordance with specific manufacturer recommendations and optimizations for speed while retaining the maximum fluorescent signal for each allele-specific primer.
[FIGURE 1 OMITTED]
The MultiCode-PLx System is based on the use of synthetic nucleobases 2'-deoxy-isoguanosine (iG) and 5-Me-isocytosine (iC), which will specifically base-pair through an altered pattern of hydrogen bonding (24). A DNA strand containing multiple iG/iC bases can be constructed that will preferentially hybridize to strands with complimentary synthetic bases and not natural DNA. These synthetic isobases are reported to increase assay specificity when incorporated into the initial PCR primers, allele-specific primers, and the proprietary tags/antitags for up to 80 microbeads (23). Primers for the initial PCR step were designed flanking SMN exon 7, which owing to sequence homology will amplify exon 7 from both SMN1 and SMN2, and the GAPDH promoter as a template control. Allele-specific extension primers were then directed toward the unique base that defines SMN1 exon 7 (C) and an invariant base in the GAPDH promoter. From postextraction to final detection on the Luminex machine, the MultiCode-PLx protocol required 2.42 h (Fig. 1A).
The F1exMAP design platform is an open-access system that allows users to independently develop multiplex assays using Tag-It technology and 100 different microbeads. PCR primers were taken from a diagnostic test that has been previously reported and involves replication of SMN exon 7 and a portion of the cystic fibrosis gene as an amplification control (21). The allele-specific primer for SMN1 again bound to the unique base in exon 7, and the primer for CFTR was specific for an invariant base at the 3' end of exon 4 (21). The Tag-It protocol requires an extra 60-min postPCR incubation, and the allele-specific reaction is 4 times longer than the corresponding step with the MultiCode-PLx System at 120 min (Fig. 1B). The microbead incubation is also 25 min longer and requires a temperature of 37 [degrees]C (Fig. 1B). This process results in an assay requiring twice as long to process from genomic template to Luminex detection.
Representative results from 96 samples using the MultiCode-PLx are presented in Fig. 2. Each 96-well plate consisted of 92 samples, 2 controls positive for SMN1 exon 7 homozygous deletions, and 2 randomly placed, no-template controls. Cluster analysis of the MultiCode-PLx assay shows 3 distinct collections including the no-template controls, which collect at the origin (Fig. 2). Thirty-nine unaffected (green diamonds) and 14 known carrier samples (yellow squares) were indistinguishable from each other and clustered together, with SMN1 MFI values ranging from 5316 to 9177 and GAPDH MFI values from 4829 to 10 569. Thirty-three affected samples (red triangles) showed clear differentiation from that group and clustered along with the positive controls (purple triangles) with SMN1 MFI values ranging from 298 to 1091 and GAPDH MFI values from 6977 to 10 285. Allele-specific reactions for SMN1 exon 7 were also conducted in the forward direction in each multiplex for further confirmation of all SMN1 genotyping calls (data not shown).
[FIGURE 2 OMITTED]
Samples were targeted for repeat analysis on the basis of either ambiguous clustering or on a lack of concordance in the genotyping calls between the SMN1 forward and reverse reactions. From this representative plate, 6 samples clearly fell outside the defined clusters, including a single affected, 2 known carriers, and 3 unaffected samples (Fig. 2). Analysis of the entire 367 blood-spot series produced a total of 7 (1.9%)specimens for repeat analysis. On repeat testing, a117 demonstrated robust signal intensity and correct genotype clustering from SMN1 forward, SMN1 reverse, and GAPDH primers (data not shown). With the MultiCode-PLx chemistry, we were able to correctly identify the SMN1 exon 7 deletion in all 164 affected samples, thus demonstrating a clinical sensitivity of 100%. Forty-six carriers and 157 unaffected samples were conversely identified as unaffected, thus demonstrating a clinical specificity of 100%.
Fig. 3 shows the comparative cluster analysis from the same data set using the Tag-It chemistry, and 3 distinct clusters are again evident. Known carrier (yellow squares) and unaffected samples (green diamonds) appear indistinguishable, with SMN1 MFI values ranging from 2108 to 4033 and CFTR MFI values from 91620 to 11573. Affected samples (red triangles) and positive controls (purple triangles) clustered together, with SMN1 MFI values ranging from 28.5 to 912 and CFTR MFI values from 9182.5 to 11779.5. Two samples, an unaffected and a carrier, clearly fall outside these clusters in Fig. 3, and a total of 4 (1.1%) were identified for repeat analysis. On reamplification, a114 samples displayed correct genotype clustering for both SMN1 and CFTR primers (data not shown).
[FIGURE 3 OMITTED]
For the 367 blood-spot series, the Tag-It chemistry demonstrated a clinical sensitivity of 100%, with the correct identification of all 164 affected samples. Correct exclusion was also made of all 157 unaffected samples and 45 of 46 of the known carrier samples. A single carrier sample (SMN1 1 copy/SMN2 1 copy) displayed a robust CFTR signal with an MFI of 9798.5 but an SMN1 MFI of only 791 (Fig. 3). This result is within the range of SMN1 values (28.5 to 912) for affected samples in this series (Fig. 3). Consequently, this carrier would have been incorrectly identified as an affected individual, resulting in a single false-positive outcome and a clinical specificity of 99.5%. For genotype confirmation, repeat analysis was conducted in duplicate, and both replicates displayed robust CFTR signals but SMN1 MFI values that were intermediary between affected and unaffected samples (Fig. 4).
