Survival Motor Neuron 1 Copy Research Articles

Alberta Spinal Muscular Atrophy Newborn Screening-Results from Year 1 Pilot Project.

Spinal muscular atrophy (SMA) is a progressive neuromuscular disease caused by biallelic pathogenic/likely pathogenic variants of the survival motor neuron 1 (SMN1) gene. Early diagnosis via newborn screening (NBS) and pre-symptomatic treatment are essential to optimize health outcomes for affected individuals. We developed a multiplex quantitative polymerase chain reaction (qPCR) assay using dried blood spot (DBS) samples for the detection of homozygous absence of exon 7 of the SMN1 gene. Newborns who screened positive were seen urgently for clinical evaluation. Confirmatory testing by multiplex ligation-dependent probe amplification (MLPA) revealed SMN1 and SMN2 gene copy numbers. Six newborns had abnormal screen results among 47,005 newborns screened during the first year and five were subsequently confirmed to have SMA. Four of the infants received SMN1 gene replacement therapy under 30 days of age. One infant received an SMN2 splicing modulator due to high maternally transferred AAV9 neutralizing antibodies (NAb), followed by gene therapy at 3 months of age when the NAb returned negative in the infant. Early data show that all five infants made excellent developmental progress. Based on one year of data, the incidence of SMA in Alberta was estimated to be 1 per 9401 live births.

Novel Alu-mediated deletions of the SMN1 gene were identified by ultra-long read sequencing technology in patients with spinal muscular atrophy

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by biallelic variants of the survival motor neuron 1 (SMN1) gene. In this study, our aim was to make a molecular diagnosis in two patients with SMA carrying only one SMN1 copy number. Using ultra-long read sequencing (Ultra-LRS), 1415 bp deletion and 3348 bp deletion of the SMN1 gene were identified in patient 1 and the father of patient 2, respectively. Ultra-LRS revealed two novel deletions, starting from the SMN1 promoter to intron 1. It also accurately provided the location of the deletion breakpoints in the SMN1 gene: chr5 g.70,924,798–70,926,212 for a 1415 bp deletion; chr5 g.70,922,695–70,926,042 for a 3348 bp deletion. By analyzing the breakpoint junctions, we identified that these genomic sequences were composed of Alu sequences, including AluJb, AluYm1, AluSq, and AluYm1, indicating that Alu-mediated rearrangements are a mechanism of SMN1 deletion events. In addition, full-length SMN1 transcripts and SMN protein in patient 1 were significantly decreased (p < 0.01), suggesting that a 1415 bp deletion that included the transcription and translation initiation sites of the SMN1 gene had severe consequences for SMN expression. Ultra-LRS can easily distinguish highly homozygous genes compared to other detection technologies, which is useful for detecting SMN1 intragenic mutations, to quickly discover structural rearrangements and to precisely present the breakpoint positions.

Simultaneous quantification of SMN1 and SMN2 copy numbers by MALDI-TOF mass spectrometry for spinal muscular atrophy genetic testing

BackgroundSpinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disorder caused by defects in the survival motor neuron 1 (SMN1) gene. Homozygous deletion of the SMN1 gene accounts for 95% of all affected SMA patients. A highly homologous gene survival motor neuron 2 (SMN2) compensates weakly with the loss of SMN1 and its copy number correlates with disease severity. MethodsWe report here the MS-CNV method combining competitive PCR and MALDI-TOF mass spectrometry for simultaneous quantification of SMN1, SMN2 and NAIP dosages. For both SMN1 and SMN2, the exon 7 and exon 8 were analyzed. MS-CNV was validated with parallel analysis by a commercial MLPA assay in two independent cohorts. ResultsIn the first cohort of 79 blood samples containing 3 SMA patients and 5 carriers, MS-CNV results were highly concordant with MLPA analysis for the copy numbers of SMN1, SMN2 and NAIP. In the second independent and blinded cohort of 62 blood samples containing 21 SMA patients and 14 carriers, MS-CNV results were also highly concordant with MLPA. Both MS-CNV and MLPA quantified SMN1 dosages without ambiguity. ConclusionsMS-CNV can be used for carrier screening and genetic diagnosis of SMA, providing dosages information for both SMN1 and SMN2 given its accuracy and high sample processing throughput by mass spectrometric analysis.

