Fang Song

Capital institute of Pediatrics, Peping, Beijing, China

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Publications (39)31.15 Total impact

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    ABSTRACT: The homozygous loss of the survival motor neuron 1 (SMN1) gene is the primary cause of spinal muscular atrophy (SMA), a neuromuscular degenerative disease. A genetically similar gene, SMN2, which is not functionally equivalent in all SMA patients, modifies the clinical SMA phenotypes. We analyzed the methylation levels of 4 CpG islands (CGIs) in SMN2 in 35 Chinese children with SMA by MassARRAY. We found that three CpG units located in CGI 1 (nucleotides (nt) −871, −735) and CGI 4 (nt +999) are significantly hypomethylated in SMA type III compared with type I or II children after receiving Bonferroni correction. In addition to the differentially methylated CpG unit of nt −871, the methylation level of the nt −290/−288/−285 unit was negatively correlated with the expression of SMN2 full-length transcripts (SMN2-fl). In addition, the methylation level at nt +938 was inversely proportional to the ratio of SMN2-fl and lacking exon 7 transcripts (SMN2-Δ7, fl/Δ7), and was not associated with the SMN2 transcript levels. Thus, we can conclude that SMN2 methylation may regulate the SMA disease phenotype by modulating its transcription.
    No preview · Article · Jan 2016 · Journal of Zhejiang University SCIENCE B
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    ABSTRACT: X‑linked ichthyosis (XLI) is an X‑linked recessive skin disorder generally restricted to males, which arises from mutations in the steroid sulfatase (STS) gene located on Xp22.3. Crigler‑Najjar syndrome (CN‑I) is a rare autosomal recessive disease caused by the homozygous or compound heterozygous mutations in the UPD‑glucuronosyltransferase 1 family, polypeptide A1 (UGT1A1) gene on chromosome 2q37. A male patient was referred to the Department of Medical Genetics with of severe icterus and ichthyosis. The patient and his family members underwent genetic tests related to XLI and CN‑I. Quantitative polymerase chain reaction on genomic DNA was performed to determine the gene copy number, while single nucleotide polymorphism array analysis was conducted to identify deletion mutations. Family pedigree analysis showed that the patient and his two cousins were all affected by ichthyosis, which was in accordance with the inheritance pattern of an X‑linked recessive disease. In addition, the patient's serum bilirubin concentration (>340 mmol/l) was markedly greater than the normal level. The patient presented with kernicterus and phenobarbital treatment was ineffective. The clinical diagnosis of XLI was confirmed molecularly by laboratory evidence of a maternal 1.61 M deletion (including the STS gene) on ChrXp22.31. Coincidentally, the male patient was also confirmed to carry a rare maternal inherited microdeletion (374 Kb) comprising the entire UGT1A1 gene combined with a paternal UGT1A1 mutation (c.1253delT), a causative event of CN‑I. To the best of our knowledge, this study reported for the first time the comorbidity of XLI and CN‑I in a male patient. The results suggested that co‑occurrence of these two recessive diseases in a patient may be incidental.
    No preview · Article · Dec 2015 · Molecular Medicine Reports
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    ABSTRACT: Kindler syndrome (KS; OMIM 173650) is a rare autosomal recessive skin disorder, which results in symptoms including blistering, epidermal atrophy, increased risk of cancer, and poor wound healing. The majority of mutations of the disease-determining gene (FERMT1 gene) are single nucleotide substitutions, including missense mutations, nonsense mutations, etc. Large deletion mutations are seldom reported. To determine the mutation in the FERMT1 gene associated with a 7-year-old Chinese patient who presented clinical manifestation of KS, we performed direct sequencing of all the exons of FERMT1 gene. For the exons 2–6 without amplicons, we analyzed the copy numbers using quantitative real-time polymerase chain reaction (qRT-PCR) with specific primers. The deletion breakpoints were sublocalized and the range of deletion was confirmed by PCR and direct sequencing. In this study, we identified a new 17-kb deletion mutation spanning the introns 1–6 of FERMT1 gene in a Chinese patient with severe KS phenotypes. Her parents were carriers of the same mutation. Our study reported a newly identified large deletion mutation of FERMT1 gene involved in KS, which further enriched the mutation spectrum of the FERMT1 gene. © 2015, Zhejiang University and Springer-Verlag Berlin Heidelberg.
