Identification of Proximal Spinal Muscular Atrophy Carriers and Patients by Analysis of SMNT and SMNC Gene Copy Number

McAndrew, Patricia; Parsons, D. Williams; Simard, Louise R.; Rochette, Camille; Ray, Peter N.; Mendell, Jerry R.; Prior, Thomas W.; Burghes, Arthur H.M.

doi:10.1086/515465

Cited by 480 publications

(501 citation statements)

References 41 publications

(71 reference statements)

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“…In their assay, SMN2 amplified somewhat better than SMN1. The precision and accuracy of the method of Feldkö tter et al 14 are similar to, or slightly lower than, those of our method ( 10,11 and data herein), which is a modification of the original method of McAndrew et al 9 All of these methods, 9 -11,14 in addition to that of Gé rard et al, 13 determine SMN1 and SMN2 copy numbers reliably. Gé rard et al 13 also demonstrated an efficiency bias in their primer-extension assay, slightly in favor of SMN2, even though they generated larger products from SMN2 (27 bp) than those from SMN1 (23 bp).…”

Section: Quantification Of Pcr Bias Between Smn1 and Smn2supporting

confidence: 73%

“…SMN1 gene dosage analysis was originally developed by McAndrew et al 9 and modified as a non-radioisotopic assay as described previously. 10 The assay has since been modified further, using 23 cycles of PCR and the ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster City, CA).…”

Section: Smn1 and Smn2 Copy Number Assay (Smn Gene Dosage Analysis)mentioning

confidence: 99%

“…We also evaluated the effect of PCR bias on our SMN gene dosage analysis, 10,11 which is based on the method of McAndrew et al 9 A priori, we assumed that the amplification efficiencies for SMN1 and SMN2 would be nearly identical since the single nucleotide difference in the segment amplified is not in the primer binding sites. The data presented herein demonstrate that this assumption was incorrect.…”

Section: Quantification Of Pcr Bias Between Smn1 and Smn2mentioning

confidence: 99%

“…16 The copy number of SMN2 correlates inversely with disease severity. 9,10,[12][13][14] Feldkö tter et al 14 found that SMN2 copy number also correlates directly with length of survival. Potential therapies for SMA include approaches to increase the expression of full-length transcripts from SMN2.…”

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confidence: 99%

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Quantification of PCR Bias Caused by a Single Nucleotide Polymorphism in SMN Gene Dosage Analysis

Ogino

Wilson

2002

The Journal of Molecular Diagnostics

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Approximately 94% of patients with spinal muscular atrophy lack both copies of SMN1 exon 7, and most carriers have only one copy of SMN1 exon 7. We described previously the effect of SMN1/SMN2 heteroduplex formation on SMN gene dosage analysis, which is a multiplex quantitative PCR assay to determine the copy numbers of SMN1 and SMN2 using DraI digestion to differentiate SMN2 from SMN1. We describe herein the quantification of PCR bias between SMN1 exon 7 and SMN2 exon 7, which differ by only one nucleotide that is not present in either primer binding site. Using samples from 272 individuals with various SMN genotypes, we found that the amplification efficiency of SMN2 was consistent only approximately 80% that of SMN1. Thus, even a single nucleotide polymorphism, not in primer binding sites, can cause reproducible PCR bias. The precision and accuracy of our SMN gene dosage analysis are high because our assay design and controls take advantage of the consistency of the PCR bias. As additional clinically significant single nucleotide polymorphisms (SNPs) are discovered, assessment of PCR bias, and judicious selection of standards and controls, will be increasingly important for quantitative PCR assays. Spinal muscular atrophy (SMA: type I, MIM no. 253300; type II, MIM no. 253550; type III, MIM no. 253400) is an autosomal recessive disorder associated with loss of motor neurons in the anterior horn of the spinal cord and caused by mutations in the Survival Motor Neuron 1 gene (SMN1; MIM no. 600354) on 5q13. 1 Coding regions of SMN1 and its centromeric homologue, SMN2 (MIM no. 601627), differ in only one base. 2 This C-to-T substitution in SMN2 exon 7 affects the activity of an exonic splice enhancer and alters the splicing pattern of SMN2 mRNA, 3 resulting in a lower level of full-length SMN transcript from SMN2 than from SMN1. 4 -6 SMN2 was shown to be unique to Homo sapiens. 7 Approximately 94% of clinically typical SMA patients lack both copies of SMN1 exon 7. 8 SMN gene dosage analysis, a method to determine the copy number of SMN1, can be used to identify SMA carriers. Exon 7 of SMN1 and SMN2 are co-amplified with genomic and internal standards. The PCR products are then digested with DraI, which cuts only SMN2 exon 7 PCR products, followed by quantification of the PCR products. 9 -11 Other methods for SMN gene dosage analysis have been described. 12-14 A single copy of SMN1 by gene dosage analysis confirms carrier status; this analysis is therefore of clinical importance. A single-copy result also supports the diagnosis of SMN1-related SMA in an affected individual, who may have one deleted allele and one allele with a small intragenic mutation. However, the final diagnosis depends largely on the index of clinical suspicion. 15 This is because the frequency of single-copy carriers in the general population (ϳ2%) approaches the frequency of individuals affected with SMN1-related SMA who have a single-copy test result (ϳ3.6% 12 ). 16 The copy number of SMN2 correlates inversely with disease severity. 9,10,[12]...

