Essential role for the tudor domain of SMN in spliceosomal U snRNP assembly: implications for spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a neurodegenerative disease of spinal motor neurons caused by reduced levels of functional survival of motor neurons (SMN) protein. SMN is part of a macromolecular complex that contains the SMN-interacting protein 1 (SIP1) and spliceosomal Sm proteins. Although it is clear that SIP1 as a component of this complex is essential for spliceosomal uridine-rich small ribonucleoprotein (U snRNP) assembly, the role of SMN and its functional interactions with SIP1 and Sm proteins are poorly understood. Here we show that the central region of SMN comprising a tudor domain facilitates direct binding to Sm proteins. Strikingly, the SMA-causing missense mutation E134K within the tudor domain severely reduced the ability of SMN to interact with Sm proteins. Moreover, antibodies directed against the tudor domain prevent Sm protein binding to SMN and abolish assembly of U snRNPs in vivo. Thus, our data show that SMN is an essential U snRNP assembly factor and establish a direct correlation between defects in the biogenesis of U snRNPs and SMA.     

Quantitative analysis of SMN1 and SMN2 genes based on DHPLC: a highly efficient and reliable carrier-screening test

Autosomal recessive spinal muscular atrophy (SMA) is a common, fatal neuromuscular disease caused by homozygous absence of the SMN1 gene in approximately 94% of patients. However, a highly homologous SMN2 gene exists in the same chromosome interval, centromeric to SMN1, and hampers detection of SMN1. We present a new, rapid, simple, and highly reliable method for detecting the SMN1 deletion/conversion and for determining the copy numbers of the SMN1 and SMN2 genes by DHPLC. We analyzed SMN1/SMN2 gene exon 7 deletion/conversion by DHPLC. A total of 25 patients with spinal muscular atrophy lacking the SMN1 gene as well as 309 control individuals from the general population and the family members of patients with SMA were analyzed. By DHPLC analysis, we could detect the SMA-affected cases efficiently just by recognizing an SMN2-only peak. Furthermore, after specific primer amplification and adjustment of the oven temperature, all of the SMA carriers with an SMN1/SMN2 ratio not equal to 1 could be identified unambiguously by this simple and efficient detection system. To calculate the total SMN1/SMN2 gene dosages further, we developed a specific multiplex competitive PCR protocol by simultaneously amplifying the CYBB gene (X-linked), the KRIT1 gene (on chromosome arm 7q), and the SMN1/SMN2 gene ratio by DHPLC. By applying this technique, we could successfully designate all of the genotypes with different SMN1/SMN2 gene copy numbers, including equal and unequal amounts of SMN1 and SMN2. We demonstrated that DHPLC is a fast and reliable tool for detection of carriers of SMA.     

Evaluation and characterization of a high-resolution melting analysis kit for rapid carrier-screening test of spinal muscular atrophy

Spinal muscular atrophy (SMA) is a common autosomal recessive disorder in humans, caused by the homozygous absence of the survival motor neuron gene 1 (SMN1). SMN2, a copy gene, influences the severity of SMA. Several assays have been described for molecular diagnosis or carrier screening of SMA. A newly developed tool based on a high-resolution melting analysis (HRMA) that enables high-throughput screening without sophisticated protocols but low costs reveals itself to be powerful. We evaluate the performance of an HRMA-based kit for a carrier-screening test of SMA that was designed to detect the substitution of a single nucleotide in SMN1 exon 7. Carriers were identified in 453 participants by quantifying the SMN1 gene and compared with denaturing high-performance liquid chromatography (DHPLC) assay. An HRMA-based kit had a higher sensitivity (100%) for carrier testing than the DHPLC assay (93%), with the added advantage that some homozygous sequence alterations could be identified. The HRMA kit is a new, fast, and highly reliable quantitative test for the SMA molecular carrier test.     

The domain encoded by exon 2 of the survival motor neuron protein mediates nucleic acid binding [published erratum appears in Hum Mol Genet 1998 Oct;7(11):1831]

Spinal muscular atrophy (SMA) is a motor neuron disorder resulting from anterior horn cell death. Survival motor neuron ( SMN ) is the SMA- determining gene and is deleted or gene converted in >95% of SMA patients. The SMN protein has a role in spliceosomal snRNP biogenesis and has therefore been implicated indirectly in general cellular RNA processing due to its unique sub-nuclear localization within structures termed 'gems', which co-localize with spliceosomal factors within coiled bodies. In this report, direct SMN RNA-binding activity, in addition to ssDNA and dsDNA binding is demonstrated. The region of SMN encoded by exon 2 is necessary and sufficient to mediate its nucleic acid-binding activities. This domain is homologous to several nucleic acid-binding factors, including several high mobility group (HMG) proteins. Additionally, previously reported SMN missense mutations isolated from SMA patients demonstrated reduced RNA-binding activity, suggesting that nucleic acid binding is functionally significant.      