Finally, we were concerned that the 99% sequence homology between SMN1 and SMN2 could lead to nonspecific binding of the SMN1 exon 7 allele-specific primer to SMN2. In mildly affected individuals with high SMN2 copy numbers, nonspecific interactions could increase the background SMN1 signal for either chemistry. This increase could lead to either ambiguous or incorrect genotype calls. In affected individuals, any signal detected by the Luminex from the SMN1 exon 7 allele-specific primer should be attributed solely to the nonspecific interaction of that oligonucleotide with the SMN2 gene. Fig. 5 shows the mean SMN1 MFI values for affected individuals with 1-5 copies of SMN2 and the mean MFI values for carriers with only 1 copy of SMN2 processed with the MultiCode-PLx or Tag-It chemistries. No dosage response is evident between SMN2 copy number and mean SMN1 MFI, and the mean background signal for affected samples is consistently 10-fold lower than the mean SMN1 signal from known carriers in both assays. These results demonstrate clear differentiation between affected and unaffected individuals or carriers regardless of SMN2 copy number in both techniques.
[FIGURE 4 OMITTED]
[FIGURE 5 OMITTED]
In conclusion, we have shown that liquid microbead arrays are a successful platform for analysis of SMA in NBS. In the course of development, we modified the MultiCode-PLx and Tag-It reactions from their standard protocol to optimize both chemistries for NBS applications. Although both chemistries are based on the same general protocol, each assay has unique methodological differences that may impact their utility in NBS. Cost (see Table 1 in the online Data Supplement that accompanies this Article at http://www.clinchem.org/vol53/issuell) and time are additional factors for consideration before adopting either assay. With a marginal increase in time and cost, new mutations for other genetic diseases can be easily implemented on the Luminex by the addition of different tagged microbeads in either multiplex reaction.
Regardless of the chemistry, for this blood-spot series both assays displayed similar accuracy with high sensitivity (100% for both chemistries) and specificity (100% for MultiCode-PLx and 99.5% for Tag-It). All positives (approximately 1 of 6000 blood spots) would require confirmation by the standard deletion test (20). Repeat analysis of the single false-positive sample from the Tag-It series displayed a consistent intermediary amplification at SMN1 exon 7, suggesting either an alteration in the template or the presence of an amplification inhibitor. Neither chemistry was influenced by the SMN2 gene, because there was no overlap between samples from affected and unaffected individuals, regardless of copy number. Additionally, carriers with a single deleted allele were not differentiated from unaffected noncarriers. Identification of carriers in NBS is problematic because it removes from an individual their ethical right of choice and necessitates the incorporation of additional resources for appropriate counseling in NBS programs.
A large pilot study examining the utility of liquid microbead arrays in an actual NBS setting (with >500 blood spots per day in the state of Ohio) becomes a necessary follow-up project to this work. By identifying SMA in the newborn period, optimal intervention before the degeneration of motor neurons may be possible. Furthermore, the results from NBS are also important for the child's family, because of the possibility for the prevention of additional cases through genetic counseling and the carrier testing of siblings and additional family members. In the future, DNA analysis from blood spots has the potential to revolutionize and expand NBS programs through technologies such as the liquid arrays validated in this report.
Grant/funding support: This research was supported by the National Institutes of Health Grant 174615.
Financial disclosures: None declared.
Acknowledgments: We would like to recognize the Families of SMA for their support and involvement in this work.
Received May 18, 2007; accepted August 29, 2007. Previously published online at DOI: 10.1373/clinchem.2007.092312
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 Nonstandard abbreviations: MS/MS tandem mass spectrometry; NBS, newborn screening; SMA, spinal muscular atrophy; MFI, median fluorescent intensity.
 Human genes: SMN1, survival of motor neuron 1, telomeric; SMN2, survival of motor neuron 2, centromeric; CFTR, cysfic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7); GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
ROBERT E. PYATT, DAVID C. MIHAL, and THOMAS W. PRIOR *
Department of Pathology, Ohio State University, Columbus, OH.
* Address correspondence to this author at: Department of Pathology, Ohio State University, Hamilton Hall 173, 1645 Neil Ave., Columbus, OH 43210. Fax 614-292-7072; e-mail Thomas.Prior@osumc.edu.
Table 1. Distribution of SMN1 copy number, SMN2 copy number, and total number for the 164 affected and 46 carrier samples in the blood spot series used in this study. SMN1 copy no. SMN2 copy no. Total no. of samples 0 1 4 0 2 34 0 3 94 0 4 30 0 5 2 1 1 10 1 2 32 1 3 4
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|Title Annotation:||Molecular Diagnostics and Genetics|
|Author:||Pyatt, Robert E.; Mihal, David C.; Prior, Thomas W.|
|Date:||Nov 1, 2007|
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