The clinical utility of a risk-modifying SNP to detect carriers for spinal muscular atrophy with increased sensitivity.

BackgroundSpinal muscular atrophy (SMA) is an autosomal recessive motor neuron disease caused by biallelic inactivation of the survival motor neuron 1 (SMN1) gene. With a prevalence of ~1 in 11,000 live births (carrier frequency of ~1:50), SMA is one of the most common severe childhood‐onset diseases; therefore, current guidelines recommend pan‐ethnic carrier screening for SMA before or during pregnancy.Routine SMN1 copy number assessment detects ~96% of all SMA carriers, but not the remaining 4% who harbor two copies of SMN1 arrayed in ‐cis [2 + 0]. The c.*3+80T>G risk‐modifying SNP positively correlates with this chromosomal configuration and may be used to modify the residual risk of being a carrier for SMA.MethodsOne year after incorporating the detection of the c.*3+80>G risk‐modifying SNP into our routine SMA carrier screen, we perform a retrospective chart review to evaluate its frequency and utilization in the prenatal clinic.ResultsIn comparison with classic carriers for SMA, study data show that individuals with two copies of SMN1 and the risk modifier were counseled less frequently about their increased risk of being a carrier for SMA.ConclusionIncorporating the c.*3+80T>G risk‐modifying SNP is important for detecting carriers for SMA with a higher clinical sensitivity.

Detection of SMN1 to SMN2 gene conversion events and partial SMN1 gene deletions using array digital PCR.

Proximal spinal muscular atrophy (SMA), a leading genetic cause of infant death worldwide, is an early-onset motor neuron disease characterized by loss of α-motor neurons and associated muscle atrophy. SMA is caused by deletion or other disabling mutations of survival motor neuron 1 (SMN1) but retention of one or more copies of the paralog SMN2. Within the SMA population, there is substantial variation in SMN2 copy number (CN); in general, those individuals with SMA who have a high SMN2 CN have a milder disease. Because SMN2 functions as a disease modifier, its accurate CN determination may have clinical relevance. In this study, we describe the development of array digital PCR (dPCR) to quantify SMN1 and SMN2 CNs in DNA samples using probes that can distinguish the single nucleotide difference between SMN1 and SMN2 in exon 8. This set of dPCR assays can accurately and reliably measure the number of SMN1 and SMN2 copies in DNA samples. In a cohort of SMA patient-derived cell lines, the assay confirmed a strong inverse correlation between SMN2 CN and disease severity. We can detect SMN1-SMN2 gene conversion events in DNA samples by comparing CNs at exon 7 and exon 8. Partial deletions of SMN1 can also be detected with dPCR by comparing CNs at exon 7 or exon 8 with those at intron 1. Array dPCR is a practical technique to determine, accurately and reliably, SMN1 and SMN2 CNs from SMA samples as well as identify gene conversion events and partial deletions of SMN1.

Spinal muscular atrophy in Venezuela: quantitative analysis of SMN1 and SMN2 genes

