Molecular analysis of the neuronal apoptosis inhibitory protein gene in families with spinal muscular atrophy.

Spinal muscular atrophy (SMA) is the second most frequent autosomal recessive disease characterized by degeneration of the anterior horn cells of the spinal cord, leading to muscular atrophy. SMA is classified into three types according to disease severity and age-onset: severe (type I), intermediate (type II) and mild (type III). Deletions in the survival motor neuron (SMN) gene, located in the chromosome region 5q11.2- 5q13.3, are major determinants of SMA phenotype. Extended deletions that include the neuronal apoptosis inhibitory protein (NAIP) gene may correlate with the severtity of SMA. SMN gene is present in two highly homologous copies, SMN1 and SMN2, but only deletions of the SMN1 gene (exons 7 and 8 or exon 7) are responsible for clinical manifestations of SMA. Here, we present the deletion profiling of SMN1 and NAIP genes in 89 children with SMA from Serbia: 52 patients with type I, 26 with type II, and 11 with type III. The homozygous deletion of the SMN1 gene was confirmed in 72 of 89 (81%) patients, being the most frequent in SMA type I (48/52): 68 patients (94.4%) with deletion of exons 7 and 8 and 4 patients (5.6%) with deletion of exon 7. The extended deletion including the NAIP gene was detected in 18 of 89 (20.2%) patients, mostly affected with type I. This study has revealed the lower incidence of deletions in the SMN1 and NAIP genes in families with SMA in Serbia and will provide important information for genetic counselling in these families.

Background:Proximal spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease characterized by symmetrical proximal muscle weakness and atrophy. According to the severity of the disease and the age of onset, SMA can be divided into three groups. The survival motor neuron (SMN) gene that is located on 5q13 is identified as the disease determining gene. Another gene in this region is neuronal apoptosis inhibitory protein (NAIP), and its functional role in the pathogenesis of SMA has not been fully elucidated. Here, we investigated the correlation between deletions in SMN and NAIP genes with clinical features of SMA patients.Materials and Methods:In the current study, 71 unrelated Iranian patients were investigated for the detection of deletions in SMN1 and NAIP genes. Polymerase chain reaction (PCR) was used to detect the deletions of exon 4 and 5 of the NAIP gene. Deletions in exon 7 and 8 of SMN1 gene were detected by RFLP-PCR with DraI and DdeI, respectively.Results:Our results showed that 51 patients have homozygous deletions in SMN1 and/or NAIP genes. Among these 51 patients, deletion in NAIP gene were found in 35 patients (65.7% of type I, 22.5% type II and 11.42% type III).Conclusion:Defect in SMN1 gene plays a major role in manifesting of the disease and NAIP (4 and 5) gene acts as a modifying factor in severity of symptoms. Correlation between NAIP gene defect and severity of the disease is confirmed. However, the exact role of NAIP gene in SMA has yet to be fully clarified.

The survival motor neuron 1 (SMN1) gene has been recognized to be responsible for spinal muscular atrophy (SMA) because it is homozygously deleted in more than 90% of SMA patients, irrespective of their clinical severity, whereas the neuronal apoptosis inhibitory protein (NAIP) gene is now considered to be a modifying factor of the severity of SMA. In Malaysia, it remains to be elucidated whether deletion of the SMN1 gene is also a main cause of SMA or whether deletion of the NAIP gene is found in the SMA patients. To clarify the pathogenesis of SMA in Malaysia, a deletion analysis of the SMN1 and NAIP genes was performed in 24 Malaysian SMA patients. Deletion analysis of exons 7 and 8 of the SMN1 gene was performed according to the method described by van der Steege et al., while deletion analysis of exon 5 of the NAIP gene was performed according to a method described by Roy et al. Homozygous deletion of SMN1 exon 7 and exon 8 were identified in 19 out of 24 patients (79%). As to the NAIP gene, deletion of exon 5 was detected in six out of 24 patients (25%). NAIP gene deletion was correlated with severity of the disease. Deletion of the SMN1 exon 7 is a major cause of SMA in Malaysia, and NAIP gene deletions are not rare in type I SMA in Malaysia. The lower percentage of the SMN1 gene deletion may be due to the possibility that the present study included some patients without SMN1 gene abnormality and/or some patients with non-deletion type mutations in the SMN1 gene.

