Identification of a novel de novo PUF60 variant causing Verheij syndrome in a fetus.

Verheij syndrome (VRJS) is a craniofacial spliceosomopathy with a wide phenotypic spectrum. Haploinsufficiency of the poly-uridine binding splicing factor 60 gene (PUF60) and its loss-of-function (LOF) variants are involved in VRJS. We evaluated a human fetus with congenital heart defects and preaxial polydactyly. Clinical data were obtained from the medical record. Whole-exome sequencing (WES) was used to explore the potential genetic etiology, and the detected variant verified using Sanger sequencing. Functional studies were performed to validate the pathogenic effects of the variant. Using trio-WES, we identified a novel PUF60 variant (NM_078480.2; c.1678 T > A, p.*560Argext*204) in the pedigree. Bioinformatic analyses revealed that the variant is potentially pathogenic, and functional studies indicated that it leads to degradation of the elongated protein and subsequently PUF60 LOF, producing some VRJS phenotypes. These findings confirmed the pathogenicity of the variant. This study implicates PUF60 LOF in the etiopathogenesis of VRJS. It not only expands the PUF60 variant spectrum, and also provides a basis for genetic counseling and the diagnosis of VRJS. Although trio-WES is a well-established approach for identifying the genetic etiology of rare multisystemic conditions, functional studies could aid in verifying the pathogenicity of novel variants.

A novel PKHD1 splicing variant identified in a fetus with autosomal recessive polycystic kidney disease.

Objective: Variants of the polycystic kidney and hepatic disease 1 (PKHD1) gene are associated with autosomal recessive polycystic kidney disease (ARPKD). This study aimed to identify the genetic causes in a Chinese pedigree with ARPKD and design a minigene construct of the PKHD1 gene to investigate the impact of its variants on splicing. Methods: Umbilical cord samples from the proband and peripheral blood samples from his parents were collected, and genomic DNA was extracted for whole-exome sequencing (WES). Bioinformatic analysis was used to identify potential genetic causes, and Sanger sequencing confirmed the existence of variants within the pedigree. A minigene assay was performed to validate the effects of an intronic variant on mRNA splicing. Results: Two variants, c.9455del (p.N3152Tfs*10) and c.2408-13C>G, were identified in the PKHD1 gene (NM_138694.4) by WES; the latter has not been previously reported. In silico analysis predicted that this intronic variant is potentially pathogenic. Bioinformatic splice prediction tools revealed that the variant is likely to strongly impact splice site function. An in vitro minigene assay revealed that c.2408-13C>G can cause aberrant splicing, resulting in the retention of 12 bp of intron 23. Conclusion: A novel pathogenic variant of PKHD1, c.2408-13C>G, was found in a fetus with ARPKD, which enriches the variant spectrum of the PKHD1 gene and provides a basis for genetic counseling and the diagnosis of ARPKD. Minigenes are optimal to determine whether intron variants can cause aberrant splicing.

Identification of a novel heterozygous missense TP63 variant in a Chinese pedigree with split-hand/foot malformation.

BACKGROUND: Tumor protein p63 is an important transcription factor regulating epithelial morphogenesis. Variants associated with the TP63 gene are known to cause multiple disorders. In this study, we determined the genetic cause of split-hand/foot malformation in a Chinese pedigree. METHODS: For this study, we have recruited a Chinese family and collected samples from affected and normal individuals of the family (three affected and two normal). Whole exome sequencing was performed to detect the underlying genetic defect in this family. The potential variant was validated using the Sanger sequencing approach. RESULTS: Using whole-exome and Sanger sequencing, we identified a novel heterozygous pathogenic missense variant in TP63 (NM_003722.5: c.921G > T; p.Met307Ile). This variant resulted in the substitution of methionine with isoleucine. Structural analysis suggested a resulting change in the structure of a key functional domain of the p63 protein. CONCLUSION: This novel missense variant expands the TP63 variant spectrum and provides a basis for genetic counseling and prenatal diagnosis of families with split-hand/foot malformation or other TP63-related diseases.