{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,12]],"date-time":"2025-10-12T05:51:05Z","timestamp":1760248265345,"version":"build-2065373602"},"reference-count":31,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2023,11,29]],"date-time":"2023-11-29T00:00:00Z","timestamp":1701216000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Ministry of Education, Culture, Sports, Science, and Technology, Japan","award":["23K07279"],"award-info":[{"award-number":["23K07279"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Genes"],"abstract":"<jats:p>The survival motor neuron 2 (SMN2) gene is a recognized modifier gene of spinal muscular atrophy (SMA). However, our knowledge about the role of SMN2\u2014other than its modification of SMA phenotypes\u2014is very limited. Discussions regarding the relationship between homozygous SMN2 deletion and motor neuron diseases, including amyotrophic lateral sclerosis, have been mainly based on retrospective epidemiological studies of the diseases, and the precise relationship remains inconclusive. In the present study, we first estimated that the frequency of homozygous SMN2 deletion was ~1 in 20 in Japan. We then established a real-time polymerase chain reaction (PCR)-based screening method using residual dried blood spots to identify infants with homozygous SMN2 deletion. This method can be applied to a future prospective cohort study to clarify the relationship between homozygous SMN2 deletion and motor neuron diseases. In our real-time PCR experiment, both PCR (low annealing temperatures) and blood (high hematocrit values and low white blood cell counts) conditions were associated with incorrect results (i.e., false negatives and positives). Together, our findings not only help to elucidate the role of SMN2, but also aid in our understanding of the pitfalls of current SMA newborn screening programs for detecting homozygous SMN1 deletions.<\/jats:p>","DOI":"10.3390\/genes14122159","type":"journal-article","created":{"date-parts":[[2023,11,29]],"date-time":"2023-11-29T03:48:24Z","timestamp":1701229704000},"page":"2159","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Real-Time PCR-Based Screening for Homozygous SMN2 Deletion Using Residual Dried Blood Spots"],"prefix":"10.3390","volume":"14","author":[{"given":"Yoshihiro","family":"Bouike","sequence":"first","affiliation":[{"name":"Faculty of Nutrition, Kobe Gakuin University, 518 Arise, Ikawadani-cho, Nishi-ku, Kobe 651-2180, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Makoto","family":"Sakima","sequence":"additional","affiliation":[{"name":"Faculty of Nutrition, Kobe Gakuin University, 518 Arise, Ikawadani-cho, Nishi-ku, Kobe 651-2180, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yuya","family":"Taninishi","sequence":"additional","affiliation":[{"name":"Faculty of Nutrition, Kobe Gakuin University, 518 Arise, Ikawadani-cho, Nishi-ku, Kobe 651-2180, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Takanori","family":"Matsutani","sequence":"additional","affiliation":[{"name":"Division of Physiology, Shinko Hospital, 1-4-47 Wakinohama-cho, Chuo-ku, Kobe 651-0072, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yoriko","family":"Noguchi","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory, Kobe University Hospital, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ryosuke","family":"Bo","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9846-4142","authenticated-orcid":false,"given":"Hiroyuki","family":"Awano","sequence":"additional","affiliation":[{"name":"Organization for Research Initiative and Promotion, Research Initiative Center, Tottori University, 86 Nishi-cho, Yonago 683-8503, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6778-4511","authenticated-orcid":false,"given":"Hisahide","family":"Nishio","sequence":"additional","affiliation":[{"name":"Department of Occupational Therapy, Faculty of Rehabilitation, Kobe Gakuin University, 518 Arise, Ikawadani-cho, Nishi-ku, Kobe 651-2180, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2023,11,29]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Nishio, H., Niba, E.T.E., Saito, T., Okamoto, K., Takeshima, Y., and Awano, H. (2023). Spinal Muscular Atrophy: The Past, Present, and Future of Diagnosis and Treatment. