Molecular analysis of SMN1, SMN2, NAIP, GTF2H2, and H4F5 genes in 157 Chinese patients with spinal muscular atrophy

Spinal muscular atrophy (SMA) is a common and lethal autosomal recessive neurodegenerative disorder, which is caused by mutations of the survival motor neuron 1 (SMN1) gene. Additionally, the phenotype is modified by several genes nearby SMN1 in the 5q13 region. In this study, we analyzed mutations in SMN1 and quantified the modifying genes, including SMN2, NAIP, GTF2H2, and H4F5 by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP), multiplex ligation-dependent probe amplification (MLPA), TA cloning, allele-specific long-range PCR, and Sanger sequencing in 157 SMA patients. Most SMA patients (94.90%) possessed a homozygous SMN1 deletion, while 10 patients demonstrated only the absence of exon 7, but the presence of exon 8. Two missense mutations (c.689 C > T and c.844 C > T) were identified in 2 patients who both carried a single copy of SMN1. We found inverse correlations between SMN2, the NAIP copy number, and the clinical severity of the disease. Furthermore, 7 severe type I patients possessed large-scale deletions, including SMN1, NAIP, and GTF2H2. We conclude that SMN1 gene conversion, SMN1 subtle mutations, SMN2 copy number, and the extent of deletion in the 5q13 region should all be considered in the genotype–phenotype analysis of SMA.     

Characterisation of SMN hybrid genes in Spanish SMA patients: de novo, homozygous and compound heterozygous cases

Autosomal recessive spinal muscular atrophy (SMA) is classified, by age of onset and maximal motor milestones achieved, into type I (severe form), type II (intermediate form) and type III (mild/moderate form). SMA is caused by mutations in the survival motor neuron telomeric gene (SMN1) and a centromeric functional copy of this gene (SMN2) exists, both genes being located at 5q13. Homozygous deletion of exons 7 and 8 of SMN1 has been detected in approx 85% of Spanish SMA patients regardless of their phenotype. Nineteen cases with the sole deletion of exon 7 but not exon 8 (2 cases of type I, 13 cases of type II, four cases of type III) were further analysed for the presence of SMN2-SMN1 hybrid genes. We detected four different hybrid structures. Most of the patients were carriers of a hybrid structure: centromeric intron 6- centromeric exon 7- telomeric exon 8 (CCT), with or without neuronal apoptosis-inhibitor protein (NAIP). In two patients, a different hybrid structure, viz. telomeric intron 6- centromeric exon 7- telomeric exon 8 (TCT), was detected with or without NAIP. A phenotype-genotype correlation comparing the different structures of the hybrid alleles was delineated. Type I cases in our series are attributable to intrachromosomal deletion with a smaller number of SMN2 copies. Most cases with hybrid genes are type II occurring by a combination of a classical deletion in one chromosome and a hybrid gene in the other. Type III cases are closely associated with homozygozity or compound heterozygozity for hybrid genes resulting from two conversion events and have more copies of hybrid genes and SMN2 than type I or II cases.     

Quantitative Analyses of SMN1 and SMN2 Based on Real-Time LightCycler PCR: Fast and Highly Reliable Carrier Testing and Prediction of Severity of Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is a common autosomal recessive disorder in humans, caused by homozygous absence of the survival motor neuron gene 1 (SMN1). SMN2, a copy gene, influences the severity of SMA and may be used in somatic gene therapy of patients with SMA in the future. We present a new, fast, and highly reliable quantitative test, based on real-time LightCycler PCR that amplifies either SMN1 or SMN2. The SMN1 copies were determined and validated in 329 carriers and controls. The specificity of the test is 100%, whereas the sensitivity is 96.2%. The quantitative analysis of SMN2 copies in 375 patients with type I, type II, or type III SMA showed a significant correlation between SMN2 copy number and type of SMA as well as duration of survival. Thus, 80% of patients with type I SMA carry one or two SMN2 copies, and 82% of patients with type II SMA carry three SMN2 copies, whereas 96% of patients with type III SMA carry three or four SMN2 copies. Among 113 patients with type I SMA, 9 with one SMN2 copy lived <11 mo, 88/94 with two SMN2 copies lived <21 mo, and 8/10 with three SMN2 copies lived 33-66 mo. On the basis of SMN2 copy number, we calculated the posterior probability that a child with homozygous absence of SMN1 will develop type I, type II, or type III SMA.     

