An update of the mutation spectrum of the survival motor neuron gene (SMN1) in autosomal recessive spinal muscular atrophy (SMA)

Spinal muscular atrophy (SMA) is characterized by degeneration of motor neurons in the spinal cord, causing progressive weakness of the limbs and trunk, followed by muscle atrophy. SMA is one of the most frequent autosomal recessive diseases, with a carrier frequency of 1 in 50 and the most common genetic cause of childhood mortality. The phenotype is extremely variable, and patients have been classified in type I-III SMA based on age at onset and clinical course. All three types of SMA are caused by mutations in the survival motor neuron gene (SMN1). There are two almost identical copies, SMN1 and SMN2, present on chromosome 5q13. Only homozygous absence of SMN1 is responsible for SMA, while homozygous absence of SMN2, found in about 5% of controls, has no clinical phenotype. Ninety-six percent of SMA patients display mutations in SMN1, while 4% are unlinked to 5q13. 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. Among the 23 different subtle mutations described so far, the Y272C missense mutation is the most frequent one, at 20%. Given this uniform mutation spectrum, direct molecular genetic testing is an easy and rapid analysis for most of the SMA patients. Direct testing of heterozygotes, while not trivial, is compromised by the presence of two SMN1 copies per chromosome in about 4% of individuals. The number of SMN2 copies modulates the SMA phenotype. Nevertheless, it should not be used for prediction of severity of the SMA.     

Genetic risk assessment in carrier testing for spinal muscular atrophy

As evidenced by the complete absence of a functionally critical sequence in exon 7, approximately 94% of individuals with clinically typical spinal muscular atrophy (SMA) lack both copies of the SMN1 gene at 5q13. Hence most carriers have only one copy of SMN1. Combining linkage and dosage analyses for SMN1, we observed unaffected individuals who have two copies of SMN1 on one chromosome 5 and zero copies of SMN1 on the other chromosome 5. By dosage analysis alone, such individuals, as well as carriers of non-deletion disease alleles, are indistinguishable from non-carrier individuals. We report that approximately 7% of unaffected individuals without a family history of SMA have three or four copies of SMN1, implying a higher frequency of chromosomes with two copies of SMN1 than previously reported. We present updated calculations for disease and non-disease allele frequencies and we describe how these frequencies can be used for genetic risk assessment in carrier testing for SMA.     

云南地区3049名育龄人群脊髓性肌萎缩症携带者筛查结果分析

目的对云南地区3049名育龄人群进行脊髓性肌萎缩症(spinal muscular atrophy,SMA)的携带者筛查,探讨本地区人群运动神经元存活基因(survival motor neuron,SMN)的拷贝数情况及携带频率。方法应用多重连接探针扩增技术(multiplex ligation-dependent probe amplification,MLPA)对SMN1及SMN2基因第7外显子的拷贝数进行检测,筛查出SMN1基因第7外显子拷贝数为1的SMA携带者。对双方均为携带者的夫妇提供产前诊断。结果在3049名育龄人群中,共检测出SMA携带者62例,携带率为1/49(2.03%)。男性携带率为1.91%(40/2094),女性携带率为2.30%(22/955),二者的差异无统计学意义(P>0.05)。SMN1杂合缺失占1.30%(41/3049),由SMN1转换为SMN2者占0.69%(21/3049)。SMN1等位基因的平均拷贝数为1.99。检出双方均为SMA携带者的夫妇2对,通过产前诊断避免了1例患病胎儿的出生。结论云南地区SMA男女携带者的频率无显著差异,符合常染色体隐性遗传模式。阐明SMA携带者的频率和SMN基因的拷贝数情况,可为遗传咨询和产前预防提供依据。     

Improving detection and genetic counseling in carriers of spinal muscular atrophy with two copies of the SMN1 gene

Spinal muscular atrophy (SMA) is an autosomal recessive disease caused by mutations in the survival motor neuron1 gene (SMN1). Global carrier frequency is around 1 in 50 and carrier detection is crucial to define couples at risk to have SMA offspring. Most SMA carriers have one SMN1 copy and are currently detected using quantitative methods. A few, however, have two SMN1 genes in cis (2/0 carriers), complicating carrier diagnosis in SMA. We analyzed our experience in detecting 2/0 carriers from a cohort of 1562 individuals, including SMA parents, SMA relatives, and unrelated individuals of the general population. Interestingly, in three couples who had an SMA child, both the parents had two SMN1 copies. Families of this type have not been previously reported. Our results emphasize the importance of performing a detailed carrier study in SMA parents with two SMN1 copies. Expanding the analysis to other key family members might confirm potential 2/0 carriers. Finally, when a partner of a known carrier presents two SMN1 copies, the study of both parents will provide a more accurate diagnosis, thus optimizing genetic counseling.     

