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Article

Liver-Targeted AAV-DJ-hCBS Therapy Achieves Long-Term Correction of Metabolic Imbalance in CBS-Deficient Mice

by
Christian P. Joschko
1,
Chih-Chieh Wang
2,
Azuwerus van Buiten
1,
Maaike Goris
1,
Femke Hoogstra-Berends
1,
Joy Wang
2,
Jan Henric Bacurio
2,
Yinxing Chen
2,
Nannan Jia
2,
June Deng
2,
Shiliang Hu
2,
Mariana Nacht
2,
Matthew J. Chiocco
2,
Robert H. Henning
1 and
Leo E. Deelman
1,*
1
Clinical Pharmacy and Pharmacology, University Medical Center Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands
2
Alexion Pharmaceuticals, AstraZeneca Rare Disease, Genomic Medicine, 100 Binney Street, Cambridge, MA 02142, USA
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2026, 27(7), 3338; https://doi.org/10.3390/ijms27073338
Submission received: 11 March 2026 / Revised: 1 April 2026 / Accepted: 4 April 2026 / Published: 7 April 2026
(This article belongs to the Section Molecular Biology)
Browse Figures
Review Reports Versions Notes

Abstract

Cystathionine β-synthase (CBS) deficiency causes classical homocystinuria with severe hyperhomocysteinemia (HHcy) that is inadequately controlled by current therapies. We tested whether liver-targeted CBS gene therapy provides durable biochemical and phenotypic rescue. Using a Cre-inducible adult mouse model of whole-body CBS loss, a single intravenous dose of AAV-DJ-hCBS (3 × 1012 or 3 × 1013 vg/kg) was administered, and the animals were followed for 12 months. Vector biodistribution showed ~100-fold hepatic enrichment over the kidney and spleen. Both doses rapidly normalized plasma homocysteine (<8 µM), maintaining correction throughout the study while preventing alopecia, weight loss, and loss of adiposity. Liver histology showed resolution of inflammation, and only 2 of 19 mice developed anti-hCBS antibodies. Liver proteomics (3998 proteins quantified) revealed CBS deficiency-associated suppression of tRNA aminoacylation and dysregulation of lipid and carbon metabolism with an HNF4A transcriptional signature, all normalized by therapy. Liver metabolomics demonstrated accumulation of S-adenosylmethionine and S-adenosylhomocysteine and disruption of phosphatidylcholine synthesis, also corrected by treatment. Plasma metabolomics revealed systemic disturbances fully normalized by hepatic CBS restoration. These findings identify the liver as the central metabolic control point in CBS deficiency and support liver-targeted gene therapy as a durable corrective strategy.

1. Introduction

Classic homocystinuria (HCU) is an autosomal recessive metabolic disorder caused by loss-of-function mutations in cystathionine β-synthase (CBS), a pyridoxal 5′-phosphate (PLP)-dependent enzyme in the transsulfuration pathway [1]. Affected individuals exhibit multisystem complications, including severe vascular disease, cognitive impairment, skeletal abnormalities, and premature mortality [2,3,4,5].
Current therapies remain limited to dietary methionine restriction, cofactor supplementation (vitamins B6, B12, and folate), and in some cases, betaine to enhance Hcy remethylation. However, these treatments only lower but do not normalize homocysteine levels, which keeps patients at risk for cardiovascular (CV) disease. Particularly pyridoxine-nonresponsive patients have high CV risk due to lower treatment efficacy [1,6,7]. Experimental approaches, such as PEGylated enzyme replacement [8], pharmacological chaperones [9], and early gene therapy strategies, have shown partial homocysteine reduction in preclinical models, but none have achieved durable or comprehensive metabolic correction [10,11].
Normally, homocysteine (Hcy) is cleared either by remethylation or transsulfuration, the latter initiated by CBS. Loss of CBS function disrupts transsulfuration, causing severe hyperhomocysteinemia (HHcy), often exceeding 100 µM. Elevated plasma Hcy contributes to endothelial dysfunction, neurocognitive deficits, and systemic metabolic disturbances, including lowered body fat mass [2,6,12,13,14,15].
Over the past decade, the view of HHcy pathophysiology has expanded from homocysteine toxicity and oxidative stress to recognition of HHcy as a multi-system metabolic disorder with tissue-specific effects. Murine and cellular models have advanced our understanding considerably. For instance, complete CBS knockout in mice produces early postnatal lethality, while models with residual enzyme activity such as the I278T mouse model revealed that CBS deficiency leads to sulfur metabolite imbalance, impaired protein persulfidation, and sphingolipid dysregulation. While in this model, methionine restriction corrects sulfur metabolite disturbances and restores protein persulfidation, thereby extending survival, it does not normalize growth, longevity, or vascular and connective tissue abnormalities [7,16,17,18,19]. Proteostasis-focused studies further show that CBS variants are unstable, with pyridoxine responsiveness linked to partial rescue of folding and stability [18,20,21]. Despite these advances, CBS deficiency alters multiple metabolic pathways beyond sulfur metabolism, including one-carbon metabolism, redox balance, and lipid homeostasis, but the links between these biochemical changes and the clinical manifestations remain poorly defined.
Because CBS is most highly expressed in the liver, this organ represents a logical target for therapeutic intervention. We hypothesized that hepatocyte-specific restoration of CBS function via adeno-associated virus DJ-type (AAV-DJ)-mediated gene therapy normalizes Hcy levels and ameliorates disease phenotypes [22]. Using a Cre-inducible Cbs-knockout adult mouse (Cbs-/-) model, we evaluated the therapeutic potential of liver-targeted delivery of human CBS (AAV-DJ-hCBS) [23,24,25]. Here, we demonstrate that a single i.v. administration of liver-targeted AAV-DJ-hCBS reverses HHcy in the long term, normalizes metabolic balance, and improves disease-associated phenotypes, highlighting liver-targeted gene therapy as a viable path toward a curative treatment for HHcy.

