Novel biallelic variants in NRROS associated with a lethal microgliopathy, brain calcifications and neurodegeneration

Abstract

Negative regulator of reactive oxygen species (NRROS) is a leucine-rich repeat protein expressed by microglia and perivascular macrophages. To date, 9 individuals have been reported with biallelic NRROS variants. Here, we report one individual with a severe neurodegenerative phenotype in which exome sequencing identified 2 novel variants in NRROS, a missense variant (c.185T>C, p.Leu62Pro) and a premature stop codon (c.310C>T, p.Gln104Ter). Pathological examination revealed both extensive grey and white matter involvement, dystrophic calcifications and infiltration of foamy macrophages. This is the first reported case of NRROS variants with an ultrastructure mitochondrial abnormality noted on electron microscopy analysis of post-mortem tissue.

INTRODUCTION

NRROS is a protein of the leucine-rich repeat family expressed by myeloid cells in the central nervous system (CNS), including microglia and perivascular macrophages [1]. While originally identified as a negative regulator of Toll-like receptor (TLR) signalling, the function of NRROS in the CNS has expanded to include negative regulation of NOX2, an NADPH oxidase responsible for reactive oxygen species (ROS) production, and activation of latent TGF- B1, a regulator of microglia activation and neuron survival cue [1-4].The overall function of NRROS is presumably in maintaining homeostasis of the CNS and preventing unwarranted oxidative stress and inflammation.

Biallelic pathogenic variants in NRROS have been reported in nine individuals with severe neurological regression, intracranial calcifications, brain atrophy, reduced myelination, and epilepsy [5, 6]. Here, we report the case of a patient with severe neurodegeneration and biallelic novel variants in NRROS and present the neuropathological findings associated with this disease, including a novel finding of mitochondrial ultrastructure abnormality.

METHODS

Detailed genetic analysis methods are provided in the supplementary information. The variants have been submitted to ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/; SCV001977605 and SCV001977606).

RESULTS

The child, female, was born to non-consanguineous parents after a normal pregnancy and had no perinatal complications. Her initial development was normal until a developmental plateau at 8 months. There were concerns of regression and decelerating head growth and at 12 months she was diagnosed with hypotonia and failure-to-thrive. At 16 months she developed myoclonic epilepsy that proved to be refractory. Initial magnetic resonance imaging (MRI) was done at 16 months and revealed global parenchymal atrophy, a thin corpus callosum and multiple T1 hyperintense foci suggestive of calcifications. Computed tomography (CT) at 18 months confirmed calcifications in the subcortical and deep white matter (Fig. 1a-c). Repeat MRI at 20 months showed a thin corpus callosum (Fig. 1d) and progressive volume loss with ventricular enlargement (Fig. 1e-f). Neurological regression was exacerbated by repeat infections and respiratory distress and the child died at 3 years and 10 months. Substantial investigations had been done to identify the etiology of her severe neurodegenerative condition. Genetic testing including array comparative genomic hybridization, mitochondrial DNA sequencing and DNA sequencing panels for Aicardi-Goutières syndrome and epilepsy, were all non-contributory. Investigations to screen for congenital infections were negative. Extensive metabolic workup (including lactate, ammonia, serum amino acids, urine organic acids, very long-chain fatty acids, acylcarnitine profile, and capillary zone electrophoresis (congenital disorders of glycosylation) were all non-contributory, as was testing for mitochondrial disorders (blue native PAGE of fibroblasts and in-gel activity staining of respiratory transport chain complexes) and lysosomal storage diseases (skin biopsy and lysosomal enzyme testing (including for NCL-1 and NCL-2). Cerebrospinal fluid testing for lactate, amino acids, 5-methyltetrahydrofolate, neopterin, and interferon-alpha were all normal. Electroencephalograms confirmed the diagnosis of myoclonic epilepsy while auditory brainstem response and electromyography were normal. NGS data re-analysis was pursued after her death, revealing the biallelic variants in NRROS. In silico modelling of the 2 variants reported here suggest both are pathogenic. The missense variant (Chr3.hg38.196659828T>C, NM_198565.3:c.185T>C, p.Leu62Pro) is absent from control population databases and causes the loss of a highly conserved leucine residue. Meanwhile, the second variant causes a premature termination codon (Chr3.hg38.196659953C>T, c.310C>T, p.Gln104Ter) resulting in a truncated protein of 104 amino acids (originally 693 residues, 85% truncation) and is predicted to cause loss-of-function due to a truncated or absent product via nonsense mediated decay. These variants, located in exon 3, were detected in the heterozygous state by trio exome sequencing with parentage confirmed.