Childhood-onset proximal spinal muscular atrophies (SMAs) are an autosomal recessive, clinically heterogeneous group of neuropathies characterised by the selective degeneration of anterior horn cells. SMA has an estimated incidence of 1 in 10,000 live births. The causative genes are survival motor neuron (SMN) gene and neuronal apoptosis inhibitory protein (NAIP) gene. Deletions of the telomeric copy of SMN gene (SMN1) have been reported in 88.5% to 95% of SMA cases, whereas the deletion rate for NAIP gene (NAIP) is between 20% and 50% depending on the disease severity. The main objective of this study was to genetically characterise the childhood onset of SMA in Iran. Molecular analysis was performed on a total of 75 patients with a clinical diagnosis of SMA. In addition to common PCR analysis for SMN1 exons 7 and 8, we analysed NAIP exons 4 and 5, along with exon 13, as a internal control, by bi-plex PCR. The homozygous-deletion frequency rate for the telomeric copy of SMN exons 7 and 8 in all types of SMA was 97%. Moreover, exons 5 and 6 of NAIP gene were deleted in approximately 83% of all SMA types. Three deletion haplotypes were constructed by using SMN and NAIP genotypes. Haplotype A, in which both genes are deleted, was seen in approximately 83% of SMA types I and II but not type III. It was also found predominantly in phenotypically severe group with an early age of onset (i.e., less than 6-month-old). We also report 34 of our prenatal diagnosis. To our knowledge, the present study is the first one giving detailed information on SMN and NAIP deletion rates in Iranian SMA patients. Our results show that the frequency of SMN1 homozygous deletions in Iran is in agreement with previous studies in other countries. The molecular analysis of SMA-related gene deletion/s will be a useful tool for pre- and postnatal diagnostic.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder characterized by degeneration of spinal cord anterior horn cells, leading to muscular atrophy. SMA is clinically classified into three subgroups based on the age of onset and severity. The majority of patients with SMA have homozygous deletions of exons 7 and 8 of the survival motor neuron (SMN) gene. The purpose of the present study was to determine the frequency of SMN and neuronal apoptosis inhibitory protein (NAIP) gene deletions in Iranian SMA patients. Experience in prenatal diagnosis of SMA in this population is also reported. To study the frequency of deletions of SMN and NAIP genes in an Iranian sample group, 75 unrelated SMA patients (54 type I, eight type II and 13 type III) were analyzed according to the methods described by van der Steege et al and Roy et al. Homozygous deletion of SMN1 exons 7 and/or 8 were identified in 68 out of 75 patients (90%). Deletion of exon 5 of the NAIP gene was found in 40/54 of type I, 2/8 of type II and 1/13 of type III patients. Deletion of the SMN1 gene is a major cause of SMA in Iran, and NAIP gene deletions were common in the present patients with type I SMA. Also, the incidence of NAIP deletion is higher in more severe SMA.

In this report we present the genetic studies in a Chinese type III spinal muscular atrophy (SMA) family. The survival motor neuron (SMN) gene exons 7 and 8 and neuronal apoptosis inhibitory protein (NAIP) gene exon 5 were amplified by sequence-specitic oligonucleotide primer and polymerase chain reaction (PCR). The SMN gene deletion was detected by restriction endonuclease digestion and single strand conformation polymorphism (SSCP) analysis of PCR-amplified products. The deletion in NAIP gene was detected directly by agarose gel electrophoresis of multiplex PCR-amplified products. All three affected siblings are homozygous for telomeric SMN gene deletion, whereas no NAIP gene deletion was found. Both parents, maternal uncle; and paternal grandmother of affected sibs showed heterozygosity for telomeric SMN deletions. Two unaffected sibs are non-carriers for SMN deletions.

