Infant With a Severe Form of GLRX5 -Related Atypical Hyperglycinemia Exhibiting Novel Cardiac and Neurologic Disease Manifestations at Autopsy

Abstract

Glutaredoxin 5 (GLRX5) is a mitochondrial protein encoded by the GLRX5 gene, which is essential for cellular redox homoeostasis, lipoic acid synthesis, and iron-sulfur cluster transfer. Rare cases of pathogenic GLRX5 mutations have been associated with sideroblastic anemia and non-ketotic hyperglycinemia with progressive spasticity and cavitating leukoencephalopathy. We report an 11-month-old child, who died following aspiration, with severe cardiomyocyte mitochondrial abnormalities and cerebral white matter degeneration in the context of a homozygous GLRX5 variant (c.208A>G, p.S70G).

Introduction

Glutaredoxin 5 (GLRX5) is a mitochondrial protein encoded by GLRX5, which is essential for cellular redox homoeostasis and lipoic acid synthesis. ¹ GLRX5, along with iron-sulfur cluster scaffold (NFU1), and bola-family-member-3 (BOLA3), are responsible for providing iron-sulfur clusters to a number of enzymes including lipoate synthase (LIAS), which is an essential cofactor for mitochondrial metabolism.^1-4

Pathogenic GLRX5 mutations were initially independently associated with isolated sideroblastic anemia^5-8 and an atypical form of non-ketotic hyperglycinemia with progressive spasticity and leukoencephalopathy but relatively preserved cognition.^2,9-14 More recently, there have been reports of more severe neurologic phenotypes in patients with GLRX5 mutation^15-17 similar to those reported in patients with LIAS, BOLA3, and NFU1 mutations.^2-3,18 We report an 11-month-old child with neurodegeneration, complex biochemical disturbances, and novel cardiomyocyte abnormalities in the context of a new homozygous GLRX5 missense variant.

Case Report

Clinical Course

This 11-month-old female of non-consanguineous Dutch-German Mennonite descent was born at term with laryngomalacia and features of Pierre-Robin sequence. This prompted a chromosomal microarray, which was unremarkable apart from a couple regions of homozygosity consistent with her parents being from a founder population. She underwent a bilateral distraction osteogenesis procedure at 2 months. Postoperatively she acutely decompensated and developed new-onset seizure activity. Physical examination demonstrated hypotonia and increased lower limb reflexes. Magnetic resonance imaging (MRI) of the brain at 2 and 4 months showed abnormal T2 signal and diffuse restricted diffusion affecting most of the white matter as well as the deep nuclei leading to a suspected diagnosis of leukoencephalopathy/leukodystrophy. Metabolic studies at that time demonstrated non-ketotic hyperglycinemia (NKH), with a plasma glycine level of 1335 µmol/L, urine glycine >13,000 µmol/L, and CSF glycine 42 µmol/L (CSF-to-plasma ratio = 0.036) prompting initiation of sodium benzoate therapy. A follow-up electroencephalogram at 9-months exhibited no epileptiform phenomenon.

By 10-months, she was experiencing progressive oral and nasogastric feeding intolerance with suspected cortical blindness, severe motor delay, and progressive spasticity. MRI showed persistent abnormality in the white matter with only mild enlargement of the lateral ventricles (Figure 1). She exhibited nystagmus, a lack of object following or fixation, poor head control, minimal hand use, and a lack of sitting or reaching. Interestingly, serology showed a new compensated lactic acidosis with pyruvate dehydrogenase and urine metabolite testing supportive of mitochondrial dysfunction. Her glycine levels were persistently elevated, although still responsive to sodium benzoate. Sequencing and deletion/duplication for AMT, GLDC, and GCSH was negative, which combined with the relatively low glycine CSF-to-plasma ratio, argued against a diagnosis of classic NKH.

Figure 1.

(A) Magnetic resonance imaging of the brain at 10 months age showed abnormal T2 signal and diffusion restriction ([B] diffusion weighted image; [C] apparent diffusion coefficient) in the deep and subcortical white matter of frontal and parietal lobes, centrum semiovale, and internal and external capsules. (D) At 11 months age (2 hours after cardiac arrest and resuscitation) CT scan also showed abnormal signal throughout the cerebral white matter, with only mild volume loss.

Trio exome sequencing was then performed, for which the proband was found to be homozygous for a novel pathogenic GLRX5 missense variant (c.208A>G, p.S70G) with both parents being heterozygous. Shortly after the sequencing was reported, the patient was admitted for clinical deterioration with marked spasticity, feeding intolerance, irritability, and continuing lactic acidosis. Following initial stabilization, she suffered cardiac arrest. CT scan of the head showed severe hypoattenuation in the cerebral white matter, which could not be accounted for by acute hypoxic-ischemic damage. She died a few hours later at 11-months of age.

Post-Mortem Examination

At autopsy, gross and microscopic examination of the lungs demonstrated changes in keeping with a significant aspiration event, which was the most likely immediate cause of death.

The heart was enlarged (87.3 g; >95th percentile for weight), with concentric left ventricular hypertrophy. Numerous diffusely-scattered cardiomyocytes contained areas of PAS- and iron-negative cytoplasmic clearing (Figure 2). Electron microscopy of affected cardiomyocytes showed intact myofibrils, pleomorphic mitochondria with predominantly tubular cristae, and a mixture of membrane-bound vacuoles and non-membrane-bound areas of cytoplasmic clearing. No hepatic abnormality was identified apart from congestion.

Figure 2.

(A) Light micrograph of cardiac muscle showing scattered cardiomyocytes with cytoplasmic clearing (arrows). (B) Electron micrograph of cardiac muscle showing pleomorphic mitochondria surrounding an area of cytoplasmic clearing most likely representing glycogen.

