Phenotypic continuum of NFU1 ‐related disorders

Abstract Bi‐allelic variants in Iron–Sulfur Cluster Scaffold (NFU1) have previously been associated with multiple mitochondrial dysfunctions syndrome 1 (MMDS1) characterized by early‐onset rapidly fatal leukoencephalopathy. We report 19 affected individuals from 10 independent families with ultra‐rare bi‐allelic NFU1 missense variants associated with a spectrum of early‐onset pure to complex hereditary spastic paraplegia (HSP) phenotype with a longer survival (16/19) on one end and neurodevelopmental delay with severe hypotonia (3/19) on the other. Reversible or irreversible neurological decompensation after a febrile illness was common in the cohort, and there were invariable white matter abnormalities on neuroimaging. The study suggests that MMDS1 and HSP could be the two ends of the NFU1‐related phenotypic continuum.


Introduction
Iron-sulfur [Fe-S] clusters are important cofactors that play a role in various cellular functions, including electron transfer along the respiratory chain, citric acid cycle, heme biosynthesis, DNA replication and repair, as well as iron homeostasis. [1][2][3] Iron-Sulfur Cluster Scaffold (NFU1) (MIM: 608100) is an [Fe-S] cluster biosynthesis factor involved in the last steps of maturation and transfer of [4Fe-4S] clusters into target proteins, including lipoic acid synthetase, mitochondrial aconitase and some subunits of respiratory chain complexes I and II. 4,5 Bi-allelic variants in NFU1 have previously been associated with multiple mitochondrial dysfunctions syndrome 1 (MMDS1) 6 characterized by early-onset leukoencephalopathy leading to a fatal outcome, typically before the age of 15 months. 6 Recently, bi-allelic variants in NFU1 have been reported in two individuals with a milder phenotype, presenting with slowly progressive spastic paraplegia with a relapsing-remitting course, long survival, and intact cognition or mild intellectual disability (ID). 7,8 Here, we report 19 affected individuals from 10 independent families with ultra-rare bi-allelic NFU1 missense variants associated with a phenotype ranging from early-onset pure to complex hereditary spastic paraplegia (HSP) characterized by a longer survival (16/19) and neurodevelopmental delay (NDD) with severe hypotonia (3/19).

Participants and clinical investigations
Exome sequencing (ES) and genome sequencing (GS), data sharing between international genetic centers, and the Gen-eMatcher platform 9 were employed to identify the 10 families reported here. Clinical data were collected via a uniform proforma. Parents/legal guardians of all affected individuals consented to the publication of genetic and clinical information. The study was approved by The Research Ethics Committee Institute of Neurology University College London (IoN UCL) (07/Q0512/26) and the local Ethics Committees of each participating center.

Exome sequencing and genome sequencing
Research/diagnostic solo or trio-ES/GS, variant filtering, and variant confirmation by Sanger sequencing and segregation analysis were performed on genomic DNA extracted from blood in different genetic laboratories following the methods previously described (Table 1). Family 10 had a mitochondrial panel test performed for the proband and targeted genotyping for familial variants for the sibling (further details in Table 1 and Supplementary  Table S1).

SDS-PAGE and western blot analysis
Human fibroblasts from an affected individual (F1-II:1) were obtained and lysed and samples were subjected to SDS-PAGE and western blotting as described previously 15 and a list of primary antibodies is provided (Supplementary Methods).
Protein modeling and characterization, Fe-S cluster transfer monitoring, and enzymatic assays Protein characterization by variable temperature circular dichroism (CD) and analytical ultracentrifugation, Fe-S cluster transfer monitored by circular dichroism, pyruvate dehydrogenase and a-ketoglutarate dehydrogenase activity assay studies were performed (Supplementary Methods).