2. Results

2.1. AAV-DJ-hCBS Normalizes Phenotype and Homocysteine Levels

In the present study, we evaluated the effects of two dose levels of a single injection of liver-targeted AAV-DJ-hCBS therapy at 3 × 1012 and 3 × 1013 vg/kg body weight, selected to achieve hepatic CBS expression within physiological range or at supraphysiological levels, respectively (Cbs-/- + hCBSlow and Cbs-/- + hCBShigh). We induced CBS knockout at adulthood (10 weeks of age) using our Cre-inducible Cbs-/- mouse, which subsequently exhibits severe HHcy typically exceeding 200 µM (<4 µM in WT), growth retardation, and facial alopecia [26]. In a long-term follow-up study, we treated Cbs-/- mice with AAV-DJ-hCBS at 12 weeks and evaluated a 48-week period by assessing effects on phenotype and metabolic profile.
Liver-targeted AAV-DJ-hCBS therapy normalizes Cbs-/- phenotypes. To confirm the efficiency and liver specificity of AAV-DJ-mediated transgene delivery, we first assessed vector distribution and hepatic CBS expression. Vector copy numbers measured by ddPCR were ~100-fold higher in the liver compared with the kidney and spleen for both doses, confirming hepatocyte-selective transduction (Figure 1H). Western blotting showed that low-dose AAV-DJ (Cbs-/- + hCBSlow) restored hepatic CBS expression, whereas the high dose (Cbs-/- + hCBShigh) resulted in a further increase (~5-fold) (Figure 1G).
Having established efficient and liver-selective CBS expression, we next examined whether this was sufficient to correct the metabolic consequences of CBS deficiency. Liver-targeted AAV-DJ-hCBS resolved HHcy in CBS-knockout mice within two weeks after injection and remained effective throughout the experiment (Figure 1A). Plasma concentrations of Hcy dropped below the detection limit (<6–8 µM) in both Cbs-/- + hCBSlow and Cbs-/- + hCBShigh, whereas HHcy persisted in Cbs-/- + GFP (AAV-DJ-GFP control) mice. Hcy levels remained undetectable in the treatment groups over the course of the study, except for one mouse in the Cbs-/- + hCBShigh (mouse 13), which reverted to HHcy at week 38 post-AAV-DJ-hCBS injection.
AAV-DJ-hCBS therapy fully corrected the growth and body composition defects characteristic of CBS deficiency. Mice in the Cbs-/- + GFP group showed impaired growth compared to wild-type Cbs+/+ mice (Figure 1B). In contrast, the Cbs-/- + hCBSlow and the Cbs-/- + hCBShigh groups showed a considerable gain in body weight immediately after AAV-DJ-hCBS injection, effectively restoring their growth curve to that of wild-type mice. Lower body weight in Cbs-/- + GFP was due to a lower body fat percentage (13.5 ± 0.7%; Figure 1C) compared to wild-type mice (27.5 ± 2.9%), which was normalized in both Cbs-/- + hCBSlow and the Cbs-/- + hCBShigh (22.1 ± 2.9 and 24.5 ± 2.5%, respectively). Consequently, AAV-DJ-hCBS therapy also normalized fluid mass and lean body weight (Figure 1D,E).
All Cbs-/- + GFP started developing facial alopecia at approximately 2–3 months after induction of HHcy, resulting in severe facial alopecia at the end of the study (Figure 1F). AAV-DJ-hCBS therapy completely prevented the development of alopecia. Moreover, prevention of alopecia was not due to CBS transgene expression in skin, as CBS expression in skin was low to undetectable in all three groups (Cbs-/- + GFP, Cbs-/- + hCBSlow and Cbs-/- + hCBShigh; Supplemental Figure S3).
In line with our previous study [26], we did not observe major abnormalities in organ weights and blood parameters after termination of the mice (Table 1). However, blood glucose was significantly decreased in Cbs-/- + GFP (9.7 ± 1.4 mM) compared to wild-type mice (13.4 ± 1.2 mM). These values were effectively corrected by AAV-DJ-hCBS treatment, as blood glucose was normalized in Cbs-/- + hCBSlow and Cbs-/- + hCBShigh (12.8 ± 3.3 and 13.2 ± 0.9 mM, respectively). In addition, the number of red blood cells (RBCs) was decreased in Cbs-/- + GFP (8.4 ± 3.3 × 1012/L) compared to wild-type mice (12.4 ± 1.4 × 1012/L). Again, AAV-DJ-hCBS treatment corrected these values, as RBC was normalized in Cbs-/- + hCBSlow and Cbs-/- + hCBShigh (11.4 ± 1.4 and 12.1 ± 1.7 × 1012/L, respectively). A similar pattern was observed for the related parameters of hematocrit and total hemoglobin and for the amount of hemoglobin in red blood cells (MCH) (Table 1). Male and female mice responded similarly to AAV-DJ-hCBS treatment, with no sex-dependent differences in HHcy, body weight, or body composition parameters. Collectively, these findings demonstrate that liver-targeted AAV-DJ-hCBS therapy restores hepatic CBS expression, thereby normalizing systemic homocysteine levels and preventing phenotype manifestation in CBS-deficient mice.

