An evaluation of carrier detection for Spinal muscular atrophy using digital PCR assay / 中华医学遗传学杂志

OBJECTIVE@#To assess the effectiveness and feasibility of carrier detection for Spinal muscular atrophy (SMA) by using digital PCR assay.@*METHODS@#Peripheral blood samples were collected from 214 pregnant women who were routinely screened for SMA carriers, of which 204 were randomly selected samples and 10 were samples with known copy numbers of SMN1 exons 7 and 8. Samples with known copy numbers of SMN1 exons 7 and 8 were randomly mixed into the experiment to validate the performance of the digital PCR assay.@*RESULTS@#The copy numbers of SMN1 exons 7 and 8 and SMN2 exons 7 and 8 in peripheral blood samples were detected by digital PCR assay. The results of SMN1 exons 7 and 8 were compared with those of the quantitative PCR method to assess the reliability and clinical performance of the digital PCR assay. Among the 204 random samples, digital PCR has detected five samples with simultaneous heterozygous deletion of SMN1 exons 7 and 8, three samples with heterozygous deletion of SMN1 exon 8 only, and 196 samples with no deletion of SMN1 exons 7 and 8. Ten samples with known SMN1 exons 7 and 8 copy numbers were detected with the expected values. The digital PCR test results were fully consistent with that of the quantitative PCR.@*CONCLUSION@#The results of digital PCR for the detection of copy number variation of SMN1 exons 7 and 8 were consistent with qPCR. Digital PCR assay was able to clearly distinguish the copy number of the target genes, therefore can be used for SMA carrier screening. Moreover, it can also detect copy number of SMN2 exons 7 and 8, which can provide more information for genetic counseling.

Results of carrier screening for Spinal muscular atrophy among 35 145 reproductive-aged individuals from Dongguan region / 中华医学遗传学杂志

OBJECTIVE@#To carry out carrier screening for Spinal muscular atrophy (SMA) in reproductive-aged individuals from Dongguan region and determine the carrier frequency of SMN1 gene mutations.@*METHODS@#Reproductive-aged individuals who underwent SMN1 genetic screening at the Dongguan Maternal and Child Health Care Hospital from March 2020 to August 2022 were selected as the study subjects. Deletions of exon 7 and 8 (E7/E8) of the SMN1 gene were detected by real-time fluorescence quantitative PCR (qPCR), and prenatal diagnosis was provided for carrier couples by multiple ligation-dependent probe amplification (MLPA).@*RESULTS@#Among the 35 145 subjects, 635 were found to be carriers of SMN1 E7 deletion (586 with heterozygous E7/E8 deletion, 2 with heterozygous E7 deletion and homozygous E8 deletion, and 47 with sole heterozygous E7 deletion). The carrier frequency was 1.81% (635/35 145), with 1.59% (29/1 821) in males and 1.82% (606/33 324) in females. There was no significant difference between the two genders (χ² = 0.497, P = 0.481). A 29-year-old woman was found to harbor homozygous deletion of SMN1 E7/E8, and was verified to have a SMN1∶SMN2 ratio of [0∶4], none of her three family members with a [0∶4] genotype had clinical symptoms. Eleven carrier couples had accepted prenatal diagnosis, and one fetus was found to have a [0∶4] genotype, and the pregnancy was terminated.@*CONCLUSION@#This study has determined the SMA carrier frequency in Dongguan region for the first time and provided prenatal diagnosis for carrier couples. The data can provide a reference for genetic counseling and prenatal diagnosis, which has important clinical implications for the prevention and control of birth defects associated with SMA.

