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.

Comparison of the accuracy of multiplex digital PCR versus multiplex ligation-dependent probe amplification in quantification of the survival of motor neuron genes copy numbers.

For over two decades, multiplex ligation-dependent probe amplification (MLPA) has served as the gold standard for genetic testing of spinal muscular atrophy. However, there is emerging evidence questioning the reliability of MLPA in determining the copy numbers (CNs) of the survival of motor neuron (SMN) gene in certain cases. Recently, digital polymerase chain reaction (dPCR) has shown potential for better performance in copy number variant detection. This study aimed to compare MLPA and dPCR in quantifying SMN1 and SMN2 CNs, identify reasons for observed discrepancies, and explore the clinical implications of false results. A total of 733 DNA samples, previously subjected to MLPA analysis, were tested using multiplex droplet dPCR assays. Samples exhibiting inconsistent results between the two methods underwent repeated dPCR assays. When inconsistencies persisted, a third method was employed for verification. Digital PCR yielded results consistent with those of MLPA in 94.4% (692/733) of samples. Forty-one cases exhibited quantitative disparities in SMN1 and/or SMN2 CNs between the two methods. Confirmatory tests revealed that 37 inaccurate results were produced by the MLPA analysis, whereas four were attributed to the dPCR method. The dPCR technique exhibits better accuracy than MLPA and is qualified for SMA genetic testing across various clinical scenarios.

Prenatal Genetic Diagnosis of Fetal Cystic Hygroma: A Retrospective Single-Center Study from China.

Fetal cystic hygroma (CH) is associated with poor prognosis and chromosomal anomalies. Recent studies have suggested that the genetic background of affected fetuses is essential for predicting pregnancy outcomes. However, the detection performance of different genetic approaches for the etiological diagnosis of fetal CH remains unclear. In this study, we aimed to compare the diagnostic efficiency of karyotyping and chromosomal microarray analysis (CMA) in a local fetal CH cohort, and tried to propose an optimized testing strategy that may help improve the cost-effectiveness of disease management. We reviewed all pregnancies that underwent invasive prenatal diagnosis between January 2017 and September 2021 at one of the largest prenatal diagnostic centers in Southeast China. We collected cases identified by the presence of fetal CH. Prenatal phenotypes and laboratory records of these patients were audited, collated, and analyzed. The detection rates of karyotyping and CMA were compared, and the concordance rate of these two methods was calculated. A total of 157 fetal CH cases were screened from 6,059 patients who underwent prenatal diagnosis. Diagnostic genetic variants were identified in 44.6% (70/157) of the cases. Karyotyping, CMA, and whole-exome sequencing (WES) identified pathogenic genetic variants in 63, 68, and 1 case, respectively. The Cohen's κ coefficient between karyotyping and CMA was 0.96, with a concordance of 98.0%. Of the 18 cases in which cryptic copy number variants <5 Mb were detected by CMA, 17 were interpreted as variants of uncertain significance, and the remaining cases were interpreted as pathogenic. Trio exome sequencing revealed a pathogenic homozygous splice site mutation in the PIGN gene in a case undiagnosed by CMA and karyotyping. Our study demonstrated that chromosomal aneuploidy abnormalities are the main genetic cause of fetal CH. Based on this, we recommend karyotyping combined with rapid aneuploidy detection as a first-tier approach for the genetic diagnosis of fetal CH. WES and CMA could improve the diagnostic yield when routine genetic tests fail to determine the cause of fetal CH.