    No preview · Article · Nov 2015 · Journal of Zhejiang University SCIENCE B
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    ABSTRACT: Proximal spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by deletion or mutation of SMN1 (survival motor neuron 1). SMN exon 7 splicing is regulated by a number of exonic and intronic regulatory sequences and the trans-factors that bind them. Variants located in or near these regulated regions should be evaluated to determine their effect on splicing. We identified the rare variant c.863G>T (r.835_*3del, p.Gly279Glufs*5) in exon 7 of SMN1 in three patients affected with type I or type II SMA. Most of the SMN1 transcripts exhibited complete loss of exon 7 in vivo. The ex vivo splicing assay demonstrated that the variant disrupts inclusion of exon 7 (~85%) in the SMN1 mRNA; replacement with various bases yielded a variety of splicing effects in SMN1 and SMN2 pre-mRNA. The c.863G>T (r.835_*3del, p.Gly279Glufs*5) variant is located in a region that includes binding sites for multiple splicing factors including Tra2β1. Thus, the variant disrupts Tra2β1 binding, but does not affect binding of hnRNP A1. These findings demonstrate how rare variants influence pre-mRNA splicing of SMN and reveal the functional influence of c.863G>T (r.835_*3del, p.Gly279Glufs*5) variant in patients with SMA.European Journal of Human Genetics advance online publication, 30 September 2015; doi:10.1038/ejhg.2015.213.
    No preview · Article · Sep 2015 · European Journal of HumanGenetics
  • Yanyan Cao · Yujin Qu · Fang Song · Jinli Bai · Yuwei Jin · Hong Wang
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    ABSTRACT: Objective: To establish a hyperphenylalaninemia related genes screening method using Ion Torrent Personal Genome Machine (PGM) for early detection and differential diagnosis of hyperphenylalaninemia (HPA). Methods: Three children with known HPA mutations and a healthy control were used for setting up the method. Ten children with HPA with known mutations were recruited for validating the method. Ion Ampliseq PCR was used to amplify the 5' and 3' untranslated region, coding sequence, and flanking introns of PAH, GCH1, PTS, QDPR, and PCBD1 genes. After the enrichment with the Ion OneTouch system, the products were sequenced by PGM. Data from the PGM were processed with Torrent Suite v2.2 software package. All variations were confirmed by Sanger sequencing. Results: For the 4 samples, the PGM output was 94.22 Mb, with approximately 99.5% of reads mapping to the target regions. Among these samples, we detected 74 variations (28 positions) including 6 known mutations. Compared with database and results of Sanger sequencing, 55 (18 positions) polymorphisms and 13 (4 positions) false positive calls were confirmed. For the 10 samples, all the known mutations were successfully identified. Conclusion: Ion Torrent PGM sequencing is suitable for screening genetic mutation underlying HPA from the perspective of metabolic pathways, which can meet the clinical demand for individualized diagnosis and treatment.