Analysis of a Chinese pedigree affected with Spinal muscular atrophy due to compound heterozygous variants of SMN gene / 中华医学遗传学杂志

OBJECTIVE@#To analyze variants of SMN gene in a Chinese pedigree affected with Spinal muscular atrophy (SMA).@*METHODS@#A Chinese pedigree diagnosed at the Nanchang First Hospital in January 2020 was selected as the study subject. Peripheral blood samples were collected for the extraction of DNA. All exons of the SMN gene were detected by multiple ligation-dependent probe amplification (MLPA). Potential variants of the SMN gene were also detected by Whole exome sequencing (WES), and the result was verified by Sanger sequencing. cDNA extracted from fresh blood sample was used as a template to verify the location of variant on the SMN genes.@*RESULTS@#The proband was found to harbor a heterozygous deletion of the SMN1 Exon7+Exon8, and a heterozygous c.81G>A variant. The SMN1 Exon7+Exon8 deletion was inherited from her father and grandmother, whilst the c.81G>A variant was inherited from her mother and maternal grandfather. Her aunt was also a carrier of the heterozygous deletion, while her paternal aunt, her husband, and their daughter were not. cDNA amplification and Sanger sequencing confirmed that the c.81G>A variant was located in the SMN1 gene.@*CONCLUSION@#MLPA combined with NGS and Sanger sequencing can identify compound heterozygous variants of the SMN gene in the SMA patients.

Expert consensus on preimplantation genetic testing for spinal muscular atrophy / 中华医学遗传学杂志

Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron degenerative disease, which is the most common fatal neuromuscular disease in pediatrics with a high carrier frequency and can lead to progressive symmetrical muscle weakness and atrophy of the trunk and limbs. Preimplantation genetic testing (PGT) can be used to prevent the birth of children with SMA effectively. To standardize PGT technologies for SMA, experts from the fields of neurology, pediatrics and reproductive genetics have discussed and drafted this consensus for guiding its clinical application.

Prenatal diagnosis of spinal muscular atrophy using multiple ligation-dependent probe amplification / 中华医学遗传学杂志

OBJECTIVE@#To carry out prenatal diagnosis for families with high risk for spinal muscular atrophy (SMA) by using multiplex ligation-dependent probe amplification (MLPA).@*METHODS@#Twenty-one families were enrolled. MLPA was used to detect copy numbers of SMN1 and SMN2 genes. Maternal contamination was excluded by using a short tandem repeat method.@*RESULTS@#For 23 fetuses from the 21 families, 14 were identified as carriers, 1 as SMA patient, and 8 as normal. By linkage analysis of parental samples, three individuals were determined as silent (2+0) carriers.@*CONCLUSION@#MLPA can determine the carrier status of SMA. The identification of three silent (2+0) carriers among the 44 parental samples indicated a risk for such families, for which genetic counseling and reproduction guidance should be provided.

Allele-specific PCR for a cost-effective & time-efficient diagnostic screening of spinal muscular atrophy.

Background & objectives: Genetic diagnosis of spinal muscular atrophy (SMA) is complicated by the presence of SMN2 gene as majority of SMA patients show absence or deletion of SMN1 gene. PCR may amplify both the genes non selectively in presence of high amount of DNA. We evaluated whether allelespecific PCR for diagnostic screening of SMA is reliable in the presence of high amount of genomic DNA, which is commonly used when performing diagnostic screening using restriction enzymes. Methods: A total of 126 blood DNA samples were tested in amounts ranging 80-200 ng, referred for the genetic diagnosis of SMA using both conventional PCR-RFLP and allele-specific PCR. Results: The results from both methods showed agreement. Further, allele-specific PCR was found to be a time-efficient and cost-effective method. Interpretation & conclusions: Our study demonstrated the accuracy of our allele-specific PCR and the results were comparable compatible with that of PCR-RFLP, indicating its practical application in SMA diagnostic screening.

Atrofia muscular espinal: Caracterización clínica, electrofisiológica y molecular de 26 pacientes / Clinical, electrophysiological and molecular study of 26 chilean patients with spinal muscular atrophy

