Presymptomatic Diagnosis of Spinal Muscular Atrophy Through Newborn Screening

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Presymptomatic Diagnosis of Spinal Muscular Atrophy Through Newborn Screening Yin-Hsiu Chien, MD, PhD1,2, Shu-Chuan Chiang, MS1, Wen-Chin Weng, MD, PhD2, Ni-Chung Lee, MD, PhD1,2, Ching-Jie Lin, MS1, Wu-Shiun Hsieh, MD2, Wang-Tso Lee, MD, PhD2, Yuh-Jyh Jong, MD, PhD3,4,5, Tsang-Ming Ko, MD6, and Wuh-Liang Hwu, MD, PhD1,2 Objective To demonstrate the feasibility of presymptomatic diagnosis of spinal muscular atrophy (SMA) through newborn screening (NBS). Study design We performed a screening trial to assess all newborns who underwent routine newborn metabolic screening at the National Taiwan University Hospital newborn screening center between November 2014 and September 2016. A real-time polymerase chain reaction (RT-PCR) genotyping assay for the SMN1/SMN2 intron 7 c.888+100A/G polymorphism was performed to detect homozygous SMN1 deletion using dried blood spot (DBS) samples. Then the exon 7 c.840C>T mutation and SMN2 copy number were determined by both droplet digital PCR (ddPCR) using the original screening DBS and multiplex ligation-dependent probe amplification (MLPA) using a whole blood sample. Results Of the 120 267 newborns, 15 tested positive according to the RT-PCR assay. The DBS ddPCR assay excluded 8 false-positives, and the other 7 patients were confirmed by the MLPA assay. Inclusion of the secondtier DBS ddPCR screening assay resulted in a positive prediction value of 100%. The incidence of SMA was 1 in 17 181 (95% CI, 1 in 8323 to 1 in 35 468). Two of the 3 patients with 2 copies of SMN2 and all 4 patients with 3 or 4 copies of SMN2 were asymptomatic at the time of diagnosis. Five of the 8 false-positives were caused by intragenic recombination between SMN1 and SMN2. Conclusion Newborn screening can detect patients affected by SMA before symptom onset and enable early therapeutic intervention. A combination of a RT-PCR and a second-tier ddPCR can accurately diagnose SMA from DBS samples with no false-positives. (J Pediatr 2017;■■:■■-■■). Trial registration ClinicalTrials.gov NCT02123186.

See editorial, p ••• and related article, p •••

S

pinal muscular atrophy (SMA), an autosomal recessive neurodegenerative disorder caused by deletions or mutations in the survival motor neuron 1 (SMN1) gene, has a reported incidence of approximately 1 in 6000-10 000 live births.1,2 SMA causes the degeneration and loss of alpha motor neurons in the anterior horn of the spinal cord, which leads to progressive muscle weakness and to respiratory failure and death in severe cases.3 Patients with type I SMA (SMA I; OMIM 253300) manifest symptoms at a mean age of 2.5 months but are typically diagnosed at age 6.3 months4; the patients progress to respiratory failure and die before age 2 years if supportive measures are not initiated.5-7 Nutrition and respiratory support prolong patient survival only marginally.8 New therapies for SMA have emerged.9 A total of 28 participants were enrolled in a Phase 1 trial in which the intrathecal administration of an antisense oligonucleotide (nusinersen) led to a significant increase in motor scores in the From the 1Department of Medical Genetics; 2Department of Pediatrics, National Taiwan University Hospital, Taipei, high-dose group,10 and a vector-based gene therapy clinical trial also has been Taiwan; 3Department of Biological Science and

DBS ddPCR MLPA NBS Rn RT-PCR SCID SMA SMN1 SMN2

Dried blood spot Droplet digital polymerase chain reaction Multiplex ligation-dependent probe amplification Newborn screening Normalized reporter fluorescence intensity Real-time polymerase chain reaction Severe combined immunodeficiency Spinal muscular atrophy Survival motor neuron 1 gene Survival motor neuron 2 gene

