High-throughput screening reveals novel mutations in spinal muscular atrophy patients

High-throughput screening reveals novel mutations in spinal muscular atrophy patients | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research article High-throughput screening reveals novel mutations in spinal muscular atrophy patients Jianbo Shu, Jingrui Wang, Yulian Fang, Zanmei Xu, Xiaowei Wang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.2.12903/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Some spinal muscular atrophy (SMA) cases are caused by either compound heterozygosity with a point mutation in one allele and a deletion in the other or compound heterozygous point mutations in SMN1 or other genes. Methods To explore more genes and mutations in the onset of SMA, 83 whole blood samples were collected from 28 core families of clinically suspected SMA, and multiplex ligation probe amplification (MLPA) was firstly performed with a SALSA MLPA Kit P021 for preliminary diagnosis. Afterwards, the complete gene sequence of SMN1 gene was detected with the high-throughput sequencing platform of Illumina HiSeq-2500 to find more mutations in the 28 core families. Furthermore, 20 SMA patients were selected from the 28 prodands, and 5 non SMA children as controls. The Life Technologies SOLiD™ technology with mate-pair chemistry was utilized to conduct the whole exome high-throughput sequencing. Results MLPA results showed that 22 probands were SMA patients, 3 probands carriers, and 3 probands normal individuals. Moreover, 2 parents from 2 SMA families were with 3 SMN1 exon7 copies. 6 SMN1 single nucleotide variants (SNVs) were identified in the 83 samples, and c.[84C>T], c.[271C>T], c.[-39A>G] and g.[70240639G>C] were novel. Compared with control group, 9102 mutation were selected out in SMA patients. SPTA1 mutation c.[-41_-40insCTCT], FUT5 SNV c.[1001A>G], and MCCC2 SNV c.[-117A>G] were the 3 most frequent mutations in SMA group (95%, 85% and 75%, respectively). Conclusions We identified some mutations in both SMN1 and other genes, and c.[271C>T], c.[-41_-40insCTCT], c.[1001A>G] and c.[-117A>G] might be associated with the onset of SMA. Neurology Molecular Genetics spinal muscular atrophy (SMA) high-throughput sequencing SMN1 onset Background Spinal muscular atrophy (SMA) is an autosomal recessive hereditary disease characterized by degeneration of spinal cord motor neurons, atrophy of skeletal muscles, and generalized weakness ( 1 ). It affects 1 in 10 000 live births, and often leads to early death ( 2 ). SMA manifests over a wide range of severity, affecting infants through adults. According to the onset time and severity of the disease, SMA is divided into 4 types (SMA1, SMA2, SMA3 and SMA4), and SMA 1, with onset before age six months; SMA 2, with onset between age 6 and 18 months; SMA 3, with onset in childhood after age 12 months; and SMA 4, with adult onset ( 3 ). Nusinersen (trade name: Spinraza) is the only approved drug to treat spinal muscular atrophy, which is administered directly to the central nervous system using an intrathecal injection ( 4 ). SMA is caused by homozygous disruption of the survival motor neuron 1 ( SMN1) gene by deletion, conversion, or mutation ( 1 ). Since SMA is one of the most common lethal genetic disorders, with a carrier frequency of 1 in 40 to 1 in 60, direct carrier dosage testing has been beneficial to many families ( 5 ). About 96% SMA are caused by a homozygous deletion of SMN1 exon7, and the remaining 4% of cases are caused either by compound heterozygosity with a point mutation in one allele and a deletion in the other or by compound heterozygous point mutations in SMN1( 6 ). SMN2 is a homologous gene of SMN1 and functions as a SMA modifier. In general, the copy number of SMN2 is substantial variation in SMA patients, and a high SMN2 copy number tends to a milder type ( 7 ). Furthermore, more and more new genes or novel mutations have been reported to be related to the morbidity, severity, treatment and prognosis of SMA with the development of gene sequencing technology. A study revealed seven different mutations of SMN1, and among them c.824G>C, and c.825–2A>T were described for the first time ( 8 ). Another study found the NAIP copy number was inversely correlated with the clinical severity of SMA ( 9 ). GTF2H2 and H4F5 have been proved to be associated with the onset and type of SMA ( 10 ). In this study, multiplex ligation probe amplification (MLPA) was firstly used for preliminary diagnosis in 28 core families of suspected SMA patients, and then the complete gene sequence of SMN1 gene was detected by high-throughput sequencing to find more mutations in the 28 core families. Afterwards, 20 children diagnosed with SMA and 5 children diagnosed with non SMA were enrolled, and the whole exome screening of other related genes was performed to explore more genes and mutations involved in the onset of SMA. Methods Patients and samples From December 2013 to May 2017, 28 probands of clinically suspected SMA, 15 males and 13 females, were accepted by our hospital (Tianjin Children Hospital, China, Tianjin) because of unstable walking, and their age ranging from 1 month to 12 years. The phenotypes of their parents were normal. 3–5 ml peripheral blood samples from the 83 enrolled cases (probands and their parents) were collected. All the subjects signed the informed consent forms for genetic testing routinely, and all procedures were in accordance with the ethical standards of the institutional and/or national research committee. MLPA was Extracted Genomic DNA from 1–2 ml peripheral blood samples with the salting out method. The nucleic acid quantitative instrument NADO DROP 2000 (Thermo Fisher Scientific Inc., Waltham, USA) was utilized to determine the quality and quantity of the extracted DNA. MLPA was performed using a SALSA MLPA Kit P021 (MRC-Holland, Amsterdam, Netherlands) according to the manufacturer’s protocol. MLPA products were run on an ABI PRISM 3130 genetic analyzer (Appliced Biosystems International Inc., California, USA). and analyzed using Gene Mapper version 3.5 software (Thermo Fisher Scientific Inc., Waltham, USA). For each sample relative peak heights were calculated and compared with 4 normal controls using the Coffalyser version 9 software (Coffalyser MLPA, Amsterdam, Netherlands). The evaluation criteria were based on the kit instructions: in normal individuals, SMN1 exon7 is 2 copies; the SMN1 gene of patients with homozygous deletion and carriers with heterozygous deletion is 0 copies and 1copy, respectively(11,12). SMN1 gene screening by high-throughput sequencing The complete genome sequence of SMN1 gene was detected from above 83 enrolled cases via high-throughput sequencing. Firstly, high-molecular-weight genomic DNAs were extracted from 2–3 ml blood samples with the DNeasy Blood and Tissue kit (QIAGEN, Dusseldorf, Germany) and 10 μg genomic