Background: Spinal Muscular Atrophy (SMA) is an autosomal recessive disorder affecting the anterior horn cells of the spinal cord resulting in muscle weakness and atrophy, linked to the homozygous disruption of the survival motor neuron 1 (SMN1) gene. It is the leading genetic cause of infant death. It has been classified into three types based on the severity of symptoms. Type I SMA is the most severe form with death within the first 2 years of life. Type II and III SMA patients show intermediate and mild forms of the disorder. Aim: To describe the clinical and electrophysiological findings of 26 Chilean patients with SMA with molecular confirmation. Patients and Methods: Retrospective multicenter analysis of patients with SMA assessed between 2003 and 2010. The diagnosis was suspected on clinical and electrophysiological criteria. Since 2006 molecular genetics confirmation was implemented in one of our centers. Results: Twenty-six patients between 2 months and 18 years of age at presentation were analyzed; 15 (58 percent) were males. SMA I, II and III clinical criteria were observed in 4 (15.4 percent), 11 (42.3 percent) and 11 (42.3 percent)patients, respectively. All had proximal muscle weakness and atrophy. Electromyography showed features of acute denervation or re-innervation with normal motor and sensory nerve conduction. Nine patients required a muscle biopsy. The genetic confirmation of the disease by PCR technique followed by restriction fragment length polymorphism method disclosed the SMN1 gene deletion in all 26 cases. All patients died secondary to respiratory failure, between eight and 14 months of life. Conclusions: An adequate clinical and molecular diagnosis of spinal muscular atrophy will help for a better management of these patients.

SMN1 and NAIP genes deletions in different types of spinal muscular atrophy in Khuzestan province, Iran

Spinal muscular atrophy [SMA] is the second most common lethal autosomal recessive disease. It is a neuromuscular disorder caused by degenerative of lower motor neurons and occasionally bulbar neurons leading to progressive limb paralysis and muscular atrophy. The SMN1 gene is recognized as a SMA causing gene while NAIP has been characterized as a modifying factor for the clinical severity and age at disease onset in SMA patients [SMA subtypes]. The relationship between NAIP deletion and type of SMA remains to be clarified; we investigated this gene alteration in all types of SMA patients. Molecular analysis was performed on fifty patients with a clinical diagnosis of SMA in Khuzestan province. In addition to common PCR-RFLP analysis for exon 7 and 8 of SMN1 gene, as an internal control we analysed NAIP deletion with PCR of exon 5 of this gene in a multiplex PCR with exon 13 of it. Homozygous-deletion frequency rate for the telomeric copy of SMN [SMN1] exon 7 in all types [type I, II, III] of SMA was approximately 90% and the frequency of deletion in exon 7 and 8 together in all types estimated about 70%. Moreover NAIP gene was deleted in about 60% of these patients and this shows deletion in 91% of type I SMA patients. The correlation between NAIP-deletion and SMN1 mutation showed a high frequency rate. In this study, high frequency of NAIP gene deletion in all type of disease shows the importance role of it in disease pathogenesis. High frequency of NAIP deletion in SMA type I, also shows the importance of the gene in type and severity of disease so it may be a modifier factor in severity of disease

Determination of SMN1 and SMN2 Copy Numbers in a Korean Population using Multiplex Ligation-dependent Probe Amplification / 대한진단검사의학회지

Determination of the copy number of the survival motor neuron (SMN) gene is important for detecting spinal muscular atrophy (SMA) carriers and compound heterozygous patients. Multiplex ligationdependent probe amplification (MLPA) assay is a simple and efficient technique used for detecting variations in the copy numbers of different genes. Race- and ethnicity-based variation in the SMA carrier frequency and the '2+0' genotype of SMN1 are important factors that should be considered when estimating the risk of being an SMA carrier. Since SMN2 plays a disease-modifying role, accurate determination of SMN2 copy numbers in SMA patients can serve as a useful prognostic tool. Therefore, information on the SMN2 genotype distributions in normal populations will be helpful in selecting appropriate reference samples for MLPA analysis. To determine SMA carrier frequencies and SMN genotype distribution, we determined the copy numbers of SMN1 and SMN2 genes using the MLPA assay in 100 unrelated Korean individuals with no family history of SMA. The frequency of SMA carriers in the Korean population appears to be 1 in 50, which indicates that the prevalence of SMA among Koreans is the same as that among individuals in the Western countries. Two of the 100 normal individuals enrolled in this study showed 3 copies of the SMN1 gene. Therefore, 1.0% of the 198 normal alleles in this population was estimated to be 2-copy alleles ('2+0' genotype). SMN2 copy numbers showed a high degree of individual variation. Our results showed that 64% of the individuals had 2 copies of SMN2, but 36% individuals had between 0, 1, or 3 copies of the gene.