2.2. AAV-DJ-hCBS Restores Liver CBS Expression and Limits Inflammation

Because the liver is the primary site of CBS expression and the target of AAV-DJ delivery, we next examined whether hepatic architecture and transgene expression were preserved following treatment. We therefore investigated liver histology and hCBS immunostaining in all groups. Liver histology revealed occasional mild perivascular inflammation in wild-type mice, with otherwise preserved architecture (Figure 2A). These lesions are commonly observed in 12-month-old C57BL/6 mice and are likely related to the normal aging process rather than a pathological condition [27]. Conversely, Cbs-/- + GFP livers displayed pronounced perivascular and focal interstitial inflammation (Figure 2C), suggesting either accelerated aging or underlying pathology. The enhanced inflammation was resolved by AAV-DJ-hCBS treatment, as both Cbs-/- + hCBSlow and Cbs-/- + hCBShigh mice demonstrated occasional mild perivascular inflammation similar to wild-type controls (Figure 2E,G). The absence of any histological changes in AAV-DJ-hCBS-treated groups relative to wild-type controls further indicates the absence of any detectable pathological effects elicited by the AAV-DJ-hCBS vector.
We next assessed hCBS transgene localization relative to endogenous hepatic CBS by immunostaining. CBS staining in wild-type livers was distributed throughout the lobules, with increased intensity in the perivenular region (Figure 2B). In Cbs-/- + GFP, CBS staining was absent (Figure 2D). The livers of Cbs-/- + hCBSlow mice showed a CBS expression pattern similar to the wild type, with staining throughout the lobules and accentuated in the perivenular region; however, staining intensity exhibited considerable heterogeneity among individual cells, with increased staining around blood vessels (Figure 2F). Cbs-/- + hCBShigh mice showed strongly increased CBS staining across the entire lobule (Figure 2H), again with considerable heterogeneity among individual cells. CBS staining revealed no clusters of adjacent CBS-expressing cells, suggesting that genomic integration of the vector DNA and clonal expansion, which has been reported at low levels in the liver for the related AAV serotype 2 [28], is unlikely. Moreover, AAV-DJ-hCBS treatment was performed at 12 weeks of age, a stage when hepatocyte proliferation has ceased [29]. Together, these findings indicate that sustained CBS expression is most likely maintained through episomal vector expression rather than genomic integration.
While AAV-DJ-hCBS treatment generally normalized liver histology in CBS-knockout mice, we noticed two exceptions, both of which were included in the quantitative analyses presented before. Mouse 8 in the Cbs-/- + hCBSlow group exhibited pronounced steatosis without inflammation, with persistent but patchy hCBS transgene expression (Supplemental Figure S1A,B). Mouse 13 in the Cbs-/- + hCBShigh group showed marked interstitial inflammation and fibrosis accompanied by complete loss of hCBS expression (Supplemental Figure S1D–F) and redevelopment of HHcy. Interestingly, both mice had developed an antibody response against human CBS (Supplemental Figure S4), which persisted until week 48. Therefore, it is likely that a sustained immune response against hCBS contributed to the abnormal histology observed in these two mice. This is supported by the observation that signs of pathology were absent in an additional five mice that also developed measurable, but transient, antibodies against hCBS, which had returned to baseline by week 48.
Nevertheless, the immunogenic risk observed in these mice likely overestimates the risk in human patients. Anti-hCBS antibody formation in CBS-knockout mice reflects a xenogeneic response to a foreign antigen. In human patients receiving gene therapy encoding the native CBS sequence, central self-tolerance is expected to substantially reduce this risk [30,31,32]. Additionally, while capsid-directed immune responses remain a recognized concern for AAV vectors, engineered capsids such as AAV-DJ have been associated with altered antigenic properties and potentially reduced immunogenicity compared to common natural serotypes [33,34], though dedicated clinical data for AAV-DJ specifically remain limited.
Together, our findings demonstrate that targeted AAV-DJ-hCBS restores liver CBS expression and resolves inflammatory changes without detectable hepatic pathology, consistent with the normalization of the phenotype.

2.3. Multi-Omics Reveals One-Carbon and Lipid Pathway Disruption, Reversed by Therapy

To further detail the impact of CBS deficiency on cellular and metabolic pathways and assess reversal by AAV-DJ-hCBS treatment, we investigated the metabolome and proteome of liver tissue in the same wild-type (WT; n = 5), Cbs-/- + GFP (n = 5), and Cbs-/- + hCBSlow (n = 8) mice. Cbs-/- + hCBSlow was selected for molecular analysis based on complete normalization of the phenotype at lower dosage.
Across all samples, we identified 3998 proteins, of which 339 were significantly altered in Cbs-/- + GFP vs. WT, which were fully restored in Cbs-/- + hCBSlow to WT levels (Figure 3B,C), signifying the complete normalization of liver protein abundances by AAV-DJ-hCBS treatment. Accordingly, principal component analysis (PCA) of liver proteome demonstrated clustering of WT and Cbs-/- + hCBSlow which was clearly separated from Cbs-/- + GFP mice (Figure 3A).
To explore the signaling routes affected by CBS knockout, we performed pathway enrichment analysis of differentially abundant proteins (DAPs), demonstrating a broad metabolic rewiring in liver of Cbs-/- + GFP. A total of 54 biological processes (GO:BP) were significantly altered (Supplemental Table S1), including suppression of tRNA aminoacylation and protein translation pathways and activation of lipid metabolism and redox/detoxification processes (Figure 3D). To distinguish opposing biological programs, we separated DAPs into 148 upregulated and 191 downregulated proteins. Downregulated proteins were enriched for translational and RNA metabolic processes, consistent with a global suppression of protein synthesis. In contrast, upregulated proteins were enriched for lipid, amino acid, and carbohydrate metabolism, consistent with a broad catabolic program (Supplemental Figure S2).
To corroborate these findings across pathway frameworks, we performed enrichment using both KEGG and Reactome. KEGG analysis identified fatty acid metabolism, carbon metabolism, PPAR signaling, and pyruvate metabolism among the most significantly upregulated pathways, whereas aminoacyl-tRNA biosynthesis was suppressed (Figure 3E). Reactome likewise highlighted amino acid, lipid, and tyrosine metabolism, while MITF–M-related transcriptional regulation was downregulated (Figure 3F). Motif enrichment of the upregulated proteins identified HNF4α as the only significantly enriched transcription factor, indicating that the metabolic pathways most affected by CBS deficiency map onto the core hepatocyte program governed by HNF4α.
Collectively, these results demonstrate that AAV-DJ-hCBS therapy normalizes the disease-associated liver proteome, reversing translational suppression and activating catabolic pathways in liver.
Next, we performed liver metabolomics to further explore rewiring of metabolism in CBS knockout. Across all samples, we detected 993 metabolites, of which 239 were retained for downstream analysis based on high-confidence annotation. AAV-DJ-hCBS therapy normalized the metabolomic profile of Cbs-/- + GFP mice, as shown by PCA, which revealed a clear separation between Cbs-/- + GFP and WT controls and full overlap of Cbs-/- + hCBSlow with WT (Figure 4A).
In total, 28 metabolites differed between Cbs-/- + GFP and WT, with 18 elevated and 10 reduced, and AAV-DJ-hCBS corrected all to WT levels. This was evidenced by hierarchical clustering, which showed that metabolite levels in Cbs-/- + hCBSlow were indistinguishable from those in WT mice (Figure 4B), and by normalization of log2 fold changes, with homocysteine showing the strongest correction (Figure 4C).
Cbs-/- + GFP were characterized by substantial increases in methionine cycle intermediates, SAM, SAH, and homocysteine and decreased betaine. Further, Cbs-/- + GFP featured alterations in other metabolites related to SAM-dependent methylation, including a decrease in several quaternary amines (stachydrine, trigonelline, carnitine) and alterations in phosphatidylethanolamine methyltransferase pathway (PEMT-pathway) intermediates (phosphocholine, CDP-ethanolamine, O-phosphorylethanolamine) reflecting impaired methylation of phosphatidylethanolamine into phosphatidylcholine. KEGG pathway analysis of significantly altered liver metabolites highlighted cysteine and methionine metabolism and glycerophospholipid metabolism as enriched (Figure 4D).
Collectively, these data show that within the cysteine and methionine pathways, Cbs-/- + GFP mice accumulated SAM, SAH, and homocysteine but maintained stable methionine, which reflects an impaired transsulfuration of homocysteine and a compensatory flux through the betaine-dependent remethylation pathway. Nevertheless, the unchanged γ-glutamylcysteine and glutathione levels indicated preserved redox balance. Further, CBS-knockout animals have impaired PEMT-mediated phosphatidylcholine synthesis, likely due to elevated SAH inhibiting SAM-dependent methylation reactions (Figure 4E,F).
Thus, CBS deficiency disrupts hepatic one-carbon and lipid methylation pathways, which are fully restored by AAV-DJ-hCBS. Together with the liver proteomic data, these metabolomic findings demonstrate that CBS deficiency drives coordinated dysregulation of protein and metabolite networks in HHcy livers, all of which are normalized by AAV-DJ-hCBS therapy.

