5qSMA: standardised retrospective natural history assessment in 268 patients with four copies of SMN2.

Newborn screening for 5qSMA offers the potential for early, ideally pre-symptomatic, therapeutic intervention. However, limited data exist on the outcomes of individuals with 4 copies of SMN2, and there is no consensus within the SMA treatment community regarding early treatment initiation in this subgroup. To provide evidence-based insights into disease progression, we performed a retrospective analysis of 268 patients with 4 copies of SMN2 from the SMArtCARE registry in Germany, Austria and Switzerland. Inclusion criteria required comprehensive baseline data and diagnosis outside of newborn screening. Only data prior to initiation of disease-modifying treatment were included. The median age at disease onset was 3.0 years, with a mean of 6.4 years. Significantly, 55% of patients experienced symptoms before the age of 36 months. 3% never learned to sit unaided, a further 13% never gained the ability to walk independently and 33% of ambulatory patients lost this ability during the course of the disease. 43% developed scoliosis, 6.3% required non-invasive ventilation and 1.1% required tube feeding. In conclusion, our study, in line with previous observations, highlights the substantial phenotypic heterogeneity in SMA. Importantly, this study provides novel insights: the median age of disease onset in patients with 4 SMN2 copies typically occurs before school age, and in half of the patients even before the age of three years. These findings support a proactive approach, particularly early treatment initiation, in this subset of SMA patients diagnosed pre-symptomatically. However, it is important to recognize that the register will not include asymptomatic individuals.

Possible implication of undescribed SMN1-SMN2 genotype in chronic EMG-pattern of SMA with transitory acute denervation.

Spinal muscular atrophy (SMA) refers to a group of genetic neuromuscular disorders affecting lower motor neurons causative of numerous phenotypes. To date, according to the age of onset, maximum muscular activity achieved, and life expectation four types of SMA are recognized, all caused by mutations in the SMN1 gene with SMN2 copy number influencing disease severity. Herein, we describe the case of a 31-year-old young male with normal psychomotor development who has experienced fatigue, cramps, and muscle fasciculations in the lower limbs for a period of 2 months. Based on electrophysiological and clinical findings we performed SMA genetic, clinical exome and RNA expression of candidate genes which led us to suggest SMN1-SMN2 genes [(2+0) and (0+0)] combination as possibly being implicated in the phenotype.

Gene Therapy for Spinal Muscular Atrophy: An Emerging Treatment Option for a Devastating Disease.

BACKGROUND: Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disease that, in most cases, involves homozygous deletion of the SMN1 gene. This causes a deficiency in survival motor neuron (SMN) protein, which plays a critical role in motor neuron development. SMA has a range of phenotype expression resulting in variable age of symptom onset, maximum motor strength achieved, and survival. Without intervention, infants with a more severe form of the disease (type 1 SMA) die before 2 years of age. Although it is rare, SMA is the most common fatal inherited disease of infancy, and until recently, treatment was primarily supportive. In 2016, a new agent, nusinersen, was approved by the FDA. Other treatments are in development, including a gene therapy, AVXS-101. These treatments are not only improving the lives of patients with SMA and their families, they are changing the disease phenotype. They have the greatest benefit when given early in the disease course. OBJECTIVES: To discuss current knowledge about SMA, provide clinical evidence for available and emerging treatment options, and present approaches for adding new therapies to hospital/health system formularies to ensure timely access to newly approved therapies for SMA. SUMMARY: Advances in clinical care have significantly extended the lives of individuals with SMA, and research into the genetic mechanisms leading to disease have revealed strategies for intervention that target the underlying cause of SMA. Nusinersen is now on the market, and other treatment options, such as AVXS-101, may soon be approved. This article provides an overview of SMA and the genetic mechanisms leading to SMN deficiency, then describes how new and emerging treatments work to overcome this deficiency and prevent associated nerve damage and disability. In addition, we discuss steps for incorporating AVXS-101 into hospital/health system formularies, along with barriers and concerns that may delay access, based in part on lessons learned with nusinersen.

Multiplex Droplet Digital PCR Method Applicable to Newborn Screening, Carrier Status, and Assessment of Spinal Muscular Atrophy.

BACKGROUND: Spinal muscular atrophy (SMA) is a progressive neuromuscular disorder with neuronal degeneration leading to muscular atrophy and respiratory failure. SMA is frequently caused by homozygous deletions that include exon 7 of the survival motor neuron gene SMN1, and its clinical course is influenced by the copy number of a nearby 5q SMN1 paralog, SMN2. Multiple ligation probe amplification (MLPA) and real-time quantitative PCR (qPCR) can detect SMN1 deletions. Yet, qPCR needs normalization or standard curves, and MLPA demands DNA concentrations above those obtainable from dried blood spots (DBSs). We developed a multiplex, droplet digital PCR (ddPCR) method for the simultaneous detection of SMN1 deletions and SMN2 copy number variation in DBS and other tissues. An SMN1 Sanger sequencing process for DBS was also developed. METHODS: SMN1, SMN2, and RPP30 concentrations were simultaneously measured with a Bio-Rad AutoDG and QX200 ddPCR system. A total of 1530 DBSs and 12 SMA patients were tested. RESULTS: Population studies confirmed 1 to 5 SMN1 exon 7 copies detected in unaffected specimens, whereas patients with SMA revealed 0 SMN1 copies. Intraassay and interassay imprecisions were <7.1% CV for individuals with ≥1 SMN1 copies. Testing 12 SMA-positive samples resulted in 100% sensitivity and specificity. CONCLUSIONS: This ddPCR method is sensitive, specific, and applicable to newborn screening and carrier status determination for SMA. It can also be incorporated with a parallel ddPCR T-cell excision circles assay for severe combined immunodeficiencies.

