NHLRC2 variants identified in patients with fibrosis, neurodegeneration, and cerebral angiomatosis (FINCA): characterisation of a novel cerebropulmonary disease

Abstract

A novel multi-organ disease that is fatal in early childhood was identified in three patients from two non-consanguineous families. These children were born asymptomatic but at the age of 2 months they manifested progressive multi-organ symptoms resembling no previously known disease. The main clinical features included progressive cerebropulmonary symptoms, malabsorption, progressive growth failure, recurrent infections, chronic haemolytic anaemia and transient liver dysfunction. In the affected children, neuropathology revealed increased angiomatosis-like leptomeningeal, cortical and superficial white matter vascularisation and congestion, vacuolar degeneration and myelin loss in white matter, as well as neuronal degeneration. Interstitial fibrosis and previously undescribed granuloma-like lesions were observed in the lungs. Hepatomegaly, steatosis and collagen accumulation were detected in the liver. A whole-exome sequencing of the two unrelated families with the affected children revealed the transmission of two heterozygous variants in the NHL repeat-containing protein 2 (NHLRC2); an amino acid substitution p.Asp148Tyr and a frameshift 2-bp deletion p.Arg201GlyfsTer6. NHLRC2 is highly conserved and expressed in multiple organs and its function is unknown. It contains a thioredoxin-like domain; however, an insulin turbidity assay on human recombinant NHLRC2 showed no thioredoxin activity. In patient-derived fibroblasts, NHLRC2 levels were low, and only p.Asp148Tyr was expressed. Therefore, the allele with the frameshift deletion is likely non-functional. Development of the Nhlrc2 null mouse strain stalled before the morula stage. Morpholino knockdown of nhlrc2 in zebrafish embryos affected the integrity of cells in the midbrain region. This is the first description of a fatal, early-onset disease; we have named it FINCA disease based on the combination of pathological features that include fibrosis, neurodegeneration, and cerebral angiomatosis.

Structural analysis of human NHLRC2, mutations of which are associated with FINCA disease

“…While NHLRC2 has a CCINC motif, structural homologues have SCINC (DipZ), WCPTC (DsbF), WCPSC (DsbE) and WCVPC (TlpA). Consistent with this difference, we have previously shown that the Trx-like domain of NHLRC2 does not contain oxidoreductase activity in a classical insulin reduction assay using either the full-length protein or the isolated Trx-like domain [ 2 ] suggesting that it is not involved in thiol-disulfide exchange.…”

“…Previously, NHLRC2 homologs were thought to be limited to animalia [ 1 , 2 ]. However, homologs can be found across different kingdoms of life, including in prokaryotes.…”

“…DNA constructs encoding for the full-length NHLRC2 and different NHLRC2 fragments were prepared as described previously and the corresponding proteins were expressed and purified as described [ 2 ]. Selenomethionine substituted NHLRC2 (221–572) was produced by inhibition of methionine biosynthesis method [ 8 ] and purified similarly to native protein.…”

“…NHLRC2 (NHL repeat containing protein 2) is an essential cytosolic protein of unknown function [ 1 ], mutation of which was recently identified in association with FINCA ( fi brosis, n eurodegeneration, c erebral a ngiomatosis) disease, a multi-organ condition which is fatal in early childhood [ 2 ]. The most prominent features of the disease were severe interstitial pulmonary fibrosis and early-onset, progressive neurodegeneration.…”

“…In addition to having an apparent role in early development, NHLRC2 has been suggested to be a novel serum biomarker for Alzheimer´s disease [ 5 ] and as having an as yet unresolved role in the regulation of ROS-induced apoptosis [ 1 ]. Homozygous deletion of the NHLRC2 gene leads to an embryonic lethality in mice [ 2 , 6 ] and a recently reported mutation in a conserved region of the β-propeller domain NHLRC2 was shown to cause neural tube defects in Angus cattle [ 7 ].…”