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Section: Quantification Of Pcr Bias Between Smn1 and Smn2supporting

confidence: 73%

Section: Smn1 and Smn2 Copy Number Assay (Smn Gene Dosage Analysis)mentioning

confidence: 99%

Section: Quantification Of Pcr Bias Between Smn1 and Smn2mentioning

confidence: 99%

mentioning

confidence: 99%

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Quantification of PCR Bias Caused by a Single Nucleotide Polymorphism in SMN Gene Dosage Analysis

Ogino

Wilson

2002

The Journal of Molecular Diagnostics

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“…For this purpose, multiplex PCR on a target sequence, a genomic reference, and internal standards for both the target and the reference sequences has been used. [2][3][4] An internal standard for a target sequence should have the same primer-binding sites as the target sequence but have an internal insertion/deletion or a different probe-binding site to allow its differentiation from the target by size or probe specificity. The target and its internal standard are amplified in the same reaction tube, so that they have approximately equal amplification efficiency.…”

Section: A Methods To Compensate For Different Amplification Efficienciesmentioning

confidence: 99%

A Method to Compensate for Different Amplification Efficiencies

Ogino¹,

Meijerink²

2001

The Journal of Molecular Diagnostics

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Molecular Analysis and Prenatal Prediction of Spinal Muscular Atrophy in Chinese Patients by the Combination of Restriction Fragment Length Polymorphism Analysis, Denaturing High-Performance Liquid Chromatography, and Linkage Analysis

Chen

Wu²,

Lin³

et al. 2007

Arch Neurol

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Background: The difficulties and incurability of spinal muscular atrophy (SMA) highlight the importance of prenatal diagnosis in families with SMA. However, the system applied in prenatal screening is far from perfect. Objectives: To optimize the molecular assays and establish a relatively perfect system for prenatal screening. Design, Setting, and Patients: A total of 87 patients and 132 parents from 77 families with SMA were screened for SMN1 mutations. Prenatal prediction was performed for 11 fetuses from 10 families with SMA. All of the samples to be tested were from the

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Identification of Proximal Spinal Muscular Atrophy Carriers and Patients by Analysis of SMNT and SMNC Gene Copy Number

Cited by 480 publications

References 41 publications

Quantification of PCR Bias Caused by a Single Nucleotide Polymorphism in SMN Gene Dosage Analysis

Quantification of PCR Bias Caused by a Single Nucleotide Polymorphism in SMN Gene Dosage Analysis

A Method to Compensate for Different Amplification Efficiencies

Molecular Analysis and Prenatal Prediction of Spinal Muscular Atrophy in Chinese Patients by the Combination of Restriction Fragment Length Polymorphism Analysis, Denaturing High-Performance Liquid Chromatography, and Linkage Analysis

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