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 allele-specific 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.

Single-sperm analysis for recurrence risk assessment of spinal muscular atrophy.

With the detection of a homozygous deletion of the survival motor neuron 1 gene (SMN1), prenatal and preimplantation genetic diagnosis (PGD) for spinal muscular atrophy has become feasible and widely applied. The finding of a de novo rearrangement, resulting in the loss of the SMN1 gene, reduces the recurrence risk from 25% to a lower percentage, the residual risk arising from recurrent de novo mutation or germline mosaicism. In a couple referred to our PGD center because their first child was affected with SMA, the male partner was shown to carry two SMN1 copies. An analysis of the SMN1 gene and two flanking markers was performed on 12 single spermatozoa, to determine whether the father carried a CIS duplication of the SMN1 gene on one chromosome and was a carrier, or if the deletion has occurred de novo. We showed that all spermatozoa that were carriers of the 'at-risk haplotype' were deleted for the SMN1 gene, confirming the carrier status of the father. We provide an original application of single germ cell studies to recessive disorders using coamplification of the gene and its linked markers. This efficient and easy procedure might be useful to elucidate complex genetic situations when samples from other family members are not available.

Assessment of liquid microbead arrays for the screening of newborns for spinal muscular atrophy.

BACKGROUND: Spinal muscular atrophy is a common neurodegenerative disorder that has recently been considered for inclusion in the next generation of newborn screening regimens. We sought to validate liquid microbead arrays for the identification of affected individuals by direct DNA analysis. METHODS: Assays were created to detect the homozygous deletions in exon 7 of the SMN1 gene found in approximately 95% of affected individuals by use of 2 different microbead chemistries on the Luminex 200: MultiCode-PLx and Tag-It. A series of 367 blood spots including 164 from affected individuals, 46 from known carriers, and 157 from unaffected individuals were then analyzed with each assay. RESULTS: The MultiCode-PLx assay required 4.2 h to perform and provided correct identification of all 164 samples from affected individuals. Correct exclusion was also made for all 46 carrier and 157 unaffected individual samples. The Tag-It assay required 6.8 h, detected all samples from affected individuals, and excluded all but 1 (99.5%) of the samples from carriers and unaffected individuals. Neither method was sensitive to increasing copy numbers of the SMN2 gene. CONCLUSIONS: Both methods showed high sensitivity and specificity for the detection of patients with spinal muscular atrophy. For both methods, ample DNA was extracted from all blood spots for analysis, and SMN2 copy numbers did not interfere. Liquid bead arrays represent a robust method for DNA analysis in newborn screening laboratories.

A feasibility study for the newborn screening of spinal muscular atrophy.

PURPOSE: The natural history of spinal muscular atrophy suggests that for maximum effect, therapeutics will need to be administered in the earliest phases of the disease. This will require the adoption of techniques for the genetic analysis of affected individuals at the newborn stage. Our objective was to examine the feasibility surrounding the newborn screening for spinal muscular atrophy. METHODS: We investigated the application of real-time polymerase chain reaction technology for newborn screening. A multiplex assay was designed to identify homozygous deletions in SMN1 exon 7 and validated using 266 samples with defined SMN1 and SMN2 copy numbers. Sensitivity and specificity were then evaluated as part of a newborn screening strategy using DNA from 153 blood spots. RESULTS: Real-time technology validation demonstrated correct exclusion of all normal and carrier samples, and identified the homozygous SMN1 exon 7 deletions in all 32 affected samples. In the series of blood spots, all 59 affected samples were correctly identified yielding an analytic sensitivity of 100%; 56 normal and 39 carrier samples were correctly excluded yielding an analytic specificity of 100% for this blood spot series. CONCLUSION: We demonstrate that effective molecular technology exists and that ethics may soon warrant the newborn screening of spinal muscular atrophy.

Implementation of SMA carrier testing in genetic laboratories: comparison of two methods for quantifying the SMN1 gene.

The degeneration and loss of motor neurons of the anterior horn characterize children affected with spinal muscular atrophy (SMA). Mutations in the survival motor neuron gene (SMN1) are determinant for the development of the disease whereas the number of copies of SMN2, the highly homologous copy of SMN1, plays a role as a phenotypic modifier factor. The detection of SMN1 homozygous deletions is the typical test for SMA diagnosis. Owing to the limitation of this test for carrier and heterozygous deletion analysis, the demand of SMN1 quantitative tests is permanently growing. The high incidence of SMA, the notable carrier frequency, the severity of the disease, and the lack of effective treatment may justify the implementation of such an analysis in DNA diagnostic labs. The advantages and disadvantages of two reliable quantitative methods were evaluated. One of these is a competitive PCR protocol using internal standards and a genomic sequence as a reference. The other method is a real-time PCR employing an external standard as a reference. Both methods present sufficient advantages for incorporation into molecular genetic diagnostic labs. The possibility of studying samples from different labs, the versatility and reproducibility of the analysis, and cost-benefit calculations must be considered in the final choice.

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.