2.4. Plasma Proteome Is Minimally Affected by CBS Deficiency, and Systemic Effects Are Driven by Metabolite Disturbances

Next, we explored whether the impact of Cbs-/- was reflected in the blood plasma proteome. LC–MS detected 379 proteins. CBS deficiency did not substantially alter the plasma proteome, and consequently, no proteins differed across groups. As a result, PCA showed strong overlap among WT, Cbs-/- + GFP, and Cbs-/- + hCBSlow mice (Figure 5A). To assess hepatic synthetic function, we curated 84 plasma proteins that are synthesized by the liver and involved in the acute-phase response, lipid and lipoprotein metabolism, coagulation, and complement pathways. Of these, only haptoglobin was increased in Cbs-/- + GFP compared with WT and Cbs-/- + hCBSlow, while all other liver-derived plasma proteins not significantly altered. These results suggest minimal impact of CBS deficiency on hepatic plasma protein synthesis and secretion.
In addition, we explored the blood plasma metabolome. In contrast to the proteome, plasma metabolomics revealed systemic alterations in Cbs-/- + GFP mice that were fully normalized by AAV-DJ-hCBS. Across all samples, 510 metabolites were detected, of which 217 were retained after high-confidence annotation. PCA showed clear separation of Cbs-/- + GFP from WT and Cbs-/- + hCBSlow (Figure 5B). Thirteen plasma metabolites differed between Cbs-/- + GFP and WT, including elevated SAM, homocysteine, and homocystine and reduced isoleucine, choline, taurine, GSSG, glucosamine, and hexose in Cbs-/-. Hierarchical clustering confirmed complete normalization in Cbs-/- + hCBSlow (Figure 5C). CBS deficiency perturbs metabolites in a compartment-specific manner, with systemic depletion of redox-related metabolites and branched-chain amino acids in plasma and localized alterations in amino acid modification and mitochondrial function in the liver. Plasma metabolites altered by CBS deficiency included choline, taurine, oxidized glutathione (GSSG), and isoleucine, whereas liver-specific changes affected N-acetylated amino acid derivatives (N-acetylhistidine, N-acetylornithine, N6-acetyllysine), carnitine, and 3-hydroxyproline.
Although direct folate and vitamin B6 species were not detected by LC-MS, 4-pyridoxic acid, the primary catabolite of vitamin B6, was detected but not significantly altered (p = 0.07), suggesting preserved vitamin B6 catabolism under CBS deficiency.