    No preview · Article · Feb 2015 · Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics
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    ABSTRACT: To explore the diversity of mutations in survival motor neuron gene 1 (SMN1) by analyzing seven cases of partial deletion of SMN1 gene. Seven patients suspected spinal muscular atrophy (SMA) were recruited from 2011 to 2013. Multiplex ligation-dependent probe amplification (MLPA) for genetic testing of SMA was based on the commercially available SALSA MLPA kit P021-A2. Then the data were analyzed by the software Coffalyser.Negative control samples were chosen with two copies of SMN1 and SMN2. Positive control samples were chosen with zero copies of SMN1 and two copies of SMN2. According to the product description (www.mlpa.com): for exon 7 and 8 of SMN1 and SMN2: a ratio of <0.7 indicates 1 copy, a ratio of 0.7-1.3 2 copies, a ratio of 1.3-1.7 3 copies and a ratio of 1.7-2.3 4 copies. For exon 1, 4, 6, 8 of SMN gene (SMN1+SMN2): a ratio <0.4 indicates 1 copy, a ratio of 4.0-0.6 2 copies, a ratio of 0.7-0.9 3 copies and a ratio of 0.9-1.1 4 copies. All samples were analyzed in duplicate. Using MLPA for clinical diagnostics, two types of partial deletions of SMN1were identified in 7 patients.Since exon 8 is not translated and has no effect on the function of SMN protein, exons 1, 4, 6, 7 were targeted.One had an isolated deletion of exon 7 while the other ones were caused by the deletions of exon 1, 4 and 7. These mutations were not detected by conventional diagnostic methods. Both types of partial deletions of SMN1 gene contained a deletion of exon 7. Two types of partial deletions of SMN1 gene indicate that the structure of SMN gene is unstable leading to a variety of mutation forms. But the major cause of SMA lies in a deletion of exon 7 of SMN1 gene.
    No preview · Article · Feb 2015 · Zhonghua yi xue za zhi
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    ABSTRACT: We evaluated survival motor neuron 2 (SMN2) and neuronal apoptosis inhibitory protein (NAIP) gene copy distribution and the association of copy number with survival in 232 Chinese spinal muscular atrophy (SMA) patients. The SMN2 and NAIP copy numbers correlated positively with the median onset age (r = 0.72 and 0.377). The risk of death for patients with fewer copies of SMN2 or NAIP was much higher than for those with more copies (P < .01). The survival probabilities at 5 years were 5.1%, 90.7%, and 100% for 2, 3, and 4 SMN2 copies and 27.9%, 66.7%, and 87.2% for 0, 1, and 2 NAIP copies, respectively. Our results indicated that combined SMN1-SMN2-NAIP genotypes with fewer copies were associated with earlier onset age and poorer survival probability. Better survival status for Chinese type I SMA might due to a higher proportion of 3 SMN2 and a lower rate of zero NAIP.
    No preview · Article · Oct 2014 · Journal of Child Neurology
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    ABSTRACT: Hyperphenylalaninemia (HPA) can be classified into phenylketonuria (PKU) and tetrahydrobiopterin deficiency (BH4D), according to the defect of enzyme activity, both of which vary substantially in severity, treatment, and prognosis of the disease. To set up a fast and comprehensive assay in order to achieve early etiological diagnosis and differential diagnosis for children with HPA, we designed a custom AmpliSeqTM panel for the sequencing of coding DNA sequence (CDS), flanking introns, 5′ untranslated region (UTR) and 3′ UTR from five HPA-causing genes (PAH, PTS, QDPR, GCH1, and PCBD1) using the Ion Torrent Personal Genome Machine (PGM) Sequencer. A standard group of 15 samples with previously known DNA sequences and a test group of 37 HPA patients with unknown mutations were used for assay validation and application, respectively. All variations were confirmed by Sanger sequencing. In the standard group, all the known mutations were detected and were consistent with the results of previous Sanger sequencing. In the test group, we identified mutations in 71 of 74 alleles, with a mutation detection rate of 95.9%. We also found a frame shift deletion p.Ile25Metfs*13 in PAH that was previously unreported. In addition, 1 of 37 in the test group was inconsistent with either the molecular diagnosis or clinical diagnosis by traditional differential methods. In conclusion, our comprehensive assay based on a custom AmpliSeqTM panel and Ion Torrent PGM sequencing has wider coverage, higher throughput, is much faster, and more efficient when compared with the traditional molecular detection method for HPA patients, which could meet the medical need for individualized diagnosis and treatment.
