Patient’s developmental history and morphological features

The patient (male) presented at birth with hypotonia, abnormally thick fingers and toes, and ichthyosis. Pruritis and facial dysmorphism were apparent since the age of 11 months (Fig. 1). He had normal statural, ponderal, and head circumference growth. Motor acquisitions during the 1st year were severely delayed with sitting acquired at 11 months and independent walking at 3 years. At 14 years, the patient was able to speak using efficient isolated words and had good communication skills. He was unable to read and presented hyperactivity and attention deficit increasing over time. To date, the patient had normal walking and has not developed neurological signs including spastic paraplegia or ataxia. Morphological studies of skeleton, abdomen, and heart were normal. Electroencephalogram showed diffuse moderate bradyrythmia. Somatosensory evoked potentials revealed prolonged latencies on the four limbs and motor evoked potentials showed the lack of cortical response. MRI at 1.5 T demonstrated early diffuse hypomyelination with coalescing and dilated Virchow–Robin spaces. 1H-MRS reveals two abnormal lipid peaks in the white matter persistent at short and long echo times (other peaks were normal, except a small increase of inositol) (Fig. 1). Positions of these peaks in our patient correspond to positions of the characteristic peaks in classic SLS but the overall profile was different. Specifically, in SLS the 1.3 ppm peak is more prominent while the 0.9 ppm peak is smaller (Fig. 1m). In our patient, the peaks were still evident at 6 years of age, the finding which in association with the leukodystrophy initially suggested the SLS diagnosis. Of note, in SLS patients both peaks are permanent through age while in our patient these peaks decreased over time and were not detected after 8 years of age (Supplementary Fig. 1).

Fig. 1 Particularities of the patient phenotype. Patient at the age of 3 years a, b and 14 years c presented with a facial dysmorphism with epicanthus, hypertelorism, broad nasal root, anteverted nares, long philtrum, thin upper lip. The written consent for publication of these photos was obtained from child’s parents. Cerebral MRI shows diffuse hypomyelination at the age of 2 years with white matter appearing respectively in hypersignal on T2-weighted sequences, in hypersignal on FLAIR sequences and in normosignal on T1-weighted sequences d, g, j. Progressive myelination and dilatation and coalescing of Virchow–Robin spaces at the age of 6 e, h, k and 14 years f, i, l. 1H-MRS in the corona radiata for classical SLS patients shows a typical major peak at 1.3 ppm and a smaller peak at 0.9 ppm (m, arrows). 1H-MRS for our patient reveals a similar pattern with two peaks at 1.3 and 0.9 ppm (arrows) at the age of 2 n and 6 years o. However, the peak at 1.3 ppm appears smaller than the peak at 0.9 ppm in our patient. We observed no decrease in N-acetyl-aspartate (NAA)/creatine (Cr) ratio, or in choline (Cho) peak suggesting normal maintenance of neuronal and myelin content but a small increase of inositol peak that may be due to some astrocytic stress. Family pedigrees p and patient’s genotype r, s Full size image

Mutations identified by whole exome sequencing

Sequencing of the ALDH3A2 exons, exon/intron junctions, and the full-length cDNA did not reveal any mutations in this gene in our patient. The following mutations were found by the whole exome analysis: (1) a de novo hemizygous mutation (c.263dup, p.Ser89Leufs*4) in the RPS6KA3 gene on the X chromosome,10,11 a deleterious frameshift mutation not carried by the patient’s mother. This finding confirmed that the patient was affected by a Coffin–Lowry syndrome8 and partially explained the patient’s features but did not explain the congenital pruritic ichthyosis, MRI or 1H-MRS features typical of SLS. (2) This patient also has compound heterozygous mutations in the ALDH1L2 gene, which encodes for a mitochondrial 10-formyltetrahydrofolate dehydrogenase.12 Discovered mutations, one intronic near splice site (c.2517-5C>T) and one frame shift (c.827del/p.Val276Glyfs*33; rs770401066, dbSNP NCBI) were not present in the homozygous state in ExAC or gnomAD. The patient’s asymptomatic parents and brother were heterozygous for one of these mutations. Segregation analysis revealed that the father harbors the Val276Glyfs*33 frameshift mutation, while the mother harbors the c.2517-5C>T intronic mutation. The presence of a mutant mRNA resulting from the frame shift was confirmed in patient’s fibroblasts by the direct sequencing. The mutated sequence predicts a truncated protein of 307 aa, including the 22 aa mitochondrial leader sequence, 253 aa of the N-terminal folate-binding domain/hydrolase catalytic center,13 and the 32 aa random peptide with no identity to known proteins resulted from the frameshift (Supplementary Fig. 2). Such truncated proteins are usually not folded properly,13 and apparently are rapidly degraded. Indeed, the truncated protein was not detected in the patient’s fibroblasts. Deficiency of the ALDH1L2 gene has not been reported and the overall consequences of the enzyme loss for the cell are not clear. We examined fibroblasts from this patient, his parents and healthy unrelated individual, and present evidence that the loss of ALDH1L2 impairs the mitochondrial function and is the likely cause of a new neuro-ichthyotic syndrome.

