Genetic Diseases Detection Solution

In China, the total incidence of birth defects is about 5.6%, and single-gene hereditary diseases account for about 22.2% of all causes. Single-gene hereditary diseases refer to genetic diseases controlled by a single pair of alleles, which have a large number of types, such as Mediterranean anemia, phenylketonuria, spinal muscular atrophy, etc. The combined incidence rate is up to 1/100, much higher than the incidence rate of Down's syndrome in China (1/680), and most single-gene hereditary diseases can lead to death, deformity or disability. Only 5% of these diseases have effective drugs, but the treatment costs are expensive. The carriers of single-gene hereditary diseases usually have no symptoms, but there is a risk of giving birth to children with genetic diseases, and they usually can not be detected by routine prenatal examination until the children are born. Therefore, it is necessary to carry out corresponding genetic testing for high-incidence single-gene hereditary diseases before marriage or pregnancy. Taking spinal muscular atrophy (SMA) as an example, it is a genetic neuromuscular disease caused by degeneration of spinal motor neurons, resulting in muscle atrophy and generalized muscle atrophy in children. The carrier rate is 1/47~1/72, and the SMA pathogenic gene is located on the telomere side of chromosome 5q13 motor neuron survival gene SMN. The SMN2 gene has two reverse repeat copies, telomere side SMN1 gene and centromere side SMN2 gene. Research by Alias L has shown that more than 90% of SMA patients have SMN1 gene homozygous deletion, while the rest are heterozygous deletion, point mutations or gene conversion. SMN2 copy number is currently recognized as a modifying factor for SMA. The more SMN2 copies a patient has, the lighter the phenotype will be. In domestic and foreign management consensus, SMN2 copy number is considered as a standard step in the diagnosis of SMA.

SMA is mainly caused by biallelic mutations in the SMN1 and SMN2 genes, and these mutations are the main basis for diagnosing SMA. Current detection techniques for SMA are mainly divided into two categories: multiplex ligation-dependent probe amplification (MLPA) and fluorescence quantitative PCR. In recent years, with the development of digital PCR technology, SMA detection techniques based on digital PCR platforms have gradually emerged. Digital PCR technology can use housekeeping gene sequences as internal controls and simultaneously perform absolute quantification of SMN1 and SMN2 genes[4]. However, currently this method mainly relies on foreign platforms, resulting in very high costs per use, which greatly limits its clinical application. In addition, current digital PCR systems have significant limitations, including being step-by-step and non-automated, where droplet generation, PCR reaction, and droplet detection are performed on different instruments. This technological approach results in cumbersome operations, non-closed sample handling that can cause contamination, and the need to manually transfer samples or chips between different instruments.

Biorain Biotechnology's self-developed fully automatic SMA detection system based on a droplet digital PCR platform allows for absolute quantification of both SMN1 and SMN2 genes simultaneously. This system achieves fully automatic reactions on the digital PCR platform, avoiding operational deviations caused by traditional digital PCR, which requires dividing the process into multiple steps such as droplet generation, amplification, and detection. Compared to MLPA method, this method has simple operation, overcomes the requirement for sample concentration, and can be used for whole blood or dried blood samples. Compared to fluorescence quantitative PCR method, this method does not rely on standard curves to achieve absolute quantification, and can simultaneously quantify SMN1 and SMN2 genes. Compared to traditional digital PCR platforms, this method can achieve the process of droplet generation, PCR amplification, and product analysis in a closed reaction system, with simple operation. The independently developed digital PCR platform of Biorui Biotechnology has controllable costs and has the potential for large-scale clinical application.

[1] Ruhno C , Mcgovern V L , Avenarius M R , et al. Complete sequencing of the SMN2 gene in SMA patients detects SMN gene deletion junctions and variants in SMN2 that modify the SMA phenotype[J]. Human Genetics, 2019.

[2] Laura A , Sara B , Maite C , et al. Utility of two SMN1 variants to improve spinal muscular atrophy carrier diagnosis and genetic counselling[J]. European Journal of Human Genetics, 2018.

[3]京医学会罕见病分会,北京医学会医学遗传学分会,北京医学会神经病学分会神经肌肉病学组,等.脊髓性肌萎缩症多学科管理专家共识[J].中华医学杂志,2019,99(19):1460-1467.

[4] Noemi V F , Dimitar G , Kimiyo R , et al. Multiplex Droplet Digital PCR Method Applicable to Newborn Screening, Carrier Status, and Assessment of Spinal Muscular Atrophy[J]. Clinical Chemistry, 2018(12):clinchem.2018.293712.