    No preview · Article · Oct 2014 · Molecular Genetics and Metabolism
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    ABSTRACT: Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder caused by mutations of the survival of motor neuron 1 (SMN1) gene. Approximately 90-95% of SMA patients have a homozygous deletion of SMN1, and 5-10% of patients are believed to have subtle mutations. The molecular diagnosis of SMN1 subtle mutations is hampered by highly homologous SMN2 gene. It is important to establish a rational molecular diagnostic procedure for SMN1 subtle mutations. We analyzed the SMN1 mutations in nine nonhomozygous patients by the following procedures: multiplex ligation-dependent probe amplification, genomic sequencing, T-A cloning on cDNA or genomic level, and/or real-time quantitative analysis. By the above molecular diagnostic procedure, six SMN1 subtle mutations, including c.5C>G(p.Ala2Gly), c.22_23 insA (p.Ser8LysfsX23), c.40G>T(p.Glu14X), c.43C>T(p.Gln15X), c.683T>A(p.Leu228X), and c.56delT(p.Val19GlyfsX21), were identified in nine Chinese patients. p.Glu14X has not been reported previously. Compared with the level of full-length SMN1 transcripts in the healthy carriers (14.1±4.5), the patient with p.Ala2Gly had no significant reduction (13.9±3.64, p=0.955). However, the levels in the patients carrying other mutations were significantly reduced (0.27±0.139 to 13.9±3.64, p=0.000-0.004). We present a reliable and rational diagnostic procedure for SMN1 subtle mutations, which would be helpful in molecular diagnosis of SMA compound heterozygotes. Our work extends the SMN1 mutation spectrum.
    No preview · Article · Jul 2014 · Genetic Testing and Molecular Biomarkers
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    ABSTRACT: Crigler-Najjar syndrome type I (CN-I) is the most severe type of hereditary unconjugated hyperbilirubinemia. It is caused by homozygous or compound heterozygous mutations of the UDP-glycuronosyltransferase gene (UGT1A1) on chromosome 2q37. Two patients clinically diagnosed with CN-I were examined in this paper. We sequenced five exons and their flanking sequences, specifically the promoter region of UGT1A1, of the two patients and their parents. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to determine the UGT1A1 gene copy number of one patient. In patient A, two mutations, c.239_245delCTGTGCC (p.Pro80HisfsX6; had not been reported previously) and c.1156G>T (p.Val386Phe), were identified. In patient B, we found that this patient had lost heterozygosity of the UGT1A1 gene by inheriting a deletion of one allele, and had a novel mutation c.1253delT (p.Met418ArgfsX5) in the other allele. In summary, we detected three UGT1A1 mutations in two CN-I patients: c.239_245delCTGTGCC (p.Pro80HisfsX6), c.1253delT (p.Met418ArgfsX5), and c.1156G>T (p.Val386Phe). The former two mutations are pathogenic; however, the pathogenic mechanism of c.1156G>T (p.Val386Phe) is unknown.
    Preview · Article · May 2014 · Journal of Zhejiang University SCIENCE B
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    ABSTRACT: To detect homozygous deletions of survival motor neuron (SMN) gene with genomic DNA sequencing, and to assess the value of genetic testing for the diagnosis of spinal muscular atrophy (SMA). Polymerase chain reaction (PCR) was used for amplifying SMN gene in 100 SMA patients and 110 controls. Four different bases (g.31957, g.32006, g.32154 and g.32269) between SMN1 and SMN2 within the amplified segments were identified with genomic DNA sequencing. Homozygous deletion of SMN1 or SMN2 was determined by the presence or absence of base peaks at such four sites. Multiplex ligation-dependent probe amplification (MLPA) was carried out to confirm the results of genomic DNA sequencing. In the 100 SMA samples, only SMN2 specific base peaks were detected at the four sites, for which the copy numbers of SMN1 and SMN2 was 0:2 or 0:3, suggesting homozygous deletion of SMN1 gene. By contrast, only SMN1 specific base peaks were detected in 5 samples, for which the ratio of SMN1:SMN2 was 2:0, indicating homozygous deletion of SMN2. At four different sites, SMN1/SMN2 heterozygous peaks were detected in the remaining 105 samples, for which SMN1:SMN2was 2:2, suggesting non-deletion of SMN1 or SMN2. The results of sequencing were consistent with those of MLPA. Genomic DNA sequencing is a rapid, accurate and economic method for the diagnosis of homozygous deletion of SMA.
