Development of a low-cost and accurate carrier screening method for spinal muscular atrophy in developing countries.

Heterozygous carriers of the survival of motor neuron 1 (SMN1) gene deletion in parents account for approximately 95% of neonatal spinal muscular atrophy cases. Given the severity of the disease, professional organizations have recommended periconceptional spinal muscular atrophy carrier screening to all couples, regardless of race or ethnicity. However, the prevalence of screening activities in mainland China remains suboptimal, mainly attributed to the limitations of the existing carrier screening methods. Herein, we aimed to develop a low-cost, accessible, and accurate carrier screening method based on duplex droplet digital PCR (ddPCR), to cover a wider population in developing countries, including China. The receiver operating characteristic curve was used to determine the cut-off value of SMN1 copy numbers. Performance validation was conducted for linearity, precision, and accuracy. In total, 482 cases were considered to validate the concordance between the developed ddPCR assay and multiplex ligation-dependent probe amplification. Linear correlations were excellent between the expected concentration of the reference gene and the observed values (R2 > 0.99). Both the intra- and inter-assay precision of our ddPCR assays were less than 6.0%. The multiplex ligation-dependent probe amplification and ddPCR results were consistent in 480 of the 482 cases (99.6%). Two cases with multiplex ligation-dependent probe amplification, suggestive of two copies of SMN1 exon 7, were classified into three copies by ddPCR analysis. The overall correct classification of the samples included in our ddPCR assay was 100%. This study demonstrates that an appropriate cut-off value is an important prerequisite for establishing a semi-quantitative method to determine the SMN1 copy numbers. Compared to conventional methods, our ddPCR assay is low-cost, highly accurate, and has full potential for application in population spinal muscular atrophy carriers screening.

Clinical Utility of a High-Resolution Melting Test for Screening Numerical Chromosomal Abnormalities in Recurrent Pregnancy Loss.

Recurrent pregnancy loss (RPL) occurs in approximately 5% of clinically identified pregnancies. Determining the cause of RPL is essential. Genetic testing, accompanied by an evidence-based workup, is the well-accepted process for evaluating RPL; however, current genetic tests have limitations in clinical practice. We, thus, developed a high-resolution melting analysis-based test (HRM test) to screen for the most common numerical chromosomal abnormalities present in the products of conception. We examined 765 products-of-conception samples with known karyotypes retrospectively using the HRM test, which showed high technical sensitivity (96.1%) and specificity (96.3%) as well as a high positive predictive value (95.9%) for the screening of chromosomal abnormalities. The cost-effectiveness of four RPL evaluation strategies that employ different genetic tests, karyotyping, chromosomal microarray/next-generation sequencing, the HRM test, and a combination of the HRM test and chromosomal microarray/next-generation sequencing, was then compared. The costs of diagnosing an explained RPL using karyotyping or the HRM test alone were similar. Performance of the HRM screening test before chromosomal microarray/next-generation sequencing analysis improved cost-effectiveness by approximately 30%. Cost-effectiveness was more prominent in the advanced maternal age group. Thus, the HRM test could be used as an initial screening tool, followed by other diagnostic methods to improve the cost-effectiveness of RPL evaluation, or as an alternative genetic test when other methods are unavailable or unaffordable.