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms241511939"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"155","DOI":"10.1016\/0092-8674(95)90460-3","article-title":"Identification and Characterization of a Spinal Muscular Atrophy-Determining Gene","volume":"80","author":"Lefebvre","year":"1995","journal-title":"Cell"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"479","DOI":"10.1006\/geno.1996.0147","article-title":"Structure and Organization of the Human Survival Motor Neurone (SMN) gene","volume":"32","author":"Lefebvre","year":"1996","journal-title":"Genomics"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1177","DOI":"10.1093\/hmg\/8.7.1177","article-title":"A single nucleotide difference that alters splicing patterns distinguishes the SMA gene SMN1 from the copy gene SMN2","volume":"8","author":"Monani","year":"1999","journal-title":"Hum. Mol. Genet."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"6307","DOI":"10.1073\/pnas.96.11.6307","article-title":"A single nucleotide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy","volume":"96","author":"Lorson","year":"1999","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"259","DOI":"10.1093\/hmg\/9.2.259","article-title":"An exonic enhancer is required for inclusion of an essential exon in the SMA-determining gene SMN","volume":"9","author":"Lorson","year":"2000","journal-title":"Hum. Mol. Genet."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"845","DOI":"10.1093\/hmg\/ddi078","article-title":"SMN\u25b37, the major product of the centromeric survival motor neuron (SMN2) gene, extends survival in mice with spinal muscular atrophy and associates with full-length SMN","volume":"14","author":"Le","year":"2005","journal-title":"Hum. Mol. Genet."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"198","DOI":"10.1002\/ajmg.1386","article-title":"SMN2-Deletion in Childhood-Onset Spinal Muscular Atrophy","volume":"101","author":"Srivastava","year":"2001","journal-title":"Am. J. Med. Genet."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"246","DOI":"10.1002\/ajmg.10354","article-title":"SMN2 Deletion in Childhood-Onset Spinal Muscular Atrophy","volume":"109","author":"Cobben","year":"2002","journal-title":"Am. J. Med. Genet."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"208","DOI":"10.1177\/0883073812445505","article-title":"Homozygous Survival Motor Neuron 2 Gene Deletion and Sporadic Lower Motor Neuron Disease in Children: Case Report and Literature Review","volume":"28","author":"Liping","year":"2013","journal-title":"J. Child. Neurol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"640","DOI":"10.1002\/ana.410430513","article-title":"Association between Centromeric Deletions of the SMN Gene And Sporadic Adult-Onset Lower Motor Neuron Disease","volume":"43","author":"Moulard","year":"1998","journal-title":"Ann. Neurol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"857","DOI":"10.1002\/ana.10378","article-title":"The Relationship of SMN to Amyotrophic Lateral Sclerosis","volume":"52","author":"Crawford","year":"2002","journal-title":"Ann. Neurol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"749","DOI":"10.1212\/WNL.56.6.749","article-title":"Homozygous deletion of the survival motor neuron 2 gene is a prognostic factor in sporadic ALS","volume":"27","author":"Veldink","year":"2001","journal-title":"Neurology"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"290","DOI":"10.1007\/s004150200007","article-title":"Homozygous exon 7 deletion of the SMN centromeric gene (SMN2): A potential susceptibility factor for adult-onset lower motor neuron disease","volume":"249","author":"Tranchant","year":"2002","journal-title":"J. Neurol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"588","DOI":"10.1038\/ejhg.2011.255","article-title":"Homozygous SMN2 deletion is a protective factor in the Swedish ALS population","volume":"20","author":"Corcia","year":"2012","journal-title":"Eur. J. Hum. Genet."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"53","DOI":"10.3349\/ymj.2012.53.1.53","article-title":"Homozygous SMN2 Deletion is a Major Risk Factor among Twenty-Five Korean Sporadic Amyotrophic Lateral Sclerosis Patients","volume":"53","author":"Lee","year":"2012","journal-title":"Yonsei Med. J."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1456","DOI":"10.1212\/01.WNL.0000032496.64510.4E","article-title":"Survival and respiratory decline are not related to homozygous SMN2 deletions in ALS patients","volume":"59","author":"Gamez","year":"2002","journal-title":"Neurology"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"24","DOI":"10.1186\/s13023-017-0671-8","article-title":"Prevalence, incidence and carrier frequency of 5q-linked spinal muscular atrophy\u2014A literature review","volume":"12","author":"Verhaart","year":"2017","journal-title":"Orphanet J. Rare Dis."