    No preview · Article · Aug 2013 · Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics
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    ABSTRACT: Angelman syndrome (AS) is a neurobehavioral disorder caused by lack of function of the maternal copy of the ubiquitin-protein ligase E3A (UBE3A) gene. In our study, 49 unrelated patients with classic AS phenotypes were confirmed by methylation-specific PCR (MS-PCR) analysis, short tandem repeat linkage analysis, and mutation screening of the UBE3A gene. Among the Chinese AS patients, 83.7% (41/49) had deletions on maternal chromosome 15q11.2-13. Paternal uniparental disomy, imprinting defects, and UBE3A gene mutations each accounted for 4.1% (2/49). Two AS patients were confirmed by MS-PCR analysis, but the pathogenic mechanism was unknown because their parents' samples were unavailable. Of the 2 described UBE3A gene mutations, i.e., p.Pro400His (c.1199C>A) and p.Asp563Gly (c.1688A>G), the latter has not been reported previously. Mutation transmission analysis showed that the p.Pro400His and p.Asp563Gly mutations originated from asymptomatic mothers. The patients with the maternal deletion showed AS clinical manifestations that were consistent with other studies. However, the incidence of microcephaly (36.7%, 11/30) was lower than that in the Caucasian population (approximately 80%), but similar to that of the Japanese population (34.5%). Our study demonstrated that the occurrence of microcephaly in AS may vary among different populations.
    No preview · Article · Apr 2013 · Clinical Genetics
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    ABSTRACT: Background Proximal spinal muscular atrophy (SMA) is a common neuromuscular disorder resulting in death during childhood. Around 81 ~ 95% of SMA cases are a result of homozygous deletions of survival motor neuron gene 1 (SMN1) gene or gene conversions from SMN1 to SMN2. Less than 5% of cases showed rare subtle mutations in SMN1. Our aim was to identify subtle mutations in Chinese SMA patients carrying a single SMN1 copy. Methods We examined 14 patients from 13 unrelated families. Multiplex ligation-dependent probe amplification analysis was carried out to determine the copy numbers of SMN1 and SMN2. Reverse transcription polymerase chain reaction (RT-PCR) and clone sequencing were used to detect subtle mutations in SMN1. SMN transcript levels were determined using quantitative RT-PCR. Results Six subtle mutations (p.Ser8LysfsX23, p.Glu134Lys, p.Leu228X, p.Ser230Leu, p.Tyr277Cys, and p.Arg288Met) were identified in 12 patients. The p.Tyr277Cys mutation has not been reported previously. The p.Ser8LysfsX23, p.Leu228X, and p.Tyr277Cys mutations have only been reported in Chinese SMA patients and the first two mutations seem to be the common ones. Levels of full length SMN1 (fl-SMN1) transcripts were very low in patients carrying p.Ser8LysfsX23, p.Leu228X or p.Arg288Met compared with healthy carriers. In patients carrying p.Glu134Lys or p.Ser230Leu, levels of fl-SMN1 transcripts were reduced but not significant. The SMN1 transcript almost skipped exon 7 entirely in patients with the p.Arg288Met mutation. Conclusions Our study reveals a distinct spectrum of subtle mutations in SMN1 of Chinese SMA patients from that of other ethnicities. The p.Arg288Met missense mutation possibly influences the correct splicing of exon 7 in SMN1. Mutation analysis of the SMN1 gene in Chinese patients may contribute to the identification of potential ethnic differences and enrich the SMN1 subtle mutation database.