Yes
No (2) Yes Yes
Prenatal features and neonatal periods were unremarkable in all subjects. No delay in early developmental milestones including head control, crawling, sitting, and speech was reported in 14/19 individuals. Cognitive function was impaired in 5/18 individuals. Gait acquisition was delayed due to either the insidious onset of limb spasticity in 13/19 individuals or spasticity precipitated by a deterioration in the context of a febrile illness in 4/19 individuals (Fig. 1B). The lower limb spasticity was detected at a mean age of 12 AE 6 months in the cohort. Spasticity was progressive leading to contractures in 13/19 persons and necessitating Achilles' tendon repair surgery in 4/19 affected individuals.
Remarkably, febrile illness leading to metabolic decompensation played a significant role in the disease course of 13/19 individuals. Thus, in F1-II:1 it led to febrile seizures followed by the onset of lower limb spasticity and reversible cognitive and motor regression, and truncal hypotonia. Similarly, F5-I:1, F6-II:3, and F8-II:5 developed reversible truncal hypotonia and/or progressive lower limb spasticity following several episodes of febrile illness. Intermittent and reversible gait deterioration associated with episodes of febrile illness was reported in F2-II:1and F9-II:1, along the course of their disease, which was described as waxing and waning. While the episodes of febrile illness had not been fatal for these persons, the other six affected individuals had regression and developed severe muscular hypotonia leading to early mortality after a febrile illness.
The mean disease duration at the most recent examination for the living individuals was 8.8 AE 5.7 years (range 1.5-24). Neuromuscular examination revealed lower limb spasticity in all persons with increased deep tendon reflexes, upgoing plantar reflexes, and mild symmetrical muscle wasting. Muscle tone from the upper limbs was uniformly intact. While two individuals were wheelchairbound, eight could ambulate using crouches or rollator walker, and three could walk independently with spastic gait. Ataxic gait was reported in 2/12 individuals, which was intermittent in one person (F2-II:1). The oldest individual in the cohort was aged 25 years and had a normal cognitive function and could perform most activities of daily living despite his slowly progressive lower limb spasticity. Seven persons had results available of metabolic screening, which were unremarkable in all.

Genetic analysis reveals ultra-rare bi-allelic variants in NFU1
A total of 9 different NFU1 missense variants were identified in this study ( Figs   conserved residues across different species (Fig. 2D) and segregated with the disease phenotype in all the families ( Fig. 2A). No other relevant variants associated with neurological or neurodevelopmental disorders were identified in the currently known monogenic disease-causing genes in the ES data. The characteristics of the variants are further summarized in Table 1 and Supplementary Table S1.

Steady-state levels of OXPHOS complexes I, II, III, and IV are decreased in homozygous p.(Val241Leu) fibroblasts
Western blot analysis on fibroblast samples showed that subunits of each complex of the electron transport chain (CI-CIV) were decreased in the affected individual F1-II:1 compared to the heterozygous mother (F1-I:1) and a pediatric control, whereas levels of complex V subunit ATP5A remained relatively unchanged (Fig. 2E).  Fig. S1C, Supplementary Results).
Additional discussion of the structural and functional biochemistry of other variants is provided in the Supplementary Material. As patient fibroblasts were available only for the p.(Val241Leu) variant, the impact of the variant on the enzymatic activities of PDH and KGDHC was measured only for this variant. Enzymatic activity assays in fibroblasts derived from F1-II:1, homozygous for p.(Val241Leu), showed decreased activity for PDH (0.50 nmol/mg protein/min; normal range 0.6-0.9 nmol/ mg protein/min), while the mean activity in the heterozygous mother was normal (0.82 nmol/mg protein/min; normal range 0.6-0.9 nmol/mg protein/min). The KGDHC activity was not impaired ( Supplementary  Fig. S2, Supplementary Results).