In this article, the correlation between the copy number of survival motor neuron 2 (SMN2) gene, neuronal apoptosis inhibitory protein (NAIP), and the phenotype of spinal muscular atrophy patients were analyzed.Forty patients with spinal muscular atrophy (SMA) were included in the study at the Department of Medical Genetics of the First People's Hospital and the Department of Neurology of the Second People's Hospital in Yunnan Province from January 2012 to September 2018. Multiplex ligation-dependent probe amplification assay was performed to determine the copy numbers of SMN2 and NAIP genes. Statistical analysis was performed to determine the correlation between copy numbers of the SMN2 and NAIP genes and the clinical phenotypes of SMA.Our results show that among the 40 SMA patients, there were 13 type I cases, 16 type II cases and 11 type III cases. A total of 37 patients possessed a homozygous deletion of SMN1 exons 7 and 8, while the other 3 SMA patients possessed a single copy of SMN1 exon 8. There was no correlation between SMA subtypes and the deletion types of SMN1 exon 7 and 8 (P = .611). The percentage of 2, 3, and 4 copies of SMN2 exon 7 was 25.0%, 62.5%, and 12.5%, respectively. The percentage of 0, 1, and 2 copies of NAIP exon 5 was 10%, 57.5%, and 32.5%, respectively. The distributions of SMN2 and NAIP copy numbers among various SMA types were significantly different (all P < .05). Five combined SMN1-SMN2-NAIP genotypes were detected, of which 0-3-1 genotype had the highest proportion than the others, accounting for 42.5%. The copy number of SMN2 and NAIP gene had synergistic effect on SMA phenotype. The combined SMN1-SMN2-NAIP genotypes with fewer copies were associated with earlier onset age, higher mortality, and smaller average age at death in SMA patients.Therefore, we conclude that the copy number variance of SMN2 and NAIP is correlated with the SMA phenotype. Analysis of the copy number structure of the SMN1-SMN2-NAIP gene is helpful for SMA typing, disease prognosis prediction, and genetic counseling.

Spinal muscular atrophy (SMA) is a disorder characterized by degeneration of lower motor neurons and occasionally bulbar motor neurons leading to progressive limb and trunk paralysis as well as muscular atrophy. Three types of SMA are recognized depending on the age of onset, the maximum muscular activity achieved, and survivorship: SMA1, SMA2, and SMA3. The survival of motor neuron (SMN) gene has been identified as an SMA determining gene, whereas the neuronal apoptosis inhibitory protein (NAIP) gene is considered to be a modifying factor of the severity of SMA. The main objective of this study was to analyze the deletion of SMN1 and NAIP genes in southern Chinese children with SMA. Here, polymerase chain reaction (PCR) combined with restriction fragment length polymorphism (RFLP) was performed to detect the deletion of both exon 7 and exon 8 of SMN1 and exon 5 of NAIP in 62 southern Chinese children with strongly suspected clinical symptoms of SMA. All the 32 SMA1 patients and 76% (13/17) of SMA2 patients showed homozygous deletions for exon 7 and exon 8, and all the 13 SMA3 patients showed single deletion of SMN1 exon 7 along with 24% (4/17) of SMA2 patients. Eleven out of 32 (34%) SMA1 patients showed NAIP deletion, and none of SMA2 and SMA3 patients was found to have NAIP deletion. The findings of homozygous deletions of exon 7 and/or exon 8 of SMN1 gene confirmed the diagnosis of SMA, and suggested that the deletion of SMN1 exon 7 is a major cause of SMA in southern Chinese children, and that the NAIP gene may be a modifying factor for disease severity of SMA1. The molecular diagnosis system based on PCR-RFLP analysis can conveniently be applied in the clinical testing, genetic counseling, prenatal diagnosis and preimplantation genetic diagnosis of SMA.

Spinal muscular atrophy (SMA), the second most common lethal autosomal recessive disorder, has an incidence of 1:10,000 newborns. SMA is divided into acute (Werdnig-Hoffmann disease, type I), intermediate (type II) and juvenile forms (Kugelberg-Welander disease, type III). The gene of all three forms of SMA maps to chromosome 5q 11.2-13.3. Two candidate genes, the survival motor neuron (SMN) gene and the neuronal apoptosis inhibitory protein (NAIP) gene, have been identified; SMN is deleted in most SMA patients. We studied both genes in 87 Brazilian SMA patients (20 type I, 14 type II and 53 type III) from 74 unrelated families, by using PCR and single strand conformation polymorphism (SSCP). Deletions of exons 7 and/or 8 of the SMN gene were found in 69% of the families: 16/20 in type I, 9/12 in type II and 26/42 in type III. Among 51 families with deletions, 44 had both exons deleted while seven had deletions only of exon 7. Deletions of exon 5 of the NAIP gene were found in 7/20 of type I, 2/12 of type II and 1/42 of type III patients. No deletion of SMN and NAIP genes was found in 112 parents, 26 unaffected sibs and 104 normal controls. No correlation between deletions of one or both genes and phenotype severity was found.