DNA was used for library generation according to the manufacturer’s recommendations. Secondly,. Briefly, the genomic DNA was mechanically sheared to an average fragment size of 1.5 kb. These size-selected fragments were then end repaired and added a special joint sequence at both ends. The probes (Roche, Basel, Switzerland) then were used to capture genomic regions including SMN gene. Secondly, the Illumina HiSeq–2500 platform (Illumina, California, USA) was used to conduct the sequencing reaction based on the manufacturer’s protocol. The average sequencing depth was 100×, and more than 96% regions was up to 20×. Thirdly, the raw data were compared with human genome (NCBI build 36, hg18), and marked repeated reads and filtered out low-quality data. Fourthly, the base quality of reads was calibrated again by the genome analysis toolkit (GATK) algorithm, and ultimately all the a single base of DNA ( point mutation) or a loss of base pairs ( deletion) were screened out with the GATK software V3.0 (Eli and Edythe L. Broad Institute, Massachusetts, USA)(13). The QC 30 of the raw data was more than 85% in all samples, and the allele frequency of all mutations was more than 20%. The whole exome screening by high-throughput sequencing Based on previous results, 20 SMA patients with homozygous deletion of SMN1 exon7 were selected from the 28 prodands, and 5 non SMA children were as controls (2 children carried with heterozygous deletion of SMN1 exon7 and 3 children with 2 SMN1 exon7copies). was utilized to perform whole exome high-throughput sequencing was performed by The Life Technologies SOLiD™ (version 3) technology with mate-pair chemistry. according to the manufacturer’s recommendations (Carlsbad, CA, USA). 20 μg high-molecular-weight genomic DNAs was used for library generation. Briefly, DNA was broken into 1.5kb fragments, repaired the ends and circularized around a long mate-pair adaptor by nicked ligation. SOLiD™ sequencing-specific sequencing adaptors were ligated to the ends of these fragments. Following PCR amplification, these mate-pair libraries were then used as templates in emulsion PCR reactions using SOLiD™ proprietary sequencing beads to generate clonal single molecule templated beads. The raw data were analyzed with GATK software. The average sequencing depth was 100×, and more than 96% regions was up to 20×. The QC 30 of the raw data was more than 85% in all samples, and the allele frequency of all mutations was more than 40%. Results MLPA MLPA results showed that 22 probands were with homozygous deletion of SMN1 exon7 (SMA patients), 3 probands carried with heterozygous deletion of SMN1 exon7 (carriers), and 3 probands with 2 SMN1 copies (normal individuals). However, the mother of a SMA patient had 3 SMN1 exon7 copies and the father was carrier, and the father of another SMA patient had 3 SMN1 exon7 copies and the mother was carrier. Here, we recorded the above 2 SMA patients as Proband–1 and Proband–2, respectively. Furthermore, 1 carrier, a 2-year old girl, had some clinical features that correspond to SMA, such as atrophy of skeletal muscles, generalized weakness and extensive neurogenic injury by electromyography. Here, the carrier was named as Proband–3. Besides, the father of Proband–3 was without heterozygous deletion of SMN1 exon7 and exon8, and the mother was a carrier. Novel mutations of SMN1 in suspected SMA family A total of 6 single nucleotide variants (SNVs) of SMN1 were identified in the 83 samples, and they were showed in Table 1. 3 SNVs located in exon, 1 in UTR5 and 2 in intron; c.[84C>T], c.[271C>T], c.[–39A>G] and g.[70240639G>C] were firstly reported here; c.[84C>T] and c.[462A>G] were synonymous mutations, and c.[271C>T] were stopgaine; c.[271C>T] caused changes in encoded amino acids. Furthermore, c.[271C>T] was found in Proband–3 and her father, and c.[462A>G] was occurred in 17 SMA patients, 2 carrier and 2 normal individuals. In addition, 1 carrier was with c.[84C>T], 1 SMA patient with c.[–39A>G], and 1 SMA patient with g.[70240639G>C]. Novel mutations occurred in only SMA patients Compared with control group, a total of 9102 mutation were selected out in SMA patients with homozygous deletion of SMN1 exon7. They were located in the exon region, and occurred only in SMA patients not in carriers and normal individuals. Among them, 2415 genes and some indefinite genes were included, and 8619 SNVs, 267 deletions and 216 inserts were contained. Here, the indefinite genes were removed, and the 30 most frequent mutations were showed in Table 2 (Frequency ≥ 50%). It was obvious from Table 2 that only the MCCC2 missense mutation c.[1001A>G] located on chromosome 5q13, which was the same location as SMN1 and SMN2. The others were unlinked to 5q13. Discussion With the development of bioinformatics, more and more mutations have been discovered in SMN1, and some of them possess significant clinical implications. Ganji et al (14) reported conducted mutation screening of SMN1 in 4 patients with 1 copy of SMN1, and identified 2 novel mutations including a single nucleotide insertion in exon 7 (c.861_862insT/p.R288X) and a deletion of nucleotide G in exon 3 (c.286delG/p.D96Tfs*53). Yamamoto et al (15) revealed 4 intragenic mutations (p.Ala2Val, p.Trp92Ser, p.Thr274TyrfsX32 and p.Tyr277Cys), and location of the mutations were associated with the clinical severity of SMA. Ronchi et al (16) described a novel SMN1 mutation that affected the donor splice site of exon 7 and resulted in an unusually severe SMA phenotype with rapid fatal outcome in an Italian infant. In this article, we found 6 SMN1 SNVs in 28 core families of suspected SMA patients, including 4 novel mutations c.[84C>T], c.[271C>T], c.[–39A>G] and g.[70240639G>C], which had never been previously reported. At present, MLPA is the gold standard for clinical diagnosis of SMA. However, MLPA can only detect the deletion of SMN1 according to the gene copy number, not detect point mutations of SMN1. It is well known that about 4% of SMA patients bear one SMN1 copy with an intragenic mutation. Therefore, some SMA patients are inevitably misdiagnosed as carriers. In this study, Proband–3 was with one SMN1 copy and the SMN1 stopgain mutation c.[271C>T], and the heterozygous deletion of SMN1 exon 7 was from her mother, and the SMN1 stopgain mutation c.[271C>T] from her father. The SMN1 stopgain mutation c.[271C>T] was never reported before, and it led to a amino acid change. Although MLPA results showed Proband–3 to be carrier, some SMA-related clinical features were occurred on her. Here, we suspected that Proband–3 might be a SMA patient caused by the heterozygous deletion of SMN1 exon 7 combined with the SMN1 stopgain mutation c.[271C>T]. Simultaneously, c.