Spinal muscular atrophy: Broad disease spectrum and sex-specific phenotypes

Spinal muscular atrophy (SMA) is one of the major genetic disorders associated with infant mortality. More than 90% of cases of SMA result from deletions of or mutations in the Survival Motor Neuron 1 (SMN1) gene. SMN2, a nearly identical copy of SMN1, does not compensate for the loss of SMN1 due to predominant skipping of exon 7. The spectrum of SMA is broad, ranging from prenatal death to infant mortality to survival into adulthood. All tissues, including brain, spinal cord, bone, skeletal muscle, heart, lung, liver, pancreas, gastrointestinal tract, kidney, spleen, ovary and testis, are directly and/or indirectly affected in SMA. Accumulating evidence on impaired mitochondrial biogenesis and defects in X chromosome-linked modifying factors, coupled with the sexual dimorphic nature of many tissues, point to sex-specific vulnerabilities in SMA. Here we review the role of sex in the pathogenesis of SMA.     

A single strand conformation polymorphism-based carrier test for spinal muscular atrophy

Spinal muscular atrophy (SMA) is an autosomal recessive disorder with a newborn prevalence of 1 in 10,000, and a carrier frequency of 1 in 40-60 individuals. The SMA locus has been mapped to chromosome 5q11.2-13. The disease is caused by a deletion of the SMN gene, often encompassing other genes and microsatellite markers. The SMN gene is present in two highly homologous copies, SMN1 and SMN2, differing at five nucleotide positions. Only homozygous SMN1 mutations cause the disease. The sequence similarity between the SMN1 and SMN2 genes can make molecular diagnosis and carrier identification difficult. We developed a sensitive and reliable molecular test for SMN1 carrier identification, by setting up a nonradioactive single strand conformation polymorphism (SSCP)-based method, which allows for the quantification of the amount of the SMN1 gene product with respect to a control gene. The assay was validated in 56 obligate (ascertained) carriers and 20 (ascertained) noncarriers. The sensitivity of the test is 96.4%, and its specificity, 98%. In addition, 6 of 7 SMA patients without homozygous deletions presented with a heterozygous deletion, suggesting a concomitant undetected point mutation on the nondeleted SMN1 allele. Therefore, the present test is effective for detecting compound hemizygote patients, for testing carriers in SMA families, and for screening for SMA heterozygotes in the general population.     

Mildly affected patients with spinal muscular atrophy are partially protected by an increased SMN2 copy number

Spinal muscular atrophy (SMA) is a recessive neuromuscular disorder caused by loss of the SMN1 gene. The clinical distinction between SMA type I to IV reflects different age of onset and disease severity. SMN2, a nearly identical copy gene of SMN1, produces only 10% of full-length SMN RNA/protein and is an excellent target for a potential therapy. Several clinical trials with drugs that increase the SMN2 expression such as valproic acid and phenylbutyrate are in progress. Solid natural history data for SMA are crucial to enable a correlation between genotype and phenotype as well as the outcome of therapy. We provide genotypic and phenotypic data from 115 SMA patients with type IIIa (age of onset <3 years), type IIIb (age of onset >3 years) and rare type IV (onset >30 years). While 62% of type IIIa patients carry two or three SMN2 copies, 65% of type IIIb patients carry four or five SMN2 copies. Three type IV SMA patients had four and one had six SMN2 copies. Our data support the disease-modifying role of SMN2 leading to later onset and a better prognosis. A statistically significant correlation for ≥4 SMN2 copies with SMA type IIIb or a milder phenotype suggests that SMN2 copy number can be used as a clinical prognostic indicator in SMA patients. The additional case of a foetus with homozygous SMN1 deletion and postnatal measurement of five SMN2 copies illustrates the role of genotypic information in making informed decisions on the management and therapy of such patients.Database: SMN1—OMIM: 600354; GeneBank: U18423, SMN2—OMIM: 601627: GeneBank: NM_022875     