3. Discussion

In this inducible adult-onset Cbs-/- mouse model, we observed the anticipated phenotype of severe HHcy (~200 µM), growth retardation, reduced adiposity, and alopecia. Liver-targeted AAV-DJ-hCBS gene therapy was well tolerated and provided sustained correction of HHcy together with rescue of these phenotypic abnormalities. Multi-omics profiling revealed extensive hepatic metabolic remodeling in untreated Cbs-/- mice, including suppression of tRNA aminoacylation and protein translation pathways, activating lipid and carbon metabolism, and marked disruptions in methyl-cycle intermediates. Unlike prior AAV-based CBS rescue studies that primarily focused on homocysteine lowering [11], our data demonstrate that selective hepatic CBS restoration is sufficient to re-establish systemic methylation flux and fully reverse coordinated metabolic remodeling in both the liver and plasma. These findings identify the liver as the dominant metabolic control point in CBS deficiency.
The extensive hepatic metabolic remodeling observed in Cbs-/- mice suggests that phenotype development is not solely a consequence of homocysteine toxicity or secondary disturbances in glutathione metabolism. Instead, our findings point to dysregulation of the methylation cycle, as evidenced by the accumulation of SAH and SAM. It is well established that SAH inhibits SAM-dependent methyltransferases at multiple levels [35,36,37]. In our dataset, this is reflected by reduced levels of glycerophospholipids, whose synthesis critically depends on SAM-mediated methylation of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) by the enzyme phosphatidylethanolamine N-methyltransferase (PEMT) [38,39,40,41]. Consistently, we previously demonstrated reduced hepatic acyl-carnitines in Cbs-/- mice [42], which derive from SAM-dependent methylation of trimethyllysine [38]. At the transcriptional level, reduced methylation is further supported by the enrichment of a HNF4A signature among upregulated proteins, in line with prior evidence that HNF4A expression can be increased through promoter demethylation [43,44,45]. Finally, at the translational level, we observed downregulation of tRNA metabolism and aminoacylation, processes dependent on methyltransferases such as NSUN2, whose loss destabilizes tRNAs and impairs aminoacylation efficiency [34,35,36,37].
The role of methylation-driven alterations in hepatic metabolism of Cbs-/- mice is further supported by observations from other methyl-cycle knockout models, which display related biochemical disturbances. SAM synthase-deficient Mat1a-/- mice show reduced hepatic SAM, impaired PEMT activity, and an altered PC/PE balance [46,47,48]. Further, GNMT-/- mice, which accumulate excess SAM due to loss of glycine N-methyltransferase, exhibit similar disturbances, including disrupted methyl flux with global DNA hypomethylation and altered carnitine and acylcarnitine metabolism [49,50,51,52]. Finally, AHCY-/- mice which are deficient for SAH hydrolase (SAHH) are embryonic lethal due to SAH-mediated inhibition of methyltransferases [53], a phenotype comparable to the embryonic lethality of Cbs-/- mice [54]. In contrast, the macrocytic anemia we observe in mice appears unique to CBS deficiency, likely reflecting diversion of folate pools into remethylation of homocysteine at the expense of thymidylate biosynthesis, resulting in impaired DNA synthesis and erythropoiesis [55,56,57]. Macrocytic anemia has only been reported in one other mouse study of Cbs-/-. The absence of this phenotype in our previous study [26] likely reflects differences in genetic background; that model was maintained on a mixed 129/SvEv × C57BL/6 background, in which strain-specific differences in erythrocyte parameters [58] and residual 129-derived genomic regions [59] may have masked this phenotype.
In humans, several case reports have shown macrocytic anemia in classical homocystinuria that could be corrected upon folate supplementation [60,61,62].
Mechanistically, SAH-driven hypomethylation and HNF4α-biased catabolic signaling converge on hepatic lipid handling and whole-body nutrient partitioning. PEMT-mediated phosphatidylcholine (PC) synthesis requires three methyl transfers and is directly inhibited by SAH, limiting PEMT flux, lowering the hepatic PC:PE ratio, and impairing VLDL assembly/secretion mechanisms tightly linked to steatohepatitis in mice and humans [63,64,65,66,67]. In parallel, HNF4A activation promotes fatty acid oxidation and acylcarnitine metabolism, enabling redistribution of fuels to peripheral tissues [68,69]. These shifts are consistent with a protein-sparing, catabolic state, supported by reduced circulating branched-chain amino acids under fasting/protein restriction [70]. Collectively, the hepatic and systemic metabolic changes provide a mechanistic basis for the hallmark phenotypes of CBS-deficient mice, including growth retardation, alopecia, and diminished fat mass [71,72,73]. Notably, selective restoration of CBS expression in the liver is sufficient to normalize these outcomes, even in the context of persistent extrahepatic deficiency, underscoring the liver as the central hub linking methylation flux to systemic metabolic homeostasis [11].
The sole restoration of liver CBS expression was sufficient to normalize systemic biochemical and organismal phenotypes. Despite persistent extrahepatic CBS loss in this model, systemic metabolic homeostasis is maintained, indicating dominance of hepatic methylation control.
More broadly, our findings support reframing hyperhomocysteinemia as a disorder of disrupted methylation flux rather than direct homocysteine toxicity, with SAH as the proximal lesion linking sulfur amino acid metabolism to systemic metabolic dysregulation.
This raises the possibility that restoring systemic methylation balance through liver-targeted CBS therapy could also mitigate complications in other conditions where hyperhomocysteinemia is observed, such as non-alcoholic fatty liver disease [74,75,76,77] and cardiovascular disease [78,79,80]. At the same time, it will be important to assess tissue-specific responses, as SAH accumulation may induce distinct gene programs—such as HNF4A activation in liver—through methylation imbalance. Whether hyperhomocysteinemia in humans likewise reflects primarily a disorder of methylation flux remains an open question. Clinical observations suggest that SAH more strongly correlates with cardiovascular disease than homocysteine [81], and emerging evidence indicates that this relationship may also apply to liver disease [82]. Definitive evidence, however, will require mapping locus-specific methylation changes and linking them to tissue dysfunction in patients. Together, these findings support the view that hyperhomocysteinemia is best understood as a methylation disorder, with SAH as the proximal lesion connecting one-carbon metabolism to systemic disease.

4. Materials and Methods

4.1. AAV Vector Design and Production

Recombinant AAV vectors encoding human CBS (AAV-DJ-hCBS) or GFP were produced in HEK293F (ThermoFisher, Waltham, MA, USA) cells by triple-plasmid transfection using pHelper, pRep/Cap (AAV-DJ) [22], and transgene plasmids at standard mass ratios. Transfections were performed using FectorVIR (101000004, Polyplus, SA, France), and cells were harvested 72 h post-transfection. AAV particles were released by detergent lysis (1× PBS, 1 mM MgCl2, 0.5% Triton X-100; ThermoFisher, Waltham, MA, USA) and purified on an ÄKTA pure 25 system (Cytiva, Marlborough, MA, USA), followed by CsCl ultracentrifugation to deplete empty capsids. Final vector preparations were formulated in buffered solution and stored at −80 °C.

4.2. Animal Treatment

All animal experiments and protocols were conducted in accordance with applicable regulations and were approved by the Institutional Animal Care and Use Committee of the University of Groningen (IVD), as well as the Dutch Central Authority for Scientific Procedures on Animals (CCD; AVD1050020186924, approved on 1 October 2019). Cbsfl/fl mice were generated as previously described [26] and subsequently backcrossed for at least 10 generations onto a C57BL/6 genetic background. Mice were bred in the animal facility of the University Medical Center Groningen (UMCG) and were allowed to acclimatize to the experimental animal facility for at least two weeks. Both sexes were included, and mice were housed individually under conventional conditions with a 12 h light/dark cycle, controlled temperature (22 ± 2 °C), and ad libitum access to food and water. Additional nesting material (cotton pads) was provided to all groups to mitigate potential heat loss associated with the reduced coat quality of mice with alopecia.
Mice received tamoxifen injections (75 mg/kg body weight, i.p.) for five consecutive days starting at 10 weeks of age to induce CBS deletion. At 11 weeks, plasma homocysteine levels were measured, and all CBS-knockout mice exhibited hyperhomocysteinemia within the target range (100–350 µM). At 12 weeks, anaesthetized mice (2% isoflurane in O2) received a single intravenous injection via the retro orbital plexus (maximum volume 100 µL) of AAV-DJ-hCBS either 3 × 1012 vg/kg; (hCBSlow), to restore hepatic CBS expression to physiological levels, or at 3 × 1013 vg/kg (hCBShigh) to assess potential effects of supraphysiological expression. Mice in the GFP group received AAV-DJ-GFP at 3 × 1013 vg/kg and served as empty vector controls. Age-matched wild-type littermates served as controls (WT) and did not receive tamoxifen or AAV treatment. All treatments were carried out in a randomized order to minimize potential confounding effects.
Cbs-/- mice were randomized into treatment groups balanced for plasma homocysteine, body weight, and sex (hCBSlow, n = 9; hCBShigh, n = 10; GFP, n = 8). Including WT mice (n = 5), a total of 32 mice were used for the experiments. Group sizes were determined by an a priori calculation assuming a 50% reduction in hyperhomocysteinemia in hCBS versus GFP, yielding n = 7 (two-tailed, effect size = 1.667, α = 0.05, power = 0.8), with 1–2 additional mice included to account for potential mortality. All animals were monitored for 48 weeks with serial measurements of plasma homocysteine, body weight, body composition, and external phenotype. Body weight was recorded weekly. Blood samples for homocysteine measurement were obtained via orbital puncture every 4 weeks from weeks 2 to 20 and every 8 weeks from week 20 to week 40. A final homocysteine measurement was conducted on blood drawn by cardiac puncture at termination (week 48). Body composition and external phenotype characterization were assessed concurrently with homocysteine measurements.
Two mice in the Cbs-/- + GFP group died of unknown causes at 1 and 37 weeks after injection. One mouse in the Cbs-/- + hCBSlow group was euthanized at week 47 due to rapid body weight loss (humane endpoint >15% body weight loss). Body composition (fat, lean, and fluid mass) was assessed by minispec LF Series (Bruker, Billerica, MA, USA).
At study termination (48 weeks), mice were anesthetized with isoflurane and euthanized by exsanguination. Blood was collected in EDTA tubes, and organs were harvested for snap-freezing in liquid nitrogen or fixation in 4% buffered formaldehyde.