Molecular characterization of SMN copy number derived from carrier screening and from core families with SMA in a Chinese population

Screening for carriers of spinal muscular atrophy (SMA) is necessary for effective clinical/prenatal diagnosis and genetic counseling. However, a population-based study of SMA prevalence in mainland China has not yet been conducted. In this study, the copy number of survival motor neuron (SMN) genes was determined in 1712 newborn cord blood samples collected from southern China and from 25 core families, which included 26 SMA patients and 44 parents, to identify SMA carriers. The results presented 13 groups with different SMN1/SMN2 ratios among 1712 newborn individuals, which corresponded to 1535 subjects with two copies of SMN1, 119 with three copies of SMN1, 17 with four copies of SMN1, and 41 with a heterozygous deletion of SMN1 exon 7. Simultaneously, two ‘2+0'' genotypes and two point mutations were found among the 44 obligate carriers in the core families, including a novel SMN1 splice-site mutation that was identified in the junction between intron 6 and exon 7 (c. 835–1G>A). These results indicated that the carrier frequency is 1/42 in the general Chinese population and that duplicated SMN1 alleles and de novo deletion mutations are present in a small number of SMA carriers. In addition, we developed and validated a new alternative screening method using a reverse dot blot assay for rapid genotyping of deletional SMA. Our research elucidated the genetic load and SMN gene variants that are present in the Chinese population, and could serve as the basis for a nationwide program of genetic counseling and clinical/prenatal diagnosis to prevent SMA in China.     

脊髓性肌萎缩症的临床实践指南

脊髓性肌萎缩症(spinal muscular atrophy,SMA)为最常见的婴幼儿致死性常染色体隐性遗传病之一,由运动神经元存活基因1(SMN1)突变所致,新生儿发病率为1/10000〜1/6000,人群携带率约为1/72〜1/47,且具有种族差异性。临床表现为进行性、对称性、以肢体近端为主的肌无力和肌萎缩,根据发病时间与临床表型又分为Ⅰ〜Ⅳ型。约95%的SMA患者是由于SMN1基因第7外显子的纯合缺失所致。患者表型差异大、SMN1基因拷贝数变化多、存在假基因干扰、人群携带率高等因素,给SMA的早期诊断、遗传咨询、治疗和预防造成了较大的闲难。本指南总结了国内外的相关研究和指南共识,并结合中国人群的实际情况,介绍了SMA患者的临床表现和发病机制,总结了诊断与遗传咨询等方面的相关经验,期望对临床医师及相关工作者有所帮助,以促进SMA的规范诊治,降低患儿的出生率。     

Identification of eight novel glucokinase mutations in Italian children with maturity-onset diabetes of the young

Maturity-onset diabetes of the young (MODY) is a clinically heterogeneous group of disorders characterized by early onset non-insulin-dependent diabetes mellitus, autosomal dominant inheritance, and primary defect in the function of the beta cells of the pancreas. Mutations in the glucokinase (GCK) gene account for 8%-56% of MODY, with the highest prevalences being found in the southern Europe. While screening for GCK mutations in 28 MODY families of Italian origin, we identified 17 different mutations (corresponding to 61% prevalence), including eight previously undescribed ones. The novel sequence variants included five missense mutations (p.Lys161Asn c.483G>C in exon 4, p.Phe171Leu c.511T>C in exon 5 and p.Thr228Ala c.682A>G, p.Thr228Arg c.683C>G, p.Gly258Cys c.772G>T in exon 7), one nonsense mutation (p.Ser383Ter c.1148C>A in exon 9), the splice site variant c.1253+1G>T in intron 9, and the deletion of 12 nucleotides in exon 10 (p.Ser433_Ile436del c.1298_1309del12). Our study indicates that mutations in the GCK/MODY2 gene are a very common cause of MODY in the Italian population and broadens our knowledge of the naturally occurring GCK mutation repertoire.     