Gross examination of the brain demonstrated an age-appropriate gyral pattern (weight 762 g; >95th percentile for age). Coronal slices through the hemispheres showed discoloration and softening of periventricular white matter with sparing of myelinated subcortical white matter. There was only minimal volume loss with slight enlargement of temporal horns of the lateral ventricles; antemortem hypoxia might have caused some brain swelling.

Light microscopic examination of the brain showed early hypoxic-ischemic neuronal damage in the neocortex, hippocampus, and cerebellum. The basal nuclei and thalami had no obvious abnormalities. In all lobes, the subcortical U-fibers extending 0.5–1 mm from the neocortex had normal myelin deposition on solochrome cyanin (+ eosin) staining and normal axon density on neurofilament immunostain. There was near-complete absence of myelin in deep white matter of the frontal and parietal lobes. Within the most severely abnormal regions, the white matter exhibited hypertrophic reactive astrocytes (immunoreactive for GFAP and alpha B crystallin), globose microglia/macrophages (immunoreactive for CD68 and HLA-DR), rare scattered oligodendrocytes (SOX10 immunoreactive nuclei), myelin debris in macrophages, a moderately reduced axon population (neurofilament immunoreactive), and scattered, bundant swollen/damaged axons (APP immunoreactive; Figure 3). These regions had a hypervascular appearance, with abundant small venous channels. Less affected regions included subcortical frontal regions, subcortical temporal regions, internal capsule, and white matter tracts within the basal nuclei. However, even seemingly normal internal capsule contained activated microglia. Long axon tracts including corticospinal tract in the brainstem and spinal cord, and posterior columns in the spinal cord exhibited vacuoles and diminished myelin staining. No aberrant hyperphosphorylated-tau or ubiquitin immunostaining was detected in any region.

Figure 3.

(A) Coronal slice of the left parietal lobe showing abnormal dark red discoloration of the deep (periventricular) white matter and normal myelin in the subcortical white matter. (B) Section of the right lateral occipital lobe stained with modified Bielschowsky shows dark staining (abundant axons) in the subcortical white matter and pallor in the deep white matter. (C) Solochrome cyanin shows absence of myelin staining in the deep white matter. (D) Immunostaining with anti-HLA-DR shows abundant reactive microglia and macrophages in the deep white matter. (E) Electron micrograph of internal capsule shows large reactive astroglial cells containing abundant mitochondria and fine granular cytoplasm between myelinated axons.

Electron microscopy of abnormal deep cerebral white matter showed hypocellular tissue with normal blood vessels, scattered macrophages, swollen/damaged axons, many small unmyelinated axons, and rare myelinated axons. Myelin lamellae were normal. Adjacent to the severely damaged tissue, in the midst of myelinated axons were scattered large glial cells that corresponded in shape to the unusual reactive astrocytes with clumped chromatin, abundant perinuclear mitochondria, and distended cytosol with uniformly-fine granular material and negligible intermediate filaments (Figure 3). Oligodendrocytes (associated with the myelin) had watery cytoplasm and clumped chromatin; these are common artifacts in autopsy tissue. Endothelial cells exhibited no obvious abnormalities. Accommodating for post-mortem changes, no abnormal mitochondria were seen.

No skeletal muscle abnormality was appreciated on light or electron microscopy.

Discussion

This is the first reported autopsy case of a child with bialleleic GLRX5 mutation and both severe neurologic and cardiac manifestations. The case highlights multiple roles of GLRX5 in glycine cleavage, iron-sulfur cluster transfer, and oxidative phosphorylation.

Our patient’s severe neurologic phenotype is characterized by what we postulate to be an oligodendrogliopathy accompanied by axonal damage preferentially affecting long axon tracts. Her non-ketotic hyperglycinemia and branched chain abnormalities are most likely due to ineffective lipoic acid synthesis and resultant glycine cleavage system defect. The exact cause of her severe white matter degeneration is unclear, but some data suggest a role for lipoic acid in iron-sulfur cluster transfer and oxidative phosphorylation. In the central nervous system, oligodendrocytes are the most reliant cell on iron-sulfur cluster transfer, which they use for myelin production. ¹⁹ However, uncertainty is raised by data in the Human Protein Atlas, ²⁰ which indicate that GLRX5 is expressed in neurons, astrocytes, and oligodendrocytes at comparable mRNA and protein levels. Since sodium benzoate therapy lowered plasma glycine levels without significantly improving the patient’s disease course, iron-sulfur cluster transfer and respiratory chain defects likely posed significant neurologic disease contributions. Lipoic acid may be a therapeutic consideration for future patients not responding to glycine scavenger treatment. Unfortunately, a lipoic acid trial was not initiated in our patient due to abrupt decompensation.

While the neurologic manifestations were likely multifactorial, the diffuse cardiomyocyte abnormalities were likely the sole result of defective oxidative phosphorylation. The cardiomyocyte pseudo-vacuolization on ultrastructure examination was favoured to represent degenerating mitochondria due to the background of abundant pleomorphic mitochondria and their resemblance to nearby swollen mitochondria, although this cannot be proven definitively. There is some evidence that mitochondria with tubular cristae are less efficient at producing ATP than those with lamellar cristae. ²¹ Given the associated cardiomegaly and concentric left ventricular hypertrophy, the patient was predisposed to acute decompensation following the terminal aspiration event.

Our report highlights the pleiotropic effects that can occur in patients with biallelic pathogenic variation in GLXR5. Further work is needed to better define genotype-phenotype correlations and elucidate underlying pathophysiology. GLXR5 mutation should be considered in the differential for any severe early onset mitochondrial disorder.