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     

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.     

Analysis of deletional damage in SMN1, SMN2, and NAIP genes in patients with spinal muscular atrophy in the northwestern region of Russia

Polymerase chain reaction with subsequent SSCP (single-strand DNA conformational polymorphism) and restriction (BselI restriction endonuclease) analyses were used to type the DNA samples of affected individuals and their relatives from 23 Russian families with high risk of spinal muscular atrophy (SMA) residing in the northwestern region of Russia. Deletions of exon 7 of the SMN gene were found in 96% of the individuals examined. The frequency of homozygous deletion of exons 7 and 8 of the SMN1 gene was 65%. The frequency of homozygous isolated deletion of the SMN1 gene exon 7 among the SMA patients was 4.3%. Homozygous deletion of exon 5 of the NAIP gene was found in 22% of SMA patients. In SMA patients, a total of seven deletion types involving the SMN1, NAIP, and SMN2 genes were detected. Deletion of exons 7 and 8 of the SMN1 gene was the most common mutation associated with SMA in patients from the northwestern Russia.     

Screening of deletions in SMN, NAIP and BTF2p44 genes in Turkish spinal muscular atrophy patients

Deletions of the spinal muscular atrophy (SMA)-determining gene, SMN1, NAIP, and a third multicopy gene, BTF2p44tel were investigated in 60 unrelated Turkish SMA patients. SMN1 was deleted for at least exons 7 and 8 in 85% of the Turkish SMA patients. The NAIP gene was deleted in 75 and 33% of type I and type II SMA patients, respectively. Analysis of the 5'end of the BTF2p44tel gene indicated the extension of deletion in 13.3% of the cases, mainly in type I patients. Deletions of the NAIP and BTF2p44tel genes were detected in 1.3 and 3.9% of carrriers, respectively, in Turkish SMA families. Two patients were detected to harbor the hybrid SMN gene, one type II with deletion of the NAIP gene, and one type III without deletion of the NAIP gene.     

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.     

Deletion analysis of the SMN and NAIP genes in Kuwaiti patients with spinal muscular atrophy

Two genes are known to be involved in spinal muscular atrophy (SMA), namely, SMN (survival motor neuron) and NAIP (neuronal apoptosis inhibitory protein). Deletion analysis of these genes has been reported for many ethnic groups. We have extended this analysis to include 15 Arabic patients (11 unrelated cases of type I, which represent practically all of the patients diagnosed within the last 2 years in Kuwait, and 4 type-II cases from a single kinship). Also, 41 healthy relatives (parents and sibs) and 44 control individuals of Arabic origin were analyzed. The homozygous deletions of exons 7 and 8 of the SMN gene were found in all SMA patients studied. Exon 5 of NAIP was homozygously absent in all type-I patients, but was retained in type-II cases. Among members of SMA families, one mother was found to be homozygously deleted for NAIP. All of the control individuals had both normal SMN and NAIP. Our results are in agreement with the general consensus that the incidence of NAIP deletion is higher in the more severe SMA cases. Furthermore, they suggest that SMA type-I chromosomes, with the dual deletion of the SMN and NAIP genes, are more common in Arabs than in patients of other ethnic origin. Received: 23 April 1996 / Revised: 17 June 1996     

Apparent gene conversions involving the SMN gene in the region of the spinal muscular atrophy locus on chromosome 5.

The survival motor neuron (SMN) gene has been described as a determining gene for spinal muscular atrophy (SMA). SMN has a closely flanking, nearly identical copy (cBCD541). Gene and copy gene can be discriminated by sequence differences in exons 7 and 8. The large majority of SMA patients show homozygous deletions of at least exons 7 and 8 of the SMN gene. A minority of patients show absence of SMN exon 7 but retention of exon 8. This is explained by results of our present analysis of 13 such patients providing evidence for apparent gene-conversion events between SMN and the centromeric copy gene. Instead of applying a separate analysis for absence or presence of SMN exons 7 and 8, we used a contiguous PCR from intron 6 to exon 8. In every case we found a chimeric gene with a fusion of exon 7 of the copy gene and exon 8 of SMN and absence of a normal SMN gene. Similar events, including the fusion counterpart, were observed in a group of controls, although in the presence of a normal SMN gene. Chimeric genes as the result of fusions of parts of SMN and cBCD541 apparently are far from rare and may partly explain the frequently observed SMN deletions in SMA patients.     

