Leukoencephalopathy with ataxia (LKPAT) is a rare autosomal recessive disorder clinically characterized by atypical neurological features including mild ataxia, cognitive impairment, psychiatric symptoms, headaches, and spasticity, along with a variable clinical presentation, which may include mild visual impairment, auditory abnormalities, and male infertility. However, specific findings on brain MRI have been recognized as symmetrical white matter signal abnormalities in the posterior limbs of the internal capsules, midbrain cerebral peduncles, and middle cerebellar peduncles. LKPAT, also known as CLCN2-related leukoencephalopathy, is associated with pathogenic variants in the CLCN2 gene encoding ClC-2, a ubiquitously expressed chloride channel protein involved in the regulation of brain ion and water homeostasis (Jentsch and Pusch, 2018). To date, only 31 pathogenic variants of CLCN2 have been identified in 48 affected individuals from 45 unrelated families worldwide (Depienne et al., 2013; Di Bella et al., 2014; Hanagasi et al., 2015; Giorgio et al., 2017; Zeydan et al., 2017; Hoshi et al., 2019; Guo et al., 2019; Ozaki et al., 2020; Parayil Sankaran et al., 2020; Ngo et al., 2020; Shi et al., 2021; Ben Mohamed et al., 2022; Abreu et al., 2023; Almasoudi et al., 2023; Holla et al., 2023; Wu et al., 2023; Cheng et al., 2023; Nóbrega et al., 2024; Garg et al., 2024; Zhou et al., 2025; Ohira et al., 2024; Orimo et al., 2025; Bavdhankar et al., 2025). In this study, we report a novel biallelic missense variant in CLCN2 in an individual with leukoencephalopathy and infertility and conduct in silico analysis to identify the effect of this variant on the ClC-2 protein structure. Furthermore, by including data from previously published CLCN2-related cases, we present a cumulative and comprehensive clinical characterization and describe the genotype–phenotype features of CLCN2-related diseases.

LKPAT was first reported in six unrelated patients who presented with variable clinical features and harbored biallelic CLCN2 variants (Depienne et al., 2013). So far, only 49 individuals with LKPAT, including our patient, have been described in detail (Depienne et al., 2013; Di Bella et al., 2014; Hanagasi et al., 2015; Giorgio et al., 2017; Zeydan et al., 2017; Hoshi et al., 2019; Guo et al., 2019; Ozaki et al., 2020; Parayil Sankaran et al., 2020; Ngo et al., 2020; Shi et al., 2021; Ben Mohamed et al., 2022; Abreu et al., 2023; Almasoudi et al., 2023; Holla et al., 2023; Wu et al., 2023; Cheng et al., 2023; Nóbrega et al., 2024; Garg et al., 2024; Zhou et al., 2025; Ohira et al., 2024; Orimo et al., 2025; Bavdhankar et al., 2025). The small number of known affected individuals may be attributed to high phenotypic variability and a mild clinical course, which may result in delayed diagnosis. Understanding the clinical characteristics and genotype–phenotype correlation is helpful for the diagnosis, genetic counseling, and development of therapeutics for LKPAT.

In this study, we reviewed the clinical and genetic features of all affected individuals reported from 2013 to date. We observed that LKPAT patients presented with a broad range of ages of onset, and adult onset was more common. Although LKPAT patients exhibited heterogeneous clinical manifestations, our literature review indicates that the majority of patients presented with ataxia. Other frequent features, including visual impairment, auditory deficits, headache, and cognitive dysfunction, were observed. Notably, most male individuals were confirmed to be infertile or failed to reproduce. Therefore, visual and auditory impairments, along with male infertility, may serve as distinctive clinical indicators of LKPAT. In this study, our patient showed atypical neurological features, including dizziness, monoparesis, and mild cerebellar ataxia. He denied any visual complaints, while the VEP test results were abnormal. In particular, he had a history of azoospermia. These characteristics were suggestive findings of LKPAT and prompted evaluation by brain MRI. In previous research studies, all cases with LKPAT showed a typical imaging appearance on brain MRI that included abnormally symmetrical and confluent hypointense signals on T1-weighted images and hyperintense signals on T2-weighted images, particularly in the posterior limbs of the internal capsules, midbrain cerebral peduncles, and middle cerebellar peduncles (Depienne et al., 2013; Guo et al., 2019). Lesions can also be observed in the central tegmental tracts, pyramidal tracts in the pons, cerebral white matter, corpus callosum, and cerebellar white matter (Nóbrega et al., 2024). LKPAT patients showed DWI hyperintensities, with or without diffusion restriction, in the affected white matter. The ADC values are associated with the size of myelin vacuoles and extracellular spaces (Depienne et al., 2013; van der Voorn et al., 2006). In our patient, the increased ADC values may be due to large vacuoles and expanded intra-myelinic water spaces in the lesions. Notably, although symmetric lesions were detected through brain MRI, some LKPAT cases, including our patient, presented with unilateral symptoms, manifesting as left-sided limb weakness, right-sided numbness, spastic paralysis of the right arm, or mild ataxia in the left upper extremity (Zeydan et al., 2017; Almasoudi et al., 2023). Accumulating pieces of evidence have indicated that the MRI changes precede the onset of neurological symptoms in LKPAT, accompanied by an extremely slow disease progression (Di Bella et al., 2014; Giorgio et al., 2017; Guo et al., 2019). As the disease progresses, patients may subsequently develop corresponding symptoms in the contralateral limbs (Almasoudi et al., 2023). In our study, the patient’s clinical manifestations may partially be attributed to the early disease stage, and bilateral weakness is likely to manifest with disease progression, for which long-term follow-up is required.