Technology, College of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan; 4Graduate Institute of Clinical Medicine, College of Medicine, Kaohsiung Medical University; 5Department of Pediatrics and Laboratory Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan; and 6 Genephile Bioscience Laboratory, Ko’s Obstetrics and Gynecology, Taipei, Taiwan Sponsored by a research grant from Biogen. Y.J.J., W.L.H., and Y.H.C. have received grants and serve on an advisory board for Biogen. W.T.L. serves on an advisory board for Biogen. The other authors declare no conflicts of interest. 0022-3476/$ - see front matter. © 2017 Elsevier Inc. All rights reserved. http://dx.doi.org10.1016/j.jpeds.2017.06.042

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THE JOURNAL OF PEDIATRICS • www.jpeds.com launched. Recently, nusinersen (Spinraza) was approved by the Food and Drug Administration for treating SMA. However, treatments are not initiated until the clinical diagnosis is made, at which point a significant portion of the motor neurons most likely are already lost.11 Rapid and irreversible loss of motor neurons begins during the first 3 months of life, and 95% of motor neurons are lost before age 6 months in patients with SMA I.11 Thus, a diagnosis of SMA in presymptomatic patients through newborn screening (NBS) could lead to better therapeutic effects.12 Humans possess 2 nearly identical SMN genes, SMN1 and SMN2. SMN2 differs from SMN1 by only 5 nucleotides, including c.840C>T, c.888+100A>G, and c.1155G>A. The exon 7 c.840C>T mutation in SMN2 results in skipping exon 7 in 90% of the splicing products. Because 95% of SMA cases are attributed to the homozygous deletion of SMN1,13-15 most diagnostic assays are designed to detect this deletion. In additionally, higher SMN2 copy numbers in patients with SMA are associated with a later onset and milder disease.16,17 Therefore, determining the SMN2 copy number is essential to provide the most informative prognosis. Owing to the new demand for newborn screening for SMA, we used a real-time polymerase chain reaction (RT-PCR) assay in a pilot SMA newborn screening trial. We had applied a similar method in our NBS program for severe combined immunodeficiency (SCID) using RT-PCR to measure the T-cell receptor excision circle copy number.18 Although several assays for SMA screening using RT-PCR,19 post-PCR high-resolution melting analysis,20 and liquid bead arrays2 have been applied to dried blood spot (DBS) samples, none of these assays has been tested in a NBS program to assess their reproducibility, false-positive rates, and false-negative rates. Here we show that the absence of homozygous SMN1 can be reliably detected from the same DBS samples used for SCID screening.

Methods This SMA pilot screening trial was integrated prospectively into the workflow of the National Taiwan University Hospital Newborn Screening Center, which routinely screens 35%37% of the newborns born in Taiwan. Newborns tested in our center in a consecutive series were eligible to participate in this study with parental consent for SMA screening. Infants from parents who had undergone SMA carrier testing were not rejected from the study. The hospital’s Institutional Review Board approved this study (201308058RIN; ClinicalTrials.gov: NCT02123186). Based on a predefined incidence of SMA of ~1:10 00021 and at least 6 affected patients identified through this screening program, the sample size for screening was estimated to be 100 000 with a 95% probability of occurrence. We performed a DNA-based method to detect the absence of SMN1 in newborns using an RT-PCR TaqMan SNP genotyping assay on a StepOnePlus RT-PCR 96-Well System (Applied Biosystems, Foster City, California). In brief, DNA was extracted from a 3.2-mm punch from each DBS sample.18 The assay mixture included TaqMan Genotyping Master Mix (Applied Biosystems), primers, 22 and probes targeting