Targeting SR Proteins Improves SMN Expression in Spinal Muscular Atrophy Cells

Spinal muscular atrophy (SMA) is one of the most common inherited causes of pediatric mortality. SMA is caused by deletions or mutations in the survival of motor neuron 1 (SMN1) gene, which results in SMN protein deficiency. Humans have a centromeric copy of the survival of motor neuron gene, SMN2, which is nearly identical to SMN1. However, SMN2 cannot compensate for the loss of SMN1 because SMN2 has a single-nucleotide difference in exon 7, which negatively affects splicing of the exon. As a result, most mRNA produced from SMN2 lacks exon 7. SMN2 mRNA lacking exon 7 encodes a truncated protein with reduced functionality. Improving SMN2 exon 7 inclusion is a goal of many SMA therapeutic strategies. The identification of regulators of exon 7 inclusion may provide additional therapeutic targets or improve the design of existing strategies. Although a number of regulators of exon 7 inclusion have been identified, the function of most splicing proteins in exon 7 inclusion is unknown. Here, we test the role of SR proteins and hnRNP proteins in SMN2 exon 7 inclusion. Knockdown and overexpression studies reveal that SRSF1, SRSF2, SRSF3, SRSF4, SRSF5, SRSF6, SRSF7, SRSF11, hnRNPA1/B1 and hnRNP U can inhibit exon 7 inclusion. Depletion of two of the most potent inhibitors of exon 7 inclusion, SRSF2 or SRSF3, in cell lines derived from SMA patients, increased SMN2 exon 7 inclusion and SMN protein. Our results identify novel regulators of SMN2 exon 7 inclusion, revealing potential targets for SMA therapeutics.     

Genetic testing and risk assessment for spinal muscular atrophy (SMA)

Spinal muscular atrophy (SMA) is one of the most common autosomal recessive diseases, affecting approximately 1 in 10,000 live births, and with a carrier frequency of approximately 1 in 50. Because of gene deletion or conversion, SMN1 exon 7 is homozygously absent in approximately 94% of patients with clinically typical SMA. Approximately 30 small intragenic SMN1 mutations have also been described. These mutations are present in many of the approximately 6% of SMA patients who do not lack both copies of SMN1, whereas SMA of other patients without a homozygous absence of SMN1 is unrelated to SMN1. A commonly used polymerase chain reaction/restriction fragment length polymorphism (PCR-RFLP) assay can be used to detect a homozygous absence of SMN1 exon 7. SMN gene dosage analyses, which can determine the copy numbers of SMN1 and SMN2 (an SMN1 homolog and a modifier for SMA), have been developed for SMA carrier testing and to confirm that SMN1 is heterozygously absent in symptomatic individuals who do not lack both copies of SMN1. In conjunction with SMN gene dosage analysis, linkage analysis remains an important component of SMA genetic testing in certain circumstances. Genetic risk assessment is an essential and integral component of SMA genetic testing and impacts genetic counseling both before and after genetic testing is performed. Comprehensive SMA genetic testing, comprising PCR-RFLP assay, SMN gene dosage analysis, and linkage analysis, combined with appropriate genetic risk assessment and genetic counseling, offers the most complete evaluation of SMA patients and their families at this time. New technologies, such as haploid analysis techniques, may be widely available in the future.     