Identification of the bruton tyrosine kinase (BTK) gene mutations in 20 Australian families with X-linked agammaglobulinemia (XLA)

X-linked agammaglobulinemia (XLA) is an immunodeficiency caused by mutations in the Bruton tyrosine kinase (BTK) gene. Twenty Australian patients with an XLA phenotype, from 15 unrelated families, were found to have 14 mutations. Five of the mutations were previously described c.83G>A (p.R28H), c.862C>T (p.R288W), c.904G>A (p.R302G), c.1535T>C (p.L512P), c.700C>T (p.Q234X), while nine novel mutations were identified: four missense c.82C>A (p.R28S), c.494G>A (p.C165Y), c.464G>A (p.C155Y), c.1750G>A (p.G584E), one deletion c.142_144delAGAAGA (p.R48_G50del), and four splice site mutations c.241-2A>G, c.839+4A>G, c.1350-2A>G, c.1566+1G>A. Carrier analysis was performed in 10 mothers and 11 female relatives. The results of this study further support the notion that molecular genetic testing represents an important tool for definitive and early diagnosis of XLA and may allow accurate carrier status and prenatal diagnosis.     

Mutational analysis of mucopolysaccharidosis type VI patients undergoing a phase II trial of enzyme replacement therapy

Mucopolysaccharidosis type VI (MPS VI; Maroteaux-Lamy syndrome) is a lysosomal storage disorder caused by mutations in the N-acetylgalactosamine-4-sulfatase (ARSB) gene. These mutations result in a deficiency of ARSB activity. Ten MPS VI patients were involved in a phase II clinical study of enzyme replacement therapy. Direct sequencing of genomic DNA from these patients was used to identify ARSB mutations. Each individual exon of the ARSB gene was amplified by PCR and subsequently sequenced. Thirteen substitutions (c.215T>G [p.L72R] c.284G>A [p.R95Q], c.305G>A [p.R102H], c.323G>T [p.G108V], c.389C>T [p.P130L], c.511G>A [p.G171S], c.904G>A [p.G302R], c.944G>A [p.R315Q], c.1057T>C [p.W353R], c.1151G>A [p.S384N], c.1178A>C [p.H393P], c.1289A>G [p.H430R] and c.1336G>C [p.G446R]), one deletion (c.238delG), and two intronic mutations (c.1213+5G>A and c.1214-2A>G) were identified. Nine of the 16 mutations identified were novel (R102H, G108V, P130L, G171S, W353R, H430R, G446R, c.1213+5G>A and c.1214-2A>G). The two common polymorphisms c.1072G>A [p.V358M] and c.1126G>A [p.V376M] were identified in some of the patients, along with the silent mutations c.972A>G and c.1191A>G. Cultured fibroblast ARSB mutant protein and residual activity were determined for each patient and, together with genotype information, used to predict the expected clinical severity of each patient.     

Novel GNE mutations in Italian families with autosomal recessive hereditary inclusion-body myopathy

The most common form of autosomal recessive (AR) hereditary inclusion-body myopathy (HIBM), originally described in Persian-Jewish families, is characterized by onset in early adult life with weakness and atrophy of distal lower limb muscles, which progress proximally and relatively spare the quadriceps. AR HIBM is associated with mutations in the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase gene (GNE) on chromosome 9p12-13. In the present study we have identified seven novel GNE mutations in patients from five unrelated Italian families with clinical and pathologic features indicative of AR HIBM. Four were missense mutations (c.1556A>G [p.N519S], c.79C>T [p.P27S], c.1798G>A [p.A600T] and c.616G>A [p.G206S]), two consisted in a single-base deletion (c.616delG [p.G206fsX4] and c.1130delT [p.I377fsX16]) and one in an intronic single-base insertion (c.1070+2dupT). These latter findings further extend the type of GNE mutations associated with HIBM. Furthermore, in one patient we also identified the c.737G>A [p.R246Q] missense mutation that corresponds to the one previously reported in a family from the Bahamas. Interestingly, in two of our families distinct mutations affected nucleotide c.616 in exon 3 (c.616delG and c.616G>A). The possibility of specific portions of the gene being more prone to mutations remains to be elucidated.     

