New clinical characteristics and novel pathogenic variants of patients with hereditary leukodystrophies

Abstract Aim Leukodystrophies are a group of inherited white matter disorders with clinical, genetic, and imaging heterogeneity, which usually pose a diagnostic challenge for physicians. We aimed to identify new clinical characteristics and novel pathogenic variants of hereditary leukodystrophies in this study. Methods Whole exome sequencing (WES) was performed in 28 unrelated patients clinically suspected with leukodystrophies. Leukocytes enzyme activity test, electroencephalogram (EEG), electromyography (EMG), and brain MRI were conducted. Functional analysis was performed, and the pathogenicity of variants was classified according to the American College of Medical Genetics and Genomics (ACMG) standards and guidelines. Results We made definite diagnosis in 8 probands with 12 pathogenic variants and reported new clinical characteristics and imaging features of these patients. Three novel pathogenic variants were identified, including a microdeletion variant c.2654_2654+3del within CSF1R, a nonsense variant c.1321C>T, and a missense variant c.166G>C within GALC. Conclusion Our results have deepened the understanding of clinical, genetic, and imaging heterogeneity of hereditary leukodystrophies, and expanded the spectrum of pathogenic variants and clinical features.

Direct sequencing of certain suspected gene associated with leukodystrophies is neither effective nor economic, while whole exome sequencing (WES) can provide convenience to screen candidate genes in a short time. 4 Discovery of causative genes leads to accurate prognosis and genetic counselling for the patients, and it also helps clinicians to get a better understanding of leukodystrophies.
Here, we performed WES in a cohort of Chinese patients with white matter abnormality of unknown etiology, dementia, or spastic paraparesis without pathogenic variants. We made definite diagnosis in 8 probands carrying 12 pathogenic variants and reported new clinical characteristics and imaging features of them.

| Whole exome sequencing
Genomic DNAs captured from peripheral blood were sequenced by WES. Details on library preparation, sequencing protocol, bioinformatics analysis, and filtering methods were conducted as described previously. 9 All filtered variants were further validated by Sanger sequencing on an ABI 3500xL Dx Genetic Analyzer (Applied Biosystems) in the probands and the available family members.

| Reverse transcription PCR
RNAs were extracted from leukocyte of proband 2 which carries the CSF1R splicing variant and normal controls by RNAiso Plus (Takara). Mutant CSF1R mRNA of proband 2, wild type CSF1R mRNA (NM_005211) of normal controls, and wild type (WT) EIF2B3 (NM_020365) mRNA were reversely transcribed into cDNA by PrimeScript™ II 1st Strand cDNA Synthesis Kit (Takara) and amplified by high-fidelity DNA polymerase KOD-Plus-Neo (TOYOBO).

| Cell culture and transfection
For analysis of eIF2B3 expression, HEK293T cells were cultured at 37°C in Dulbecco's modified Eagle's medium (DMEM) (HyClone) supplemented with 10% fetal bovine serum (GIBCO) and cotransfected with WT or mutant EIF2B3 plasmids with pEGFP-N1 as exogenous control using Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions. Simultaneously, since the activity of eIF2B holocomplex could be inhibited by phosphorylated α subunit of eIF2 at Ser51 (eIF2αP), 12,13 this experiment was also used to detect eIF2α phosphorylation level to evaluate the effects of mutant EIF2B3 on eIF2B indirectly. For study on the ATF4-5′UTR-linked report, HEK293T cells were cotransfected with pEGFP-ATF4-5′UTR and p3 × flag-EIF2B3. (Santa Cruz biotechnology), rabbit anti-eIF2α (Cell signaling technology), rabbit anti-eIF2αP (Cell signaling technology), and mouse antiactin (Santa Cruz biotechnology) followed by horseradish peroxidase (HRP)-conjugated secondary antibody (Merck Millipore). Then, the protein was visualized by enhanced chemiluminescent substrates (Thermo Scientific).

| Genetic findings and pathogenicity classification of variants
After variant screening via WES and verification by Sanger sequencing, we found 12 distinct variants (Table 1) in eight unrelated patients ( Figure 1A). Among these 12 variants, three variants ( Figure 1B,C) are novel and absent in dbSNP, gnomAD, and ExAC.
All of 12 variants are absent in our WES database that contain 500 Chinese controls. As shown in Table 1  In the remaining nine known variants, eight variants (Figure 2A right, Figure S1) were previously reported as pathogenic variants, except EIF2B3 c.22A>T (Figure 2A, left). Given that EIF2B3 c.1037T>C ( Figure 2A, right) was a known pathogenic variant, we investigated whether the functional influence of c.22A>T was similar to that of c.1037T>C. From our study, neither c.22A>T nor c.1037T>C was identified to appreciably destabilize the protein ( Figure 2B). Both c.22A>T and c.1037T>C enhanced eIF2α phosphorylation without increase of total eIF2α ( Figure 2C) and increased the level of expression of EGFP regulated by the uORFs (Figure 2D). In addition, this patient had the onset of cognitive deterioration and ataxia at 57, with a bilateral, diffuse and symmetric involvement of the cerebral white matter ( Figure 2E). She was bedridden with two fractures because of her unsteady walk. Her disease lasted 4 years, and she died at 61. Combined the results of functional experiment with diagnostic clinical manifestations, we deduced that EIF2B3 c.22A>T was also a disease-causing variant.

| Novel clinical and imaging features
Ultimately  Leukoencephalopathy with VWM is an autosomal recessive neurodegenerative disease due to pathogenic variants in the five genes (EIF2B1 to 5) encoding the five subunits of the eucaryotic initiation factor 2B (eIF2B). 16 The variation in disease severity is extremely wide with stress-provoked episodes of rapid deterioration. 1 Survival increased with increasing age of onset and 80% of patients with an age of onset over 5 years were expected to be without severe disability at 14 years of disease evolution. 16 In this study, we identified c.22A>T and c.1037T>C variants within EIF2B3 in the proband of family 4 and firstly confirmed the pathogenicity of c.22A>T, which was never reported to be associated with VWM. Both of c.22A>T and c.1037T>C were indirectly proved to inhibit eIF2B activity. [11][12][13] However, the proband had the onset symptom at 57 and died at 61.

ACK N OWLED G M ENTS
We sincerely thank the participants for their cooperation and willingness to participate in this study. We thank Ms Wan-Qing Xu for editing the manuscript.