Detection of spinal muscular atrophy carriers by nested polymerase chain reaction of single sperm cells

Spinal muscular atrophy (SMA) is an autosomal recessive disorder with a carrier frequency of approximately 1 in 40. Approximately 95% of patients have homozygous deletions of exon 7 and/or 8 of the SMN1 gene. Carrier testing for SMA is relatively complex and requires quantitative polymerase chain reaction (PCR) of genomic DNA to determine SMN1 copy number. The purpose of this study was to assess the feasibility of carrier testing for SMA in males, by nested PCR analysis of SMN1 deletions in single sperm cells. A nested PCR method was developed to amplify SMN1 exon 7 in single cells. Restriction enzyme digestion with DraI was used to differentiate between the highly homologous SMN1 and SMN2 genes. Single sperm cells from five known SMA carriers and six noncarriers were analyzed. Among the five carriers, a total of 132 single sperm cells were analyzed and SMN1 exon 7 deletion was detected in 68 cells (51.5%). In contrast, among the six noncarriers, a total of 136 single sperm cells were analyzed. Of these, an apparent SMN1 exon 7 deletion was detected in four sperm cells. This was interpreted as an allele dropout (ADO) rate of 2.9%. We conclude that nested PCR of SMN1 exon 7 is an accurate and reproducible method for detection of SMA male carriers with a SMN1 deletion.     

Intragenic telSMN mutations: frequency, distribution, evidence of a founder effect, and modification of the spinal muscular atrophy phenotype by cenSMN copy number.

The autosomal recessive neuromuscular disorder proximal spinal muscular atrophy (SMA) is caused by the loss or mutation of the survival motor neuron (SMN) gene, which exists in two nearly identical copies, telomeric SMN (telSMN) and centromeric SMN (cenSMN). Exon 7 of the telSMN gene is homozygously absent in approximately 95% of SMA patients, whereas loss of cenSMN does not cause SMA. We searched for other telSMN mutations among 23 SMA compound heterozygotes, using heteroduplex analysis. We identified telSMN mutations in 11 of these unrelated SMA-like individuals who carry a single copy of telSMN: these include two frameshift mutations (800ins11 and 542delGT) and three missense mutations (A2G, S262I, and T274I). The telSMN mutations identified to date cluster at the 3' end, in a region containing sites for SMN oligomerization and binding of Sm proteins. Interestingly, the novel A2G missense mutation occurs outside this conserved carboxy-terminal domain, closely upstream of an SIP1 (SMN-interacting protein 1) binding site. In three patients, the A2G mutation was found to be on the same allele as a rare polymorphism in the 5' UTR, providing evidence for a founder chromosome; Ag1-CA marker data also support evidence of an ancestral origin for the 800ins11 and 542delGT mutations. We note that telSMN missense mutations are associated with milder disease in our patients and that the severe type I SMA phenotype caused by frameshift mutations can be ameliorated by an increase in cenSMN gene copy number.     

Molecular analysis of survival motor neuron (SMN) and neuronal apoptosis inhibitory protein (NAIP) genes of spinal muscular atrophy patients and their parents

We have assayed deletions of two candidate genes for spinal muscular atrophy (SMA), the survival motor neuron (SMN) and neuronal apoptosis inhibitory protein (NAIP) genes, in 101 patients from 86 Chinese SMA families. Deletions of exons 7 and 8 of the telomeric SMN gene were detected in 100%, 78.6%, 96.6%, and 16.7%, in type I, II, III, and adult-onset SMA patients, respectively. Deletion of exon 7 only was found in eight type II and one type III patient. One type II patient did not have a deletion of either exon 7 or 8. The prevalence of deletions of exons 5 and 6 of the NAIP gene were 22.5% and 2.4% in type I and II SMA patients, respectively. We also examined four polymorphisms of SMN genes and found that there were only two, SMN-2 and ^CBCD541-2, in Chinese subjects. In our study, analysis of the ratio of the telomeric to centromeric portion (T/C ratio) of the SMN gene after enzyme digestion was performed to differentiate carriers, normals, and SMA patients. We found the T/C ratio of exon 7 of the SMN gene differed significantly among the three groups, and may be used for carrier analysis. An asymptomatic individual with homozygous deletion of exons 7 and 8 of the SMN gene showed no difference in microsatellite markers in the SMA-related 5q11.2–5q13.3. In conclusion, SMN deletion in clinically presumed child-onset SMA should be considered as confirmation of the diagnosis. However, adult-onset SMA, a heterogeneous disease with phenotypical similarities to child-onset SMA, may be caused by SMN or other gene(s). Received: 13 November 1996 / Accepted: 13 May 1997     