BackgroundSpinal muscular atrophy (SMA) is mostly caused by homozygous deletions in the survival motor neuron 1 (SMN1) gene. SMN2, its paralogous gene, is a genetic modifier of the disease phenotype, and its copy number is correlated with SMA severity. The purpose of the study was to investigate the number of copies of the SMN1 and SMN2 genes in a Venezuelan population control sample and in patients with a presumptive diagnosis of SMA, besides estimating the frequency of mutation carriers in the population.ResultsSMN1 and SMN2 gene copies were assessed in 49 Venezuelan dweller unrelated normal individuals and in 94 subjects from 29 families with a SMA presumptive diagnosis, using the quantitative PCR method. A SMN1 deletion carrier frequency of 0.01 and 0.163 of homozygous absence of the SMN2 gene were found in the Venezuelan control sample. Deletion of SMN1 exon 7 was confirmed in 15 families; the remaining 14 index cases had two SMN1 copies and a heterogeneous phenotype not attributable to SMN deletions. Based on clinical features of the index cases and the SMN2 copy number, a positive phenotype-genotype correlation was demonstrated. No disease geographical aggregation was found in the country.ConclusionThe frequency of carriers of the deletion of exon 7 in SMN1 in the Venezuelan control population was similar to that observed in populations worldwide, while the frequency of 0 copies of the SMN2 gene (16.3 %) seems to be relatively high. All these findings have pertinent implications for the diagnosis and genetic counseling on SMA in Venezuela.

Spinal Muscular Atrophy in the Black South African Population: A Matter of Rearrangement?

Spinal muscular atrophy (SMA) is a neuromuscular disorder, characterized by muscle atrophy and impaired mobility. A homozygous deletion of survival motor neuron 1 (SMN1), exon 7 is the main cause of SMA in ~94% of patients worldwide, but only accounts for 51% of South African (SA) black patients. SMN1 and its highly homologous centromeric copy, survival motor neuron 2 (SMN2), are located in a complex duplicated region. Unusual copy number variations (CNVs) have been reported in black patients, suggesting the presence of complex pathogenic rearrangements. The aim of this study was to further investigate the genetic cause of SMA in the black SA population. Multiplex ligation-dependent probe amplification (MLPA) testing was performed on 197 unrelated black patients referred for SMA testing (75 with a homozygous deletion of SMN1, exon 7; 50 with a homozygous deletion of SMN2, exon 7; and 72 clinically suggestive patients with no homozygous deletions). Furthermore, 122 black negative controls were tested. For comparison, 68 white individuals (30 with a homozygous deletion of SMN1, exon 7; 8 with a homozygous deletion of SMN2, exon 7 and 30 negative controls) were tested. Multiple copies (>2) of SMN1, exon 7 were observed in 50.8% (62/122) of black negative controls which could mask heterozygous SMN1 deletions and potential pathogenic CNVs. MLPA is not a reliable technique for detecting carriers in the black SA population. Large deletions extending into the rest of SMN1 and neighboring genes were more frequently observed in black patients with homozygous SMN1, exon 7 deletions when compared to white patients. Homozygous SMN2, exon 7 deletions were commonly observed in black individuals. No clear pathogenic CNVs were identified in black patients but discordant copy numbers of exons suggest complex rearrangements, which may potentially interrupt the SMN1 gene. Only 8.3% (6/72) of clinically suggestive patients had heterozygous deletions of SMN1, exon 7 (1:0) which is lower than previous SA reports of 69.5%. This study emphasizes the lack of understanding of the architecture of the SMN region as well as the cause of SMA in the black SA population. These factors need to be taken into account when counseling and performing diagnostic testing in black populations.

Enhanced Carrier Screening for Spinal Muscular Atrophy: Detection of Silent (SMN1: 2 + 0) Carriers Utilizing a Novel TaqMan Genotyping Method.

Individuals whose copies of the survival motor neuron 1 (SMN1) gene exist on the same chromosome are considered silent carriers for spinal muscular atrophy (SMA). Conventional screening for SMA only determines SMN1 copy number without any information regarding how those copies are arranged. A single nucleotide variant (SNV) rs143838139 is highly linked with the silent carrier genotype, so testing for this SNV can more accurately assess risk to a patient of having an affected child. Using a custom-designed SNV-specific Taqman genotyping assay, we determined and validated a model for silent-carrier detection in the laboratory. An initial cohort of 21 pilot specimens demonstrated results that were 100% concordant with a reference laboratory method; this cohort was utilized to define the reportable range. An additional 177 specimens were utilized for a broader evaluation of clinical validity and reproducibility. Allelic-discrimination analysis demonstrated tight clustering of genotype groupings and excellent reproducibility, with a coefficient of variation for all genotypes ranging from 1% to 4%. The custom-developed Taqman SNV genotyping assay we tested provides a rapid, accurate, and cost-effective method for routine SMA silent-carrier screening and considerably improves detection rates of residual risk for SMA carriers.