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Shinohara, M., Niba, E.T.E., Wijaya, Y.O.S., Takayama, I., Mitsuishi, C., Kumasaka, S., Kondo, Y., Takatera, A., Hokuto, I., and Morioka, I. (2019). A Novel System for Spinal Muscular Atrophy Screening in Newborns: Japanese Pilot Study. Int. J. Neonatal Screen, 5.","DOI":"10.3390\/ijns5040041"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"985","DOI":"10.1016\/S0140-6736(95)90732-7","article-title":"PCR-based DNA test to confirm clinical diagnosis of autosomal recessive spinal muscular atrophy","volume":"345","author":"Grootscholten","year":"1995","journal-title":"Lancet"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"358","DOI":"10.1086\/338627","article-title":"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","volume":"70","author":"Schwarzer","year":"2002","journal-title":"Am. J. Hum. Genet."},{"key":"ref_22","unstructured":"Institute of Medicine (US) (2010). Challenges and Opportunities in Using Residual Newborn Screening Samples for Translational Research: Workshop Summary, National Academies Press (US)."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Noguchi, Y., Bo, R., Nishio, H., Matsumoto, H., Matsui, K., Yano, Y., Sugawara, M., Ueda, G., Wijaya, Y.O.S., and Niba, E.T.E. (2022). PCR-Based Screening of Spinal Muscular Atrophy for Newborn Infants in Hyogo Prefecture, Japan. Genes, 13.","DOI":"10.3390\/genes13112110"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"485","DOI":"10.1128\/JCM.39.2.485-493.2001","article-title":"Purification And Characterization of PCR-Inhibitory Components in Blood Cells","volume":"39","year":"2001","journal-title":"J. Clin. Microbiol."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Connelly, C.M., Porter, L.R., and TerMaat, J.R. (2014). PCR amplification of a triple-repeat genetic target directly from whole blood in 15 minutes as a proof-of-principle PCR study for direct sample analysis for a clinically relevant target. BMC Med. Genet., 15.","DOI":"10.1186\/s12881-014-0130-5"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"2569","DOI":"10.1007\/s00216-018-0931-z","article-title":"Inhibition mechanisms of hemoglobin, immunoglobulin G, and whole blood in digital and real-time PCR","volume":"410","author":"Sidstedt","year":"2018","journal-title":"Anal. Bioanal. Chem."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"574","DOI":"10.1016\/j.nmd.2021.03.007","article-title":"Newborn screening programs for spinal muscular atrophy worldwide: Where we stand and where to go","volume":"31","author":"Dangouloff","year":"2021","journal-title":"Neuromuscul. Disord."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"88","DOI":"10.1016\/j.clinbiochem.2011.10.019","article-title":"Spinal muscular atrophy: Clinical validation of a single-tube multiplex real time PCR assay for determination of SMN1 and SMN2 copy numbers","volume":"45","author":"Maranda","year":"2012","journal-title":"Clin. Biochem."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"124","DOI":"10.1016\/j.jpeds.2017.06.042","article-title":"Presymptomatic Diagnosis of Spinal Muscular Atrophy Through Newborn Screening","volume":"190","author":"Chien","year":"2017","journal-title":"J. Pediatr."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1038\/s41431-019-0476-4","article-title":"High-throughput genetic newborn screening for spinal muscular atrophy by rapid nucleic acid extraction from dried blood spots and 384-well qPCR","volume":"28","author":"Czibere","year":"2020","journal-title":"Eur. J. Hum. Genet."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"293","DOI":"10.1089\/gtmb.2020.0312","article-title":"Dried Blood Spot Screening System for Spinal Muscular Atrophy with Allele-Specific Polymerase Chain Reaction and Melting Peak Analysis","volume":"25","author":"Wijaya","year":"2021","journal-title":"Genet. Test. Mol. Biomark."}],"container-title":["Genes"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4425\/14\/12\/2159\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T21:33:20Z","timestamp":1760132000000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4425\/14\/12\/2159"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,11,29]]},"references-count":31,"journal-issue":{"issue":"12","published-online":{"date-parts":[[2023,12]]}},"alternative-id":["genes14122159"],"URL":"https:\/\/doi.org\/10.3390\/genes14122159","relation":{},"ISSN":["2073-4425"],"issn-type":[{"type":"electronic","value":"2073-4425"}],"subject":[],"published":{"date-parts":[[2023,11,29]]}}}