Identification of the molecular defects in Spanish and Argentinian mucopolysaccharidosis VI (Maroteaux-Lamy syndrome) patients, including 9 novel mutations

Maroteaux-Lamy syndrome, or mucopolysaccharidosis VI (MPS VI), is an autosomal recessive lysosomal storage disorder caused by a deficiency of N-acetylgalactosamine-4-sulfatase or arylsulfatase B (ARSB). We aimed to analyze the spectrum of mutations responsible for the disorder in Spanish and Argentinian patients, not previously studied. We identified all the ARSB mutant alleles, nine of them novel, in 12 Spanish and 4 Argentinian patients. The new changes were as follows: six missense mutations: c.245T>G [p.L82R], c.413A>G [p.Y138C], c.719C>T [p.S240F], c.922G>A [p.G308R], c.1340G>T [p.C447F] and c.1415T>C [p.L472P]; one nonsense mutation: c.966G>A [p.W322X]; and two intronic changes involving splice sites: c.1142+2T>A, in the donor splice site of intron 5, which promotes skipping of exon 5, and c.1143-1G>C, which disrupts the acceptor site of intron 5, resulting in skipping of exon 6. We also report 10 previously described mutations as well as several non-pathogenic polymorphisms. Haplotype analysis indicated a common origin for most of the mutations found more than once. Most of the patients were compound heterozygotes, whereas only four of them were homozygous. These observations confirm the broad allelic heterogeneity of the disease, with 19 different mutations in 16 patients. However, the two most frequent mutations, c.1143-1G>C and c.1143-8T>G, present in both populations, accounted for one-third of the mutant alleles in this group of patients.     

Analysis of mutations in the tudor domain of the survival motor neuron protein SMN.

Autosomal recessive childhood onset spinal muscular atrophy (SMA) is a leading cause of infant mortality caused by mutations in the survival motor neuron (SMN) gene. The SMN protein is involved in RNA processing and is localised in structures called GEMs in the nucleus. Nothing is yet understood about why mutations in SMN gene result in the selective motor neuron loss observed in patients. The SMN protein domains conserved across several species may indicate functionally significant regions. Exon 3 of SMN contains homology to a tudor domain, where a Type I SMA patient has been reported to harbour a missense mutation. We have generated missense mutants in this region of SMN and have tested their ability to form GEMs when transfected into HeLa cells. Our results show such mutant SMN proteins still localise to GEMs. Furthermore, exon 7 deleted SMN protein appears to exert a dominant negative effect on localisation of endogenous SMN protein. However, exon 3 mutant protein and exon 5 deleted protein exert no such effect.     

Determination of SMN1 and SMN2 copy number using TaqMan technology

Infantile spinal muscular atrophy (SMA) is a neuromuscular disease caused by homozygous deletion of the SMN1 gene in more than 90% of patients. Identification of carriers for the SMN1 deletion is important for diagnostic purposes and for genetic counseling. The current practical implications of SMN2 copy number determination are limited but may be important, for example, for future drug trials. Here we present a new rapid and reliable approach to determine the copy numbers of the SMN1 and SMN2 genes: For differentiation of the two genes, we developed a quantitative test on the basis of TaqMan technology using minor groove binder (MGB) probes. To evaluate the approach in respect to detection of SMN1 deletion carriers, we tested 40 putative carriers as well as 100 controls. We confirmed the carrier status in all individuals; furthermore, the distribution of SMN1 and SMN2 copies in the control cohort corresponded to that published previously. In total, a clear-cut differentiation between the different copy number ranges could be observed for both genes. This distinct differentiation is based on the exact specificity of MGB probes and the parallel analysis of an external reference locus that circumvents the problem of unavoidable deviations in DNA concentrations. The simplicity and reproducibility of the TaqMan assay presented here should facilitate its establishment in molecular diagnostic laboratories. Nevertheless, the applicability of quantitative analyses of SMN copy numbers requires knowledge about its options and limitations, based on the complex nature of the SMN region and the clinical variability of SMA. Therefore, determination of SMN1 and SMN2 copy numbers should only be offered after careful consideration in each case.     