Volume ■■ c.888+100A (5’-FAM-CAGATGTTAAAAAGTTG-3’ MGB) and c.888+100G (5’-VIC-CAGATGTTAGAAAGTTG-3’ MGB). The quality controls in each 96-well plate included a water blank, a filter paper blank, and 3 DNA samples with known SMN1:SMN2 copy numbers: 0:2 (affected), 1:2 (carrier), and 2:2 (normal). The RNase P copy number, which was derived from the SCID screening assay,18 was used to assess DNA quality. This screening method does not detect SMN1 point mutations. The results were interpreted using the gene amplification efficiency, which is represented by DRn. Rn (the normalized reporter fluorescence intensity) was obtained from each assay, and DRn was the difference between the Rn at the end point and the starting points. Samples with an SMN1 DRn lower than the cutoff (indicating no amplification of SMN1) but with a normal SMN2 DRn or a normal RNase P copy number were defined as abnormal. Samples with a low SMN1 DRn but both a low SMN2 DRn and a low RNase P copy number were defined as unsatisfactory samples, and a repeat DNA extraction and RT-PCR assay were performed. The cutoffs were defined based on DRn values of known patients and normal newborns. The positive screening results were confirmed by both a droplet digital PCR (ddPCR) assay (Bio-Rad, Hercules, California) 23 using the original screening DBS sample and a multiplex ligation-dependent probe amplification (MLPA) assay24 using DNA extracted from recalled whole blood samples. MLPA has been shown to be a versatile and fast technique for determining different nucleic acid sequences in a single reaction.25 A homozygous deletion of SMN1 exon 7 (with or without deletion of exon 8) confirmed the diagnosis of SMN-associated SMA (5q SMA).26 Affected patients with 3 or fewer copies of SMN2 were followed by a pediatric neurologist. Patients with 4 or more copies of SMN2 were advised to consult a neuromuscular expert if signs of muscle weakness occurred. Genetic counseling was provided to the parents to explain the inheritance, risk of recurrence, and future birth plan options for SMA. To study the etiology of the false-positives, we used allelespecific PCR to amplify the c.840C- or c.840T-led SMN1/ SMN2 exon 7 to exon 8 sequences. The amplicons from falsepositive screening cases were sequenced to determine the nucleotide variations. To study the incidence of SMN1/ SMN2 hybrid genes, we performed an RT-PCR genotyping assay targeting c.888+100 using the amplified DNA fragments. The occurrence of SMN1/SMN2 hybrid genes was confirmed by Sanger sequencing.

Results The screening method was validated by testing 2937 anonymous newborn DBS samples and 9 DNA samples with known SMN1 and SMN2 copy numbers. SMA carriers (with 1 copy of SMN1 and variable copies of SMN2) and patients (zero copies of SMN1) could be separated by plotting the DRn of SMN1 (y-axis) against the DRn of SMN2 (x-axis) (Figure 1). The mean SMN1 DRn of the 2937 samples was 2.64 ± 0.39 (range, 1.09-4.17), and the SMN1 DRn values of the patients

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Figure 1. The c.888+100 RT-PCR genotyping assay results. A, Genotyping assay using purified DNA with known SMN1 and SMN2 copy numbers. B, Results from a typical 96-well SMA newborn screening assay. The green dots indicate samples with 1 or more copies each of SMN1 and SMN2. The blue dots indicate samples with zero copies of SMN2. The red dots indicate positive control DNA samples with zero copies of SMN1. The remaining area contains unsatisfactory samples with low DRn values for both SMN1 and SMN2, as no template control (NTC), represented by the dark dot. The cutoffs were <1 for SMN1 and <0.5 for SMN2.

with SMA were close to 0. The cutoff for screening was set arbitrarily at SMN1 DRn <1. Seventy-seven additional DNA samples with a known SMA affected status were tested using this method, and the results were perfectly matched, making the sensitivity and specificity both 100%. A total of 120 267 newborns were screened from November 2014 to September 2016 (Figure 2). The analysis was completed for more than 98% of the samples within 3 calendar days after arrival of the sample at our center. Fifty samples revealed unsatisfactory results, and a repeat DNA extraction and RT-PCR assay excluded SMA. Fifteen samples had a positive RT-PCR screening result (Figure 3; available at www.jpeds.com). The mean SMN1 DRn of the 120 267 samples was 2.65 ± 0.41 (range, 1.01-4.59), and the SMN1 DRn values of the 15 RTPCR screen-positives ranged from 0.11 to 0.33. A ddPCR assay using the original NBS DBS revealed that 7 patients had a homozygous deletion of SMN1, whereas the other 8 cases had 1 copy of SMN1 (Figure 2); the ddPCR was used to determine the patients’ SMN2 copy numbers. Using recalled whole blood samples, the MLPA assay revealed exactly the same SMN1 and SMN2 copy numbers as the ddPCR assay (Table I); therefore, the accuracy of this screening program was 100%. The incidence of SMA in the present study was 1 in 17 181 cases (95% CI, 1 in 8323 to 1 in 35 468). The specificity of the RTPCR screening was 99.99%, and the positive prediction rate by RT-PCR only was 47%. Adding ddPCR as a second-tier assay eliminated the false-positive screening results, and the positive predictive value was 100%. The sensitivity of SMA NBS using the RT-PCR method was estimated as 95%, because this method detected only the absence of homozygous SMN1, although we were not aware of any patients with false-negative screening results during this period.

