Clinical, Radiological, and Genetic Profile of Patients with FA2H-Associated Neurodegeneration: Eight Cases from India and a Review of the Literature

Clinical details

Walking difficulty (8/8) was the most common presenting symptom. Patients also experienced speech slurring (6/8), upper-limb clumsiness (3/8), cognitive symptoms (2/8), and mild motor global developmental delay (2/8). A positive family history of similar clinical findings and consanguineous parentage was noted in 1 and 6 patients, respectively.

On examination, all patients had lower-limb-predominant pyramidal involvement, including spasticity, hyperreflexia, and extensor plantar responses. In addition, cerebellar signs were present in five patients, all of whom had lower-limb and gait-predominant ataxia; four also had upper-limb and speech involvement, and three had eye involvement in the form of hypermetric saccades or nystagmus. Cognition was impaired in two patients, mild optic atrophy in two patients, oculomotor abnormalities in six patients (nystagmus: 3, broken pursuit: 2, upgaze restriction: 1, exotropia: 1), dysarthria in seven patients, foot weakness in one patient, and a normal sensory examination in all. Four patients were able to walk without support, three with support, and one was wheelchair dependent. Among the seven ambulant patients, spastic-ataxic gait was noted in five and pure spastic gait in two. Additionally, one patient had hand dystonia, two had pes cavus, and five had ankle contracture.

Electrophysiology and imaging

Electrophysiological evaluation revealed sensory axonal neuropathy (1/4, others 3 normal), prolonged p100 latency on visual evoked potential (VEP) (2/3, others were normal), normal brainstem auditory evoked potentials (BAER) (4/4), and normal lower limb somatosensory evoked potentials (SSEP) (5/5). MRI brain was available in all eight patients and showed callosal atrophy (8/8), cerebellar atrophy (8/8), periventricular white matter T2 and fluid attenuated inversion sequence (FLAIR) hyperintensity (8/8), hypointensity of globus pallidus interna (GPi) on T2/FLAIR (4/6), and optic atrophy (4/8) (Figure 1 and Table 1).

Genetic testing

FA2H has two isoforms, and the canonical isoform (NM_024306.5) encodes a 372-amino acid protein that has a Cytochrome b5 heme-binding domain (8–86), a transmembrane domain (168–188), and a fatty acid hydroxylase domain (219–361) (Figure 2). Exome sequencing was performed in all eight patients, revealing seven unique pathogenic or likely pathogenic variants in FA2H, all in a homozygous state. None of the patients carried any additional homozygous variant relevant to the phenotype. Of the seven variants, two variants have been previously described (c.703C>T;p.Arg235Cys and c.379C>T;p.Arg127Ter) (4), and the other five variants were novel (c.200_202del;p.His67del, c.130C>A;p.Pro44Thr, c.536delT;p.Leu179ArgfsTer62, c.83G>C;p.Arg28Pro, c.86_97dup;p.Arg29_Arg32dup) (Figure 2). Missense variants were the most frequent (3 variants). In addition, there was one variant each of in-frame deletion (c.200_202del;p.His67del), in-frame duplication (c.86_97dup;p.Arg29_Arg32dup), stop-gain (c.379C>T;p.Arg127Ter), and frameshift deletion (c.536delT;p.Leu179ArgfsTer62). One novel variant (c.200_202del;p.His67del) was seen in two unrelated patients.

Figure 2

Patient-1 and Patient-7, both born to consanguineous parents, were identified with a 3-bp deletion within exon 1 of the FA2H (NC_000016.10:g.74774554_74774556del;c.200_202del;p.His67del), leading to an in-frame deletion of the histidine amino acid at the 67^th position. The variant is rare, observed in 0.0007% in gnomAD v4.0 Genomes and 0.001% in the in-house database, with no homozygous variants reported. The deleted sequence TGT is highly conserved, with a PhyloP100 score of 7.07 for this variant. The deletion of histidine at the 67^th codon is expected to shorten the Cyt-b5 heme-binding domain, thereby altering its structure. The Sanger sequencing in Case-1 showed this variant as homozygous in the patient and heterozygous in the unaffected parents. Sanger sequencing could not be performed in Patient-7.

