High-throughput genetic newborn screening for spinal muscular atrophy by rapid nucleic acid extraction from dried blood spots and 384-well qPCR

Abstract

Establishing nucleic acid-based assays for genetic newborn screening (NBS) provides the possibility to screen for genetically encoded diseases like spinal muscular atrophy (SMA), best before the onset of symptoms. Such assays should be easily scalable to 384-well reactions that make the screening of up to 2000 samples per day possible. We developed a test procedure based on a cleanup protocol for dried blood spots and a quantitative (q)PCR to screen for a homozygous deletion of exon 7 of the survival of motor neuron 1 gene (SMN1) that is responsible for >95% of SMA patients. Performance of this setup is evaluated in detail and tested on routine samples. Our cleanup method for nucleic acids from dried blood spots yields enough DNA for diverse subsequent qPCR applications. To date, we have applied this approach to test 213,279 samples within 18 months. Thirty patients were identified and confirmed, implying an incidence of 1:7109 for the homozygous deletion. Using our cleanup method, a rapid workflow could be established to prepare nucleic acids from dried blood spot cards. Targeting the exon 7 deletion, no invalid, false-positive, or false-negative results were reported to date. This allows timely identification of the disease and grants access to the recently introduced treatment options, in most cases before the onset of symptoms. Carriers are not identified, thus, there are no concerns of whether to report them.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1

Schematic overview of the SMN-coding genes. SMN1 is encoded on chromosome 5 by nine exons (exons 2 and 3 are usually referred to as exon 2a and 2b, respectively [12]). Exon 7 (the eighth exon) is missing in most SMA patients, and is missing from SMN2 transcripts as well. SMN1 and SMN2 only differ by five nucleotides as indicated [c.835-44G>A (rs1454173648); c.840C>T (rs1164325688); c.888+100A>G (rs212214); c.888+215A>G (rs1244569826); c.1155G>A (rs1208416968)]. All variants shown are described using the NM_000344.3 transcript reference sequence based on NG_008691.1 for intron positions. Primers and probes for the c.840 site are indicated in gray; the site itself is indicated by a gray square in the forward primer

Fig. 2

Performance comparison of the CXCE-buffer cleanup vs. extraction of nucleic acids on a MagnaPure96. The amplification curves show the performance of nucleic acids of both extraction methods from the same sample (gray lines: MagnaPure96 extraction, black lines: CXCE-buffer cleanup) in the SMN1 exon 7 deletion qPCR

Fig. 3

Schematic contamination patterns on a 96-well plate. Plate A shows the results of automated punching, Plate M from manual punching with white wells: no amplification, black wells: expected amplification, and gray wells: late amplification, i.e., contamination including the respective Cq-values