Ten novel MSH2 and MLH1 germline mutations in families with HNPCC

Hereditary nonpolyposis colorectal cancer (HNPCC) is one of the most common hereditary cancer-susceptibility syndromes. Germline mutations in mismatch repair genes are associated with the clinical phenotype of HNPCC. We report ten novel germline mutations, three in MSH2 and seven in MLH1. All but one mutation have been found in families fulfilling criteria of the Bethesda guidelines; four of them additionally fulfilled the Amsterdam criteria I or II. Eight mutations were considered pathogenic and predictive diagnostics in healthy family members at risk shall be undertaken; these include five frameshift mutations leading to premature stop codons, in MSH2: c.1672delT (p.S558Xfs) and c.2466_2467delTG (p.C822X) and in MLH1: c.1023delG (p.R341Xfs), c.1127_1128dupAT (p.K377Xfs) and c.1310delC (p.P437Xfs); three mutations leading to splice aberrations, in MSH2: c.1661G>C (r.1511_1661del) and in MLH1: c.677+3A>C (r.589_677del) and c.1990-2A>G predicted to result in a splice site defect. The remaining two mutations are unclassified variants with assumed pathogenicity: one missense mutation in the highly conserved ATPase domain of MLH1 (c.122A>G [p.D41G]) and one in-frame insertion of twelve nucleotides in MLH1 (c.2155_2156insATGTGTTCCACA [p.I719delinsNVFHI]). These two mutations were not found in 102 alleles of healthy control individuals. The corresponding tumors from all patients showed a high level of microsatellite instability (MSI-H). Immunohistochemistry (IHC) revealed complete loss of expression of the affected protein in the tumor cells from all but three patients. The tumors from the patients with the mutations c.1127_1128dupAT and c.1990-2A>G showed a reduction of expression of the MLH1-protein, rather than complete loss. In the tumor from the patient with the missense mutation c.122A>G [p.D41G] a normal expression of the proteins coded by MLH1 and MSH2 was noticed.     

Identification of two novel SMN1 point mutations associated with a very severe SMA-I phenotype

Spinal muscular atrophy (SMA) is a common autosomal recessive genetic disorder characterized by degeneration of motor neurons and weakness and muscle atrophy. Approximately 95% of SMA patients are caused by homozygous deletions of the SMN1 gene, whereas the remaining 5% of patients harbor compound heterozygous mutations such as an SMN1 deletion allele and an intragenic mutation (insertions, deletions, or point mutations) in the other SMN1 allele. Although analysis for the SMN1/SMN2 copy number is relatively easy, molecular genetic testing for patients with subtle mutations is still compromised due to the presence of a highly homologous SMN2 gene. Herein, we analyzed the SMN1/SMN2 copy number by multiplex ligation-dependent probe amplification (MLPA) and subtle mutations by long-range PCR (LR-PCR) for two “nondeletion” SMA patients. We identified a missense mutation (c.280G > T, p. (Val94Phe)) and a splicing mutation c.*3+3A > T in SMN1 gene not previously described in the scientific literature. Giving the severe phenotype of the two patients, we speculated that these two point mutations could significantly affect the function of SMN proteins. Our results provide important information for genetic counseling and prenatal diagnosis in these families and enrich the SMN1 mutation database.     

Novel mutations in the human MCCA and MCCB gene causing methylcrotonylglycinuria

Methylcrotonylglycinuria (MCG) is an inborn error of leucine catabolism and has a recessive pattern of inheritance that results from the deficiency of 3-methylcrotonyl-CoA carboxylase (MCC). The clinical phenotypes are highly variable ranging from neonatal onset with severe neurological involvement to asymptomatic adults. Here we identified two novel MCCA (exon 3: c.137G>A; p.46G>E), (IVS7-1G>A splice site mutation), and four novel MCCB (exon 11: c.1065A>T; p.355L>F), (exon 15: c.1430A>G; p.477Q>R), (exon 16: c.1549G>A; p.517G>R), (exon 16: c.1559A>C; p.520Y>S) mutant alleles from five MCC-deficient patients.     

一例脊肌萎缩症患者及其家系的SMN基因突变分析

目的 对1例脊肌萎缩症(spinal muscular atrophy,SMA)患者及其家系成员的SMN基因行突变分析.方法 采用多重连接依赖性探针扩增(multiplex ligation-dependent probe amplification,MLPA)技术、逆转录聚合酶链反应及T克隆-测序技术对患者SMN基因进行拷贝数分析和点突变的鉴定,应用MLPA和针对点突变区域的SMN基因第5外显子PCR-直接测序法对患者父母SMN基因进行拷贝数分析和点突变的证实,同时用200名正常人外周血进行相关位点对照研究.结果 患者具有1个拷贝的SMNl基因和1个拷贝的SMN2基因,在这个SMNl基因第230位密码子上存在1个未见报道的错义突变S230L(TCA→TTA);父亲具有两个SMN1和两个SMN2拷贝,其中1个SMNl基因存在S230L突变;母亲具有1个SMN1拷贝,而SMN2基因是纯合缺失的.200名对照中未发现该位点突变.结论 在1个SMA家系中发现了1个新的SMNI基因点突变,即S230L,并准确分析了此家系各成员的SMN基因型.