Population screening and cascade testing for carriers of SMA

Spinal muscular atrophy (SMA) is one of the most common autosomal-recessive diseases, caused by absence of both copies of the survival motor neuron 1 (SMN1) gene. Identification of SMA carriers has important implications for individuals with a family history and the general population. SMA carriers are completely healthy and most are unaware of their carrier status until they have an affected child. A total of 422 individuals have been studied to identify SMA carriers. This cohort included 117 parents of children homozygously deleted for SMN1 (94% were carriers and 6% had two copies of SMN1; of these individuals, two in seven had the '2+0' genotype, two in seven were normal but had children carrying a de novo deletion and three in seven were unresolved), 158 individuals with a significant family history of SMA (47% had one copy, 49% had two copies and 4% had three copies of SMN1) and 146 individuals with no family history of SMA (90% had two copies, 2% had one copy and 8% had three copies of SMN1). The SMA carrier frequency in the Australian population appears to be 1/49 and the frequency of two-copy SMN1 alleles and de novo deletion mutations are both at least 1.7%. A multimodal approach involving quantitative analysis, linkage analysis and genetic risk assessment (GRA), facilitates the resolution of SMA carrier status in individuals with a family history as well as individuals of the general population, providing couples with better choices in their family planning.     

Quantitative analysis of SMN1 gene and estimation of SMN1 deletion carrier frequency in Korean population based on real-time PCR

Spinal muscular atrophy (SMA) is an autosomal recessive disorder, caused by homozygous absence of the survival motor neuron gene (SMN1) in approximately 94% of patients. Since most carriers have only one SMN1 gene copy, several SMN1 quantitative analyses have been used for the SMA carrier detection. We developed a reliable quantitative real-time PCR with SYBR Green I dye and studied 13 patients with SMA and their 24 parents, as well as 326 healthy normal individuals. The copy number of the SMN1 gene was determined by the comparative threshold cycle (Ct) method and albumin was used as a reference gene. The homozygous SMN1 deletion ratio of patients was 0.00 and the hemizygous SMN1 deletion ratio of parents ranged from 0.39 to 0.59. The deltadelta Ct ratios of 7 persons among 326 normal individuals were within the carrier range, 0.41-0.57. According to these data, we estimated the carrier and disease prevalence of SMA at 1/47 and 1/8,496 in Korean population, respectively. These data indicated that there would be no much difference in disease prevalence of SMA compared with western countries. Since the prevalence of SMA is higher than other autosomal recessive disorders, the carrier detection method using real-time PCR could be a useful tool for genetic counseling.     

Six unaffected livebirths following preimplantation diagnosis for spinal muscular atrophy

Spinal muscular atrophy (SMA) is a severe neurodegenerativeautosomal recessive disorder, second only in frequency to cysticfibrosis. In its most severe form, SMA type I (Werdnig–Hoffman),death invariably ensues before age 2 years from respiratoryfailure or infection. Around 98% of clinical cases of SMA arecaused by the homozygous absence of a region of exons 7 and8 of the telomeric copy of the SMN gene (SMN1) on chromosome5. We have developed a novel means of preimplantation diagnosisof SMA using a nested polymerase chain reaction (PCR) amplificationof exon 7 of SMN, followed by a HinfI restriction digest ofthe PCR product enabling the important SMN1 gene to be distinguishedfrom the centromeric SMN2 gene which has no clinical phenotype.This method was designed to reduce the likelihood of misdiagnosis.Five couples were treated using this method. Four proceededto embryo transfer which resulted in six liveborns (one singleton,one twin and one triplet), all free of SMA. Embryo transferwas not performed in one cycle because of PCR contamination. preimplantation genetic diagnosis/SMN1/spinal muscular atrophy     

Screening for spinal muscular atrophy mutation carriers among 4931 pregnant women from Liuzhou region of GuangxiCSCD

Objective: To screen for carriers of SMN1 gene mutation, which underlies spinal muscular atrophy (SMA), in 4931 pregnant women from Liuzhou region of Guangxi, and to determine the carrier rate. Methods: Combined denaturing high-performance liquid chromatography (DHPLC) and multiple PCR techniques were used to detect the copy number of SMN1 gene. The carrier frequency was calculated. The spouse of the carrier was also screened, and prenatal diagnosis was provided to the couples who were both positive. Results: Among the 4931 pregnant women, 61 were found to harbor only one copy of the SMN1 gene, which yielded a carrier rate of 1. 2%. Subsequent testing has identified 1 fetus carrying homozygous deletions of the SMN1 gene. Conclusion: The carrier rate of SMA mutation in Liuzhou region is slightly lower than that of other regions of southern China. DHPLC can effectively screen the carriers of SMA mutation and provide a basis for genetic counseling and prenatal diagnosis. © 2018 MeDitorial Ltd. All rights reserved.     