Carrier frequency of spinal muscular atrophy in Thailand.

Spinal muscular atrophy (SMA) is one of the leading causes of death in infants and young children from heritable diseases. Patients diagnosed with SMA develop symmetrical progressive muscle weakness and atrophy from degeneration of alpha motor neurons. Approximately 95% of patients have a homozygous deletion of survival motor neuron 1 (SMN1) gene in exon 7 and inherited in autosomal recessive pattern. Considering the high prevalence of SMA carrier in many population, it is possible that SMA is one of the most common autosomal recessive disorders in Thailand and Southeast Asia. In this study, we analyzed DNA from peripheral blood of 505 healthy Thai adults using quantitative PCR-based for SMN1 gene exon 7 copy number analysis. Individual samples with heterozygous deletion of SMN1 gene were confirmed with MLPA. The result identified 9 samples (1.78%) with heterozygous deletion and 39 samples as more than 2 copies of SMN1. No homozygous deletion was detected in the samples. In conclusion, we established carrier frequency of SMA in selected Thai population at 1.8% from 505 participants. The prevalence coincides with prevalence in East Asia and Caucasian population. The result could be implemented for SMA carrier screening in couples at risk in the region.

Complete sequencing of the SMN2 gene in SMA patients detects SMN gene deletion junctions and variants in SMN2 that modify the SMA phenotype.

Spinal muscular atrophy (SMA) is a progressive motor neuron disease caused by loss or mutation of the survival motor neuron 1 (SMN1) gene and retention of SMN2. We performed targeted capture and sequencing of the SMN2, CFTR, and PLS3 genes in 217 SMA patients. We identified a 6.3 kilobase deletion that occurred in both SMN1 and SMN2 (SMN1/2) and removed exons 7 and 8. The deletion junction was flanked by a 21bp repeat that occurred 15 times in the SMN1/2 gene. We screened for its presence in 466 individuals with the known SMN1 and SMN2 copy numbers. In individuals with 1 SMN1 and 0 SMN2 copies, the deletion occurred in 63% of cases. We modeled the deletion junction frequency and determined that the deletion occurred in both SMN1 and SMN2. We have identified the first deletion junction where the deletion removes exons 7 and 8 of SMN1/2. As it occurred in SMN1, it is a pathogenic mutation. We called variants in the PLS3 and SMN2 genes, and tested for association with mild or severe exception patients. The variants A-44G, A-549G, and C-1897T in intron 6 of SMN2 were significantly associated with mild exception patients, but no PLS3 variants correlated with severity. The variants occurred in 14 out of 58 of our mild exception patients, indicating that mild exception patients with an intact SMN2 gene and without modifying variants occur. This sample set can be used in the association analysis of candidate genes outside of SMN2 that modify the SMA phenotype.

Mechanism of Splicing Regulation of Spinal Muscular Atrophy Genes.

Spinal muscular atrophy (SMA) is one of the major genetic disorders associated with infant mortality. More than 90% cases of SMA result from deletions or mutations of 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. However, correction of SMN2 exon 7 splicing has proven to confer therapeutic benefits in SMA patients. The only approved drug for SMA is an antisense oligonucleotide (Spinraza™/Nusinersen), which corrects SMN2 exon 7 splicing by blocking intronic splicing silencer N1 (ISS-N1) located immediately downstream of exon 7. ISS-N1 is a complex regulatory element encompassing overlapping negative motifs and sequestering a cryptic splice site. More than 40 protein factors have been implicated in the regulation of SMN exon 7 splicing. There is evidence to support that multiple exons of SMN are alternatively spliced during oxidative stress, which is associated with a growing number of pathological conditions. Here, we provide the most up to date account of the mechanism of splicing regulation of the SMN genes.