Patient-2, born to consanguineous parents, was identified with a homozygous missense variant in exon-1 of the FA2H (NC_000016.10:g.74774626G>T;c.130C>A;p.Pro44Thr). The variant is rare, observed in 0.00007% in gnomAD v4.0 Exomes and not reported in the in-house database, with no homozygous variants reported. The PhyloP conservation score for this variant is 5.65; SIFT and Polyphen2 scores predict it as deleterious. The variant has a Combined Annotation Dependent Depletion (CADD) score of 32 and a Rare Exome Variant Ensemble Learner (REVEL) score of 0.9. It is located adjacent to the heme binding residue His43. Previously, a cytosine-to-thymine variant resulting in the same p.Pro44Ser is reported to be associated with FA2H-related HSP conditions in Turkish and Czech patients [6, 14]. Sanger sequencing confirmed the variant as homozygous in the patient, heterozygous in the unaffected parents, and absent in the unaffected sibling.

Patient-3, born to consanguineous parents, was identified with a novel homozygous 1-bp deletion in the exon 4 of the FA2H (NC_000016.10:g.74726302del;c.536delT;p Leu179Argfs*62) that predictively results in a frameshift and premature truncation of the protein. The variant is rare, observed in 0.00007% in gnomAD v4.0 Exomes and not reported in the in-house database, with no homozygous variants reported. The resulting frameshift introduces a premature termination codon 62 codon downstream of 179 codon, is located in exon 4 of 7, is neither in the last exon nor near the start codon, and is positioned more than 50–55 nucleotides upstream of the final exon–exon junction. Accordingly, it does not meet established nonsense-mediated decay escape criteria, and in-silico tools (aenmd and MutationTaster) predict nonsense-mediated mRNA decay. The premature truncation of the protein results in the loss of transmembrane domains and zinc-binding sites. Sanger sequencing revealed that this variant was homozygous in the patient and heterozygous in the mother. The father’s sample was not available for the Sanger sequencing.

Patient-4, born to consanguineous parents, was identified with a novel homozygous missense variant in exon-1 of the FA2H (NC_000016.10:g.74774673C>G;c.83G>C;p.Arg28Pro). This variant is neither reported in the gnomAD database nor in the in-house database. The variant is predicted to be deleterious by Polyphen and MutationTaster and is located in the Cytochrome b5 heme-binding domain. The variant has a CADD score of 30 and a REVEL score of 0.66. Sanger sequencing confirmed that this variant was homozygous in the patient and heterozygous in the mother; the father’s sample was unavailable for segregation analysis (Figure 2).

Patient-5, born to consanguineous parents, was identified with a homozygous nonsense variant in the exon-3 of the FA2H (NC_000016.10:g.74727371G>A;c.379C>T;p.Arg127*). The variant is rare, observed in 0.0006% in gnomAD v4.0 and not reported in the in-house database, with no homozygous variants reported. It is reported as pathogenic in ClinVar (rs1210045384) and causes a stop-gain mutation, resulting in a premature protein truncation and loss of transmembrane domains and zinc-binding sites. The resulting premature termination codon is located in exon 3 of 7, is neither in the last exon nor near the start codon, and is positioned more than 50–55 nucleotides upstream of the final exon–exon junction. Accordingly, it does not meet established nonsense-mediated decay escape criteria, and in-silico tools (aenmd and MutationTaster) predict nonsense-mediated mRNA decay. Sanger sequencing showed this variant as homozygous in the patient and heterozygous in the unaffected parents.

Patient-6, born to non-consanguineous parents, was identified with a homozygous missense variant in exon 5 of the FA2H (NC_000016.10:g.74719071G>A;c.703C>T;p.Arg235Cys). The variant is rare, observed in 0.0009% in gnomAD with no homozygous variants reported. It is reported as pathogenic in ClinVar (rs387907039) and has been previously reported [4]. The PhyloP conservation score for this variant is 8.79; SIFT and Polyphen2 scores predict it as deleterious, has a CADD score of 32 and a REVEL score of 0.85. Although the parents were reportedly unrelated, the possibility of distant unreported consanguinity cannot be excluded, given the patient’s homozygosity for the variant and a positive family history in the brother. We could not perform Sanger sequencing in the parents and the brother.