4.3. Homocysteine

Approximately 50 µL of blood was collected from the retro-orbital sinus of mice under isoflurane anesthesia (2%). Samples were collected in EDTA tubes, and plasma was separated by centrifugation at 2500 g for 10 min. For automated homocysteine measurement, 15 µL of plasma was diluted in 185 µL of multi-assay diluent (Abbott, Abbott Park, IL, USA) and analyzed by chemiluminescent microparticle immunoassay on an Alinity analyzer (Abbott, Abbott Park, IL, USA). The assay provided a lower limit of detection of 6–8 µM.

4.4. Western Blot Analysis

Liver tissue samples were homogenized in ice-cold RIPA buffer (Igepal CA-630, sodium deoxycholate, and 20% SDS in PBS) supplemented with a protease inhibitor cocktail (Roche Diagnostics, Rotkreuz, Switzerland), sodium orthovanadate (Sigma-Aldrich, St. Louis, MO, USA), and β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA). Total protein concentration was determined using the Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of protein (25 µg in 25 µL) were resolved by SDS–PAGE (Bio-Rad Laboratories, Hercules, CA, USA) and transferred to nitrocellulose membranes using the Trans-Blot Turbo system (Bio-Rad Laboratories, Hercules, CA, USA). Membranes were blocked with 5% skim milk (Sigma-Aldrich, St. Louis, MO, USA) for 20 min and incubated overnight at 4 °C with anti-CBS antibody (CBS D8F2P Rabbit Monoclonal Antibody #14782, 1:1000; Cell Signaling Technology, Danvers, MA, USA). After three washes with TBST, membranes were incubated with HRP-conjugated goat anti-rabbit secondary antibody (1:2000; Dako, Glostrup, Denmark) for 2 h at room temperature. Chemiluminescent signals were detected using Western Lightning Ultra (PerkinElmer, Waltham, MA, USA). Blots were reprobed with beta-actin antibody (beta-actin C-7 HRP, 1:1000, Santa Cruz sc-47778, Santa Cruz Biotechnology, Dallas, TX, USA), and CBS expression was expressed as the ratio of CBS over beta-actin.

4.5. Droplet Digital PCR (ddPCR)

DNA copy number was determined using the QX200 Droplet Digital PCR system (Bio-Rad Laboratories, Hercules, CA, USA). Reactions (22 μL) contained 11 μL 2× ddPCR Supermix for Probes (no dUTP; Bio-Rad Laboratories, Hercules, CA, USA, Cat. No. 10026868), 0.4 μL of forward primer (100 μM, 5′-GCCAAGTGTGAGTTCTTCAAC-3′), 0.4 μL of reverse primer (100 μM, 5′-CGGATGTCGGCTCGATAAT-3′), 0.2 μL of probe (100 μM, 5′-CCTGCGGATGATTGAGGATGCTGA-3′), 5 μL of nuclease-free H2O, and 5 μL of DNA template. Droplets were generated with a QX200 Droplet Generator and amplified under the following conditions: 95 °C for 10 min; 40 cycles of 94 °C for 30 s and 60 °C for 1 min; 98 °C for 10 min; 4 °C hold. Droplets were analyzed with a QX200 Droplet Reader, and copy number was calculated using QuantaSoft Analysis Pro version 1.7.4.0917 (Bio-Rad Laboratories, Hercules, CA, USA).

4.6. Metabolomics

Sample analysis was carried out by MS-Omics as follows. The analysis was carried out using a Thermo Scientific Vanquish LC coupled to an Orbitrap Exploris 240 MS (Thermo Fisher Scientific, Waltham, MA, USA). An electrospray ionization interface was used as ionization source. Analysis was performed in positive and negative ionization mode under polarity switching. The UPLC was performed using a slightly modified version of the protocol described by Doneanu et al. (UPLC/MS Monitoring of Water-Soluble Vitamin Bs in Cell Culture Media in Minutes, Water Application note 2011, 720004042en). Peak areas were extracted using Compound Discoverer 3.3 (Thermo Fisher Scientific, Waltham, MA, USA). Compound identification was performed at four levels as follows. Level 1: identification by retention times (compared against in-house authentic standards), accurate mass (with an accepted deviation of 3 ppm), and MS/MS spectra. Level 2a: identification by retention times (compared against in-house authentic standards) and accurate mass (with an accepted deviation of 3 ppm). Level 2b: identification by accurate mass (with an accepted deviation of 3 ppm) and MS/MS spectra. Level 3: identification by accurate mass alone (with an accepted deviation of 3 ppm).
For downstream analyses, only compounds annotated at Levels 1, 2a, or 2b were retained. Data were normalized and subjected to multivariate analysis, differential testing, and pathway enrichment. KEGG pathway enrichment was performed using MetaboAnalyst 6.0 (https://www.metaboanalyst.ca, accessed December 2025 through March 2026).