    Preview · Article · Sep 2012 · BMC Medical Genetics
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    ABSTRACT: Mutations in fumarylacetoacetate hydrolase (FAH) gene can lead to tyrosinemia type 1 (HT1), a relatively rare autosomal recessive disorder. To date, no molecular genetic defects of HT1 in China have been described. We investigated a Chinese family with a HT1 child to identify mutations in FAH. DNA sequencing was used for mutations screening in FAH gene. Real-time polymerase chain reaction (PCR) was performed to determine the FAH gene expression level. To confirm the presence of degradation by the nonsense-mediated mRNA decay pathway (NMD), the fragments containing R237X mutations were analyzed by primer introduced restriction analysis-polymerase chain reaction (PIRA-PCR) and cDNA sequencing. Finally, the effects of the mutations reported in this study were predicted by online softwares. A boy aged 3 years and 8 months was diagnosed clinically with HT1 based on his manifestations and biochemical abnormalities. Screening of FAH gene revealed two heterozygous mutations R237X and L375P transmitted from his mother and father respectively. In this pedigree, the amount of FAH mRNA relative to a healthy control was 0.44 for the patient, 0.77 for his mother and 1.07 for his father. Moreover, both PIRA-PCR and cDNA sequencing showed significant reduction of the FAH mRNA with R237X nonsense mutation. The missense mutation of L375P was not reported previously and prediction software showed that this mutation decreased the stability of protein structure and affected protein function. This is the first case of HT1 analyzed by molecular genetics in China. The R237X mutation in FAH down- regulates the FAH gene expression, and the L375P mutation perhaps interrupts the secondary structure of FAH protein.
    No preview · Article · Jun 2012 · Chinese medical journal
  • Yu-wei Jin · Yu-jin Qu · Hong Wang · Jin-li Bai · Fang Song
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    ABSTRACT: To explore the applicability and limitation of PCR-restriction fragment length polymorphism (PCR-RFLP) method for genetic diagnosis of spinal muscular atrophy (SMA). PCR-RFLP was applied to detect potential deletion in exons 7 and 8 of SMN1 gene in 935 suspected cases with SMA. Multiplex ligation-dependent probe amplification(MLPA) was carried out to analyze dosage alteration of SMN1 gene in 339 of such cases. To confirm the accuracy of PCR-RFLP method for homozygous and heterozygous deletions detection, the consistency of PCR-RFLP and MLPA results were assessed with a Pearson Chi-square test. Homozygous deletion of exon 7 of SMN1 was detected in 590 suspected cases. The rate of diagnosis was therefore 63.1% (590/935). For the 339 suspected cases, PCR-RFLP and MLPA respectively identified 194 and 196 homozygous deletions in the exon 7 of SMN1 gene, suggesting a good consistency (98.9%)(Chi-square = 0.2, P = 0.88). However, only 4 of 339 cases was found to carry a heterozygous deletion of SMN1 exon 7 by PCR-RFLP, in contrast with 17 detected by MLPA. The consistency only reached 23.5%, for which statistical significance was detected (Chi-square = 8.29, P< 0.01). Although PCR-RFLP is a simple, specific and efficient method for SMA diagnosis, it has obvious limitation for the diagnosis of 5%-10% SMA patients who have carried a compound heterozygous mutation.
