Update on leukodystrophies and developing trials

At the cellular level, due to the crucial role of the different CNS cells in generating and supporting the integrity of myelin, leukodystrophies are not only caused by defects in the myelin but also by dysfunction in other CNS structures [3, 9]. Similarly, at the molecular level, disruptions in one pathway can have far-reaching consequences on other pathways (Table 1) [10]. An in-depth understanding of the pathomechanisms underlying leukodystrophies is crucial for the development of innovative therapies.

Clinical manifestations of leukodystrophies vary depending on the specific aetiology and age of onset, which can range from birth to adulthood. However, there is a spectrum of disease presentation across all age groups, with symptoms and signs changing accordingly (Table 2). Hypomyelinating diseases tend to present earlier than demyelinating forms and early age of onset is usually associated with more severe manifestations. Leukodystrophies are progressive disorders, although some rare cases show significant improvements over time, both clinically and radiologically [9, 30]. Differentiating neurological and non-neurological symptoms can be helpful in suspecting a specific diagnosis.

Non-neurological symptoms can involve different systems and can be typical of a specific type of leukodystrophy (Table 2).

The diagnostic process for leukodystrophies begins with a thorough patient history and a comprehensive general and neurological examination. Because of the similarities in clinical findings among different types of leukodystrophies, it is crucial to combine these findings with specific brain MRI patterns. Additional diagnostic tools, including biochemical and metabolic tests, electrophysiological studies, ophthalmology consultation, neuroimaging, and genetic studies, can be used in parallel to enhance the accuracy of diagnosis in heritable white matter disorders. The integration of MRI, careful clinical evaluation, and next-generation genetic sequencing shows promise in reducing the number of unsolved cases (Fig. 3). Newborn screening can also play a pivotal role in early diagnosis and treatment [41].

Hypomyelinating leukodystrophies exhibit mild white matter abnormalities on T2-weighted imaging with iso- or hyperintense T1 signals, while demyelinating forms show prominent T2-weighted abnormalities and significant T1 hypointensity (Fig. 1). Adequate brain imaging protocols should be followed, including axial and sagittal T1-weighted, axial T2-weighted, and FLAIR sequences. Post-contrast T1-weighted images can provide additional information for certain conditions. Multiple MRI scans at intervals of at least 6 months may be necessary to better evaluate the progression and differential diagnosis.

While specific MRI findings can vary depending on the type and stage of the leukodystrophy, some typical findings may be observed. Cystic changes in the anterior temporal lobe are associated with several leukodystrophies such as AGS [39] and megalencephalic leukoencephalopathy with subcortical cysts (MLC) [33]. The corpus callosum is typically generally affected in MLD, Krabbe disease [35], and X-ALD [40], while the involvement of the inner rim of the anterior corpus callosum is suggestive of VWM [32]. Cystic degeneration is also observed in VWM [32] and AxD [31]. Prominent hypomyelination of the brainstem and the corticospinal tract is indicative of PMD [37]. Regions of demyelination alternating with relatively preserved myelinated areas give rise to a “tigroid pattern”, which is indicative of MLD [42].

While MRI is a sensitive technique for detecting brain white matter abnormalities, it has limitations and may not always provide conclusive results.

Genetics testing when considering the approach to genetic testing for leukodystrophies, several factors come into play. If a specific diagnosis is strongly suspected based on clinical symptoms and imaging findings, targeted genes or appropriate gene panels may be an initial step; however, if the diagnosis is uncertain or there is a suspicion of a broader genetic aetiology, WES or WGS becomes the test of choice [43]. WES has significantly improved the diagnostic yield of leukodystrophies, increasing the percentage of resolved cases by around 10% in recent years [4, 44]. Compared with the traditional step-by-step diagnostic approach, WES is more cost effective and delivers results within weeks or months, and should be commonly considered in clinical practice. It is important to note that WES may not detect all genetic variants, and an additional challenge associated with WES is the interpretation of variants of uncertain significance (VUS). VUS are genetic variants that lack sufficient evidence to determine their pathogenicity or benign nature. Further functional studies, segregation analysis within families, or the accumulation of additional evidence over time may be necessary to classify VUS as either pathogenic or benign. Negative findings on WES may indicate that the patient does not have a genetic disease, but clinical and MRI recognition of the disease along with targeted genetic testing may still be necessary. While WES-based gene-panel analysis is faster than open WES, it shares similar limitations. Genetic testing, including WES and WGS, should not be viewed as a standalone diagnostic tool.

Timely and accurate diagnosis of leukodystrophies is crucial for appropriate treatment options and participation in clinical trials.