4.7. Proteomics

Liver lysates were mixed with LDS loading buffer (Thermo Fisher Scientific, Waltham, MA, USA) to a final protein concentration of 10 µg, incubated for 10 min at 70 °C, and briefly electrophoresed for approximately 5 min at 100 V on 4–12% Bis-Tris gels (Thermo Fisher Scientific, Waltham, MA, USA). After Coomassie staining (Westburg, Leusden, The Netherlands) and destaining with Milli-Q water, the single protein band was excised, diced, and washed sequentially with 30% and 50% (v/v) acetonitrile in 100 mM of ammonium bicarbonate for 30 min each at 500 rpm, followed by 100% acetonitrile for 5 min at 37 °C. Proteins were reduced with 10 mM of dithiothreitol for 30 min at 55 °C and alkylated with 55 mM of iodoacetamide for 30 min at room temperature in the dark. After dehydration, proteins were digested overnight with sequencing-grade trypsin (5 ng/µL; Promega, Madison, WI, USA) at 37 °C. Peptides were extracted with 75% acetonitrile containing 5% formic acid for 20 min at room temperature, dried under vacuum, and resuspended in 0.1% formic acid to a final concentration of 0.05 µg/µL.
Discovery-based proteomic analyses were performed on an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) coupled to an Evosep One LC system equipped with a 15 cm × 150 µm, 1.5 µm column (Evosep, Odense, Denmark) using the 30SPD gradient. Mobile phases consisted of 0.1% formic acid in water (buffer A) and 0.1% formic acid in acetonitrile (buffer B). The instrument was operated in positive-ion HRMS1-direct data-independent acquisition (DIA) mode across a 400–1000 m/z range with 16 m/z isolation windows and FAIMS compensation voltages of −45 V and −60 V.
Raw DIA data were processed with Spectronaut v19.1 (Biognosys, Schlieren, Switzerland) using the directDIA workflow against the Mus musculus UniProt database (17,141 entries). Quantification was performed at the MS1 level with and without local normalization.
Duplicate protein identifiers were collapsed to single entries, and proteins with more than 70% missing values across all samples were excluded from further analysis. At the sample level, those with fewer than 30% of total proteins detected were also excluded. Remaining missing values were imputed per protein, and intensities were log2-transformed to yield a normalized dataset.
Data were subjected to multivariate analysis, differential testing, and pathway enrichment. Pathway enrichment was carried out in R using the gprofiler2 package, with all detected proteins as background. To evaluate hepatic synthetic function, a curated plasma protein panel was assembled encompassing acute-phase response, lipid and lipoprotein metabolism, coagulation, and complement pathways, based on liver-derived proteins and Reactome pathway sets (“COMPLEMENT,” “COAGULATION,” and “LIPOPROTEIN|CHYLOMICRON”).

4.8. Histology and Immunohistochemistry

Liver samples fixed in 4% buffered formaldehyde were embedded in paraffin, and 4 µm sections were prepared using a microtome (HistoCore AUTOCUT, Leica Microsystems, Wetzlar, Germany). Sections were mounted on glass slides (StarFrost, Ahrensburg, Germany), deparaffinized, and rehydrated through a graded ethanol series (100%, 96%, and 70%; 10 min each).
For hematoxylin and eosin (H&E) staining, sections were stained with Mayer’s hematoxylin (5 min; Sigma-Aldrich, St. Louis, MO, USA), rinsed in tap water, counterstained with eosin (5 min), and mounted with DePeX resin (Serva, Heidelberg, Germany).
For Sirius Red staining, sections were deparaffinized, rehydrated, and stained with 0.1% Sirius Red F3B (Merck KGaA, Darmstadt, Germany, Direct Red 80) in saturated picric acid for 1 h, followed by washing in acidified water (0.5% glacial acetic acid), dehydration through 3 changes in 100% ethanol, and mounting with DePeX.
For CBS immunohistochemistry, sections were deparaffinized and subjected to heat-induced epitope retrieval overnight at 60 °C in citrate buffer (pH 6.0; Sigma-Aldrich, St. Louis, MO, USA). After cooling to room temperature and washing in PBS (Klinipath, Duiven, The Netherlands), endogenous peroxidase activity was quenched with 0.3% hydrogen peroxide (30 min; Sigma-Aldrich, St. Louis, MO, USA). Sections were blocked with 10% goat serum in 1% BSA (Dako, Glostrup, Denmark) for 30 min, followed by overnight incubation at 4 °C with rabbit anti-CBS antibody (1:200, Cell Signaling Technology, Danvers, MA, USA, for liver and skin; 1:5000, Proteintech, Rosemont, IL, USA, for kidney) in PBS containing 1% BSA and 1% goat serum. After three PBS washes, slides were incubated for 1 h at room temperature with HRP-conjugated goat anti-rabbit secondary antibody (1:100, Dako, Glostrup, Denmark), developed with DAB chromogen (Abcam, Cambridge, UK) for 10 min, counterstained with hematoxylin (30 s), dehydrated, and mounted with DePeX.

4.9. Data Analysis

Due to the nature of the intervention, blinding during treatment administration was not feasible; however, outcome assessment and data analysis were performed by investigators blinded to group allocation. Reduction in plasma homocysteine levels was defined as the primary outcome and served as the basis for sample size calculation. Secondary outcomes comprised phenotypic changes (including body weight and alopecia), as well as molecular alterations assessed by proteomic and metabolomic analyses.
Data integration and statistical analyses were performed in R (version 4.4.2; RStudio version 2025.09.0 + 387) and Python (version 3.12.11). Data preprocessing, normalization, and visualization were conducted using the tidyverse, ggplot2, and ComplexHeatmap packages in R and with pandas, numpy, and matplotlib in Python. Principal component analysis (PCA) and hierarchical clustering were applied to assess multivariate structure and group separation across proteomic, metabolomic, and biochemical datasets. PCA was performed using scaled and centered log2-transformed data (FactoMineR and factoextra in R), and cluster dendrograms were generated from Euclidean distance matrices with complete linkage.
Univariate testing was performed using one-way ANOVA or Student’s t-test, depending on the experimental design, implemented in both R (stats and car packages) and Python (scipy.stats). p values were corrected for multiple testing using the Benjamini–Hochberg false discovery rate (FDR) method, and features with FDR < 0.05 were considered significantly different.
Graphical representations were finalized in R and Python and prepared for publication using Adobe Illustrator version 30.2 (Adobe Inc., San Jose, CA, USA).
All data are presented as the mean ± standard error of the mean, unless otherwise specified.