Fig. 1. Neuroimaging findings.

CT scan of the brain performed at the age of 18 months, (a) parasagittal and (b,c) axial cuts, showing cerebral atrophy and punctate calcification in the subcortical and deep white matter. Brain MRI at the age of 20 months showing a (d) thin corpus callosum and (e,f) mild to moderate cerebral atrophy

Detailed pathological examination was performed. At death, brain weight was 500g, 9 SD below the expected for age and sex (1150 ± 70 g). Macroscopically, there was severe reduction of white matter, with almost none in the centrum semiovale, and the white matter was discoloured and spongy (Fig. 2a). The corpus callosum was thin, there was marked dilatation of the ventricles, shrinkage of the cerebellar folia and atrophy of the brainstem. Histologically, the brain was diffusely gliotic with severe reduction of white matter, including no myelin staining in the hemispheric white matter and hardly any residual axons (Fig. 2b-c). There were dystrophic calcifications in the white matter, both along the grey-white matter junction and in deeper white matter regions (Fig. 2d). Notably, there was widespread infiltration of microglia and clusters of foamy macrophages both diffusely and in the perivascular regions, with some cytoplasmic staining for Luxol Fast Blue, suggestive of myelin debris (Fig. 2e-g). Electron microscopy of post-mortem brain tissue showed occasional rounded mitochondria with aberrantly organized cristae (Fig. 2f).

Fig. 2. Neuropathological findings.

(a) Coronal section of the cerebral hemisphere at the level of the basal ganglia showing hemispheric white matter that is shrunken, spongy and discolored, a thin corpus callosum and marked ventricular enlargement. (b) Low-power section through the frontal lobe immunostained for neurofilaments showing expected staining of neurites in the cortex (CX) but an absence of axons in the white matter (WM). (c) H&E-LFB staining of the same frontal lobe section showing an intact CX but shrunken WM, devoid of myelin. (d) Calcifications present in the hemispheric WM (H&E). (e) CD68 imunostain showing diffuse infiltration of macrophages in the WM and minimal involvement in the CX, (f) with abundant neutral lipids in the macrophages infiltrating the hemispheric WM (arrows, Oil Red O staining). (g) Section of the cerebral peduncle stained with H&E-LFB showing almost no myelin staining and heavy infiltration of foamy macrophages (black arrows), with one containing LFB-positive cytoplasmic material, consistent with myelin-debris (green arrow). (h) Electron microscopy analysis of post-mortem WM tissue. The single arrow shows a normal mitochondrion whereas the double arrow points to an enlarged, globular mitochondrion with concentrically arranged cristae (25 000X)

DISCUSSION

Two recent publications have reported a total of 6 variants in NRROS associated with early-onset progressive neurodegenerative disease in 9 individuals (OMIM:615322, PMIM:618875).^6,7 Previously reported variants include one missense, one nonsense and four deletions causing either an elongated or truncated protein.^6,7 The individual here showed clinical and neuroradiological overlap with previous cases (Table 1).