Correlation between genotype and phenotype in Korean patients with spinal muscular atrophy

The goal of this study was to define the correlation between genotype and phenotype in Korean patients with spinal muscular atrophy (SMA). The SMA can be classified into three groups based on the age of onset and the clinical course. The candidate genes, survival motor neuron (SMN) gene, neuronal apoptosis inhibitory protein (NAIP) gene, and p44 gene were mapped and duplicated with telomeric and centromeric. The loss of the telomeric SMN occurs by a different mechanism. That is the deletion or conversion of telomeric SMN to centromeric SMN, in which case the conversion could produce a mild phenotype and deletion could produce a severe one. It has been known that there may be a balance between the numbers of copies expressed by the centromeric and telomeric SMN genes. In our study, ten patients with type I SMA and two type II patients were identified by their clinical findings and DNA studies. The major deletion of SMA candidate genes, deletion of the SMN gene, NAIP gene, and p44 gene were identified in six patients with type I SMA, while the rest of type I and all the type II patients showed the deletion of the SMN gene only. Allele numbers of the C212 marker were compared in patients and normal controls in order to find the correlation between the copy numbers and the clinical severity. The result was that type I patients had 2-5 alleles and the normal controls had 4-6. This suggests that the deletion is a major determining factor in the clinical phenotype. However, two type I patients with telomeric NAIP gene deletion notably had 4-5 alleles, as in the normal controls. This result implies that the correlation between the copy numbers and the severity is uncertain as opposed to the previous hypothesis. One type I patient showed the conversion of the centromeric SMN gene to the telomeric, which supports the conclusion that gene conversion is an important molecular mechanism for SMA. In the study of one hundred normal newborns, two physically normal newborns showed deletion of the centromeric SMN gene, suggesting frequent rearrangement in the locus.     

Analysis of the SMN and NAIP genes in slovak spinal muscular atrophy patients

We identified homozygous absence of exon 7 of the telomeric copy of the survival motor neuron gene (telSMN) in 88.4% (38/43) of spinal muscular atrophy (SMA) patients from Slovakia. Additional deletions within the neuronal apoptosis inhibitory protein (NAIP) gene were found in 38.5% of type I, 12.5% of type II and never in type III SMA patients. Neither the SMN nor the NAIP gene was deleted in 81 healthy relatives and 25 controls tested. In one family, pseudodominant inheritance was identified. Both the type III SMA father and type II SMA son carried the homozygous deletion of the telSMN gene. One SMA I patient showed an SMN hybrid gene, probably created by intrachromosomal deletion. In two haploidentical type II SMA sibs, the telSMN exon 7 was absent on one chromosome, while the other carried an A-->G transition 96 bp upstream of exon 7 of the telSMN gene, a potential disease-causing mutation in these patients.     

Gene deletion patterns in spinal muscular atrophy patients with different clinical phenotypes

Spinal muscular atrophy (SMA) is an autosomal recessive disorder characterized by degeneration of lower motor neurons. We have assayed deletions in two candidate genes, the survival motor neuron (SMN) and neuronal apoptosis inhibitory protein (NAIP) genes, in 108 samples, of which 46 were from SMA patients, and 62 were from unaffected subjects. The SMA patients included 3 from Bahrain, 9 from South Africa, 2 from India, 5 from Oman, 1 from Saudi Arabia, and 26 from Kuwait. SMN gene exons 7 and 8 were deleted in all type I SMA patients. NAIP gene exons 5 and 6 were deleted in 22 of 23 type I SMA patients. SMN gene exon 7 was deleted in all type II SMA patients while exon 8 was deleted in 19 of 21 type II patients. In 1 type II SMA patient, both centromeric and telomeric copies of SMN exon 8 were deleted. NAIP gene exons 5 and 6 were deleted in only 1 type II SMA patient. In 1 of the 2 type III SMA patients, SMN gene exons 7 and 8 were deleted with no deletion in the NAIP gene, while in the second patient, deletions were detected in both SMN and NAIP genes. None of the 62 unaffected subjects had deletions in either the SMN or NAIP gene. The incidence of biallelic polymorphism in SMN gene exon 7 (BsmAI) was found to be similar (97%) to that (98%) reported in a Spanish population but was significantly different from that reported from Taiwan (0%). The incidence of a second polymorphism in SMN gene exon 8 (presence of the sequence ATGGCCT) was markedly different in our population (97%) and those reported from Spain (50%) and Taiwan (0%).     

