Development and validation of a 4-color multiplexing spinal muscular atrophy (SMA) genotyping assay on a novel integrated digital PCR instrument

Jiang, Lingxia; Lin, Robert; Gallagher, Steve; Zayac, Andrew; Butchbach, Matthew E.R.; Hung, Paul J.

doi:10.1038/s41598-020-76893-7

Cited by 20 publications

(18 citation statements)

References 46 publications

(68 reference statements)

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“…The droplet digital PCR is another emerging technique for SMN1 copy number assay 47 – 51 . Although, the improved digital PCR technology has been reported 52 , the most are operational complexity and the related equipment and reagents are expensive. At present, it is mostly used as a validation method for positive results 22 , 53 and has not been adopted extensively for carrier screening of SMA.…”

Section: Discussionmentioning

confidence: 99%

Next generation sequencing is a highly reliable method to analyze exon 7 deletion of survival motor neuron 1 (SMN1) gene

Zhao

Wang

Xin

et al. 2022

Sci Rep

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Spinal muscular atrophy (SMA) is one of the most common and severe genetic diseases. SMA carrier screening is an effective way to identify couples at risk of having affected children. Next-generation sequencing (NGS)-based expanded carrier screening could detect SMN1 gene copy number without extra experiment and with high cost performance. However, its performance has not been fully evaluated. Here we conducted a systematic comparative study to evaluate the performance of three common methods. 478 samples were analyzed with multiplex ligation probe amplification (MLPA), real-time quantitative polymerase chain reaction (qPCR) and NGS, simultaneously. Taking MLPA-based results as the reference, for 0 copy, 1 copy and ≥ 2 copy SMN1 analysis with NGS, the sensitivity, specificity and precision were all 100%. Using qPCR method, the sensitivity was 100%, 97.52% and 94.30%, respectively; 98.63%, 95.48% and 100% for specificity; and 72.72%, 88.72% and 100% for precision. NGS repeatability was higher than that of qPCR. Moreover, among three methods, NGS had the lowest retest rate. Thus, NGS is a relatively more reliable method for SMN1 gene copy number detection. In expanded carrier screening, compared with the combination of multiple methods, NGS method could reduce the test cost and simplify the screening process.

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Section: Discussionmentioning

confidence: 99%

Next generation sequencing is a highly reliable method to analyze exon 7 deletion of survival motor neuron 1 (SMN1) gene

Zhao

Wang

Xin

et al. 2022

Sci Rep

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“…Genome sequencing data from the 37 SMA patients and the two healthy individuals have been previously published by the Motor Neuron Diseases Research Laboratory (MNDRL, Wilmington, DE, USA) in collaboration with Illumina, Inc. (San Diego, CA, USA) [ 41 ]. The SMA status as well as SMN1 and SMN2 copy numbers for the MNDRL cohort were confirmed by digital droplet PCR ( Table 2 ) [ 42 , 43 , 44 ]. We also analyzed the reference SMN1 sequence (gene ID: ENSG00000172062, human genome assembly GRCh38, p13) that is available in Ensemble.…”

Section: Methodsmentioning

confidence: 98%

Comprehensive In Silico Analysis of Retrotransposon Insertions within the Survival Motor Neuron Genes Involved in Spinal Muscular Atrophy

Pinto

Cunha

Chaves

et al. 2022

Biology

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Transposable elements (TEs) are interspersed repetitive and mobile DNA sequences within the genome. Better tools for evaluating TE-derived sequences have provided insights into the contribution of TEs to human development and disease. Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disease that is caused by deletions or mutations in the Survival Motor Neuron 1 (SMN1) gene but retention of its nearly perfect orthologue SMN2. Both genes are highly enriched in TEs. To establish a link between TEs and SMA, we conducted a comprehensive, in silico analysis of TE insertions within the SMN1/2 loci of SMA, carrier and healthy genomes. We found an Alu insertion in the promoter region and one L1 element in the 3′UTR that may play an important role in alternative promoter as well as in alternative transcriptional termination. Additionally, several intronic Alu repeats may influence alternative splicing via RNA circularization and causes the presence of new alternative exons. These Alu repeats present throughout the genes are also prone to recombination events that could lead to SMN1 exons deletions and, ultimately, SMA. TE characterization of the SMA genomic region could provide for a better understanding of the implications of TEs on human disease and genomic evolution.

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“…The master mix was loaded onto the Absolute Q microfluidic array partitioning plate, and the isolation buffer (Thermo Fisher Scientific) was overlaid. The dPCR plate was run in the Applied Biosystems QuantStudio Absolute Q dPCR System with a program of 96°C for 10 min, followed by 45 cycles at 96°C for 5 s and 60°C for 30 s. This dPCR system has been described in detail previously ( 107 ). Primer and probe information for dPCR are listed in table S1.…”

Section: Methodsmentioning

confidence: 99%

Disruption of the circadian clock drives Apc loss of heterozygosity to accelerate colorectal cancer

et al. 2022

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An alarming rise in young onset colorectal cancer (CRC) has been reported; however, the underlying molecular mechanism remains undefined. Suspected risk factors of young onset CRC include environmental aspects, such as lifestyle and dietary factors, which are known to affect the circadian clock. We find that both genetic disruption and environmental disruption of the circadian clock accelerate Apc- driven CRC pathogenesis in vivo. Using an intestinal organoid model, we demonstrate that clock disruption promotes transformation by driving Apc loss of heterozygosity, which hyperactivates Wnt signaling. This up-regulates c-Myc , a known Wnt target, which drives heightened glycolytic metabolism. Using patient-derived organoids, we show that circadian rhythms are lost in human tumors. Last, we identify that variance between core clock and Wnt pathway genes significantly predicts the survival of patients with CRC. Overall, our findings demonstrate a previously unidentified mechanistic link between clock disruption and CRC, which has important implications for young onset cancer prevention.

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Development and validation of a 4-color multiplexing spinal muscular atrophy (SMA) genotyping assay on a novel integrated digital PCR instrument

Cited by 20 publications

References 46 publications

Next generation sequencing is a highly reliable method to analyze exon 7 deletion of survival motor neuron 1 (SMN1) gene

Next generation sequencing is a highly reliable method to analyze exon 7 deletion of survival motor neuron 1 (SMN1) gene

Comprehensive In Silico Analysis of Retrotransposon Insertions within the Survival Motor Neuron Genes Involved in Spinal Muscular Atrophy

Disruption of the circadian clock drives Apc loss of heterozygosity to accelerate colorectal cancer

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