[271C>T] might be involved in the onset of SMA. In addition, [2+0] genotype carriers are two SMN1 copies on one chromosome and with deletion of SMN1 on the other chromosome (17). In this article, we found 2 patients (Proband–1 and Proband–2) whose one parent was carriers and the other parent with 3 SMN1 exon7 copies (Proband–1’s mother and Proband–2’s father). Based on our results we suspected that Proband–1’s mother and Proband–2’s father might be [2+0] genotype carriers. SMN1 and SMN2 present on chromosome 5q13, and of the 5q13-linked SMA patients, 96.4% show homozygous absence of SMN1 exons 7 and 8 or exon 7 only, whereas 3.6% present a compound heterozygosity with a subtle mutation on one chromosome and a deletion/gene conversion on the other chromosome ( 6 ). Here, we identified more mutations combined with homozygous absence of SMN1 exons7 (Table 2). The 3 most frequent mutations were the insertion mutation c.[–41_–40insCTCT] in SPTA1 exon1 (rs111674514), the SNV c.[1001A>G] in FUT5 exon2 (rs778984), and the SNV c.[–117A>G] in MCCC2 exon1 (rs11746722). SPTA1 encodes the human erythroid alpha-spectrin, which is an actin crosslinking and molecular scaffold protein that links the plasma membrane to the actin cytoskeleton, and functions in the determination of cell shape, arrangement of transmembrane proteins, and organization of organelles (18, 19). Mutations in SPTA1 can lead to a variety of hereditary red blood cell disorders, including elliptocytosis type 2, pyropoikilocytosis, and spherocytic hemolytic anemia (20, 21). FUT5 encodes alpha1,3-fucosyltransferase in human (22). The down-regulation of FUT5 reduces the expression of sialyl-Lewis antigens and the adhesion and binding capacities of gastric cancer cells (23). Gene transfer of alpha1,3-fucosyltransferase increased tumor growth of the PC–3 human prostate cancer cell line through enhanced adhesion to prostatic stromal cells (24). Methylcrotonyl CoA carboxylase β (MCCβ) is encoded by MCCC2, and point mutations and deletion events in MCC2 can lead to MCC deficiency (25, 26). MCC deficiency is a rare autosomal recessive genetic disorder whose clinical presentations range from benign to profound metabolic acidosis and death in infancy, which is has something in common with SMA in some ways. MCCC2 locates on chromosome 5q13, which was the same as SMN1. Some studies indicated that SMN1 was the causative gene, and other genes on 5q13 region acted as modifier gene (such as SMN2, NAIP and GTF2H2), , which were associated with disease severity (27). The mutations rs111674514 in SPTA1, rs778984 in FUT5 and rs11746722 in MCCC2 have been identified previously, but the clinical significance remains uncertain. In this article, we found they were widely prevalent in SMA patients, and almost nonexistent in non-patients. Therefore, it suggested they might be involved in the morbidity of SMA. Conclusions We found more mutations in both SMN1 and other genes, and some of them were associated with the onset of SMA, such as the SMN1 stopgain mutation c.[271C>T], the SPTA1 insertion mutation c.[–41_–40insCTCT], the FUT5 SNV c.[1001A>G], and the MCCC2 SNV c.[–117A>G]. Abbreviations SMA: spinal muscular atrophy; MLPA: multiplex ligation probe amplification; SNVs: single nucleotide variants; SMN: survival of motor neuron; NAIP: neuronal apoptosis inhibitory protein; GATK: genome analysis toolkit; MCCβ: Methylcrotonyl CoA carboxylase β Declarations Acknowledgements We would like to thank all the members of our research group for their enthusiastic participation in this study. Authors’ contributions CC designed the study and drafted the initial manuscript, and revised the manuscript. YM participated in the design of study and critically reviewed the manuscript. JS and YF collected samples and critically reviewed the manuscript. JW, ZX and XW analyzed and interpreted data and reviewed the manuscript. All authors read and approved the final manuscript. Funding This work was supported by the Program of Tianjin Science and Technology Plan (No. 18ZXDBSY00170), the Key Project of Tianjin Health Care Professionals (Grant No.16KG166) and the National Natural Science Foundation of China (Grant No.81771589). The authors declare that they have no conflict of interest with the organizations that sponsored the research, and the funding bodys were not involved in study design, data collection, analysis and writing of the study. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Ethics approval and consent to participate All the subjects signed the informed consent forms for genetic testing routinely, and the study was approved by the medical ethics committee of Tianjin Children’s Hospital. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. References 1.Lunn MR, Wang CH. Spinal muscular atrophy. Lancet. 2008;371:2120–33. 2.Sheng-Yuan Z, Xiong F, Chen YJ, et al. Molecular characterization of SMN copy number derived from carrier screening and from core families with SMA in a Chinese population. Eur J Hum Genet. 2010;18:978–84. 3.Kolb S J, Kissel J T. Spinal muscular atrophy. Neurol Clin. 2015;33:831–46. 4.Ottesen EW. ISS-N1 makes the First FDA-approved Drug for Spinal Muscular Atrophy. Transl Neurosci. 2017;8:1–6. 5.Prior TW, Nagan N. Spinal Muscular Atrophy: Overview of Molecular Diagnostic Approaches. Curr Protoc Hum Genet. 2016;88:9–27. 6.Wirth B. 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Tables Table 1 Mutations in SMN1 Position MT Ex-In Function REF>ALT dbSNP cHGVS AAChange Frequency 70234668 snv exon2 synonymous C>T NA c.[84C>T] NA 3.61% (3/83) 70220892 snv exon3 stopgain C>T NA c.[271C>T] p.[Gln91X] 2.41% (2/83) 70238373 snv exon4 synonymous A>G rs4915 c.[462A>G] NA 83.13% (69/83) 70220892 snv UTR5 unknown A>G NA c.[-39A>G] NA 4.82% (4/83) 70240639 snv intron unknown G>C NA g.[70240639G>C] NA 8.43% (7/83) 70247937 snv intron unknown A>C rs200563560 g.[70247937A>C] NA 1.20% (1/83) MT, mutation type; Ex-In, exon or intron; snv, single nucleotide variant; NA, no report or no change; X, unknown amino acids; Frequency, frequency in all samples. Due to technical limitations, table 2 is only available as a download in the supplemental files section Supplementary Files Table2.