Patient-8, born to non-consanguineous parents, was identified with a homozygous in-frame duplication variant in the exon 1 of the FA2H (NC_000016.10:g.74774658A>AGGCGGGCCCCGC;c.86_97dup;p.Arg29_Arg32dup). The variant is rare, observed in 0.0007% in gnomAD v4.0 Genomes, in a heterozygous state, and absent in the in-house database. It is expected to lengthen the protein in the Cyt-b5 heme-binding domain, thereby altering the protein’s structure. Sanger sequencing confirmed the variant as homozygous in the patient and heterozygous in the unaffected parents. Although the parents were reportedly unrelated, the possibility of unreported consanguinity cannot be excluded, given that the variant is homozygous in the patient.

Review of literature

On searching PubMed with the keyword “FA2H”, we identified a total of 209 articles as of 12-05-2025. Among them, 43 articles described patients with FAHN, including clinical, radiological, and/or genetic details. Two reports duplicated the same patient [15, 16]. Hence, we were left with 42 articles comprising 130 patients. Including the current study, there are a total of 138 cases.

Clinical features

As per the available literature, the majority of patients have a first-decade onset (Supplementary Table-1) [8]. Global developmental delay has been reported, but is observed in a minority of patients. Conversely, cognitive impairment appears to be common, with severity ranging from mild to severe impairment. Additional psychiatric and behavioural abnormalities have also been reported rarely. Eye abnormalities, including optic atrophy, strabismus, ophthalmoplegia, nystagmus, saccadic abnormalities, and oculomotor apraxia, have been reported in multiple cases. However, owing to missing data in the majority, the actual prevalence of these in FAHN is difficult to comment upon. Dysarthria is almost universal, but its character is not reported uniformly; it appears spastic in the majority. Additional dysphagia was observed in a minority of cases. There were no reports of any pseudobulbar effect.

Based on the literature review, pyramidal signs appear to be a sine qua non feature in patients with FAHN. It can be in the form of spasticity, brisk deep tendon reflexes, and/or extensor plantar response. The spasticity and hyperreflexia are often present predominantly in the lower limb, and hence, FAHN is classified under the SPG group as SPG35 [4]. In addition to pyramidal signs, additional movement disorder phenomenologies are not infrequent, with ataxia being the most common, followed by dystonia. Both ataxia and dystonia tend to be lower-limb predominant and symmetrical. Rigidity, bradykinesia, rest tremor, and myoclonus have also been rarely reported [8].

Imaging

Radiologically, all patients had callosal and cerebellar atrophy and periventricular white matter hyperintensity, while mineralization was demonstrated in only half of the cases (Supplementary Table-2) [8, 18]. In a separate investigation conducted in India, Sait et al. documented eight cases of FAHN, all of which manifested during childhood, with an average onset age of five years. Consistent with our observations, the MRI findings in all these patients showed periventricular white matter involvement in the bilateral parietal regions. Additionally, varying extents of brainstem and cerebellar atrophy, along with corpus callosum thinning, were consistently observed across all individuals. However, only one patient in their cohort showed iron accumulation in the brain, whereas iron accumulation is observed in half of our cases [19].

Electrophysiology

Electrophysiologically, motor evoked conduction time appears to be the most sensitive test, with three-fourths of the tested showing abnormality, followed by visual pathway impairment in more than half of the patients. Peripheral nerve abnormality is observed in approximately one-fourth of cases, with all cases showing sensory impairment, with or without motor abnormality. The electrophysiological findings are in line with those observed in cases of HSP except for a prolonged VEP. Visual pathway abnormalities, including a pale optic disc, MRI optic nerve atrophy and hyperintensity, and prolonged or absent VEPs, may help differentiate HSP due to FA2H from other HSPs.