Molecular prenatal diagnosis of autosomal recessive childhood spinal muscular atrophies (SMAs)

Autosomal recessive childhood spinal muscular atrophy (SMAs) is the second most common neuromuscular disorder and a common cause of infant disability and mortality. SMA patients are classified into three clinical types based on age of onset, and severity of symptoms. About 94% of patients have homozygous deletion of exon 7 in survival motor neuron (SMN1) gene. The neuronal apoptosis inhibitory protein (NAIP) gene was found to be more frequently deleted in the severest form of the disease. This study aimed to comment on the implementation of genetic counseling and prenatal diagnosis of SMAs for 85 fetuses from 75 Egyptian couples at risk of having an affected child. The homozygous deletion of exon 7 in SMN1 gene and the deletion of exon 5 of the NAIP gene were detected using PCR-REFLP and multiplex PCR methods respectively. Eighteen fetuses showed homozygous deletion of exon 7 in SMN1 gene and deletion of exon 5 in NAIP gene. In conclusion prenatal diagnosis is an important tool for accurate diagnosis and genetic counseling that help decision making in high risk families.     

Hybrid survival motor neuron genes in patients with autosomal recessive spinal muscular atrophy: new insights into molecular mechanisms responsible for the disease.

Spinal muscular atrophy (SMA) is a frequent autosomal recessive neurodegenerative disorder leading to weakness and atrophy of voluntary muscles. The survival motor-neuron gene (SMN), a strong candidate for SMA, is present in two highly homologous copies (telSMN and cenSMN) within the SMA region. Only five nucleotide differences within the region between intron 6 and exon 8 distinguish these homologues. Independent of the severity of the disease, 90%-98% of all SMA patients carry homozygous deletions in telSMN, affecting either exon 7 or both exons 7 and 8. We present the molecular analysis of 42 SMA patients who carry homozygous deletions of telSMN exon 7 but not of exon 8. The question arises whether in these cases the telSMN is truncated upstream of exon 8 or whether hybrid SMN genes exist that are composed of centromeric and telomeric sequences. By a simple PCR-based assay we demonstrate that in each case the remaining telSMN exon 8 is part of a hybrid SMN gene. Sequencing of cloned hybrid SMN genes from seven patients, as well as direct sequencing and single-strand conformation analysis of all patients, revealed the same composition in all but two patients: the base-pair differences in introns 6 and 7 and exon 7 are of centromeric origin whereas exon 8 is of telomeric origin. Nonetheless, haplotype analysis with polymorphic multicopy markers, Ag1-CA and C212, localized at the 5' end of the SMN genes suggests different mechanisms of occurrence, unequal rearrangements, and gene conversion involving both copies of the SMN genes. In approximately half of all patients, we identified a consensus haplotype, suggesting a common origin. Interestingly, we identified a putative recombination hot spot represented by recombination-stimulating elements (TGGGG and TGAGGT) in exon 8 that is homologous to the human deletion-hot spot consensus sequence in the immunoglobulin switch region, the alpha-globin cluster, and the polymerase alpha arrest sites. This may explain why independent hybrid SMN genes show identical sequences.     

Analysis of Deletions in SMN1, SMN2, and NAIPGenes in Spinal Muscular Atrophy Patients from the Northwestern Region of Russia

Polymerase chain reaction with subsequent SSCP (single-strand DNA conformational polymorphism) and restriction (BselI restriction endonuclease) analyses were used to type the DNA samples of affected individuals and their relatives from 23 Russian families with high risk of spinal muscular atrophy (SMA) residing in the northwestern region of Russia. Deletions of exon 7 of the SMN1gene were found in 96% of the individuals examined. The frequency of homozygous deletion of exons 7 and 8 of the SMN1gene was 65%. The frequency of homozygous isolated deletion of the SMN1gene exon 7 among the SMA patients was 4.3%. Homozygous deletion of exon 5 of the NAIPgene was found in 22% of SMA patients. In SMA patients, a total of seven deletion types involving the SMN1, NAIP, and SMN2genes were detected. Deletion of exons 7 and 8 of the SMN1gene was the most common mutation associated with SMA in patients from the northwestern Russia.     