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3630","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research article","associatedPublications":[],"authors":[{"id":129816,"identity":"8da7f3d7-844c-42b4-bda1-e5b7cac69e74","order_by":1,"name":"Jianbo Shu","email":"","orcid":"","institution":"Tianjin Children's Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jianbo","middleName":"","lastName":"Shu","suffix":""},{"id":129817,"identity":"086bbf44-9d53-4e1e-8fb2-571f9092b3be","order_by":2,"name":"Jingrui Wang","email":"","orcid":"","institution":"Tianjin Marvel Medical Laboratory Co., Ltd","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jingrui","middleName":"","lastName":"Wang","suffix":""},{"id":129818,"identity":"08045ae6-111f-49f1-8fb1-ba1da4c5fd43","order_by":3,"name":"Yulian Fang","email":"","orcid":"","institution":"Tianjin Children's Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yulian","middleName":"","lastName":"Fang","suffix":""},{"id":129819,"identity":"ee64c85f-5ab4-4b9c-99da-e4721d129ffb","order_by":4,"name":"Zanmei Xu","email":"","orcid":"","institution":"Tianjin Marvel Medical Laboratory Co., Ltd","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zanmei","middleName":"","lastName":"Xu","suffix":""},{"id":129820,"identity":"3f4cc7b8-2f48-459a-99dc-321455946b37","order_by":5,"name":"Xiaowei Wang","email":"","orcid":"","institution":"Tianjin Marvel Medical Laboratory Co., Ltd","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiaowei","middleName":"","lastName":"Wang","suffix":""},{"id":129821,"identity":"8f589cb0-c5c5-463b-b697-3713bdf90ca7","order_by":6,"name":"Yingtao Meng","email":"","orcid":"","institution":"Tianjin Children's Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yingtao","middleName":"","lastName":"Meng","suffix":""},{"id":129822,"identity":"2965adc1-a8c5-4250-8135-8979dfc965e9","order_by":7,"name":"Chunquan Cai","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4klEQVRIiWNgGAWjYFACxgZmJJ4NDz9/A2la0mQkZxwgbA+ylsM2Bg0J+JXztx9u/FzYdkd2w/GzBz/8KDvPY8BwgPHDxxzcWiTOJDZLz2x7ZrzhTF6yZM+52zzmzA3MkjO34dZiwJDYxszbdjhxw4EcM2bGtts8lg0H2Jh58WnhfwjVcv4NSMs5HoMDCQS0SMBsuQG25QBhLRI3HjZL85w7bDzzxhtjoF+SeSRnHGzG6xf+/vSHn3nKDsv2nc8xBIaYnT0/f/PBDx/xaIEBxgYwxYbEJkXLKBgFo2AUjAJUAAAWjlRZBijyhgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-9784-5250","institution":"Tianjin Children's Hospital","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Chunquan","middleName":"","lastName":"Cai","suffix":""}],"badges":[],"createdAt":"2019-08-09 15:05:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.2.12903/v1","doiUrl":"https://doi.org/10.21203/rs.2.12903/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":13470877,"identity":"d62741fc-efa8-44f8-8e3a-1828ad0091d1","added_by":"auto","created_at":"2021-09-16 21:07:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":281223,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3630/v1/8c110422-1029-4e01-8d3d-e06048414774.pdf"},{"id":147920,"identity":"9b563c9b-463d-4d50-a07a-d085a6465cc5","added_by":"auto","created_at":"2019-11-04 12:32:35","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":19986,"visible":true,"origin":"","legend":"","description":"","filename":"Table2.docx","url":"https://assets-eu.researchsquare.com/files/a8ee7e93-1963-436b-aee3-11c7f3772021/v1/Table 2.docx"}],"financialInterests":"","formattedTitle":"High-throughput screening reveals novel mutations in spinal muscular atrophy patients","fulltext":[{"header":"Background","content":"\u003cp\u003eSpinal muscular atrophy (SMA) is an autosomal recessive hereditary disease characterized by degeneration of spinal cord motor neurons, atrophy of skeletal muscles, and generalized weakness (\u003ca href=\"#_ENREF_1\"\u003e1\u003c/a\u003e). It affects 1 in 10 000 live births, and often leads to early death (\u003ca href=\"#_ENREF_2\"\u003e2\u003c/a\u003e). SMA manifests over a wide range of severity, affecting infants through adults. According to the onset time and severity of the disease, SMA is divided into 4 types (SMA1, SMA2, SMA3 and SMA4), and SMA 1, with onset before age six months; SMA 2, with onset between age 6 and 18 months; SMA 3, with onset in childhood after age 12 months; and SMA 4, with adult onset (\u003ca href=\"#_ENREF_3\"\u003e3\u003c/a\u003e). Nusinersen (trade name: Spinraza) is the only approved drug to treat spinal muscular atrophy, which is administered directly to the central nervous system using an intrathecal injection (\u003ca href=\"#_ENREF_4\"\u003e4\u003c/a\u003e). SMA is caused by homozygous disruption of the survival motor neuron 1 (\u003cem\u003eSMN1)\u003c/em\u003e gene by deletion, conversion, or mutation (\u003ca href=\"#_ENREF_1\"\u003e1\u003c/a\u003e). Since SMA is one of the most common lethal genetic disorders, with a carrier frequency of 1 in 40 to 1 in 60, direct carrier dosage testing has been beneficial to many families (\u003ca href=\"#_ENREF_5\"\u003e5\u003c/a\u003e). About 96% SMA are caused by a homozygous deletion of \u003cem\u003eSMN1\u003c/em\u003e exon7, and the remaining 4% of cases are caused either by compound heterozygosity with a point mutation in one allele and a deletion in the other or by compound heterozygous point mutations in \u003cem\u003eSMN1(\u003c/em\u003e\u003ca href=\"#_ENREF_6\"\u003e6\u003c/a\u003e). \u003cem\u003eSMN2\u003c/em\u003e is a homologous gene of \u003cem\u003eSMN1\u003c/em\u003e and functions as a SMA modifier. In general, the copy number of \u003cem\u003eSMN2\u003c/em\u003e is substantial variation in SMA patients, and a high \u003cem\u003eSMN2\u003c/em\u003e copy number tends to a milder type (\u003ca href=\"#_ENREF_7\"\u003e7\u003c/a\u003e). Furthermore, more and more new genes or novel mutations have been reported to be related to the morbidity, severity, treatment and prognosis of SMA with the development of gene sequencing technology. A study revealed seven different mutations of \u003cem\u003eSMN1,\u003c/em\u003e and among them c.824G\u0026gt;C, and c.825–2A\u0026gt;T were described for the first time (\u003ca href=\"#_ENREF_8\"\u003e8\u003c/a\u003e). Another study found the \u003cem\u003eNAIP \u003c/em\u003ecopy number was inversely correlated with the clinical severity of SMA (\u003ca href=\"#_ENREF_9\"\u003e9\u003c/a\u003e). \u003cem\u003eGTF2H2\u003c/em\u003e and \u003cem\u003eH4F5\u003c/em\u003e have been proved to be associated with the onset and type of SMA (\u003ca href=\"#_ENREF_10\"\u003e10\u003c/a\u003e). In this study, multiplex ligation probe amplification (MLPA) was firstly used for preliminary diagnosis in 28 core families of suspected SMA patients, and then the complete gene sequence of \u003cem\u003eSMN1\u003c/em\u003e gene was detected by high-throughput sequencing to find more mutations in the 28 core families. Afterwards, 20 children diagnosed with SMA and 5 children diagnosed with non SMA were enrolled, and the whole exome screening of other related genes was performed to explore more genes and mutations involved in the onset of SMA.\u003c/p\u003e\n"},{"header":"Methods","content":"\u003ch2\u003ePatients and samples\u003c/h2\u003e\n\u003cp\u003eFrom December 2013 to May 2017, 28 probands of clinically suspected SMA, 15 males and 13 females, were accepted by our hospital (Tianjin Children Hospital, China, Tianjin) because of unstable walking, and their age ranging from 1 month to 12 years. The phenotypes of their parents were normal. 3–5 ml peripheral blood samples from the 83 enrolled cases (probands and their parents) were collected. All the subjects signed the informed consent forms for genetic testing routinely, and all procedures were in accordance with the ethical standards of the institutional and/or national research committee.