Genetic details

Genetic analysis of all eight families identified three missense variants (c.130C>A; p.Pro44Thr, c.83G>C;.Arg28Pro, and c.703C>T; p.Arg235Cys), one in frame deletion (c.200_202del; p.His67del), one frameshift variant (c.536delT;p.Leu179Argfs*62), one stop-gain variant (c.379C>T;p.Arg127Ter) and one in-frame duplication variant (c.86_97dup; p.Arg29_Arg32dup). Variants c.200_202del, c.130C>A, c.83G>C, and c.86_97dup identified in Case-1, Case-7, Case-2, Case-4, and Case-8 are situated within exon 1, which encompasses the cytochrome b5-heme binding domain responsible for intramolecular electron transport during catalytic activity [2]. The variant c.200_202del exhibits an in-frame deletion of a histidine residue at the 67^th position, which is directly associated with the heme group. This variant was identified in two unrelated patients in our study. Case 2 had a novel missense variant c.130C>A; p.Pro44Thr. Previously, a variant with a different nucleotide change at the same loci (c.130C>T) that results in a different amino acid substitution at the same codon (p.Pro44Ser) has been reported in a Turkish and a Czech male associated with the disease conditions [6, 14]. Additionally, another variant, c.131C>A, which results in an amino acid substitution at codon 44 (p.Pro44Gln), has also been documented as disease-causing, and, interestingly, it was reported to be caused by uniparental disomy [20]. These variants are located adjacent to the heme-binding site at codon 43, which encodes histidine. The novel missense variant c.83G>C; p.Arg28Pro that substitutes the conserved arginine residue at the 28^th position and the in-frame duplication variant c.86_97dup; p.Arg29_Arg32dup that extends the protein by 4 amino acids are both situated within the cytochrome b5-heme binding region. These variants are predicted to affect the function of the protein’s cytochrome b5 domain. The importance of the heme-binding domain is supported by a comparative lipidomic analysis of the patient and control groups, which reveals notable differences in lipid metabolites resulting from the deletion of the heme-binding domain of FA2H. In our study, three out of five variants lie in the first exon, specifically in the Cytochrome b5-heme binding domain, which is important for the catalytic activity of the FA2H enzyme [2, 12].

Case 3 and Case 5 present with truncating variants c.536delT; p.Leu179ArgfsTer62 and c.379C>T; p.Arg127Ter, respectively. Deletion of T at position 536 is anticipated to induce a frameshift variation in the codon, resulting in premature termination of the protein with only 241 amino acids, consequently leading to the loss of the fatty acid hydroxylase domain. In Case 5, the variant c. 379C>T introduces a premature stop codon UGA at 127^th position, resulting in the loss of the hydroxylase domain. In Case 6, a previously known variant was identified at c.703C>T, resulting in an amino acid substitution at the 235^th position. This position lies between the histidine-containing zinc binding sites 234 and 239 within the fatty acid hydroxylase domain. The myelin sheath is crucial for the transmission of electrical signals in both the central and peripheral nervous systems. FA2H is the sole enzyme identified in eukaryotes capable of hydroxylating linear fatty acids at the C-2 position, a step important for myelin synthesis. Variants in FA2H are associated with hereditary leukodystrophy and spastic paraparesis in humans, underscoring its important role in the nervous system [2, 21]. Alterations in these amino acids caused by the reported variants are predicted to impair catalytic activity, leading to disruptions in lipid metabolism and potentially contributing to the development of neurological disorders such as FAHN.

FAHN is a relatively rapidly progressive SPG type with loss of ambulation noted over 5–10 years of disease progression. Therapeutically, symptomatic treatment and rehabilitative measures remain the mainstay, with no studies on disease-modifying therapy. The chelation therapy tried in PKAN has not been studied in FAHN patients. Symptomatic therapy in the form of baclofen or other anti-spastic medications for pyramidal signs, botulinum toxin injections for spasticity, regular physiotherapy and monitoring for visual dysfunction is advised for patients with FAHN. Genetic counselling is important to make the parents aware of the risk of the disease in the subsequent pregnancy and options to prevent it.

This study has several limitations. Owing to its retrospective design, complete investigative data were not available for all patients. Sanger sequencing to confirm segregation of the identified variants could not be performed in some parents and extended family members. Additionally, functional studies to validate the pathogenicity of the novel variants identified in our cohort could not be undertaken.

In conclusion, we present the clinical and genetic profiles of eight patients from unrelated families, and report five novel variants in FA2H. FAHN should be considered in the first decade onset of chronic progressive SPG syndromes, either isolated or with associated ataxia, optic atrophy, neuropathy, dystonia and seizures. Although classified as an NBIA, iron deposition is not universal. Our results highlight the importance of genetic and radiological investigations in the diagnosis and management of FAHN. Leukodystrophy in young children without any detectable biochemical abnormality should be considered for FAHN diagnosis.