Among the 7 patients, 3 (patients 3, 4, and 6) had 2 SMN2 copies, 2 (patients 2 and 7) had 3 SMN2 copies, and 2 (patients 1 and 5) had 4 SMN2 copies (Table I). All of these patients’ parents were informed of the screening results, and all patients were brought to the doctor by age 11 days (Table II). Among the 3 patients with 2 SMN2 copies, patient 3 had symptoms at birth and patients 4 and 6 had no symptoms at the time of diagnosis but were seen to have decreased muscle power during the follow-up period (Table II). The parents of patient 4 refused investigational treatment, and patient 6 participated in a clinical trial at age 3 weeks. Among the 2 patients with 3 SMN2 copies, patient 2 was noted to have a motor development delay at age 1 year. The 2 patients with 4 SMN2 copies were normal at the latest follow-up visits. False-Positive Screening Cases In the 8 false-positive screening cases, the screening RT-PCR genotyping assay revealed the absence of homozygous SMN1 at c.888+100, but the ddPCR assay revealed the presence of 1 copy of SMN1 at c.840; none of these 8 patients was affected. We next amplified the SMN1-specific sequences from these 8 patients using c.840C-based primers. We found a c.888+102A>C mutation in 2 patients. This nucleotide change prevents the annealing of the RT-PCR probe and causes false-positives. One patient had a relatively low SMN2 DRn and RNase P copy number but no nucleotide changes in the SMN1-specific sequence. In the other 5 patients, c.840C was followed by c.888+100G and c.1155A (ie, the 3′ portion after c.840 of SMN1 was converted to SMN2) (Figure 4; available at www.jpeds.com). We also performed allele-specific PCR followed by the c.888+100 genotyping assay on 1463 anonymous DBS samples

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Figure 2. The workflow and outcomes of the participants throughout the study.

Table I. Newborn screening results for spinal muscular atrophy Cases Patient 1 2 3 4 5 6 7 False positive at first screen 1 2† 3† 4† 5† 6† 7‡ 8‡

Sex

SMN1 (c.888+100A) RT-PCR DRn

SMN1:SMN2 copies by ddPCR

SMN1:SMN2 copies by MLPA

M M F F F M M

0.24 0.16 0.17 0.11 0.29 0.29 0.11

0:4 0:3 0:2 0:2 0:4 0:2 0:3

0:4 0:3 0:2 0:2 0:4 0:2 0:3

F M* M M* M F M F

0.33 0.23 0.22 0.26 0.25 0.19 0.25 0.15

1:2 1:1 1:2 1:1 1:2 1:3 1:3 1:3

1:2 1:1 1:2 1:1 1:2 1:3 1:3 1:3

DRn, difference in the reporter and baseline fluorescence intensities; ddPCR, droplet digital PCR; MLPA, multiplex ligation-dependent probe amplification; RT-PCR, real-time polymerase chain reaction. *Siblings. †SMN1 hybrid. ‡SMN1 c.888+102A>C mutation.

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Table II. Outcomes of patients identified by newborn screening SMN2 copy number

Age at diagnosis, d

1 2

4 3

11 4

3

2

4

4 5 6 7

2 4 2 3

7 11 8 11

Patients

Condition at diagnosis Normal Normal but with imperforate anus Poor sucking and swallowing, no movement, on ventilator Normal Normal Normal Normal

to estimate the incidence of hybrid genes. We found that 5 DBS samples had a heterozygous SMN1 hybrid gene (defined by c.840C, c.888+100G, and c.1155A), and another 5 DBS samples had a heterozygous SMN2 hybrid gene (defined by c.840T, c.888+100A, and c.1155G) (Figure 4). Therefore, the allele frequencies of the SMN1 and SMN2 hybrid genes were both 1:585.