\u003c/p\u003e\n\u003ch2\u003eMLPA\u003c/h2\u003e\n\u003cp\u003ewas Extracted Genomic DNA from 1–2 ml peripheral blood samples with the salting out method. The nucleic acid quantitative instrument NADO DROP 2000 (Thermo Fisher Scientific Inc., Waltham, USA) was utilized to determine the quality and quantity of the extracted DNA. MLPA was performed using a SALSA MLPA Kit P021 (MRC-Holland, Amsterdam, Netherlands) according to the manufacturer’s protocol. MLPA products were run on an ABI PRISM 3130 genetic analyzer (Appliced Biosystems International Inc., California, USA). and analyzed using Gene Mapper version 3.5 software (Thermo Fisher Scientific Inc., Waltham, USA). For each sample relative peak heights were calculated and compared with 4 normal controls using the Coffalyser version 9 software (Coffalyser MLPA, Amsterdam, Netherlands). The evaluation criteria were based on the kit instructions: in normal individuals, SMN1 exon7 is 2 copies; the \u003cem\u003eSMN1\u003c/em\u003e gene of patients with homozygous deletion and carriers with heterozygous deletion is 0 copies and 1copy, respectively(11,12).\u003c/p\u003e\n\u003ch2\u003eSMN1 gene screening by high-throughput sequencing\u003c/h2\u003e\n\u003cp\u003eThe complete genome sequence of \u003cem\u003eSMN1 \u003c/em\u003egene was detected from above 83 enrolled cases via high-throughput sequencing. Firstly, high-molecular-weight genomic DNAs were extracted from 2–3 ml blood samples with the DNeasy Blood and Tissue kit (QIAGEN, Dusseldorf, Germany) and 10 μg genomic DNA was used for library generation according to the manufacturer’s recommendations. Secondly,. Briefly, the genomic DNA was mechanically sheared to an average fragment size of 1.5 kb. These size-selected fragments were then end repaired and added a special joint sequence at both ends. The probes (Roche, Basel, Switzerland) then were used to capture genomic regions including \u003cem\u003eSMN \u003c/em\u003egene. Secondly, the Illumina HiSeq–2500 platform (Illumina, California, USA) was used to conduct the sequencing reaction based on the manufacturer’s protocol. The average sequencing depth was 100×, and more than 96% regions was up to 20×. Thirdly, the raw data were compared with human genome (NCBI build 36, hg18), and marked repeated reads and filtered out low-quality data. Fourthly, the base quality of reads was calibrated again by the genome analysis toolkit (GATK) algorithm, and ultimately all the a single base of DNA ( point mutation) or a loss of base pairs ( deletion) were screened out with the GATK software V3.0 (Eli and Edythe L. Broad Institute, Massachusetts, USA)(13). The QC\u003csub\u003e30\u003c/sub\u003e of the raw data was more than 85% in all samples, and the allele frequency of all mutations was more than 20%.\u003c/p\u003e\n\u003ch2\u003eThe whole exome screening by high-throughput sequencing\u003c/h2\u003e\n\u003cp\u003eBased on previous results, 20 SMA patients with homozygous deletion of \u003cem\u003eSMN1\u003c/em\u003e exon7 were selected from the 28 prodands, and 5 non SMA children were as controls (2 children carried with heterozygous deletion of \u003cem\u003eSMN1\u003c/em\u003e exon7 and 3 children with 2 \u003cem\u003eSMN1\u003c/em\u003e exon7copies). was utilized to perform whole exome high-throughput sequencing was performed by The Life Technologies SOLiD™ (version 3) technology with mate-pair chemistry. according to the manufacturer’s recommendations (Carlsbad, CA, USA). 20 μg high-molecular-weight genomic DNAs was used for library generation. Briefly, DNA was broken into 1.5kb fragments, repaired the ends and circularized around a long mate-pair adaptor by nicked ligation. SOLiD™ sequencing-specific sequencing adaptors were ligated to the ends of these fragments. Following PCR amplification, these mate-pair libraries were then used as templates in emulsion PCR reactions using SOLiD™ proprietary sequencing beads to generate clonal single molecule templated beads. The raw data were analyzed with GATK software. The average sequencing depth was 100×, and more than 96% regions was up to 20×. The QC\u003csub\u003e30\u003c/sub\u003e of the raw data was more than 85% in all samples, and the allele frequency of all mutations was more than 40%.\u003c/p\u003e"},{"header":"Results","content":"\u003ch2\u003eMLPA\u003c/h2\u003e\n\u003cp\u003eMLPA results showed that 22 probands were with homozygous deletion of \u003cem\u003eSMN1\u003c/em\u003e exon7 (SMA patients), 3 probands carried with heterozygous deletion of\u003cem\u003e SMN1\u003c/em\u003e exon7 (carriers), and 3 probands with 2 \u003cem\u003eSMN1 \u003c/em\u003ecopies (normal individuals). However, the mother of a SMA patient had 3\u003cem\u003e SMN1\u003c/em\u003e exon7 copies and the father was carrier, and the father of another SMA patient had 3\u003cem\u003e SMN1\u003c/em\u003e exon7 copies and the mother was carrier. Here, we recorded the above 2 SMA patients as Proband–1 and Proband–2, respectively. Furthermore, 1 carrier, a 2-year old girl, had some clinical features that correspond to SMA, such as atrophy of skeletal muscles, generalized weakness and extensive neurogenic injury by electromyography. Here, the carrier was named as Proband–3. Besides, the father of Proband–3 was without heterozygous deletion of \u003cem\u003eSMN1 \u003c/em\u003eexon7 and exon8, and the mother was a carrier.\u003c/p\u003e\n\u003ch2\u003eNovel mutations of SMN1 in suspected SMA family\u003c/h2\u003e\n\u003cp\u003eA total of 6 single nucleotide variants (SNVs) of SMN1 were identified in the 83 samples, and they were showed in Table 1. 3 SNVs located in exon, 1 in UTR5 and 2 in intron; c.[84C\u0026gt;T], c.[271C\u0026gt;T], c.[–39A\u0026gt;G] and g.[70240639G\u0026gt;C] were firstly reported here; c.[84C\u0026gt;T] and c.[462A\u0026gt;G] were synonymous mutations, and c.[271C\u0026gt;T] were stopgaine; c.[271C\u0026gt;T] caused changes in encoded amino acids. Furthermore, c.[271C\u0026gt;T] was found in Proband–3 and her father, and c.[462A\u0026gt;G] was occurred in 17 SMA patients, 2 carrier and 2 normal individuals. In addition, 1 carrier was with c.[84C\u0026gt;T], 1 SMA patient with c.[–39A\u0026gt;G], and 1 SMA patient with g.[70240639G\u0026gt;C].\u003c/p\u003e\n\u003ch2\u003eNovel mutations occurred in only SMA patients\u003c/h2\u003e\n\u003cp\u003eCompared with control group, a total of 9102 mutation were selected out in SMA patients with homozygous deletion of \u003cem\u003eSMN1\u003c/em\u003e exon7. They were located in the exon region, and occurred only in SMA patients not in carriers and normal individuals. Among them, 2415 genes and some indefinite genes were included, and 8619 SNVs, 267 deletions and 216 inserts were contained. Here, the indefinite genes were removed, and the 30 most frequent mutations were showed in Table 2 (Frequency ≥ 50%). It was obvious from Table 2 that only the \u003cem\u003eMCCC2\u003c/em\u003e missense mutation c.