    No preview · Article · Feb 2012 · Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics
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    ABSTRACT: To investigate the type and frequency of gene conversion from SMN1 to SMN2 in Chinese patients affected with spinal muscular atrophy (SMA), and to explore the relationship between gene conversion and clinical phenotype. Non-homozygous deletion of SMN1 gene exon 8 was screened among 417 patients with SMN1 exon 7 homozygous deletions. To analyze and verify the types of gene conversion, genomic DNA sequencing, multiplex ligation-dependent probe amplification (MLPA), and gene subcloning and sequencing were carried out. Thirty-one patients (7.4% of all) with non-homozygous deletions of SMN1 exon 8 were detected. Through series of experiments, the fusion genes SMN1/SMN2 in all cases were delineated. Five types of gene conversions were identified, which included SMN2-I7b/SMN1 E8, SMN2-I7a/SMN1 I7b, SMN2-E7/SMN1 I7a, SMN1 I6/SMN2 E7/SMN1 I7a and SMN2-E7/SMN1 I7a/SMN2 I7b. Such conversions were found in the type I-III patients. For 10 patients with type I-III SMA and 3 copies of SMN2 gene produced by conversion, the average survival age was 5 year and 4 months. Partial conversions of SMN1 gene have been found among Chinese SMA patients. The type of conversion and frequency seem to be different from those of other races. Gene conversion to some extent may impact on survival time and rate of SMA patients, especially type I SMA.
    No preview · Article · Dec 2011 · Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics
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    Xiushan Ge · Jinli Bai · Yanyu Lu · Yujin Qu · Fang Song
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    ABSTRACT: The authors retrospectively studied the natural history of 237 patients with infantile spinal muscular atrophy in China. The onset ages (mean ± SD) for types I to III were 3.1 ± 2.7, 8.7 ± 3.8, and 21.1 ± 11.7 months, respectively. The survival probabilities for type I patients at 1, 2, and 5 years were 44.9%, 38.1%, and 29.3%, respectively, and for type II patients, the probabilities were 100%, 100%, and 97%, respectively. All type III patients were alive. Type I patients with onset age after 2 months had significantly increased survival than those with onset before 2 months (P < .05). It should be noticed that survival probability at 2 years in type I patients in our study was close to that in other Asian samples of spinal muscular atrophy, but slightly better than that among whites. Patients accepted minimal proactive interventions other than antibiotics for pulmonary infection, so our study provides reliable baseline data of natural history of spinal muscular atrophy in China.
    Full-text · Article · Sep 2011 · Journal of child neurology
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    ABSTRACT: Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder. About 80% - 90% of SMA patients are missing both copies of SMN1, and 5% - 10% of patients are compound heterozygotes. In the present study, we aimed to analyze survival motor neuron 1 (SMN1) gene mutation in three patients with spinal muscular atrophy and their families to explore the effect of mutation on SMN protein function and the relationship between mutation and clinical phenotype. According to the international criterion, all patients were diagnosed by a neurologist. Patient 1 is a 5 years old boy with SMA type II. Patient 2, female, 2.5 years old, was SMA type II. Patient 3, female, 9 years old, was SMA type III. The brother of patient 3 was SMA type II, too. The age at last examination was 14 years. Genomic DNA was extracted from peripheral blood leukocytes by using standard phenol/chloroform method and total RNA was extracted from whole blood with QIAamp RNA Blood Mini Kit. PCR/RFLP was used to detect the homozygosis deletion of the SMN1 exon 7, and multiplex ligation-dependent probe amplification (MLPA) were performed to analyze the gene dosage of SMN1 and SMN2 for each patient and his/her family members; reverse transcriptase (RT)-PCR and clone sequencing were conducted for identifying the point mutation of SMN1 in three patients. The sequencing of genomic DNA and MLPA were carried out in the 3 families members to confirm the transition of mutation. No homozygous deletion of the SMN1 exon 7 was observed in any member of the 3 families. Case 1 and case 2 had one SMN1 copy compound with c.400G > A (p.Glu134Lys) mutation on it and SMN2 was two copies, respectively. Case 3 and her brother also had one copy of SMN1 and two copies of SMN2, and a mutation c.689C > T (p.Ser230Leu) occurred on the retained SMN1. All point mutations were from their fathers and deletion come from their mothers for SMN1 gene. In this work, p.Glu134Lys and p.Ser230Leu mutations were identified in three unrelated families and p.Glu134Lys from two patients was first discovered in Chinese SMA. The p.Glu134Lys mutation within the SMN Tudor domain prevents the binding of SMN and Sm. The fact that p.Ser230Leu results in a polar amino acid substituted for non-polar amino acid possibly affects the structure of SMN and then damages its function. SMN1 point mutation analysis is not only advantageous to the diagnosis of those patients with heterozygous deletion of SMN1, but will be beneficial to the prenatal diagnosis and genetic counseling for their families.