Discussion The present SMA pilot NBS trial successfully detected patients with SMA shortly after birth. Six of the 7 patients that we detected were asymptomatic at the time of diagnosis, but 3 patients developed symptoms during the follow-up period. Because a significant portion of motor neurons may be lost before symptoms appear,11 presymptomatic diagnosis and initiation of treatment before symptom onset is an important goal to achieve the best therapeutic effects.12 The incidence of SMA in the current NBS program was 1:17 181, whereas the incidence of SMA in a carrier screening program in Taiwan was approximately 1:9000.21 We are not aware of any patients during this period who were screen falsenegatives, although we will continue to monitor for missed cases of late-onset SMA. All patients diagnosed with symptomatic SMA are requested to register with the rare disease registration system maintained by the National Health Authority before they can use the resources covered by the Rare Disease Act and national health insurance in Taiwan. The discrepancy in incidence that we found could be due to chance, given that our determination of incidence and that of the previous carrier screening program lie within the confidence interval of our point estimate. The difference could be real and perhaps due to the utility of SMA carrier screening in the general population in Taiwan. A portion of carrier testing-negative parents still participated in the SMA NBS, whereas the number of terminated affected fetuses from the carrier test-positive parents was unknown. NBS for SMA is still valuable in such conditions due to false-negative carrier testing resulting from the occurrence of 2 copies of the SMN1 gene on chromosome 5. In the present study, we used a RT-PCR assay to detect the SMN1 homozygous deletion. Other assays, such as highresolution melting analysis, also can be used to measure SMN1 and SMN2 copy numbers.20 However, RT-PCR assays are

Age at latest visit, mo 25 23

Condition at latest visit

3

Normal Walk with bilateral support at 13 mo, tongue fasciculation since 13 mo, unable to walk at 17 mo Died at age 3 mo

1.5 2.5 8 6

Unable to kick since 1 month of age Normal (older brother has SMA III diagnosed at age 2.5 y) On trial treatment at age 3 wk, decrease in muscle power at 3 wk Normal, on trial treatment since age 1.5 mo

widely used in NBS for SCID, and RT-PCR assays can be multiplexed to reduce screening costs.22 In the present study, we used an RT-PCR genotyping assay that targeted the c.888+100A/G polymorphism and showed that this assay robustly differentiated c.888+100A from c.888+100G. However, we did find false-positive screening results caused by SMN1 hybrids in 5 cases and by probe binding site mutations in 2 cases. The existence of SMN hybrid genes has been described previously,15,27,28 but a false-positive c.888+100 screening test result can be readily excluded using the ddPCR assay as a second-tier test. The cost for our SMA NBS was $3 US per test, including the equipment, reagents, and personnel. The cost per test could be as low as $1.50 if DNA extraction were available from other screening tests, such as SCID NBS. Only 15 samples from the total of ~120 000 samples required the second-tier ddPCR test; therefore, the add-on cost per case from the additional test is very low and can be neglected. Falsenegative screening results caused by a SMN2 hybrid (SMN1 homozygous deletion in the presence of a SMN2 hybrid) also can occur, although the risk is negligible compared with the 5% false-negative results caused by point mutations, which cannot be detected by any current screening method. An RTPCR genotyping assay targeting the c.840C>T mutation is also possible16; however, there are 8 reported single nucleotide polymorphisms flanking c.840, which can interfere with probe binding and cause false-positive screening results in the c.840 RT-PCR assay. The reliability of different c.840 RT-PCR assays in large-scale population screening requires further elucidation. As for the remaining 5% of patients with SMA who cannot be detected by our current NBS methods, complete sequencing of SMN1 will be needed. It may be conducted by next-generation sequencing (NGS)29; however, applying highthroughput NGS to NBS is still impractical. When coupled with a second-tier ddPCR assay, the current SMA newborn screening revealed no false-positive screening results. We do not report the carrier status because the current RT-PCR assay does not detect SMA carriers; however, early diagnosis of some patients with late-onset disease is an ethical concern in NBS. SMA NBS detects all types of SMA, but predicting the age of onset in patients with 4 or more copies of SMN2 is less certain. Therefore, we tried our best not to disturb patients with late-onset SMA and asked them to report only when symptoms appeared. Disease progression in late-onset SMA is slow; thus, initiation of treatment after symptom onset