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.     

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.     

Prenatal diagnosis of spinal muscular atrophy in Macedonian families

Spinal muscular atrophy (SMA) is the second most common lethal autosomal recessive disorder of childhood, affecting approximately 1 in 6,000-10,000 births, with a carrier frequency of 1 in 40-60. There is no effective cure or treatment for this disease. Thus, the availability of prenatal testing is important. The aim of this study was to establish an efficient and rapid method for prenatal diagnosis of SMA and genetic counseling in families with risk for having a child with SMA. In this paper we present the results from prenatal diagnosis in Macedonian SMA families using direct analysis of fetal DNA. The probands of these families were previously found to be homozygous for a deletion of exons 7 and 8 of SMN1 gene. DNA obtained from chorionic villas samples and amniocytes was analyzed for deletions in SMN gene. SMN exon 7 and 8 deletion analysis was performed by polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP). Of the 12 prenatal diagnoses, DNA analysis showed normal results in eight fetuses. Four of the fetuses were homozygote for a deletion of exons 7 and 8 of SMN1. After genetic counseling, the parents of the eight normal fetuses decided to continue the pregnancy, while in the four families with affected fetuses, the pregnancy was terminated. The results were confirmed after birth.     

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.     

An analysis of chimeric SMN genes--new possibilities in the study of the molecular genetic nature of mutations and in the diagnosis of spinal muscular atrophy (SMA)]

Results of analysis of chimeric SMN genes among some high SMA-risk families from Ukraine using the EcoRV and DdeI restriction enzyme hydrolysis of PCR products is presented. Chimeric cen/telSMN gene was detected in probands with homozygous deletions of telSMN exon 7 only, as well in proband with absent of homozygous deletion of exons 7 and/or 8 of the SMN gene. Effectivity of approach of detection of chimeric SMN genes based on the EcoRV and DdeI restriction enzyme analysis of PCR products and mechanisms of formation of chimeric SMN genes are discussed.     

Allele-specific amplification of exon 7 in the survival motor neuron (SMN) genes for molecular diagnosis of spinal muscular atrophy

There are two highly homologous survival motor neuron (SMN) genes in humans but molecular defects in the SMN1 gene cause spinal muscular atrophy (SMA). More than 90% of SMA patients are shown to have a homozygous deletion of exon 7 in the SMN1 gene. Therefore, a simple test for exon 7 deletion would be very useful in the molecular diagnosis of SMA. However, limited methods are available, and most of these methods utilize expensive instruments and consumables. Here, we describe a simple allele-specific PCR test, which can be performed using standard equipment in DNA laboratories. The principle of the test is based on a single nucleotide difference (C versus T) between the exon 7 of SMN1 and SMN2 genes. Using allele-specific primers, two PCR amplifications are performed for each sample to amplify a 404-bp diagnostic fragment, and consequent electrophoresis of PCR products on agarose gel provides definitive information concerning the exon 7 deletion To rule out false negatives, a 500-bp fragment from the N-acetyltransferase gene was coamplified as an internal control in each test. We have, so far, analyzed 41 SMA samples with our method, and tested the validity of results using an independent restriction fragment length polymorphism (RFLP) method. Genotyping results obtained by both methods were in complete agreement for all of the samples analyzed. Our method can also be used to detect heterozygous deletion of exon 7 in SMN genes, if the relative intensities of the diagnostic and internal control bands are determined.     

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.     

Deletions of the survival motor neuron gene in unaffected siblings of patients with spinal muscular atrophy.

DNA studies in 103 spinal muscular atrophy (SMA) patients from The Netherlands revealed homozygosity for a survival motor neuron (SMN) deletion in 96 (93%) of 103. Neuronal apoptosis inhibitory protein deletions were found in 38 (37%) of 103 and occurred most frequently in SMA type I. SMN deletions have not yet been described to occur in healthy subjects. In this study, however, four unaffected sibs from two SMA families showed homozygosity for SMN deletions. Homozygosity for an SMN deletion in unaffected persons seems to be very rare. Therefore, demonstration of a homozygous SMN deletion in a clinically presumed SMA patient should be considered as a confirmation of the diagnosis, whether or not SMN is in fact the causal gene for SMA.