    No preview · Article · Jun 2011 · Zhonghua er ke za zhi. Chinese journal of pediatrics
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    ABSTRACT: To identify the point mutations in survival motor neuron gene 1 SMN1 gene and confirm the existence of compound heterozygous mutations in Chinese patients with spinal muscular atrophy (SMA). Three unrelated patients were diagnosed and clinically typed according to the criteria of proximal SMA established by the International SMA Consortium. Multiplex ligation-dependent probe amplification (MLPA) analysis was carried out to measure the copy numbers of SMN1, SMN2 and neuronal apoptosis inhibitory protein gene (NAIP)in the patients. The point mutation analysis of SMN1 gene was performed by reversed transcript-polymerase chain reaction (RT-PCR) and cloning sequencing. The MLPA assay and point mutation analysis were also performed in the family members to confirm the transmission of the mutations. Two point mutations were identified in the present study, i.e., the p.Leu228X in one patient and p.Arg288Met in two patients. The mutation p.Arg288Met was first reported in Chinese and p.Leu228X was first reported in Mainland Chinese. The case carrying p.Leu228X mutation was diagnosed as SMA I with 2 copies of SMN2, and the cases with p.Arg288Met were diagnosed as SMA I and SMA II , respectively, with 3 copies of SMN2 gene. The mutations p.Leu228X and p.Arg288Met caused severe clinical phenotypes, SMA I or SMA II. This study suggested that the compound heterozygous mutations of SMN1 existed in Chinese SMA patients, which was rarely reported previously in Chinese. It was necessary to detect the point mutation in SMN1 for genetic diagnosis of those patients with heterozygous deletion of SMN1, which would be beneficial to prenatal diagnosis and genetic counseling in these families.
    No preview · Article · Apr 2011 · Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics
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    ABSTRACT: Infantile proximal spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder. Approximately 90% - 95% cases of SMA result from homozygous deletion of survival motor neuron gene 1 (SMN1) and 5% cases are caused by compound heterozygous mutation (a SMN1 deletion on one allele and a subtle mutation on the other allele). In this research, two unrelated patients were clinically diagnosed according to the criteria of proximal SMA. Genetic diagnosis was performed to detect the homozygous deletion of exon 7 of SMN1 by PCR-restriction fragment length polymorphism (RFLP) and genomic sequencing. Multiplex ligation-dependent probe amplification (MLPA) analysis was carried out to measure copy numbers of SMN1, SMN2 and neuronal apoptosis inhibitor protein (NAIP) in the patients. Further sequencing of SMN1 allele-specific PCR (AS-PCR) and SMN1 clones were also performed to analyze the point mutation of SMN1 gene. Additionally, the pedigree analysis of these two families was carried out to identify the transmission of the mutation. The inconsistent results using PCR-RFLP and genomic sequencing showed homozygous deletion of exon 7 of SMN1 and heterozygous deletion accompanied with a suspicious mutation in SMN1 gene, respectively. MLPA analysis of these two cases exhibited one SMN1 copy deletion. One identical c.863G > T (p.Arg288Met) mutation was found in two cases by sequencing the SMN1 clones, which confirmed that both cases were SMA compound heterozygotes. One case showed partial conversion to form hybrid SMN (SMN2 I7/SMN1 E8) identified by clones sequencing and another case carrying 3 SMN2 implied complete conversion from SMN1 to SMN2. p.Arg288Met is more a disease-causing mutation than a polymorphism variation, and children with this mutation may have more severe phenotypes.
    Preview · Article · Feb 2011 · Chinese medical journal