Combination of valproic acid and morpholino splice-switching oligonucleotide produces improved outcomes in spinal muscular atrophy patient-derived fibroblasts

Spinal muscular atrophy (SMA), the leading genetic cause of infant mortality worldwide, is characterised by the homozygous loss of the survival motor neuron 1 (SMN1) gene. The consequent degeneration of spinal motor neurons and progressive atrophy of voluntary muscle groups results in paralysis and eventually premature infantile death. Humans possess a second nearly identical copy of SMN1, known as SMN2. However, SMN2 produces only 10–20% functional SMN protein due to aberrant splicing of its pre-mRNA that leads to the exclusion of exon 7. This level of SMN is insufficient to rescue the phenotype. Recently developed splice-switching antisense oligonuclotides (SSO) have shown great promise in correcting the aberrant splicing of SMN2 towards producing functional SMN protein. Several FDA approved drugs are being repurposed for SMA treatment including valproic acid (VPA), a histone deacetylase inhibitor, which has been shown to increase overall SMN2 expression. In this study, we have characterised the effects of single and combined treatment of VPA and a SSO based on phosphorodiamidate morpholino oligomer (PMO) chemistry. We conjugated both VPA and PMO to a single cell-penetrating peptide (Apolipoprotein E (ApoE)) for their simultaneous intracellular delivery. Treatment of SMA Type I patient-derived fibroblasts with the conjugates showed no additive increase in the level of full-length SMN2 mRNA expression over both 4 and 16 h treatments indicating that conjugation of VPA to ApoE-PMO has limited benefit. However, treatment with a combination of VPA and ApoE-PMO induced more favourable splice switching activity than either agent alone, promoting exon 7 inclusion in SMN2 transcripts. Our results suggest that combination therapy of VPA and ApoE-PMO is superior in upregulating SMN2 production in vitro, as compared to singular treatment of each compound at both transcriptional and protein levels. This study provides the first indication of a novel dual therapy approach for the potential treatment of SMA.

A novel human-specific splice isoform alters the critical C-terminus of Survival Motor Neuron protein.

Spinal muscular atrophy (SMA), a leading genetic disease of children and infants, is caused by mutations or deletions of Survival Motor Neuron 1 (SMN1) gene. SMN2, a nearly identical copy of SMN1, fails to compensate for the loss of SMN1 due to skipping of exon 7. SMN2 predominantly produces SMNΔ7, an unstable protein. Here we report exon 6B, a novel exon, generated by exonization of an intronic Alu-like sequence of SMN. We validate the expression of exon 6B-containing transcripts SMN6B and SMN6BΔ7 in human tissues and cell lines. We confirm generation of SMN6B transcripts from both SMN1 and SMN2. We detect expression of SMN6B protein using antibodies raised against a unique polypeptide encoded by exon 6B. We analyze RNA-Seq data to show that hnRNP C is a potential regulator of SMN6B expression and demonstrate that SMN6B is a substrate of nonsense-mediated decay. We show interaction of SMN6B with Gemin2, a critical SMN-interacting protein. We demonstrate that SMN6B is more stable than SMNΔ7 and localizes to both the nucleus and the cytoplasm. Our finding expands the diversity of transcripts generated from human SMN genes and reveals a novel protein isoform predicted to be stably expressed during conditions of stress.

Copy Number Variations in the Survival Motor Neuron Genes: Implications for Spinal Muscular Atrophy and Other Neurodegenerative Diseases