The specific phenotype (for example, ataxia, visual impairment, and male infertility) combined with highly typical imaging abnormalities may be a strong diagnostic clue of LKPAT. Notably, it is important to consider FXTAS and NIID as differential diagnoses due to the overlapping phenotypes with LKPAT (Jacquemont et al., 2003; Sone et al., 2019). FXTAS is an X-linked neurodegenerative disorder caused by short CGG expansion (55–200 repeats) in the UTR of the FMR1 gene (Jacquemont et al., 2003; Verkerk et al., 1991). Similar to LKPAT, the core clinical manifestations of FXTAS are intention tremor and cerebellar ataxia (Jacquemont et al., 2003). However, FXTAS is a late-onset disorder usually presenting at the mean age of 62 and commonly manifests with executive dysfunction, Parkinsonism, and neuropathy (Cabal-Herrera et al., 2020). Although brain MRI of FXTAS typically shows symmetrical white matter abnormalities with hyperintense signals on T2-weighted images in the middle cerebellar peduncles, no signal abnormalities have been reported in the posterior limbs of the internal capsules and midbrain cerebral peduncles (Cohen et al., 2006; Filley et al., 2015). Moreover, generalized brain atrophy, corpus callosum thinning, and ventricular enlargement are also present in patients with FXTAS, which are not observed in LKPAT cases (Cohen et al., 2006; Filley et al., 2015; Hashimoto et al., 2011). Thus, FXTAS can be differentiated from LKPAT by these features. NIID is a rare neurodegenerative disease caused by expanded GGC repeats in the NOTCH2NLC gene (Sone et al., 2019). NIID and LKPAT share overlapping clinical features, including ataxia, cognitive impairment, muscle weakness, and seizures, which cause diagnostic confusion in the early stages (Sone et al., 2016). However, NIID typically shows bilateral hyperintensity at the corticomedullary junction on DWI images, a feature that is absent in LKPAT (Nóbrega et al., 2024; Garg et al., 2024; Sone et al., 2016; Yu et al., 2019). In this study, the results of FMR1 and NOTCH2NLC gene analysis in our patient showed normal repeat expansions. Based on the patient’s clinical features and MRI findings, the diagnoses of FXTAS and NIID can both be excluded.