Table 1:

Molecular, clinical and neuroradiological features of individuals with NRROS variants

	Dong et al 2020 6						Smith et al 2020 7			Current case
Variant / family info	c.1981delC c.1981delC (family 1)	c.1981delC c.1981delC (family 1)	c.1981delC c.1981delC (family 1)	c.1981delC c.1981delC (family 2)	c.1644delG c.1644delG (family 3)	c.190delC, c.29T>C (family 4)	c.1777C>T c..1777C>T (family 5)	c.1777C>T c.1777C>T (family 5)	c.1257del c.1257del (family 6)	c.185C>T c.310T>C (family 7)
Age of onset	7mo	10mo	9mo	Birth	12mo	6mo	6mo	18mo	14mo	8mo
Age of death	4yr 2mo	3yr 3mo	Alive at 20mo	26mo	Alive at 9yr 3mo	Alive at 4yr, 6mo	3yrs	29mo	27mo	3yr 10mo
Hypotonia (axial)	+	+	+	+	+	+	N.A.	N.A.	N.A.	+
Refractory seizures	+	+	+	+	+	+	+	+	+	+
Developmental Delay	+	+	−	+ (mild)	−	+ (mild)	−	−	−	−
Developmental regression	+	+	+	+	+	+	+	+	+	+
Cerebral atrophy	−	−	−	+	+	+	+	+	+	+
Reduced white matter	+	+	+	+	+	+	N.A.	N.A.	N.A.	+
Corpus callosum thinning	+	+	+	−	+	+	N.A.	N.A.	N.A.	+
Calcifications	N.A.	N.A.	+	N.A.	+	+	+	+	+	+
Head circumference (at time of death)	N.A.	N.A.	N.A.	N.A.	N.A.	N.A.	10th percentile	10th percentile	<1st percentile	<3rd percentile

Abbreviations: +, present; −, absent.

Functionally, NRROS is considered a crucial factor in the pathogenesis of neuroinflammation and microglia-mediated neurotoxicity [3, 7, 8]. Presumably, loss of NRROS function leads to excess oxidative stress and neuroinflammation, a situation intricately linked to neurodegeneration [9]. ROS has also been linked to epilepsy, as the increased levels of ROS following acute cerebral injuries are thought to underlie the greater susceptibility of these individuals to develop epilepsy [10]. Notably, Aicardi-Goutières Syndrome is an interferonopathy form of leukodystrophy in which intracranial calcifications are a cardinal feature, suggesting neuroinflammation could underlie the brain calcifications seen here [11].

NRROS is also required to activate TGF-Β1 in macrophages and microglia [2]. Biallelic loss-of-function variants in TGFB1 cause a phenotype akin to NRROS deficiency, including hypotonia, either global developmental delay or normal development followed by neurological regression, epilepsy (refractory in 1 individual), and neuroimaging findings of cortical atrophy, reduced myelination, and a thin corpus callosum [12]. TGF-Β1 in the CNS has been implicated in both microglia homeostasis and neuron survival [4, 13]. Accordingly, neonatal Tgfb1^−/− mice show a significant increase in apoptotic neurons and reactive microglia compared to littermate controls and in Tgfb1^−/+ mice, excitotoxic injury increased apoptotic neuron staining and coincided with prominent microgliosis. Overall, the human and mouse data support a non-redundant role for TGF-Β1 and a crucial role for NRROS through its unique ability to activate TGF-Β1.

Finally, the presence of an ultrastructure mitochondrial abnormality identified here is of interest as this has not been previously reported. Mitochondria structure and function are frequently altered in neurodegeneration [14]. While mitochondrial abnormalities can originate from a variety of genomic or environmental insults, oxidative stress is one factor known to interfere with mitochondria and could underlie the ultrastructure defect seen here [14]. However, previous studies on mitochondria and oxidative stress demonstrate ultrastructural changes that do not resemble the alterations seen in this case [15]. Further pathological examination and functional studies are necessary for a definitive conclusion about the role of NRROS in mitochondria homeostasis.

In summary, we report the clinical, neuroradiological, and pathological features of one case with two novel variants in NRROS and further support NRROS deficiency as associated with an ultra-rare form of severe pediatric neurodegeneration, brain calcifications and intractable epilepsy. Finally, we report the first case in which biallelic pathogenic NRROS variants are associated with a mitochondria ultrastructure abnormality.

Date availability statement

The data supporting this study are included within the report.