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THE JOURNAL OF PEDIATRICS • www.jpeds.com is less likely to compromise the outcome. More critical consideration and natural history studies are needed to weigh the potential psychological harm against the potential benefits for those with ≥4 copies of SMN. The current screening method does not detect point mutations in the SMN1 gene that are present in 5% of patients with SMA; this issue also needs to be clarified during the informed consent process. The current pilot program proves that NBS for SMA is feasible. A combination of a RT-PCR genotyping assay and a second-tier ddPCR assay can accurately diagnose SMA from DBS samples with no false-positives. The benefits of a RTPCR assay include the potential to multiplex with the current SCID screening test. Presymptomatic diagnosis of earlyonset SMA offers patients the opportunity to receive timely treatment, but genetic counseling and management of the early diagnosis of late-onset SMA are also important. ■ We thank Chien-Hao Huang (Genephile Bioscience Laboratory) for technical support, Chao Sun and Mei Liu (Biogen) for designing the primers and probe, and John Staropoli (Biogen) for helpful discussions. We also gratefully acknowledge the support from the parents and all physicians and nurses who helped in this program through our newborn screening system. Submitted for publication Feb 3, 2017; last revision received May 15, 2017; accepted Jun 16, 2017 Reprint requests: Wuh-Liang Hwu, MD, PhD. Department of Medical Genetics, National Taiwan University Hospital, 8 Chung-Shan South Road, Taipei 10041, Taiwan. E-mail: [email protected]

References 1. Pearn J. Incidence, prevalence, and gene frequency studies of chronic childhood spinal muscular atrophy. J Med Genet 1978;15:409-13. 2. Prior TW, Snyder PJ, Rink BD, Pearl DK, Pyatt RE, Mihal DC, et al. Newborn and carrier screening for spinal muscular atrophy. Am J Med Genet A 2010;152A:1608-16. 3. Crawford TO, Pardo CA. The neurobiology of childhood spinal muscular atrophy. Neurobiol Dis 1996;3:97-110. 4. Lin CW, Kalb SJ, Yeh WS. Delay in diagnosis of spinal muscular atrophy: a systematic literature review. Pediatr Neurol 2015;53:293-300. 5. Dubowitz V. Chaos in the classification of SMA: a possible resolution. Neuromuscul Disord 1995;5:3-5. 6. Ignatius J. The natural history of severe spinal muscular atrophy– further evidence for clinical subtypes. Neuromuscul Disord 1994;4:5278. 7. Finkel RS, McDermott MP, Kaufmann P, Darras BT, Chung WK, Sproule DM, et al. Observational study of spinal muscular atrophy type I and implications for clinical trials. Neurology 2014;83:810-7. 8. Oskoui M, Levy G, Garland CJ, Gray JM, O’Hagen J, De Vivo DC, et al. The changing natural history of spinal muscular atrophy type 1. Neurology 2007;69:1931-6. 9. Tisdale S, Pellizzoni L. Disease mechanisms and therapeutic approaches in spinal muscular atrophy. J Neurosci 2015;35:8691-700. 10. Chiriboga CA, Swoboda KJ, Darras BT, Iannaccone ST, Montes J, De Vivo DC, et al. Results from a phase 1 study of nusinersen (ISIS-SMN(Rx)) in children with spinal muscular atrophy. Neurology 2016;86:890-7. 11. Swoboda KJ, Prior TW, Scott CB, McNaught TP, Wride MC, Reyna SP, et al. Natural history of denervation in SMA: relation to age, SMN2 copy number, and function. Ann Neurol 2005;57:704-12.

