Background: Developmental disorders (DDs) are early onset disorders affecting 5%–10% of children worldwide. Chromosomal microarray analysis detecting CNVs is currently recommended as the first-tier test for DD diagnosis. However, this analysis omits a high percentage of disease-causing single nucleotide variations (SNVs) that warrant further sequencing. Currently, next-generation sequencing can be used in clinical scenarios detecting CNVs, and the use of exome sequencing in the DD cohort ahead of the microarray test has not been evaluated. Methods: Clinical exome sequencing (CES) was performed on 1090 unrelated Chinese DD patients who were classified into five phenotype subgroups. CNVs and SNVs were both detected and analysed based on sequencing data. Results: An overall diagnostic rate of 41.38% was achieved with the combinational analysis of CNV and SNV. Over 12.02% of patients were diagnosed based on CNV, which was comparable with the published CMA diagnostic rate, while 0.74% were traditionally elusive cases who had dual diagnosis or apparently homozygous mutations that were clarified. The diagnostic rates among subgroups ranged from 21.82% to 50.32%. The top three recurrent cytobands with diagnostic CNVs were 15q11.2-q13.1, 22q11.21 and 7q11.23. The top three genes with diagnostic SNVs were: MECP2, SCN1A and SCN2A. Both the diagnostic rate and spectrums of CNVs and SNVs showed differences among the phenotype subgroups. Conclusion: With a higher diagnostic rate, more comprehensive observation of variations and lower cost compared with conventional strategies, simultaneous analysis of CNVs and SNVs based on CES showed potential as a new first-tier choice to diagnose DD.

SMYD4 belongs to a family of lysine methyltransferases. We analyzed the role of smyd4 in zebrafish development by generating a smyd4 mutant zebrafish line (smyd4^L544Efs*1) using the CRISPR/Cas9 technology. The maternal and zygotic smyd4^L544Efs*1 mutants demonstrated severe cardiac malformations, including defects in left-right patterning and looping and hypoplastic ventricles, suggesting that smyd4 was critical for heart development. Importantly, we identified two rare SMYD4 genetic variants in a 208-patient cohort with congenital heart defects. Both biochemical and functional analyses indicated that SMYD4(G345D) was pathogenic. Our data suggested that smyd4 functions as a histone methyltransferase and, by interacting with HDAC1, also serves as a potential modulator for histone acetylation. Transcriptome and bioinformatics analyses of smyd4^L544Efs*1 and wild-type developing hearts suggested that smyd4 is a key epigenetic regulator involved in regulating endoplasmic reticulum-mediated protein processing and several important metabolic pathways in developing zebrafish hearts. SMYD4 belongs to a SET and MYND domain-containing lysine methyltransferase. In zebrafish, smyd4 is ubiquitously expressed in early embryos and becomes enriched in the developing heart at 48 hours post-fertilization (hpf). We generated a smyd4 mutant zebrafish line (smyd4^L544Efs*1) using the CRISPR/Cas9 technology. The maternal and zygotic smyd4^L544Efs*1 mutants demonstrated a strong defect in cardiomyocyte proliferation, which led to a severe cardiac malformation, including left-right looping defects and hypoplastic ventricles. More importantly, two rare genetic variants of SMYD4 were enriched in a 208-patient cohort with congenital heart defects. Both biochemical and functional analyses indicated that SMYD4(G345D) was highly pathogenic. Using mass spectrometric analysis, SMYD4 was shown to specifically interact with histone deacetylase 1 (HDAC1) via its MYND domain. Altered di- and tri-methylation of histone 3 lysine 4 (H3K4me2 and H3K4me3) and acetylation of histone 3 in smyd4^L544Efs*1 mutants suggested that smyd4 plays an important role in epigenetic regulation. Transcriptome and pathway analyses demonstrated that the expression levels of 3,856 genes were significantly altered, which included cardiac contractile genes, key signaling pathways in cardiac development, the endoplasmic reticulum-mediated protein processing pathway, and several important metabolic pathways. Taken together, our data suggests that smyd4 is a key epigenetic regulator of cardiac development.

Congenital heart disease (CHD) is a common birth defect, and most cases occur sporadically. Mutations in key genes that are responsible for cardiac development could contribute to CHD. To date, the genetic causes of CHD remain largely unknown. In this study, twenty-nine candidate genes in CHD were sequenced in 106 patients with Tetralogy of Fallot (TOF) using target exome sequencing (TES). The co-immunoprecipitation (CO-IP) and luciferase reporter gene assays were performed in HEK293T cells, and wild-type and mutant mRNA of ZFPM2 were microinjected into zebrafish embryos. Rare variants in key cardiac transcriptional factors and JAG1 were identified in the patients. Four patients carried multiple gene variants. The novel E1148K variant was located at the eighth Zinc-finger domain of FOG2 protein. The CO-IP assays in the HEK293T cells revealed that the variant significantly damaged the interaction between ZFPM2/FOG2 and GATA4. The luciferase reporter gene assays revealed that the E1148K mutant ZFPM2 protein displayed a significantly greater inhibition of the transcriptional activation of GATA4 than the wild-type protein. The wild-type mRNA and the E1148K mutant mRNA of ZFPM2 were injected into zebrafish embryos. At 48 hpf, in the mutant mRNA injection group, the number of embryos with an abnormal cardiac chamber structure and a loss of left–right asymmetry was increased. By 72 hpf, the defects in the chamber and left–right asymmetry became obvious. We performed TES in sporadic TOF patients and identified rare variants in candidate genes in CHD. We first validated the E1148 K variant in ZFPM2, which is likely involved in the pathogenesis of CHD via GATA4. Moreover, our results suggest that TES could be a useful tool for discovering sequence variants in CHD patients.

Introduction and aim: Metformin has potential in treating patients with psoriasis, and this meta-analysis aims to explore the impact of metformin supplementation on treatment efficacy for psoriasis. Material and methods: The PubMed, EMbase, Web of science, EBSCO, and Cochrane library...