Prenatal Diagnosis and Functional Analysis of Two Compound Heterozygous Variants in the KLHL40 Gene Causing Nemaline Myopathy 8

BACKGROUND: Nemaline myopathy (NEM) is a rare congenital muscular disorder characterized by slow progression or static neuromuscular symptoms, which is mainly caused by variants in genes encoding the myofilament protein of skeletal muscle sarcomere. This study aimed to conduct prenatal diagnosis and functional analysis of variants in the KLHL40 gene causing NEM 8. METHODS: A Chinese family who experienced multiple pregnancies with recurrent prenatal ultrasound anomalies (such as clubfoot and polyhydramnios) and subsequent neonatal death was enrolled in this study. Etiology diagnosis was conducted using karyotype, chromosomal microarray analysis (CMA), and exome sequencing (ES). Quantitative real-time reverse transcription polymerase chain reaction (qPCR) and western blotting were performed to investigate the transcription and expression levels of the defective gene. Hematoxylin-eosin (HE) stain and modified Gomori staining were used for further pathological examination. RESULTS: No chromosomal abnormalities were detected by karyotype and CMA in the proband of this family. However, the ES results revealed two compound heterozygous variants in KLHL40 gene NM_152393.4:c.[1516A> C p.(T506P)]; [1327G> A p.(G443S)] in the fetus. The c.1516A> C p.(T506P) variant was interpreted as pathogenic, whereas the p.G443S variant was rarely reported and classified as a variant of uncertain significance. Interestingly, the subsequent western blotting analysis elicited a significantly decreased expression level of KLHL40 protein in fetal skeletal muscle tissues compared with the controls. In addition, the HE and modified Gomori stains demonstrated nemaline bodies in the fetus using skeletal muscle tissue. CONCLUSION: Combining prenatal ultrasound and ES testing increases diagnostic yield among NEM-affected fetuses. Our results extend the mutational spectrum of the pathogenic KLHL40 variant c.1327G> A p.(G443S) linked to NEM8 and aid genotype-phenotype correlation analysis.