Results of a Pilot External Quality Assessment Scheme for Genetic Testing of Newborns with Spinal Muscular Atrophy.

BACKGROUND: This study aims to evaluate the ability of laboratories to perform spinal muscular atrophy (SMA) genetic testing in newborns based on dried blood spot (DBS) samples, and to provide reference data and advance preparation for establishing the pilot external quality assessment (EQA) scheme for SMA genetic testing of newborns in China. METHODS: The pilot EQA scheme contents and evaluation principles of this project were designed by National Center for Clinical Laboratories (NCCL), National Health Commission. Two surveys were carried out in 2022, and 5 batches of blood spots were submitted to the participating laboratory each time. All participating laboratories conducted testing upon receiving samples, and test results were submitted to NCCL within the specified date. RESULTS: The return rates were 75.0% (21/28) and 95.2% (20/21) in the first and second surveys, respectively. The total return rate of the two examinations was 83.7% (41/49). Nineteen laboratories (19/21, 90.5%) had a full score passing on the first survey, while in the second survey twenty laboratories (20/20, 100%) scored full. CONCLUSIONS: This pilot EQA survey provides a preliminary understanding of the capability of SMA genetic testing for newborns across laboratories in China. A few laboratories had technical or operational problems in testing. It is, therefore, of importance to strengthen laboratory management and to improve testing capacity for the establishment of a national EQA scheme for newborn SMA genetic testing.

Result of a Pilot External Quality Assessment Scheme for Clinical Diagnosis of Inherited Metabolic Disorders in China.

BACKGROUND: We aimed to evaluate the diagnostic capabilities of Chinese laboratories for inherited metabolic disorders (IMDs) using gas chromatography-mass spectrometry (GC-MS) on urine samples. Meanwhile, based on the result of the pilot external quality assessment (EQA) scheme, we hope to establish a standardized and reliable procedure for future EQA practice. METHODS: We recruited laboratories that participated in the EQA of quantitative analysis of urinary organic acids with GC-MS before joining the surveys. In each survey, a set of five real urine samples was distributed to each participant. The participants should analyze the sample by GC-MS and report the "analytical result", "the most likely diagnosis", and "recommendation for further tests" to the NCCL before the deadline. RESULTS: A total of 21 laboratories participated in the scheme. The pass rates were 94.4% in 2020 and 89.5% in 2021. For all eight IMDs tested, the analytical proficiency rates ranged from 84.7% - 100%, and the interpretational performance rate ranged from 88.2% - 97.0%. The performance on hyperphenylalaninemia (HPA), 3-methylcrotonyl-CoA carboxylase deficiency (MCCD), and ethylmalonic encephalopathy (EE) samples were not satisfactory. CONCLUSIONS: In general, the participants of this pilot EQA scheme are equipped with the basic capability for qualitative organic acid analysis and interpretation of the results. Limited by the small size of laboratories and samples involved, this activity could not fully reflect the state of clinical practice of Chinese laboratories. NCCL will improve the EQA scheme and implement more EQA activities in the future.

Plant immunity suppressor SKRP encodes a novel RNA-binding protein that targets exon 3' end of unspliced RNA.

The regulatory roles of RNA splicing in plant immunity are emerging but still largely obscure. We reported previously that Phytophthora pathogen effector Avr3c targets a soybean protein SKRP (serine/lysine/arginine-rich protein) to impair soybean basal immunity by regulating host pre-mRNA alternative splicing, while the biochemical nature of SKRP remains unknown. Here, by using Arabidopsis as a model, we studied the mechanism of SKRP in regulating pre-mRNA splicing and plant immunity. AtSKRP confers impaired plant immunity against Phytophthora capsici and associates with spliceosome component PRP8 and splicing factor SR45, which positively and negatively regulate plant immunity, respectively. Enhanced crosslinking and immunoprecipitation followed by high-throughput sequencing (eCLIP-seq) showed AtSKRP is a novel RNA-binding protein that targets exon 3' end of unspliced RNA. Such position-specific binding of SKRP is associated with its activity in suppressing intron retention, including at positive immune regulatory genes UBP25 and RAR1. In addition, we found AtSKRP self-interact and forms oligomer, and these properties are associated with its function in plant immunity. Overall, our findings reveal that the immune repressor SKRP is a spliceosome-associated protein that targets exon 3' end to regulate pre-mRNA splicing in Arabidopsis.

ALBA proteins confer thermotolerance through stabilizing HSF messenger RNAs in cytoplasmic granules.

High temperature is one of the major environmental stresses affecting plant growth and fitness. Heat stress transcription factors (HSFs) play critical roles in regulating the expression of heat-responsive genes. However, how HSFs are regulated remains obscure. Here, we show that ALBA4, ALBA5 and ALBA6, which phase separate into stress granules (SGs) and processing bodies (PBs) under heat stress, directly bind selected messenger RNAs, including HSF mRNAs, and recruit them into SGs and PBs to protect them from degradation under heat stress in Arabidopsis. The alba456 triple mutants, but not single and double mutants, display pleiotropic developmental defects and hypersensitivity to heat stress. Mutations in XRN4, a cytoplasmic 5' to 3' exoribonuclease, can rescue the observed developmental and heat-sensitive phenotypes of alba456 seedlings. Our study reveals a new layer of regulation for HSFs whereby HSF mRNAs are stabilized by redundant action of ALBA proteins in SGs and PBs for plant thermotolerance.