Volume ■■ 12. Phan HC, Taylor JL, Hannon H, Howell R. Newborn screening for spinal muscular atrophy: anticipating an imminent need. Semin Perinatol 2015;39:217-29. 13. Wirth B. An update of the mutation spectrum of the survival motor neuron gene (SMN1) in autosomal recessive spinal muscular atrophy (SMA). Hum Mutat 2000;15:228-37. 14. Mailman MD, Heinz JW, Papp AC, Snyder PJ, Sedra MS, Wirth B, et al. Molecular analysis of spinal muscular atrophy and modification of the phenotype by SMN2. Genet Med 2002;4:20-6. 15. Alias L, Bernal S, Fuentes-Prior P, Barceló MJ, Also E, MartinezHernández R, et al. Mutation update of spinal muscular atrophy in Spain: molecular characterization of 745 unrelated patients and identification of four novel mutations in the SMN1 gene. Hum Genet 2009;125:2939. 16. Feldkötter M, Schwarzer V, Wirth R, Wienker TF, Wirth B. Quantitative analyses of SMN1 and SMN2 based on real-time lightCycler PCR: fast and highly reliable carrier testing and prediction of severity of spinal muscular atrophy. Am J Hum Genet 2002;70:358-68. 17. Chen TH, Tzeng CC, Wang CC, Wu SM, Chang JG, Yang SN, et al. Identification of bidirectional gene conversion between SMN1 and SMN2 by simultaneous analysis of SMN dosage and hybrid genes in a Chinese population. J Neurol Sci 2011;308:83-7. 18. Chien YH, Chiang SC, Chang KL, Yu HH, Lee WI, Tsai LP, et al. Incidence of severe combined immunodeficiency through newborn screening in a Chinese population. J Formos Med Assoc 2015;114:12-6. 19. Pyatt RE, Prior TW. A feasibility study for the newborn screening of spinal muscular atrophy. Genet Med 2006;8:428-37. 20. Dobrowolski SF, Pham HT, Downes FP, Prior TW, Naylor EW, Swoboda KJ. Newborn screening for spinal muscular atrophy by calibrated shortamplicon melt profiling. Clin Chem 2012;58:1033-9. 21. Su YN, Hung CC, Lin SY, Chen FY, Chern JP, Tsai C, et al. Carrier screening for spinal muscular atrophy (SMA) in 107,611 pregnant women during the period 2005-2009: a prospective population-based cohort study. PLoS One 2011;6:e17067. 22. Taylor JL, Lee FK, Yazdanpanah GK, Staropoli JF, Liu M, Carulli JP, et al. Newborn blood spot screening test using multiplexed real-time PCR to simultaneously screen for spinal muscular atrophy and severe combined immunodeficiency. Clin Chem 2015;61:412-9. 23. Zhong Q, Bhattacharya S, Kotsopoulos S, Olson J, Taly V, Griffiths AD, et al. Multiplex digital PCR: breaking the one target per color barrier of quantitative PCR. Lab Chip 2011;11:2167-74. 24. Huang CH, Chang YY, Chen CH, Kuo YS, Hwu WL, Gerdes T, et al. Copy number analysis of survival motor neuron genes by multiplex ligationdependent probe amplification. Genet Med 2007;9:241-8. 25. Passon N, Dubsky de Wittenau G, Jurman I, Radovic S, Bregant E, Molinis C, et al. Quick MLPA test for quantification of SMN1 and SMN2 copy numbers. Mol Cell Probes 2010;24:310-4. 26. van der Steege G, Grootscholten PM, van der Vlies P, Draaijers TG, Osinga J, Cobben JM, et al. PCR-based DNA test to confirm clinical diagnosis of autosomal recessive spinal muscular atrophy. Lancet 1995;345:9856. 27. Cuscó I, Barceló MJ, del Rio E, Martin Y, Hernández-Chico C, Bussaglia E, et al. Characterisation of SMN hybrid genes in Spanish SMA patients: de novo, homozygous and compound heterozygous cases. Hum Genet 2001;108:222-9. 28. Hahnen E, Schönling J, Rudnik-Schöneborn S, Zerres K, Wirth B. Hybrid survival motor neuron genes in patients with autosomal recessive spinal muscular atrophy: new insights into molecular mechanisms responsible for the disease. Am J Hum Genet 1996;59:1057-65. 29. Feng Y, Ge X, Meng L, Scull J, Li J, Tian X, et al. The next generation of population-based spinal muscular atrophy carrier screening: comprehensive pan-ethnic SMN1 copy-number and sequence variant analysis by massively parallel sequencing. Genet Med 2017. doi:10.1038/gim.2016.215. [Epub ahead of print].

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Figure 4. Graphic illustration of the SMN hybrid genes. SMN1 is shown in black, and SMN2 is shown in gray. The polymorphic nucleotides are indicated. The positions of the primers used in the allele-specific PCR are depicted.

Figure 3. Cumulative results of the SMA newborn screening. The screening-positive and screening-negative newborns are grouped separately. The mean SMN1 DRn for the screening-negative newborns was 2.67 ± 0.4 (range, 1.014.59). The box shows the 25th-75th percentiles, and the whiskers represent the 1st-99th percentiles. The cutoff for SMN1 is <1.

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