Characterization of patient’s fibroblasts

Compared to fibroblasts from a healthy individual (control, C cells), patient’s fibroblasts (R cells) have barely detectable ALDH1L2 protein (Fig. 2a–c and Supplementary Fig. 3). Levels of ALDH1L2 mRNA were also significantly lower in patient’s cells (Fig. 2d and Supplementary Fig. 4). Since one of the alleles of the ALDH1L2 gene in the patient has mutation near the splice site, we attributed the decrease in the mRNA level to the impaired transcription caused by the mutation. Indeed, splice site mutations are known to cause loss of gene expression.14 Levels of the ALDH3A2 protein were not different between the two cell lines (Fig. 2a and Supplementary Fig. 3), an indication that FALDH deficiency was not the primary cause of the patient’s symptoms. The ALDH1L2 enzyme catalyzes the conversion of 10-formyl-THF to THF and CO 2 simultaneously producing NADPH from NADP+ (Fig. 2e).12,15,16 Therefore, the ALDH1L2 activity is likely to affect folate metabolism but the extent of the enzyme contribution to the maintenance of reduced folate pools is not clear. The total folate levels were not significantly different between patient’s (R) and control (C) fibroblasts (Fig. 2f), only 10-formyl-THF was noticeably and significantly different between two fibroblast cultures (Fig. 2f). ALDH1L1, the cytosolic homolog of ALDH1L2 and a major user of 10-formyl-THF17,18 was not present in either fibroblast culture (Supplementary Fig. 5). Therefore, the three-fold increase of this folate upon the ALDH1L2 loss (Fig. 2f) indicates that the enzyme is a major user of 10-formyl-THF. The ratio of NADPH/NADP+, metabolites also involved in ALDH1L2 catalysis, was more than four-fold lower in patient versus control fibroblasts (Fig. 2g and Supplementary Fig. 6), supporting the role of ALDH1L2 as the main source of NADPH generation.19 Furthermore, patient’s fibroblasts have much lower ATP levels in mitochondria as well as in whole cells (Fig. 2h and Supplementary Figs. 7 and 8) with the ATP/ADP ratio indicating a very low energy status in patient’s fibroblasts (Fig. 3b, d). Another characteristic feature of ALDH1L2-deficient fibroblasts is a decreased proliferation rate (Fig. 2i), which was not responsive to the increase of folate in media (10 μM leucovorin or 20 μM folic acid). In fact, the metabolomics analysis demonstrated differences between the patient and control fibroblasts beyond folate metabolism (Fig. 2j) with statistically significant (p < 0.05) differences for 250 out of 475 assigned metabolites.

Fig. 2 Difference between fibroblasts from the patient (R cells) and fibroblasts from healthy individual (C cells). a–c R cells have much lower levels of ALDH1L2 protein as evaluated by Western blot assay (a, ratios of averaged band intensities are indicated; statistics is shown in Supplementary Fig. 3) and confocal microscopy b in cells or by Western blot assay in isolated mitochondria (c). In panel a, samples were from different plates (biological replicates) with 20 μg of the total protein loaded per well. d Levels of ALDH1L2 mRNA are lower in R cells (mean ± SE of three biological replicates). e Cytosolic and mitochondrial folate pathways. f Levels of folate coenzymes (FA, folic acid; THF, tetrahydrofolate; 5-MTHF, 5-methyl-THF; 10-CHO-THF, 10-formyl-THF) in C and R cells (only 10-CHO-THF was noticeably and significantly different between the two cell lines). For each cell type mean ± SE of three independent experiments (each done in quadruplicate) is shown (3 biological replicates each includes 4 technical replicates). g Ratio of NADPH/NADP+ in C and R cells (mean ± SE of four biological replicates). h Levels of ATP in C and R cells (mean ± SE of four biological replicates). For panels d, g, h, p values were below 0.001 (Student’s t-test) for the comparison of R and C cells (detailed statistical analysis for these panels is shown in Supplementary Figs. 4 and 6–8). i Proliferation rate of C and R cells measured in real-time (xCelligence); samples with three densities of cells were monitored for each cell type. In each case, experiments were done in duplicate with automated averaging of data points. j PCA for metabolites (475 total) measured in C and R cells (n = 4; samples are biological replicates) Full size image

