Loss of Nexilin function leads to a recessive lethal fetal cardiomyopathy characterized by cardiomegaly and endocardial fibroelastosis

Abstract

The Nexilin F-Actin Binding Protein (Nexilin) encoded by NEXN is a cardiac Z-disc protein important for cardiac function and development in humans, zebrafish, and mice. Heterozygote variants in the human NEXN gene have been reported to cause dilated and hypertrophic cardiomyopathy. Homozygous variants in NEXN cause a lethal form of human fetal cardiomyopathy, only described in two patients before. In a Swedish, four-generation, non-consanguineous family comprising 42 individuals, one female had three consecutive pregnancies with intrauterine fetal deaths caused by a lethal form of dilated cardiomyopathy. Whole-exome sequencing and variant analysis revealed that the affected fetuses were homozygous for a NEXN variant (NM_144573:c.1302del;p.(Ile435Serfs*3)). Moreover, autopsy and histology staining declared that they presented with cardiomegaly and endocardial fibroelastosis. Immunohistochemistry staining for Nexilin in the affected fetuses revealed reduced antibody staining and loss of striation in the heart, supporting loss of Nexilin function. Clinical examination of seven heterozygote carriers confirmed dilated cardiomyopathy (two individuals), other cardiac findings (three individuals), or no cardiac deviations (two individuals), indicating incomplete penetrance or age-dependent expression of dilated cardiomyopathy. RNA sequencing spanning the variant in cDNA blood of heterozygote individuals revealed nonsense-mediated mRNA decay of the mutated transcripts. In the current study, we present the first natural course of the recessively inherited lethal form of human fetal cardiomyopathy caused by loss of Nexilin function. The affected family had uneventful pregnancies until week 23-24, followed by fetal death at week 24-30, characterized by cardiomegaly and endocardial fibroelastosis.

Keywords: NEXN; Nexilin; cardiomyopathy; lethal.

FIGURE 1

A four‐generation family affected with three recurrent intrauterine fetal deaths (IUFD) were investigated for a pathogenetic variant in NEXN (NM_144573:c.1302del). (a) This family consists of 42 individuals. Seventeen individuals were included in the clinical and genetic study. Heterozygote carriers with confirmed dilated cardiomyopathy (DCM) are highlighted in gray. Homozygous fetuses with lethal fetal dilated cardiomyopathy (lfDCM) are highlighted in black. LBBB = left bundle branch block. (b) Pictures of the fetal hearts (IV:5–7) with cardiomegaly. (c) The NEXN variant segregated in the family with obligate carriers being heterozygous and affected fetuses homozygous. Sequencing result using the reverse primer is shown

FIGURE 2

Functional analysis of the NEXN variant reveals reduced Nexilin expression, degraded muscle fibers, and loss of striation in the heart. (a) Schematic of the NEXN transcripts and the predicted protein domains of the human Nexilin protein. In gray and white are the exons with little to no expression in adult heart respectively. All previously reported cardiomyopathy patients associated with NEXN variants of autosomal dominant inheritance (black) and autosomal recessive inheritance (red) are included in the schematic. IGcam = immunoglobulin superfamily class domain, * = compound heterozygote variants. (a, b) RNA studies reveal two new isoforms of NEXN transcripts (yellow and pink), identified in both cases and controls, suggesting novel transcripts of the general population. The region spanned by the PCR are highlighted with a dotted square. (c) RNA studies of heterozygote parents could not identify the c.1302del variant (NM_144573), suggesting that the variant introduces nonsense‐mediated mRNA decay. The chromatogram in the figure was performed using forward primer. (d) Immunohistochemistry of paraffin‐embedded heart tissues of affected fetuses (case) and age‐matched controls using the NEXN antibody (NEXN‐TA590405) indicated loss of Nexilin function by reduced Nexilin expression, degraded muscle fibers, and loss of cross striations in the heart

FIGURE 3

Endocardial fibroelastosis (EFE) as a characteristic for lethal fetal dilated cardiomyopathy (lfDCM) caused by a homozygous variant in NEXN. (a) Histology staining of heart sections from three affected fetuses (cases) and two age‐matched controls revealed increased deposition of collagen and elastin in cases compared to controls. Staining was performed with Hematoxylin & Eosin (H&E), Masson's trichrome (MAT), Sirius red (SR), and Verhoeff‐Van Gieson (VVG). The scale bars in the top images are 200 μm, and 50 μm in the zoomed in images below. (b) Barplots visualizing the increase in width of connective tissue and elastic fibers in the above heart sections of the same individuals. The values are mean values of the width (pixels) from 10 representative measurements per individual in histology‐stained slides with 20× of magnitude (C) and 4× of magnitude