[1001A\u0026gt;G] located on chromosome 5q13, which was the same location as \u003cem\u003eSMN1\u003c/em\u003e and \u003cem\u003eSMN2.\u003c/em\u003e The others were unlinked to 5q13.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWith the development of bioinformatics, more and more mutations have been discovered in \u003cem\u003eSMN1,\u003c/em\u003e and some of them possess significant clinical implications. Ganji et al (14) reported conducted mutation screening of \u003cem\u003eSMN1\u003c/em\u003e in 4 patients with 1 copy of \u003cem\u003eSMN1,\u003c/em\u003e and identified 2 novel mutations including a single nucleotide insertion in exon 7 (c.861_862insT/p.R288X) and a deletion of nucleotide G in exon 3 (c.286delG/p.D96Tfs*53). Yamamoto et al (15) revealed 4 intragenic mutations (p.Ala2Val, p.Trp92Ser, p.Thr274TyrfsX32 and p.Tyr277Cys), and location of the mutations were associated with the clinical severity of SMA. Ronchi et al (16) described a novel \u003cem\u003eSMN1\u003c/em\u003e mutation that affected the donor splice site of exon 7 and resulted in an unusually severe SMA phenotype with rapid fatal outcome in an Italian infant. In this article, we found 6\u003cem\u003e SMN1\u003c/em\u003e SNVs in 28 core families of suspected SMA patients, including 4 novel mutations c.[84C\u0026gt;T], c.[271C\u0026gt;T], c.[–39A\u0026gt;G] and g.[70240639G\u0026gt;C], which had never been previously reported. At present, MLPA is the gold standard for clinical diagnosis of SMA. However, MLPA can only detect the deletion of \u003cem\u003eSMN1 \u003c/em\u003eaccording to the gene copy number, not detect point mutations of \u003cem\u003eSMN1.\u003c/em\u003e It is well known that about 4% of SMA patients bear one \u003cem\u003eSMN1\u003c/em\u003e copy with an intragenic mutation. Therefore, some SMA patients are inevitably misdiagnosed as carriers. In this study, Proband–3 was with one \u003cem\u003eSMN1\u003c/em\u003e copy and the\u003cem\u003e SMN1\u003c/em\u003e stopgain mutation c.[271C\u0026gt;T], and the heterozygous deletion of \u003cem\u003eSMN1\u003c/em\u003e exon 7 was from her mother, and the\u003cem\u003e SMN1\u003c/em\u003e stopgain mutation c.[271C\u0026gt;T] from her father. The\u003cem\u003e SMN1\u003c/em\u003e stopgain mutation c.[271C\u0026gt;T] was never reported before, and it led to a amino acid change. Although MLPA results showed Proband–3 to be carrier, some SMA-related clinical features were occurred on her. Here, we suspected that Proband–3 might be a SMA patient caused by the heterozygous deletion of \u003cem\u003eSMN1\u003c/em\u003e exon 7 combined with the\u003cem\u003e SMN1\u003c/em\u003e stopgain mutation c.[271C\u0026gt;T]. Simultaneously, c.[271C\u0026gt;T] might be involved in the onset of SMA. In addition, [2+0] genotype carriers are two \u003cem\u003eSMN1\u003c/em\u003e copies on one chromosome and with deletion of \u003cem\u003eSMN1\u003c/em\u003e on the other chromosome (17). In this article, we found 2 patients (Proband–1 and Proband–2) whose one parent was carriers and the other parent with 3 \u003cem\u003eSMN1\u003c/em\u003e exon7 copies (Proband–1’s mother and Proband–2’s father). Based on our results we suspected that Proband–1’s mother and Proband–2’s father might be [2+0] genotype carriers.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSMN1\u003c/em\u003e and \u003cem\u003eSMN2\u003c/em\u003e present on chromosome 5q13, and of the 5q13-linked SMA patients, 96.4% show homozygous absence of \u003cem\u003eSMN1\u003c/em\u003e exons 7 and 8 or exon 7 only, whereas 3.6% present a compound heterozygosity with a subtle mutation on one chromosome and a deletion/gene conversion on the other chromosome (\u003ca href=\"#_ENREF_6\"\u003e6\u003c/a\u003e). Here, we identified more mutations combined with homozygous absence of \u003cem\u003eSMN1\u003c/em\u003e exons7 (Table 2). The 3 most frequent mutations were the insertion mutation c.[–41_–40insCTCT] in \u003cem\u003eSPTA1\u003c/em\u003e exon1 (rs111674514), the SNV c.[1001A\u0026gt;G] in \u003cem\u003eFUT5\u003c/em\u003e exon2 (rs778984), and the SNV c.[–117A\u0026gt;G] in \u003cem\u003eMCCC2\u003c/em\u003e exon1 (rs11746722). \u003cem\u003eSPTA1\u003c/em\u003e encodes the human erythroid alpha-spectrin, which is an actin crosslinking and molecular scaffold protein that links the plasma membrane to the actin cytoskeleton, and functions in the determination of cell shape, arrangement of transmembrane proteins, and organization of organelles (18, 19). Mutations in \u003cem\u003eSPTA1\u003c/em\u003e can lead to a variety of hereditary red blood cell disorders, including elliptocytosis type 2, pyropoikilocytosis, and spherocytic hemolytic anemia (20, 21). \u003cem\u003eFUT5\u003c/em\u003e encodes alpha1,3-fucosyltransferase in human (22). The down-regulation of \u003cem\u003eFUT5\u003c/em\u003e reduces the expression of sialyl-Lewis antigens and the adhesion and binding capacities of gastric cancer cells (23). Gene transfer of alpha1,3-fucosyltransferase increased tumor growth of the PC–3 human prostate cancer cell line through enhanced adhesion to prostatic stromal cells (24). Methylcrotonyl CoA carboxylase β (MCCβ) is encoded by\u003cem\u003e MCCC2,\u003c/em\u003e and point mutations and deletion events in \u003cem\u003eMCC2 \u003c/em\u003ecan lead to MCC deficiency (25, 26). MCC deficiency is a rare autosomal recessive genetic disorder whose clinical presentations range from benign to profound metabolic acidosis and death in infancy, which is has something in common with SMA in some ways. \u003cem\u003eMCCC2\u003c/em\u003e locates on chromosome 5q13, which was the same as \u003cem\u003eSMN1.\u003c/em\u003e Some studies indicated that \u003cem\u003eSMN1\u003c/em\u003e was the causative gene, and other genes on 5q13 region acted as modifier gene (such as \u003cem\u003eSMN2,\u003c/em\u003e \u003cem\u003eNAIP\u003c/em\u003e and \u003cem\u003eGTF2H2),\u003c/em\u003e, which were associated with disease severity (27). The mutations rs111674514 in \u003cem\u003eSPTA1,\u003c/em\u003e rs778984 in \u003cem\u003eFUT5\u003c/em\u003e and rs11746722 in \u003cem\u003eMCCC2\u003c/em\u003e have been identified previously, but the clinical significance remains uncertain. In this article, we found they were widely prevalent in SMA patients, and almost nonexistent in non-patients. Therefore, it suggested they might be involved in the morbidity of SMA.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eWe found more mutations in both \u003cem\u003eSMN1\u003c/em\u003e and other genes, and some of them were associated with the onset of SMA, such as the\u003cem\u003e SMN1\u003c/em\u003e stopgain mutation c.[271C\u0026gt;T], the\u003cem\u003e SPTA1\u003c/em\u003e insertion mutation c.[–41_–40insCTCT], the\u003cem\u003e FUT5\u003c/em\u003e SNV c.[1001A\u0026gt;G], and the \u003cem\u003eMCCC2 \u003c/em\u003eSNV c.[–117A\u0026gt;G].