Fig. 3 Patient fibroblasts have lower ATP and energy index, and altered mitochondrial morphology in comparison with fibroblasts from parents or SLS patient. a Western blots assays of ALDH1L2 and ALDH3A2 in fibroblasts isolated from the patient (R), both parents (mother, M; father, F) and from an SLS patient (SLS). LR denotes patient’s fibroblasts transduced for ALDH1L2 expression. Numbers on the bottom of each panel indicate band intensity (arbitrary units) for ALDH1L2 and ALDH3A2 relative to the intensity of corresponding actin band. b Doubling time and energy index of different fibroblast cultures (cell labeling as in panel a). c ATP levels measured by a colorimetric assay in different fibroblast cultures. Three different samples (biological replicates) were used in this experiment; for each sample, 4 measurements (technical replicates) were performed and the average of these measurements were used to calculate mean ± SE. d TMRM (tetramethylrhodamine) to MitoTracker Green ratio in different fibroblasts. Six samples (biological replicates) were analyzed for each cell type. e Levels of ROS evaluated by confocal microscopy after DCF (2′,7′-dichlorodihydrofluorescein diacetate) staining in patient (R cells) and control (C cells) fibroblasts (values were calculated from the analysis of 10 cells for each cell type; laser power was kept uniform for all measurements). f Confocal images (108×) of different fibroblast cultures (as in panel a). Live cells were stained with Hoechst (nucleus staining, light-blue), MitoTracker Green (mitochondrial staining, green), or TMRM (mitochondrial staining, red); scale bars, 10 µM. For panels c, d, *p < 0.05; **p < 0.01; ***p < 0.001 Full size image

Comparison of our patient’s fibroblasts with fibroblasts derived from parents

Fibroblast cultures generated from both parents (mother, M, heterozygous for the intronic mutation; father, F, heterozygous for the mutation causing a premature stop codon) showed ALDH1L2 protein expression though its levels were noticeably higher in father’s cells (Fig. 3a). Both cell lines have similar levels of ALDH3A2 proteins comparable with those of patient’s fibroblasts (Fig. 3a), and both demonstrated a much faster proliferation rate than R cells as indicated by the doubling time (Fig. 3b). ATP levels in the father’s cells were remarkably higher than in R cells (Fig. 3c). Furthermore, mitochondria from R cells have lower membrane potential (Fig. 3d) and showed increased levels of reactive oxygen species (Fig. 3e). Correspondingly, metabolomic analysis has shown increased levels (fold-change > 2; p < 0.05) of several oxidative stress biomarkers such as methionine sulfoxide, 5-oxoproline, and ophthalmate, in patient’s cells (Supplementary Data File 1). Confocal microscopy has shown differences in mitochondrial morphology in patient’s fibroblasts with the appearance of rounded isolated mitochondria, which were not seen in healthy control or father’s fibroblasts (Fig. 3f). The morphology of mitochondria in mother’s fibroblasts was similar to the morphology of the control and father’s fibroblasts though mother’s cells were smaller in size compared to fibroblasts from other individuals (Fig. 3f). These cells as well have normal doubling time (Fig. 3b) and mitochondrial membrane potential (Fig. 3d).

Electron microscopy further confirmed altered mitochondrial morphology in R cells compared to father’s cells (Supplementary Fig. 9). In contrast to mitochondria of father’s cells, which are filamentous as commonly seen in cultured fibroblasts,20 mitochondria of R cells appear to be shorter and distorted. Another noticeable feature of R cells was the presence of large vesicles not seen in F cells (Supplementary Fig. 9). We suggest that these alterations are linked to the metabolic effects caused by the ALDH1L2 deficiency. Thus, significant differences in metabolic profile in R cells were associated with amino acid, nucleotide, and lipid pathways (Fig. 4 and Supplementary Data File 1). Of note, accumulation of all common amino acids is indicative of decreased protein biosynthesis,21 which is in line with decreased proliferation and low energy status in R cells. Strong changes in the lipid profiles were seen in R cells with the most dramatic increase of acylcarnitine metabolites (Fig. 4) and the reduction of mono- and diglycerides as well as all classes of phospholipids (Supplementary Data File 1).