Metabolic testing and biomarkers Biochemical and metabolic tests can provide guidance on the most appropriate genetic testing approach and help identify treatable entities, improving the accuracy of the final diagnosis in numerous leukodystrophies [5, 45, 46]. Recommended biochemical tests include very long-chain fatty acid (VLCFA) profiles, which are elevated in the plasma and tissues of patients with X-ALD [40, 47‐50], and measurements of leukocyte lysosomal enzyme activity are used to investigate deficiencies in galactocerebrosidase (GALC) (Krabbe disease) [51] and arylsulfatase A (ARSA) activity (MLD) [52]. In some cases, enzymatic studies must be supported by biochemical assessments of substrate accumulation, for instance, elevated urine sulfatide levels are used as biomarkers to provide additional evidence for the diagnosis of MLD [53‐55]. Furthermore, CTX is characterized biochemically by elevated levels in plasma cholestanol concentration (five- to ten-fold greater than normal), urine bile alcohol concentration, as well as plasma bile alcohol concentration. Disease-specific metabolic profiles can be used as biomarkers to help with early and definitive diagnosis, better disease prognostication, elucidating disease mechanisms, as well as monitoring therapeutic outcomes from specific treatments, especially with advancing therapeutic options [56‐60]. For instance, a correlation between residual ARSA enzyme activity and the severity of clinical presentation was reported in MLD patients [61], and longitudinal assessments showed that there were decreases in dried blood spot (DBS) concentrations of psychosine, a substrate of the GALC enzyme, associated with natural disease progression and treatment with HSCT in Krabbe disease patients [62, 63]. Moreover, non-disease-specific and non-invasive biomarkers of neuroaxonal and astroglial injury, such as neurofilament light chain (NfL) and glial fibrillary acidic protein (GFAP) levels, can be widely applicable across leukodystrophies. Blood NfL levels were correlated with abnormalities on brain MRI in MLD [64], GFAP levels in the CSF of AxD patients were consistently elevated [65], and the potential of plasma NfL as a biomarker of spinal cord degeneration in adrenoleukodystrophy has been illustrated [66]. Additionally, mathematical models can be implemented to identify novel biomarkers for leukodystrophies, where an in silico systems biology-based modelling approach was used to identify a panel of 16 potential biomarker candidates for future validation in clinical cohorts to aid in prediction of early damage in MLD [67].

Currently, there are no disease-modifying treatments available for the majority of leukodystrophies. The treatment is mainly symptomatic and often palliative. Symptomatic treatment typically involves a multidisciplinary team of specialists across paediatrics, neurology, rehabilitation, orthopaedics, physical therapy, speech-language therapy, occupational therapy, social work, nutrition, pulmonary, sleep medicine, ophthalmology, audiology, and palliative care. Managing spasticity, a prevalent problem in leukodystrophies, can be accomplished through oral medications, such as baclofen, tizanidine, or diazepam, plus botulinum toxin. Anti-seizure medications can be used for treating seizures. It is important to pay close attention to possible respiratory complications and to diet and feeding methods to ensure sufficient caloric intake and to prevent aspiration. Furthermore, patients with leukodystrophies often require specific interventions (Table 3).

Gene therapy is a therapeutic approach that involves introducing a functional copy of a gene into cells to achieve therapeutic effects [69]. Gene transfer can be performed ex vivo or in vivo. In ex vivo gene therapy, cells are taken from the patient's body and modified outside the body (in vitro), while in in vivo gene therapy, the gene transfer is performed directly inside the patient's body. Both approaches can be undertaken using various techniques, such as viral vectors (e.g. lentiviruses or adenoviruses) or non-viral methods (e.g. plasmid DNA or mRNA). Directly targeting the brain with gene therapy is becoming a viable option [70].

Bone marrow transplants or hematopoietic stem cell transplantation (HSCT) has shown successful outcomes in attenuating disease progression in leukodystrophies, characterized by the presence of cytotoxic metabolites in the brain (X-ALD, CTX, Krabbe) (Table 3). The rationale behind HSCT in these diseases involves the ability of hematopoietic cells to infiltrate the CNS and express the missing gene or enzyme necessary for the degradation of cytotoxic metabolites.

Lentiviruses have been used as a treatment in patients with pre-symptomatic MLD [71] with clinically relevant benefits [38, 72].

Gene therapy using adeno-associated viruses (AAVs) [73, 74] has several advantages over lentiviruses, including a broader tropism for CNS cell populations and the ability to cross the blood–brain barrier (for specific serotypes). An AAV1-GALC gene therapy study demonstrated reduced psychosine levels in the brain of a mouse model, indicating that delivery of the viral vector via the cerebroventricular system can decrease the rate of the disease progression in Krabbe disease [75]. Another study using a viral vector carrying the ASPA gene in patients with Canavan disease demonstrated safety and resulted in some clinical benefits [76]. However, it is possible that gene delivery alone may not be sufficient and may need to be combined with immunomodulation or silencing of the mutant gene.

Antisense oligonucleotides (ASOs) are RNA-based therapies that can alter protein expression (Canavan disease and PMD) [77].

CRISPR/Cas9 technology has not been studied on leukodystrophies, but it is a promising method for precise gene editing through homology-dependent repair mechanisms and can be used to repair disease-causing alleles by changing the DNA at the desired location of the chromosome.