\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eSMA: spinal muscular atrophy; MLPA: multiplex ligation probe amplification; SNVs: single nucleotide variants; \u003cem\u003eSMN:\u003c/em\u003e survival of motor neuron; \u003cem\u003eNAIP:\u003c/em\u003e neuronal apoptosis inhibitory protein; GATK: genome analysis toolkit; MCCβ: Methylcrotonyl CoA carboxylase β\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eWe would like to thank all the members of our research group for their enthusiastic participation in this study.\u003c/p\u003e\n\u003ch2\u003eAuthors’ contributions\u003c/h2\u003e\n\u003cp\u003eCC designed the study and drafted the initial manuscript, and revised the manuscript. YM participated in the design of study and critically reviewed the manuscript. JS and YF collected samples and critically reviewed the manuscript. JW, ZX and XW analyzed and interpreted data and reviewed the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis work was supported by the Program of Tianjin Science and Technology Plan (No. 18ZXDBSY00170), the Key Project of Tianjin Health Care Professionals (Grant No.16KG166) and the National Natural Science Foundation of China (Grant No.81771589). The authors declare that they have no conflict of interest with the organizations that sponsored the research, and the funding bodys were not involved in study design, data collection, analysis and writing of the study.\u003c/p\u003e\n\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\n\u003cp\u003eAll the subjects signed the informed consent forms for genetic testing routinely, and the study was approved by the medical ethics committee of Tianjin Children’s Hospital.\u003c/p\u003e\n\u003ch2\u003eConsent for publication\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n"},{"header":"References","content":"\u003cp\u003e1.Lunn MR, Wang CH. Spinal muscular atrophy. Lancet. 2008;371:2120–33.\u003c/p\u003e\n\u003cp\u003e2.Sheng-Yuan Z, Xiong F, Chen YJ, et al. Molecular characterization of SMN copy number derived from carrier screening and from core families with SMA in a Chinese population. Eur J Hum Genet. 2010;18:978–84.\u003c/p\u003e\n\u003cp\u003e3.Kolb S J, Kissel J T. Spinal muscular atrophy. Neurol Clin. 2015;33:831–46.\u003c/p\u003e\n\u003cp\u003e4.Ottesen EW. ISS-N1 makes the First FDA-approved Drug for Spinal Muscular Atrophy. Transl Neurosci. 2017;8:1–6.\u003c/p\u003e\n\u003cp\u003e5.Prior TW, Nagan N. Spinal Muscular Atrophy: Overview of Molecular Diagnostic Approaches. Curr Protoc Hum Genet. 2016;88:9–27.\u003c/p\u003e\n\u003cp\u003e6.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.\u003c/p\u003e\n\u003cp\u003e7.Stabley DL, Harris AW, Holbrook J, et al. SMN1 and SMN2 copy numbers in cell lines derived from patients with spinal muscular atrophy as measured by array digital PCR. Mol Genet Genomic Med. 2015;3:248–57.\u003c/p\u003e\n\u003cp\u003e8.Zabnenkova VV, Dadali EL, Artemieva SB, Sharkova IV, Rudenskaya GE, Polyakov AV. SMN1 gene point mutations in type I–IV proximal spinal muscular atrophy patients with a single copy of SMN1. Genetika. 2015;51:1075–82.\u003c/p\u003e\n\u003cp\u003e9.He J, Zhang QJ, Lin QF, et al. Molecular analysis of SMN1, SMN2, NAIP, GTF2H2, and H4F5 genes in 157 Chinese patients with spinal muscular atrophy. Gene. 2013;518:325–9.\u003c/p\u003e\n\u003cp\u003e10.Zeng G, Zheng H, Cheng J, et al. Analysis and carrier screening for copy numbers of SMN and NAIP genes in children with spinal muscular atrophy. Chinese J Med Genet. 2014;31:152–5.\u003c/p\u003e\n\u003cp\u003e11.Zhidai Liu, Penghui Zhang, Xiaoyan He, et al. New Multiplex real-time PCR approach to detect gene mutations for spinal muscular atrophy. BMC Neurology. 2016, 16:141.\u003c/p\u003e\n\u003cp\u003e12.Jayesh Sheth, Mehui Mistri, Riddhi Bhavsar, et al. Batten disease: biochemical and molecular characterization revealing novel PPT1 and TPP1 gene mutations in Indian patients. BMC Neurology. 2018, 18:203.\u003c/p\u003e\n\u003cp\u003e13.Xi Chen, Yusheng Tong, Zhifeng Shi, et al. Noninvasive molecular diagnosis of craniopharvngioma with MRI-based radiomics approach. BMC Neurology. 2019, 19:6.\u003c/p\u003e\n\u003cp\u003e14.Ganji H, Nouri N, Salehi M, et al. Detection of intragenic SMN1 mutations in spinal muscular atrophy patients with a single copy of SMN1. J Child Neurol. 2015;30:558–62.\u003c/p\u003e\n\u003cp\u003e15.Yamamoto T, Sato H, Lai PS, et al. Intragenic mutations in SMN1 may contribute more significantly to clinical severity than SMN2 copy numbers in some spinal muscular atrophy (SMA) patients. Brain Dev. 2014;36:914–20.\u003c/p\u003e\n\u003cp\u003e16.Ronchi D, Previtali SC, Sora MG, et al. Novel splice-site mutation in SMN1 associated with a very severe SMA-I phenotype. J Mol Neurosci. 2015;56:212–5.\u003c/p\u003e\n\u003cp\u003e17.Ar Rochmah M, Awano H, Awaya T, et al. Spinal muscular atrophy carriers with two SMN1 copies. Brain Dev. 2017;39:851–60.\u003c/p\u003e\n\u003cp\u003e18.Govindaraj V, Rao AJ. Proteomic identification of non-erythrocytic alpha-spectrin–1 down-regulation in the pre-optic area of neonatally estradiol–17beta treated female adult rats. Horm Mol Biol Clin Investig. 2016;26:165–72.\u003c/p\u003e\n\u003cp\u003e19.Speicher DW. Structural and functional features of the alpha–1 domain from human erythrocyte spectrin. Prog Clin Biol Res. 1984;165:441–56.\u003c/p\u003e\n\u003cp\u003e20.Niss O, Chonat S, Dagaonkar N, et al. Genotype-phenotype correlations in hereditary elliptocytosis and hereditary pyropoikilocytosis. Blood Cells Mol Dis. 2016;61:4–9.\u003c/p\u003e\n\u003cp\u003e21.Han E, Kim A, Park J, et al. Spectrin Tunis (Sp alpha (I/78)) in a Korean family with hereditary elliptocytosis. Ann Lab Med. 2013;33:386–9.\u003c/p\u003e\n\u003cp\u003e22.Weston BW, Nair RP, Larsen RD, Lowe JB. Isolation of a novel human alpha (1,3)fucosyltransferase gene and molecular comparison to the human Lewis blood group alpha (1,3/1,4)fucosyltransferase gene. Syntenic, homologous, nonallelic genes encoding enzymes with distinct acceptor substrate specificities. J Biol Chem. 1992;267:4152–60.\u003c/p\u003e\n\u003cp\u003e23.Padro M, Cobler L, Garrido M, de Bolos C. Down-regulation of FUT3 and FUT5 by shRNA alters Lewis antigens expression and reduces the adhesion capacities of gastric cancer cells. Biochim Biophys Acta. 2011;1810:1141–9.\u003c/p\u003e\n\u003cp\u003e24.Inaba Y, Ohyama C, Kato T, et al. Gene transfer of alpha1,3-fucosyltransferase increases tumor growth of the PC–3 human prostate cancer cell line through enhanced adhesion to prostatic stromal cells. Int J Cancer. 2003;107:949–57.\u003c/p\u003e\n\u003cp\u003e25.Morscher RJ, Grunert SC, Burer C, et al. A single mutation in MCCC1 or MCCC2 as a potential cause of positive screening for 3-methylcrotonyl-CoA carboxylase deficiency. Mol Genet Metab. 2012;105:602–6.\u003c/p\u003e\n\u003cp\u003e26.Nguyen KV, Naviaux RK, Patra S, Barshop BA, Nyhan WL. Novel mutations in the human MCCA and MCCB gene causing methylcrotonylglycinuria. Mol Genet Metab. 