Proximal spinal muscular atrophy (SMA), a leading genetic cause of infant death worldwide, is an early-onset, autosomal recessive neurodegenerative disease characterized by the loss of spinal α-motor neurons. This loss of α-motor neurons is associated with muscle weakness and atrophy. SMA can be classified into five clinical grades based on age of onset and severity of the disease. Regardless of clinical grade, proximal SMA results from the loss or mutation of SMN1 (survival motor neuron 1) on chromosome 5q13. In humans a large tandem chromosomal duplication has lead to a second copy of the SMN gene locus known as SMN2. SMN2 is distinguishable from SMN1 by a single nucleotide difference that disrupts an exonic splice enhancer in exon 7. As a result, most of SMN2 mRNAs lack exon 7 (SMNΔ7) and produce a protein that is both unstable and less than fully functional. Although only 10–20% of the SMN2 gene product is fully functional, increased genomic copies of SMN2 inversely correlates with disease severity among individuals with SMA. Because SMN2 copy number influences disease severity in SMA, there is prognostic value in accurate measurement of SMN2 copy number from patients being evaluated for SMA. This prognostic value is especially important given that SMN2 copy number is now being used as an inclusion criterion for SMA clinical trials. In addition to SMA, copy number variations (CNVs) in the SMN genes can affect the clinical severity of other neurological disorders including amyotrophic lateral sclerosis (ALS) and progressive muscular atrophy (PMA). This review will discuss how SMN1 and SMN2 CNVs are detected and why accurate measurement of SMN1 and SMN2 copy numbers is relevant for SMA and other neurodegenerative diseases.

SMN1 and SMN2 copy numbers in cell lines derived from patients with spinal muscular atrophy as measured by array digital PCR.

Proximal spinal muscular atrophy (SMA) is an early-onset motor neuron disease characterized by loss of α-motor neurons and associated muscle atrophy. SMA is caused by deletion or other disabling mutation of survival motor neuron 1 (SMN1). In the human genome, a large duplication of the SMN-containing region gives rise to a second copy of this gene (SMN2) that is distinguishable by a single nucleotide change in exon 7. Within the SMA population, there is substantial variation in SMN2 copy number; in general, those individuals with SMA who have a high SMN2 copy number have a milder disease. Because SMN2 functions as a disease modifier, its accurate copy number determination may have clinical relevance. In this study, we describe the development of an assay to assess SMN1 and SMN2 copy numbers in DNA samples using an array-based digital PCR (dPCR) system. This dPCR assay can accurately and reliably measure the number of SMN1 and SMN2 copies in DNA samples. In a cohort of SMA patient-derived cell lines, the assay confirmed a strong inverse correlation between SMN2 copy number and disease severity. Array dPCR is a practical technique to determine, accurately and reliably, SMN1 and SMN2 copy numbers from SMA samples.

G.P.231: A novel splice-site mutation in SMN1 resulting in a very severe SMA1 phenotype

Spinal muscular atrophy (SMA) is an autosomal recessive genetic motor neuron disorder caused by a defect in the survival motor neuron 1 (SMN1) gene resulting from deletions or other mutations. The number of copies of SMN2, a gene paralogous to SMN1, modifies clinical course. The homozygous deletion of exon 7 is the most recurrent molecular defect but heterozygous deletions in compound with single nucleotide intragenic mutations have also been reported. We describe the clinical and molecular features of a child born to not consanguineous parents who developed a severe muscle hypotonia at 10 days of age. Clinical diagnosis was compatible with SMA type I (Werdnig–Hoffmann) phenotype. SMA locus genotype by quantitative PCR analysis revealed one copy of both SMN1 and SMN2 genes. Sequence analysis in the proband disclosed the heterozygous novel c.888+1G>C substitution affecting the donor splice site between exon 7 and intron 7. The deletion and the point mutation are independently inherited from the parents. Standard and quantitative RT-PCR experiments on proband’s cDNA disclosed a severe reduction of the full-length/delta7 ratio of SMN isoforms as well as the presence of a transcript originating from SMN1 but lacking exon 7. Approximately 3–5% of patients with SMA retain at least one copy of the SMN1 gene carrying pathogenic insertions, deletions, or point mutations on the other allele. The number of intragenic mutations affecting splicing is even narrower. Our findings demonstrate that the novel c.888+1G>C mutation is highly deleterious, resulting in the almost total exclusion of exon 7. Sequence analysis is helpful to identify these defects whose effects might be highly detrimental on SMN stability and function.