We evaluated survival motor neuron 2 (SMN2) and neuronal apoptosis inhibitory protein (NAIP) gene copy distribution and the association of copy number with survival in 232 Chinese spinal muscular atrophy (SMA) patients. The SMN2 and NAIP copy numbers correlated positively with the median onset age (r = 0.72 and 0.377). The risk of death for patients with fewer copies of SMN2 or NAIP was much higher than for those with more copies (P < .01). The survival probabilities at 5 years were 5.1%, 90.7%, and 100% for 2, 3, and 4 SMN2 copies and 27.9%, 66.7%, and 87.2% for 0, 1, and 2 NAIP copies, respectively. Our results indicated that combined SMN1-SMN2-NAIP genotypes with fewer copies were associated with earlier onset age and poorer survival probability. Better survival status for Chinese type I SMA might due to a higher proportion of 3 SMN2 and a lower rate of zero NAIP.

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%).

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%).

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).

Spinal muscular atrophy is an autosomal recessive disorder characterized by degeneration of lower motor neurons. We have investigated the presence of survival motor neuron gene and neuronal apoptosis inhibitory protein gene deletions in 17 Arab and 1 Indian families with spinal muscular atrophy (15 type I and 3 type II). Homologous deletions were detected in exons 7 and 8 of the survival motor neuron gene and exon 5 of the neuronal apoptosis inhibitory protein gene in all patients with type I spinal muscular atrophy. Exon 13 of the neuronal apoptosis inhibitory protein gene was deleted in only one patient with type I spinal muscular atrophy. In two patients with type II spinal muscular atrophy, only exons 7 and 8 of the survival motor neuron gene were deleted whereas exons 5 and 13 of the neuronal apoptosis inhibitory protein gene were present. In another patient with spinal muscular atrophy type II, exons 7 and 8 of the survival motor neuron gene and exon 5 of the neuronal apoptosis inhibitory protein gene were deleted. This latter patient also had the Pierre Robin syndrome. No deletion was detected in healthy siblings or the parents. The deletions found in our patients are similar to those reported in other population groups.

Objective To investigate the value of multiplex ligation⁃dependent probe amplification (MLPA) method in the prenatal diagnosis of spinal muscular atrophy (SMA). Methods Six SMA pedigrees, which included 7 patients, 12 parents and 6 fetuses, were admitted in our hospital. MLPA was used to detect the survival motor neuron (SMN) and other modifier genes, according to steps of hybridization, ligation, PCR reaction, fragment separation by capillary electrophoresis and peak pattern evaluation. Synchronously, the deletion of SMN1 gene was detected by polymerase chain reaction⁃restriction fragment length polymorphism (PCR⁃RFLP). The DNA samples of fetuses were collected by centrifuging the amniotic fluid as well as derived from amniotic cell culture. Results According to MLPA, 7 patients and 1 fetus were detected to carry homozygous deletion of survival motor neuron 1 (SMN1) gene, which was also detected by PCR⁃PFLP. In addition, 11 parents and 5 fetuses carried one copy of SMN1 gene, while 1 parent who was also a carrier of SMA carried two copies of SMN1 gene. Furthermore, after being analyzed by MLPA, 10 cases carried one copy of SMN2 gene, while 15 cases had two copies of SMN2 gene. After detecting the neuronal apoptosis inhibitory protein (NAIP) gene, 3 cases had the deletion of NAIP gene while others showed normal. Conclusion MLPA can detect the deletion and quantify the copy numbers of SMN and other modifier genes, improving the efficiency and stability of genetic diagnosis. It is adequate for detecting patients and carriers of SMA, as well as providing reliable evidence for genetic counseling. DOI：10.3969/j.issn.1672⁃6731.2012.03.012