2011;102:218–21.\u003c/p\u003e\n\u003cp\u003e27.Theodorou L, Nicolaou P, Koutsou P, et al. Genetic findings of Cypriot spinal muscular atrophy patients. Neurol Sci. 2015;36:1829–34.\u003c/p\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 Mutations in \u003cem\u003eSMN1\u003c/p\u003e\n\u003ctable style=\"border-collapse: collapse; border: none;\" width=\"699\"\u003e\n\u003ctbody\u003e\n\u003ctr style=\"height: 14.35pt;\"\u003e\n\u003ctd style=\"width: 56.75pt; border-top: solid windowtext 1.0pt; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: none; padding: .75pt 5.4pt .75pt 5.4pt; height: 14.35pt;\" width=\"76\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003ePosition\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 33.1pt; border-top: solid windowtext 1.0pt; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: none; padding: .75pt 5.4pt .75pt 5.4pt; height: 14.35pt;\" width=\"44\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003eMT\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 37.8pt; border-top: solid windowtext 1.0pt; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: none; padding: .75pt 5.4pt .75pt 5.4pt; height: 14.35pt;\" width=\"50\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003eEx-In\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 70.9pt; border-top: solid windowtext 1.0pt; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: none; padding: .75pt 5.4pt .75pt 5.4pt; height: 14.35pt;\" width=\"95\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003eFunction\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 42.5pt; border-top: solid windowtext 1.0pt; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: none; padding: .75pt 5.4pt .75pt 5.4pt; height: 14.35pt;\" width=\"57\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003eREF\u0026gt;ALT\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 71.75pt; border-top: solid windowtext 1.0pt; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: none; padding: .75pt 5.4pt .75pt 5.4pt; height: 14.35pt;\" width=\"96\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003edbSNP\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 91.25pt; border-top: solid windowtext 1.0pt; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: none; padding: .75pt 5.4pt .75pt 5.4pt; height: 14.35pt;\" width=\"122\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003ecHGVS\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 63.8pt; border-top: solid windowtext 1.0pt; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: none; padding: .75pt 5.4pt .75pt 5.4pt; height: 14.35pt;\" width=\"85\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003eAAChange\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 56.7pt; border-top: solid windowtext 1.0pt; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: none; padding: .75pt 5.4pt .75pt 5.4pt; height: 14.35pt;\" width=\"76\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"font-family: 'Times New Roman',serif;\"\u003eFrequency\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 14.35pt;\"\u003e\n\u003ctd style=\"width: 56.75pt; border: none; padding: .75pt 5.4pt .75pt 5.4pt; height: 14.35pt;\" width=\"76\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; 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border: none; border-bottom: solid windowtext 1.0pt; padding: .75pt 5.4pt .75pt 5.4pt; height: 11.5pt;\" width=\"76\"\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cspan style=\"font-family: 'Times New Roman',serif;\"\u003e1.20% (1/83)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style=\"line-height: 150%;\"\u003e\u003cspan style=\"font-family: 'Times New Roman',serif;\"\u003eMT, mutation type; Ex-In, exon or intron; snv,\u003c/span\u003e \u003cspan style=\"font-family: 'Times New Roman',serif;\"\u003esingle nucleotide variant; NA, no report or no change; X, unknown amino acids; Frequency, frequency in all samples.\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003eDue to technical limitations, table 2 is only available as a download in the supplemental files section\u003c/p\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"spinal muscular atrophy (SMA), high-throughput sequencing, SMN1, onset","lastPublishedDoi":"10.21203/rs.2.12903/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.2.12903/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Background Some spinal muscular atrophy (SMA) cases are caused by either compound heterozygosity with a point mutation in one allele and a deletion in the other or compound heterozygous point mutations in SMN1 or other genes. Methods To explore more genes and mutations in the onset of SMA, 83 whole blood samples were collected from 28 core families of clinically suspected SMA, and multiplex ligation probe amplification (MLPA) was firstly performed with a SALSA MLPA Kit P021 for preliminary diagnosis. Afterwards, the complete gene sequence of SMN1 gene was detected with the high-throughput sequencing platform of Illumina HiSeq-2500 to find more mutations in the 28 core families. Furthermore, 20 SMA patients were selected from the 28 prodands, and 5 non SMA children as controls. The Life Technologies SOLiD™ technology with mate-pair chemistry was utilized to conduct the whole exome high-throughput sequencing. Results MLPA results showed that 22 probands were SMA patients, 3 probands carriers, and 3 probands normal individuals. Moreover, 2 parents from 2 SMA families were with 3 SMN1 exon7 copies. 6 SMN1 single nucleotide variants (SNVs) were identified in the 83 samples, and c.[84C\u003eT], c.[271C\u003eT], c.[-39A\u003eG] and g.[70240639G\u003eC] were novel. Compared with control group, 9102 mutation were selected out in SMA patients. SPTA1 mutation c.[-41_-40insCTCT], FUT5 SNV c.[1001A\u003eG], and MCCC2 SNV c.[-117A\u003eG] were the 3 most frequent mutations in SMA group (95%, 85% and 75%, respectively). Conclusions We identified some mutations in both SMN1 and other genes, and c.[271C\u003eT], c.[-41_-40insCTCT], c.[1001A\u003eG] and c.[-117A\u003eG] might be associated with the onset of SMA.","manuscriptTitle":"High-throughput screening reveals novel mutations in spinal muscular atrophy patients","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2019-08-15 16:47:48","doi":"10.21203/rs.2.12903/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7858f90c-9173-4a4f-af21-61c05487ad75","owner":[],"postedDate":"August 15th, 2019","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":21379,"name":"Neurology"},{"id":21380,"name":"Molecular Genetics"}],"tags":[],"updatedAt":"","versionOfRecord":[],"versionCreatedAt":"2019-08-15 16:47:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3630","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"identity":"rs-3630","version":["v1"]},"buildId":"ehx78VzkSd0WSzXnipQa-","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}