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%).     

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.     

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.     

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     

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.     

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.     

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.     

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 in the SMN gene in infantile and adult spinal muscular atrophy patients from the same family

Recently, a gene determining spinal muscular atrophy (SMA), termed survival motor neuron (SMN) gene, has been isolated from the 5g13 region. This gene has been found to be deleted in most patients with childhood-onset SMA. We have studied the SMN gene in a clinically heterogeneous family, including one patient affected by infantile chronic SMA and three subjects with mild adult-onset muscle weakness. Deletions in the SMN gene were detected in all of these patients, indicating that the childhood and adult SMAs are genetically homogeneous in this family. Genotyping of the family members established that the three mildly affected individuals were homozygous for the same haplotype from the SMA region, whereas the more severely affected patient was heterozygous with one different haplotype.     

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.     

Cloning and characterization of rat neuronal apoptosis inhibitory protein cDNA

The human neuronal apoptosis inhibitory protein (NAIP) gene was originally discovered because of its deletion in infantile spinal muscular atrophy (SMA), a childhood genetic disorder characterized by motor neuron loss and progressive paralysis with muscular atrophy. Although SMA is now known to be caused by deletions of survival motor neuron (SMN), the fact that NAIP is an anti-apoptotic protein is consistent with the NAIP gene modifying SMA severity. Here we report the cloning of a 1.5 kb rat NAIP cDNA fragment which contains BIR-3 (third baculovirus inhibitory repeat) domain. This fragment shows 78% homology to the human NAIP and 86% homology to the murine counterpart. We have investigated the distribution of NAIP mRNA expressing neurons by in situ RT-PCR technique in the rat central nervous system (CNS). Although all of the neurons appeared to express NAIP mRNA ubiquitously, pronounced elevation of NAIP mRNA expression was observed in the areas innervated by glutamatergic neurons after kainic acid (KA) injection. We have raised an anti-rat NAIP antiserum in rabbits using NAIP cDNA and recombinant rat NAIP, and carried out an immunohistological investigation. We observed highly immunoreactive neuronal subpopulations in the retinal ganglion, cerebral cortex, hippocampus, basal forebrain, thalamus, areas of midbrain, Purkinje cells of the cerebellum, and motor neurons in the spinal cord. Increased immunoreactivity of glutamatergic neurons was also observed broadly in the CNS after KA treatment. This study provides additional evidence that expression of mRNA and gene products of NAIP seem to be regulated in response to excessive stimuli or injuries in rat CNS, and these results are compatible with an anti-apoptotic role of NAIP in acute SMA as well as in brain injuries.     

Mutation update of spinal muscular atrophy in Spain: molecular characterization of 745 unrelated patients and identification of four novel mutations in the SMN1 gene

Spinal muscular atrophy (SMA) is caused by mutations in the SMN1 gene. We have studied the molecular pathology of SMA in 745 unrelated Spanish patients using PCR-RFLP, SMN gene dosage analysis, linkage studies, long-range PCR and direct sequencing. Our systematic approach allowed us to complete genetic testing and risk assessment in 736 SMA patients (98.8%). Females were more frequently affected by the acute form of the disease (type I), whereas chronic forms (type II–III) predominated in males (p < 0.008). Absence of the SMN1 gene was detected in 671 patients (90%), and hybrid SMN1–SMN2 genes were observed in 37 cases (5%). Furthermore, we detected 13 small mutations in 28 patients (3.8%), four of which were previously identified in other populations (c.91dupT; c.770_780dup11; p.Tyr272Cys and p.Thr274Ile), while five mutations were found to date only in Spanish patients (c.399_402delAGAG, p.Ile116Phe, p.Gln136Glu, c.740dupC and c.834+2T>G). The c.399_402delAGAG mutation accounted for 1.9% of all Spanish SMA patients. Finally, we discovered four novel mutations: c.312dupA, c.411delT, p.Trp190X and p.Met263Thr. Our results confirm that most SMA cases are due to large genetic rearrangements in the repetitive region of the SMA locus, resulting in absence-dysfunction of the SMN1 gene. By contrast, ancestrally inherited small mutations are responsible for only a small number of cases. Four prevalent changes in exons 3 and 6 (c.399_402delAGAG; c.770_780dup11; p.Tyr272Cys; p.Thr274Ile) accounted for almost 70% of our patients with these subtle mutations. An SMN–SMN dimer model featuring tight hydrophobic-aromatic interactions is proposed to explain the impact of mutations at the C-terminal end of the protein.     

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     

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.     

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.     

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.