Unusual molecular findings in autosomal recessive spinal muscular atrophy.

All three types of autosomal recessive spinal muscular atrophy map to chromosome 5q11.2-q13.3 and are associated with deletions or mutations of the SMN (survival motor neurone) gene. The availability of a test to distinguish between the SMN gene and its nearly identical centromeric copy cBCD541 allows molecular diagnosis. We have analysed patients from 24 Belgian and 34 Turkish families for the presence or absence of a deletion in the SMN gene. A homozygous deletion in the SMN gene was seen in 90% of unrelated SMA patients. A non-radioactive SSCP assay allows for a semiquantitative analysis of the copy number of the centromeric and SMN genes. Hence, direct carrier detection has become feasible under certain conditions. We observed a phenotypically normal male, father of an SMA type I patient, presenting with only a single copy of the SMN gene and lacking both copies of the cBCD541 gene. This illustrates that a reduction of the total number of SMN and cBCD541 genes to a single SMN copy is compatible with normal life. In another SMA type I family, there is evidence for a de novo deletion of the centromeric gene in a normal sib. This observation illustrates the susceptibility of the SMA locus to de novo deletions and rearrangements.     

Molecular analysis of the SMN and NAIP genes in Spanish spinal muscular atrophy (SMA) families and correlation between number of copies of cBCD541 and SMA phenotype [published erratum appears in Hum Mol Genet 1996 May;5(5):710]

Spinal muscular atrophy is an autosomal recessive disorder which affects about 1 in 10,000 individuals. The three clinical forms of SMA were mapped to the 5q13 region. Three candidate genes have been isolated and shown to be deleted in SMA patients: the Survival Motor Neuron gene (SMN), the Neuronal Apoptosis Inhibitory Protein gene (NAIP) and the XS2G3 cDNA. In this report we present the molecular analysis of the SMN exons 7 and 8 and NAIP exon 5 in 65 Spanish SMA families. NAIP was mostly deleted in type I patients (67.9%) and SMN was deleted in 92.3% of patients with severe and milder forms. Most patients who lacked the NAIP gene also lacked the SMN gene, but we identified one type II patient deleted for NAIP exon 5 but not for SMN exons 7 and 8. Two other patients carried deletions of NAIP exon 5 and SMN exon 7 but retained the SMN exon 8. Three polymorphic variants from the SMN gene, showing changes on the sequence of the centromeric (cBCD541) and telomeric copies of the SMN gene, were found. In addition, we show several genetic rearrangements of the telomeric SMN gene, which include duplication of this gene in one normal chromosome, and putative gene conversion events in affected and normal chromosomes. Altogether these results corroborate the high genetic variability of the SMA region. Finally, we have determined the ratio between the number of centromeric and telomeric copies of the SMN gene in parents of SMA patients, showing that the majority of parents of types II and III patients carried three or more copies of the cBCD541 gene; we suggest a relationship between the number of copies of cBCD541 and the disease phenotype.      

Spinal Muscular Atrophy: From Gene Discovery to Clinical Trials

Spinal muscular atrophy (SMA) is a common neuromuscular disorder with autosomal recessive inheritance, resulting in the degeneration of motor neurons. The incidence of the disease has been estimated at 1 in 6000–10,000 newborns with a carrier frequency of 1 in 40–60. SMA is caused by mutations of the SMN1 gene, located on chromosome 5q13. The gene product, survival motor neuron (SMN) plays critical roles in a variety of cellular activities. SMN2, a homologue of SMN1, is retained in all SMA patients and generates low levels of SMN, but does not compensate for the mutated SMN1. Genetic analysis demonstrates the presence of homozygous deletion of SMN1 in most patients, and allows screening of heterozygous carriers in affected families. Considering high incidence of carrier frequency in SMA, population‐wide newborn and carrier screening has been proposed. Although no effective treatment is currently available, some treatment strategies have already been developed based on the molecular pathophysiology of this disease. Current treatment strategies can be classified into three major groups: SMN2‐targeting, SMN1‐introduction, and non‐SMN targeting. Here, we provide a comprehensive and up‐to‐date review integrating advances in molecular pathophysiology and diagnostic testing with therapeutic developments for this disease including promising candidates from recent clinical trials.     

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.     