SMN Protein Can Be Reliably Measured in Whole Blood with an Electrochemiluminescence (ECL) Immunoassay: Implications for Clinical Trials

Spinal muscular atrophy (SMA) is caused by defects in the survival motor neuron 1 (SMN1) gene that encodes survival motor neuron (SMN) protein. The majority of therapeutic approaches currently in clinical development for SMA aim to increase SMN protein expression and there is a need for sensitive methods able to quantify increases in SMN protein levels in accessible tissues. We have developed a sensitive electrochemiluminescence (ECL)-based immunoassay for measuring SMN protein in whole blood with a minimum volume requirement of 5μL. The SMN-ECL immunoassay enables accurate measurement of SMN in whole blood and other tissues. Using the assay, we measured SMN protein in whole blood from SMA patients and healthy controls and found that SMN protein levels were associated with SMN2 copy number and were greater in SMA patients with 4 copies, relative to those with 2 and 3 copies. SMN protein levels did not vary significantly in healthy individuals over a four-week period and were not affected by circadian rhythms. Almost half of the SMN protein was found in platelets. We show that SMN protein levels in C/C-allele mice, which model a mild form of SMA, were high in neonatal stage, decreased in the first few weeks after birth, and then remained stable throughout the adult stage. Importantly, SMN protein levels in the CNS correlated with SMN levels measured in whole blood of the C/C-allele mice. These findings have implications for the measurement of SMN protein induction in whole blood in response to SMN-upregulating therapy.     

Evaluation of muscle strength and motor abilities in children with type II and III spinal muscle atrophy treated with valproic acid

Background  

Spinal muscular atrophy (SMA) is an autosomal recessive disorder that affects the motoneurons of the spinal anterior horn, resulting in hypotonia and muscle weakness. The disease is caused by deletion or mutation in the telomeric copy of SMN gene (SMN1) and clinical severity is in part determined by the copy number of the centromeric copy of the SMN gene (SMN2). The SMN2 mRNA lacks exon 7, resulting in a production of lower amounts of the full-length SMN protein. Knowledge of the molecular mechanism of diseases has led to the discovery of drugs capable of increasing SMN protein level through activation of SMN2 gene. One of these drugs is the valproic acid (VPA), a histone deacetylase inhibitor.     

VPAC2 receptor agonist BAY 55-9837 increases SMN protein levels and moderates disease phenotype in severe spinal muscular atrophy mouse models

Background

Spinal Muscular Atrophy (SMA) is one of the most common inherited causes of infant death and is caused by the loss of functional survival motor neuron (SMN) protein due to mutations or deletion in the SMN1 gene. One of the treatment strategies for SMA is to induce the expression of the protein from the homologous SMN2 gene, a rescuing paralog for SMA.

Methods and results

Here we demonstrate the promise of pharmacological modulation of SMN2 gene by BAY 55-9837, an agonist of the vasoactive intestinal peptide receptor 2 (VPAC2), a member of G protein coupled receptor family. Treatment with BAY 55-9837 lead to induction of SMN protein levels via activation of MAPK14 or p38 pathway in vitro. Importantly, BAY 55-9837 also ameliorated disease phenotype in severe SMA mouse models.

Conclusion

Our findings suggest the VPAC2 pathway is a potential SMA therapeutic target.