Genetics of low spinal muscular atrophy carrier frequency in sub‐Saharan Africa

Spinal muscular atrophy (SMA) is one of the most common severe hereditary diseases of infancy and early childhood in North America, Europe, and Asia. SMA is usually caused by deletions of the survival motor neuron 1 (SMN1) gene. A closely related gene, SMN2, modifies the disease severity. SMA carriers have only 1 copy of SMN1 and are relatively common (1 in 30-50) in populations of European and Asian descent. SMN copy numbers and SMA carrier frequencies have not been reliably estimated in Malians and other sub-Saharan Africans. We used a quantitative polymerase chain reaction assay to determine SMN1 and SMN2 copy numbers in 628 Malians, 120 Nigerians, and 120 Kenyans. We also explored possible mechanisms for SMN1 and SMN2 copy number differences in Malians, and investigated their effects on SMN mRNA and protein levels. The SMA carrier frequency in Malians is 1 in 209, lower than in Eurasians. Malians and other sub-Saharan Africans are more likely to have ≥3 copies of SMN1 than Eurasians, and more likely to lack SMN2 than Europeans. There was no evidence of gene conversion, gene locus duplication, or natural selection from malaria resistance to account for the higher SMN1 copy numbers in Malians. High SMN1 copy numbers were not associated with increased SMN mRNA or protein levels in human cell lines. SMA carrier frequencies are much lower in sub-Saharan Africans than in Eurasians. This finding is important to consider in SMA genetic counseling in individuals with black African ancestry.

SMN1 gene duplications are more frequent in patients with progressive muscular atrophy

Survival Motor Neuron 1 (SMN1) is a causative gene for autosomal recessive infantile and juvenile proximal spinal muscular atrophy. SMN1 duplications have recently been found to increase susceptibility to amyotrophic lateral sclerosis. The role of centromeric SMN copy (SMN2) has been postulated in progressive muscular atrophy (PMA). The aim of this study was to analyse the SMN1 and SMN2 copy number variations in patients with PMA. SMN1 and SMN2 genotype was studied in 87 patients with PMA, diagnosed at the Department of Neurology, Medical University of Warsaw, between 1992 and 2012 and in 600 healthy controls. Results demonstrated that three copies of SMN1 were found in 8.1% of PMA patients and in 24% of PMA patients with disease duration above 48 months compared to 4.6% of the general population. Patients with three SMN1 copies had a limb onset, lower median age of onset and longer disease duration compared to patients with two SMN1 copies. There were no significant differences in the SMN2 copy numbers. In conclusion, the increased copy number of SMN1 may be a susceptibility factor to PMA and influence the clinical phenotype.

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.

Homozygous SMN2 deletion is a protective factor in the Swedish ALS population

Abnormal survival motor neuron 1 (SMN1)-copy number has been associated with an increased risk of amyotrophic lateral sclerosis (ALS) in French and Dutch population studies. The aim of this study was to determine whether SMN gene copy number increases the risk of ALS or modulates its phenotype in a cohort of Swedish sporadic ALS (SALS) patients. In all, 502 Swedes with SALS and 502 Swedish controls matched for gender and age were enrolled. SMN1 and SMN2 gene copy numbers were studied by a semi-quantitative PCR method. A genotype-phenotype comparison was performed in order to determine whether SMN genes modulate the phenotype of ALS. The results were also compared with our previously reported French cohort of ALS patients. There was no difference between Swedish patients and controls in the frequency of SMN1 and SMN2 copy numbers. The frequency of SMN1 gene copies differed significantly between the French and Swedish ALS populations. The duration of the disease was significantly longer in the Swedish cohort with homozygous deletions of SMN2 when compared with the French cohort. Abnormal SMN1 gene copy number cannot be considered as a universal genetic susceptibility factor for SALS and this result underlines the importance of reproducing association gene studies in groups from different origins. We also suggest that SMN2 gene copy number might have different effects on ALS progression in disparate human populations.