Carrier frequency of SMN1‐related spinal muscular atrophy in north Indian population: The need for population based screening program

Chromosome 5q related Spinal muscular atrophy (SMA) is an autosomal recessive, progressive, neuromuscular disorder most commonly caused by homozygous deletion of exon 7 or exon 7 and 8 of SMN1 gene. Being the leading genetic cause of infant mortality, studies of its prevalence and incidence are necessary. Carrier testing for the common pathogenic variant for SMA is offered to the couples visiting our tertiary care hospital in North India. Subjects were tested for SMA carrier status by Multiplex Ligation‐dependent Probe amplification (MLPA) technique for deletion of exons 7 and 8 of SMN1 gene. The retrospective data of individuals tested for SMA carrier status in last 4 years (2016–2019) was evaluated. Six hundred and six individuals without family history of SMA or carrier of SMA who were subjected to MLPA based screening for SMA carrier status were included in the study. The carrier frequency of SMN1 deletion (deletion of exon 7 and/or exon 8) was found to be 1 in 38 (16 out of 606). The catchment area of our medical genetics clinic covering the state of Uttar Pradesh (16.5% of Indian population according to censusindia.gov.in , 2011) and neighboring states, showing SMA carrier frequency of 1:38 in a cohort with no prior positive family history has important significance for policy making.     

Somatic mosaicism for a heterozygous deletion of the survival motor neuron (SMN1) gene

Infantile spinal muscular atrophy (SMA) is a common autosomal recessive disease with a high demand for carrier testing. The disease is caused by homozygous deletions of the survival motor neuron (SMN)1 gene on chromosome 5q13 in more than 90% of cases. Meanwhile, several reliable quantitative methods for carrier detection in the general population have been implemented with a risk of at least 5% for false negative results. Linkage analyses with chromosome 5 markers can be used for complementary information, but they are restricted to risk estimation of close relatives in affected families. Here, we present the first observation of a somatic mosaicism in an SMA carrier. Molecular genetic studies gave evidence that the SMN1 deletion of an SMA type I patient most probably arose from somatic mosaicism in the paternal grandmother. The patient's father and his two brothers were shown to be carriers of three different maternal haplotypes in 5q13. Final conclusions for genetic counselling were only possible after both linkage analysis and quantitative real-time PCR analysis of SMN1 copy numbers.     

Inverse correlation between SMN1 and SMN2 copy numbers: evidence for gene conversion from SMN2 to SMN1

Most carriers of autosomal recessive spinal muscular atrophy (SMA) have only one copy of SMN1 because of SMN1 gene deletions or gene conversions from SMN1 to SMN2, which has only one base difference in coding sequence from SMN1. Using SMN gene dosage analysis, we determined the copy numbers of SMN1 and SMN2 in the general population as well as in SMA patients and carriers. Increased SMN1 copy number is associated with decreased SMN2 copy number in the general population; that is, SMN2 copy number was decreased to one or zero copies in 11 of 13 individuals with three or four copies of SMN1, whereas only 71 of 164 individuals with two copies of SMN1 had one or zero copies of SMN2 (P<0.01). SMN2 copy number was increased to three or four in a subset of SMN1 deletion/conversion carriers, and in most SMA patients with a milder phenotype. In conclusion, our data provide evidence that gene conversion from SMN2 to SMN1 occurs, and that SMN1 converted from SMN2 is present in the general population.     

Implementation of SMA carrier testing in genetic laboratories: comparison of two methods for quantifying the SMN1 gene

The degeneration and loss of motor neurons of the anterior horn characterize children affected with spinal muscular atrophy (SMA). Mutations in the survival motor neuron gene (SMN1) are determinant for the development of the disease whereas the number of copies of SMN2, the highly homologous copy of SMN1, plays a role as a phenotypic modifier factor. The detection of SMN1 homozygous deletions is the typical test for SMA diagnosis. Owing to the limitation of this test for carrier and heterozygous deletion analysis, the demand of SMN1 quantitative tests is permanently growing. The high incidence of SMA, the notable carrier frequency, the severity of the disease, and the lack of effective treatment may justify the implementation of such an analysis in DNA diagnostic labs. The advantages and disadvantages of two reliable quantitative methods were evaluated. One of these is a competitive PCR protocol using internal standards and a genomic sequence as a reference. The other method is a real-time PCR employing an external standard as a reference. Both methods present sufficient advantages for incorporation into molecular genetic diagnostic labs. The possibility of studying samples from different labs, the versatility and reproducibility of the analysis, and cost-benefit calculations must be considered in the final choice.     

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.     

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.     

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。     

Proximal and distal spinal muscular atrophy in one family: molecular genetic studies provide further evidence for the non-allelic origin of both diseases.

We present the results of clinical and molecular genetic investigations of a family in which the father suffers from distal spinal muscular atrophy and the younger son is affected by infantile autosomal recessive SMA type I. The molecular analysis of the SMN gene showed homozygous deletions of telSMN exons 7 and 8 in the son only. This was probably the result of a new mutation in the paternal haplotype, since the affected boy did not inherit one copy of the marker Ag1-CA. These results indicate that distal and proximal SMA in this family are not caused by the same gene on chromosome 5q.