Clinical Validation of Droplet Digital Polymerase Chain Reaction (ddPCR) for SMN1 and SMN2 Copy Number Determination in Newborn Screening
Clinical Genetics and Therapeutics
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Primary Categories:
- Clinical Genetics
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Secondary Categories:
- Clinical Genetics
Introduction:
Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by loss of α-motor neurons in the spinal cord and has been a leading genetic cause of infant death. It results from a homozygous deletion or mutation of the SMN1 gene on chromosome 5q13, which encodes for the majority of functional SMN protein. The SMN2 gene, a paralog of SMN1 on chromosome 5q, produces about ~5-10% of functional SMN protein. Most SMA cases involve deletion of exon 7 of the SMN1 gene. When SMN1 gene is absent, if multiple copies of the SMN2 gene are present, this may alleviate the severity of SMA. California began screening newborns for SMA in June 2020. Bio-Rad® ddPCR kits were used to determine SMN1 and SMN2 gene copy numbers in the newborn screening specimens. This abstract discusses the clinical validation of ddPCR copy number determination for SMN1 and SMN2 genes for the purposes of newborn screening.
Methods:
This study measured accuracy of the Bio-Rad® ddPCR kits in determining SMN1 and SMN2 gene copy numbers by analyzing samples with known copy numbers and observing if results matched. Known SMA positive control samples were also tested for concordance with ddPCR analysis. A precision study for the SMN1 gene was carried out using CDC SMA positive, CDC carrier, cord blood, blanks, and Bio-Rad® controls. A precision study for the SMN2 gene was performed as well on known controls. Since June 2020, 89 newborns screened positive for SMA on California newborn screening, and their SMN2 copy numbers were determined using in-house ddPCR assay. These numbers were compared to the copy numbers found by the reference laboratories performing confirmatory testing. ddPCR uses the concentration of SMN1, SMN2 & RPP30 (reference gene) in dried blood spots (DBS) to make copy number determinations. DNA is extracted from a 3.2 mm disc punch from a DBS card using a custom-made robot designed by Hamilton Company (Reno, NV). The 3.2 mm disc is first washed by shaking for 10 minutes with 100 µL of QuantaBio® Extracta DBS Buffer (Beverly, MA) and discarding the initial buffer. Then 40 µL of the same brand buffer is added to the washed 3.2 mm disc, incubating for 30 minutes at 95°C. An aliquot of 4 µL is taken from this incubated mixture for ddPCR preparation and added to 18 µL of mastermix included in the Bio-Rad® SMN1 & SMN2 Copy Number Determination kits. This final mixture is placed in a droplet generator and the resulting droplets transferred to a standard 96 well plate. The plate is then placed in a thermocycler for a two-hour PCR run followed by concentration determination using a specialized ddPCR droplet reader.
Results:
All copy number results obtained using the Bio-Rad® ddPCR kits were within the expected whole copy number integer ±0.5 of the known copy number samples. The ddPCR analysis of known SMA positive controls also matched. For the precision study on SMN1, comparison of expected and observed values was very close, with maximum CV% below 10 and linear regression analysis showing an R2 value closer to 1. The precision study on SMN2 for known controls showed maximum CV% below 8 with R2 value of 0.99. The ddPCR assay of SMN2 in the 89 known SMA positives showed the same copy numbers as the reference laboratories.
Conclusion:
Clinical validation of Bio-Rad® QX200 ddPCR for SMN1 and SMN2 copy number determination has demonstrated excellent accuracy and precision. As further clinical validation of ddPCR, the SMN2 copy numbers of all SMA positive specimens identified by California newborn screening since June 2020 matched those found by reference laboratories.
Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by loss of α-motor neurons in the spinal cord and has been a leading genetic cause of infant death. It results from a homozygous deletion or mutation of the SMN1 gene on chromosome 5q13, which encodes for the majority of functional SMN protein. The SMN2 gene, a paralog of SMN1 on chromosome 5q, produces about ~5-10% of functional SMN protein. Most SMA cases involve deletion of exon 7 of the SMN1 gene. When SMN1 gene is absent, if multiple copies of the SMN2 gene are present, this may alleviate the severity of SMA. California began screening newborns for SMA in June 2020. Bio-Rad® ddPCR kits were used to determine SMN1 and SMN2 gene copy numbers in the newborn screening specimens. This abstract discusses the clinical validation of ddPCR copy number determination for SMN1 and SMN2 genes for the purposes of newborn screening.
Methods:
This study measured accuracy of the Bio-Rad® ddPCR kits in determining SMN1 and SMN2 gene copy numbers by analyzing samples with known copy numbers and observing if results matched. Known SMA positive control samples were also tested for concordance with ddPCR analysis. A precision study for the SMN1 gene was carried out using CDC SMA positive, CDC carrier, cord blood, blanks, and Bio-Rad® controls. A precision study for the SMN2 gene was performed as well on known controls. Since June 2020, 89 newborns screened positive for SMA on California newborn screening, and their SMN2 copy numbers were determined using in-house ddPCR assay. These numbers were compared to the copy numbers found by the reference laboratories performing confirmatory testing. ddPCR uses the concentration of SMN1, SMN2 & RPP30 (reference gene) in dried blood spots (DBS) to make copy number determinations. DNA is extracted from a 3.2 mm disc punch from a DBS card using a custom-made robot designed by Hamilton Company (Reno, NV). The 3.2 mm disc is first washed by shaking for 10 minutes with 100 µL of QuantaBio® Extracta DBS Buffer (Beverly, MA) and discarding the initial buffer. Then 40 µL of the same brand buffer is added to the washed 3.2 mm disc, incubating for 30 minutes at 95°C. An aliquot of 4 µL is taken from this incubated mixture for ddPCR preparation and added to 18 µL of mastermix included in the Bio-Rad® SMN1 & SMN2 Copy Number Determination kits. This final mixture is placed in a droplet generator and the resulting droplets transferred to a standard 96 well plate. The plate is then placed in a thermocycler for a two-hour PCR run followed by concentration determination using a specialized ddPCR droplet reader.
Results:
All copy number results obtained using the Bio-Rad® ddPCR kits were within the expected whole copy number integer ±0.5 of the known copy number samples. The ddPCR analysis of known SMA positive controls also matched. For the precision study on SMN1, comparison of expected and observed values was very close, with maximum CV% below 10 and linear regression analysis showing an R2 value closer to 1. The precision study on SMN2 for known controls showed maximum CV% below 8 with R2 value of 0.99. The ddPCR assay of SMN2 in the 89 known SMA positives showed the same copy numbers as the reference laboratories.
Conclusion:
Clinical validation of Bio-Rad® QX200 ddPCR for SMN1 and SMN2 copy number determination has demonstrated excellent accuracy and precision. As further clinical validation of ddPCR, the SMN2 copy numbers of all SMA positive specimens identified by California newborn screening since June 2020 matched those found by reference laboratories.