Fig. 4 Metabolomic analysis of R, F, and LR fibroblasts. a Lentivirus-based expression of ALDH1L2 in LR fibroblasts restores levels of the enzyme seen in control or parent’s cells (Western blot assay of isolated mitochondria and confocal image of fibroblasts stained with ALDH1L2-specific antibody, St indicates lane with molecular weight standards, VDAC is shown as mitochondrial marker; green fluorescence indicates ALDH1L2; nuclei were co-stained with DAPI). b PCA (principal component analysis, performed with SIMCA Version 15.0.2, Sartorius Stedim Data Analytics AB, Umeå, Sweden) of metabolomic data (total of 516 metabolites) for R, F, and LR fibroblasts (n = 5 biological replicates in each case). c Heat map representation of the metabolite comparison between R, F, and LR cells (performed with Qlucore Omics Explorer v.3.4 software, Qlucore, Lund, Sweden; data were filtered by p value ≤ 0.05). d Schematic depicting the TCA cycle and its connection to carnitine pathway. e, f Levels of Krebs cycle metabolites and carnitine and most acylcarnitine derivatives are similar in F and LR cells but compared to R cells are much lower in both cell lines. Statistically significant differences (n = 5) are highlighted in green (p < 0.05, decreased metabolites), red (p < 0.05, increased metabolites), or light red (p < 0.1, increased metabolites). g Proposed mechanism for the effect of the ALDH1L2 loss Full size image

Restoration of biochemical properties by expression of wild-type ALDH1L2

Re-expression of ALDH1L2 (via viral transduction) in patient fibroblasts (LR cells, Fig. 4a) restored the morphological features of father’s cells, including re-appearance of filamentous mitochondria (Fig. 3f) and disappearance of large vesicles (Supplementary Fig. 9); decreased the doubling time (Fig. 3b); increased levels of ATP (Fig. 3c) and mitochondrial membrane potential (Fig. 3d) thus improving the energy status of these cells and making them more similar to father’s fibroblasts. Overall, the metabotype of patient cells with restored ALDH1L2 expression was shifted towards the metabotype of father cells (Fig. 4b, c) indicating that the metabotype of patient cells is associated with the loss of ALDH1L2. Specifically, Krebs cycle intermediates and acyl carnitines were similar in LR and F fibroblasts (Fig. 4d–f). These data indicate that the ALDH1L2 loss affects fatty acid metabolism. In fact, several reports implicated the enzyme in β-oxidation and fatty acid metabolism22,23,24 though underlying mechanisms are not clear. Alternatively, alterations in fatty acid metabolism could be a cellular response to oxidative stress associated with the ALDH1L2 loss.25 The phenotype rescue by transduction of ALDH1L2 indicates that the metabolic changes and mitochondrial dysfunction were not caused by Coffin–Lowry syndrome.

Comparison of our patient’s fibroblast with fibroblasts derived from an SLS patient

We have also compared our patient’s fibroblasts with the fibroblast culture from a patient with classical SLS caused by the loss of ALDH3A2 enzyme due to a homozygous mutation (c.471+1delG in intron 3) associated with the splicing abnormality of the ALDH3A2 gene. Levels of ALDH1L2 appeared normal in the SLS patient and were similar to the protein level in father’s fibroblasts (Fig. 3a). These cells have a standard doubling time and ATP levels similar to father’s cells characterized in this study (Fig. 3b, d). Confocal microscopy has shown that SLS fibroblasts have a typical mitochondrial morphology (Supplementary Fig. 10a) distinct from that observed in R cells (Fig. 3f). The metabotype of SLS fibroblasts was different from the metabotype of fibroblasts from our patient as well as metabotypes of healthy control or asymptomatic parents of our patient (Supplementary Figs. 10b, 10c, and 11; and Supplementary Data File 1). These differences suggest that our patient has a distinct biochemical basis for SLS-like symptoms.