Discussion
The bioenergetic function of the mitochondrion is dependent on Fe-S cluster-containing proteins. Three distinctly organized biosynthetic pathways are involved in the maturation of Fe-S clusters in mammals with the last step involving NUBPL, NFU1, BOLA3, IBA57, ISCA2, and ISCA1. Bi-allelic pathogenic variants in five of them, starting from NFU1 in the above-mentioned list, are currently associated with MMDS types 1 to 5 respectively, and typically present with severe and fatal early onset encephalopathy with multiple biochemical abnormalities. 6 An atypical presentation of MMDS has been reported in several individuals, mainly encompassing a slightly milder disease course with a longer survival [17][18][19] for NFU1 and ISCA2, or a complex HSP phenotype for NFU1 and IBA57. 7,8,20 Our report expands the number of individuals with bi-allelic NFU1 variants presenting with complex HSP and also describes previously unreported pure HSP phenotype, thereby suggesting that MMDS1 and HSP could be the two ends of the NFU1-associated phenotypic continuum.
Pyramidal symptoms in the form of spastic tetraparesis have frequently been reported in individuals with typical MMDS1 presentation, highlighting the role of mitochondria and axonal transport in the corticospinal tract function. 6 Currently, due to a large overlap between HSP and other inherited neurological disorders, at least 28/81 genetic forms of HSP are assigned alternative phenotypes on the Online Mendelian Inheritance in Man (OMIM) database, resulting in a diagnostic challenge. 21 Therefore, delineating the full phenotypic spectrum of diseaseassociated genes and accurate clinical classification are important for diagnostic rates.
Another remarkable aspect of the present cohort is the presence of neurological decompensation after a febrile illness. Interestingly, three distinct responses to episodes of febrile illness were observed in our cohort: (1) fatal outcome after the first or consecutive episode in six individuals; (2) onset of spasticity with variably reversible motor and cognitive regression in three individuals; and (3) intermittent and fully reversible gait deterioration in four other individuals. The episodes of neurological deterioration, most likely caused by acute illness-induced metabolic decompensation, is common in mitochondrial diseases 22 but have rarely been reported in the mitochondrial causes of HSP. 23,24 A remarkable inter-and intrafamilial phenotypic variability was observed in the present cohort and the same amino acid substitutions of the NFU1 protein have been observed in both MMDS1 and HSP cases (Supplementary discussion for details). The relatively milder course and phenotypic variability of the most commonly reported MMDS1-associated  8 These include white matter hyperintensity, which was present in most of the individuals in the present series, cystic degeneration and cavitation in the frontal regions, which was noted in individual F10-II:2, and areas of restricted diffusion involving the bilateral subcortical and periventricular white matter, noted in three out of four individuals with available diffusion-weighted imaging.
Apart from the possible epigenetic factors, 7,8 we suspect that the variants observed in the present study might be causing milder defects for the NFU1 structure and function compared to the MMDS1-linked variants. The different variants might affect, for example, partner protein binding in a distinct way leading to high phenotypic variability connected to NFU1 variants (Supplementary Discussion for further details). The residual PDH activity and the normal KGDHC activity in F1-II:1 might be consistent with a less severe form of the disease with prolonged survival. The decreased OXPHOS protein levels and the only slightly affected PDH and normal KGDHC activity results in F1-II:1 fibroblasts might be explained by the changed affinity of the mutated NFU1 for the different partner proteins (Supplementary Discussion for further details). The disparity between plasma and CSF metabolites levels has been shown in an individual with MMDS, suggesting the manifestation of biallelic NFU1 variants with tissue-specific phenotypes. 25 In summary, we report 16 affected individuals with an HSP presentation of bi-allelic NFU1 variants and 3 affected individuals with GDD, hypotonia, longer survival, and fatal response to metabolic decompensation, which contrasts with the typical MMDS1 presentation of NFU1 deficiency and highlights the NFU1-associated disease continuum. Figure 2. Overview of the genetic and biochemical characteristics of NFU1 variants. (A) Pedigrees and segregation results of the ten families included in this study. (B) NFU1 gene structure with the localization of all previously known mutations (black, above) and mutations reported in this cohort (colored, below). The corresponding amino acid changes is shown in (C) with known protein domains in NFU1 (NifU N-terminal domain, residue 59 to 155, and NifU C-terminal domain, residue 162 to 247) depicted in the illustration. The variant, p.(Val241Leu), was the only mutation discovered in this study to have been located outside of the NFU1 protein domains. Regions are not drawn to scales and both illustrations were created from the program. 16