The definitive diagnosis of LKPAT is confirmed by the identification of biallelic pathogenic variants in CLCN2 through molecular genetic testing. The ClC-2 protein is expressed in the plasma membrane, where it transports chloride ions and performs critical functions in cellular homeostasis (Thiemann et al., 1992; Wang et al., 2017). Each subunit of the ClC-2 homodimer is composed of 18 α-helices (named helix A-R) and 2 CBS domains (Figure 4A). Helix B–R within the transmembrane domain containing an ion transport pathway is involved in chloride conduction, and the CBS domains play a modulating role associated with voltage-dependent channel activation (Wang et al., 2017; Yusef et al., 2006). At present, a total of 32 pathogenic missense variants of CLCN2 in LKPAT patients have been described in the literature. More than half of the variants were truncation variants, which can cause premature stop codons and consequently lead to nonsense-mediated mRNA decay or protein truncation (Jentsch and Pusch, 2018). These pathogenic variants may directly decrease ClC-2 expression or affect the formation of a functional channel, thereby leading to loss of ClC-2 function, which is closely associated with LKPAT (Jentsch and Pusch, 2018; Gaitan-Penas et al., 2017). In addition, nearly half were missense variants, most of which were located in the transmembrane domain around the ion permeation pathway of ClC-2 (Figures 4A–C). Recently, researchers have identified three chloride-binding sites along the channel in human ClC-2, including pore filter residues S162 and Y553 and gating glutamate E205 (Ma et al., 2023; McKiernan et al., 2020). The opening and closing of the ClC-2 transporter are controlled by the gating glutamate through swinging the side chain of E205, which regulates chloride ion selectivity and permeability (Park et al., 2017; Dutzler et al., 2003). In the present study, we reported a novel homozygous CLCN2 A506V variant located near the ion permeation pathway and analyzed its potential pathogenic mechanism through in silico analysis. We observed that substitution of A506 with V506 altered hydrogen bond formation in nearby amino acids, particularly in chloride-binding sites in the crystal structural model of human ClC-2. As hydrogen bonds are important for protein spatial conformation and stability (Cao et al., 2024; Dong et al., 2022), the subtle changes between E205 and Y553 and between S162 and K188 may interfere with chloride conduction and impair the gating properties of ClC-2. Furthermore, GlialCAM, as a ClC-2-binding partner, has been reported to target ClC-2 to astrocyte–astrocyte junctions and glia limitans in white matter, stabilize ClC-2 expression at the plasma membrane, and increase its channel activity (Jeworutzki et al., 2012; Gaitan-Penas et al., 2017). Reduced ClC-2 expression in the plasma membrane and chloride currents caused by the CLCN2 A500V variant located around the ion permeation pathway can be partially rescued by co-expression with GlialCAM in glial cells (Gaitan-Penas et al., 2017). In this study, our results showed that hydrogen bonds and π-interactions between the interface residues of ClC-2 and GlialCAM were changed in the predicted protein interaction model by the ZDOCK program. This altered interaction of ClC-2 and GlialCAM may contribute to the abnormal formation of a complex and disrupt the stability and biophysical properties of ClC-2. Therefore, we speculate that the CLCN2 A506V variant located around the ion permeation pathway may have a negative effect on chloride conduction and GlialCAM–ClC-2 interaction in glial cells, which fail to compensate for an action-potential-induced excess of potassium in myelinated axons and, consequently, lead to osmotic intra-myelinic edema (Goppner et al., 2021; Blan et al., 2007; Rash, 2010). Moreover, impaired transportation of chloride ions may change the ionic environment of germ cells, thus causing testicular degeneration and infertility (Blan et al., 2007; Bosl et al., 2001).

Notably, some previously reported missense variants have been shown to cause gain-of-function of ClC-2 channels, which is associated with familial hyperaldosteronism type II (FH-II) (Scholl, 2022). In contrast to the loss-of-function CLCN2 variants, all pathogenic variants identified in patients with FH-II so far are located in the cytosolic domains (predominantly in the N-terminal inactivation domain) or near the cytosolic ends of transmembrane helices that are hypothesized to interact with the N-terminal inactivation domain of ClC-2 (Jordt and Jentsch, 1997) (Figure 4A). These missense variants could open the ClC-2 channel, thus resulting in increased chloride efflux and continuous depolarization in zona glomerulosa cells (Jordt and Jentsch, 1997; Scholl et al., 2018; Stowasser et al., 2019; Grunder et al., 1992). Cellular depolarization activates voltage-dependent calcium channels, enhances calcium influx, and ultimately increases aldosterone production by inducing the expression of aldosterone synthase, a rate-limiting enzyme for aldosterone biosynthesis (Scholl et al., 2018; Stowasser et al., 2019). Therefore, we provide the potential genotype–phenotype features of CLCN2-related diseases as follows: CLCN2 pathogenic variants identified in human beings, in the form of loss-of-function and gain-of-function variants, are associated with LKPAT and FH-II, respectively. LKPAT-related pathogenic variants are mostly located in transmembrane domains near the chloride ion transport pathway or cause truncated proteins, whereas variants of FH-II mainly affect the N-terminal inactivation domain. However, a broad range of ages of onset and phenotypic variability were observed among individuals with LKPAT carrying the same CLCN2 variants. Further studies are needed to analyze the genotype–phenotype correlation of LKPAT. A limitation of this study is the lack of family validation. We conducted a combination of multiple bioinformatics and functional analyses to provide reliable evidence supporting the pathogenicity of the identified variant. In-depth mechanistic studies are required to analyze the effect of the A506V variant in CLCN2-related leukoencephalopathy.

We identified a novel CLCN2 homozygous pathogenic variant in a patient presenting with LKPAT. Slow progression and non-specific and highly variable clinical symptoms make the diagnosis of CLCN2-related leukoencephalopathy challenging. Individuals with ataxia, auditory or visual impairment, and male individuals with infertility, coupled with the characteristic white matter changes in MRI, should be tested for CLCN2-related leukoencephalopathy. Currently, there are no specific treatments for this disease. Prenatal genetic testing is essential to prevent the birth of affected children. Our study expands the genotypic spectrum of LKPAT, indicates the potential pathogenesis of the CLCN2 A506V variant, and provides valuable insights for further studies toward the therapeutics of CLCN2-related leukoencephalopathy.