4.10. Generative AI Statement

Large language model-based generative AI tools (ChatGPT, OpenAI, GPT-5.2; accessed 2026) were used for language editing and for debugging data analysis code. No AI tools were used for data generation, analysis decisions, or interpretation. All scientific content and conclusions were verified by the authors.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms27073338/s1.

Author Contributions

C.P.J.: conceptualization, methodology, software, formal analysis, visualization, writing—original draft; C.-C.W.: conceptualization, methodology, investigation, writing—review and editing; A.v.B.: investigation; M.G.: investigation; F.H.-B.: investigation; J.W.: investigation; J.H.B.: investigation; Y.C.: investigation; N.J.: investigation; J.D.: investigation; S.H.: investigation; M.N.: conceptualization, methodology, project administration, writing—review and editing; M.J.C.: methodology, project administration, writing—review and editing; R.H.H.: supervision, writing—review and editing; L.E.D.: conceptualization, methodology, formal analysis, investigation, data curation, supervision, project administration, writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by GUIDE and University Medical Center Groningen (M.D./Ph.D. grant to C.P.J.; No. 124533). This work was supported by Alexion Pharmaceuticals, Astra Zeneca Rare Disease (Research agreement No. 117008).

Institutional Review Board Statement

The animal study protocol was approved by the Institutional Review Board of Dutch Animal Experiments Committee (CCD) (protocol code AVD1050020186924 and approved on 1 October 2019).

Informed Consent Statement

Not applicable.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors on request.

Acknowledgments

We thank J.C. Wolters and the Interfaculty Mass Spectrometry Center (IMSC), University Medical Center Groningen, for performing the LC–MS–based proteomics analyses and for providing guidance during the initial setup and data processing stages.

Conflicts of Interest

Authors C.-C.W., J.W., J.H.B., Y.C., N.J., J.D., S.H., M.N., and M.J.C. are presently or were previously employed by Alexion Pharmaceuticals, AstraZeneca Rare Disease, and may be Astra Zeneca shareholders. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Abbreviations

The following abbreviations are used in this manuscript:
AAVAdeno-associated virus
CBSCystathionine β-synthase
DAPDifferentially abundant protein
ddPCRDroplet digital polymerase chain reaction
GFPGreen fluorescent protein
GO:BPGene Ontology: Biological Process
GSSGOxidized glutathione
H&EHematoxylin and eosin
hCBSHuman cystathionine β-synthase
HCTHematocrit
HCUClassic homocystinuria
HcyHomocysteine
HGBHemoglobin
HHcyHyperhomocysteinemia
HNF4αHepatocyte nuclear factor 4 alpha
KEGGKyoto Encyclopedia of Genes and Genomes
LC–MSLiquid chromatography–mass spectrometry
MCHMean corpuscular hemoglobin
PCPhosphatidylcholine
PCAPrincipal component analysis
PEPhosphatidylethanolamine
PEMTPhosphatidylethanolamine N-methyltransferase
PLPPyridoxal 5′-phosphate
RBCRed blood cell
SAHS-adenosylhomocysteine
SAMS-adenosylmethionine
WTWild type

References

  1. Morris, A.A.M.; Kožich, V.; Santra, S.; Andria, G.; Ben-Omran, T.I.M.; Chakrapani, A.B.; Crushell, E.; Henderson, M.J.; Hochuli, M.; Huemer, M.; et al. Guidelines for the Diagnosis and Management of Cystathionine Beta-Synthase Deficiency. J. Inherit. Metab. Dis. 2017, 40, 49–74. [Google Scholar] [CrossRef] [Scilit]
  2. Mudd, S.H.; Skovby, F.; Levy, H.L.; Pettigrew, K.D.; Wilcken, B.; Pyeritz, R.E.; Andria, G.; Boers, G.H.; Bromberg, I.L.; Cerone, R.; et al. The Natural History of Homocystinuria Due to Cystathionine Beta-Synthase Deficiency. Am. J. Hum. Genet. 1985, 37, 1–31. [Google Scholar]
  3. Orendác, M.; Kozich, V.; Zeman, J.; Hyánek, J.; Bzdúch, V.; Misovicová, N.; Marklová, E.; Vad’urová, L.; Pijácková, A. Clinical picture of homocystinuria with cystathionine beta-synthase deficiency in 19 Czech and Slovak patients. Cas. Lek. Cesk 2000, 139, 500–507. [Google Scholar] [PubMed]
  4. Yap, S.; Boers, G.H.; Wilcken, B.; Wilcken, D.E.; Brenton, D.P.; Lee, P.J.; Walter, J.H.; Howard, P.M.; Naughten, E.R. Vascular Outcome in Patients with Homocystinuria Due to Cystathionine Beta-Synthase Deficiency Treated Chronically: A Multicenter Observational Study. Arter. Thromb. Vasc. Biol. 2001, 21, 2080–2085. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  5. Yap, S.; Naughten, E.R.; Wilcken, B.; Wilcken, D.E.; Boers, G.H. Vascular Complications of Severe Hyperhomocysteinemia in Patients with Homocystinuria Due to Cystathionine Beta-Synthase Deficiency: Effects of Homocysteine-Lowering Therapy. Semin. Thromb. Hemost. 2000, 26, 335–340. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  6. Skovby, F.; Gaustadnes, M.; Mudd, S.H. A Revisit to the Natural History of Homocystinuria Due to Cystathionine β-Synthase Deficiency. Mol. Genet. Metab. 2010, 99, 1–3. [Google Scholar] [CrossRef] [Scilit]
  7. Majtan, T.; Olsen, T.; Sokolova, J.; Krijt, J.; Křížková, M.; Ida, T.; Ditrói, T.; Hansikova, H.; Vit, O.; Petrak, J.; et al. Deciphering Pathophysiological Mechanisms Underlying Cystathionine Beta-Synthase-Deficient Homocystinuria Using Targeted Metabolomics, Liver Proteomics, Sphingolipidomics and Analysis of Mitochondrial Function. Redox Biol. 2024, 73, 103222. [Google Scholar] [CrossRef] [Scilit]
  8. Bublil, E.M.; Majtan, T.; Park, I.; Carrillo, R.S.; Hůlková, H.; Krijt, J.; Kožich, V.; Kraus, J.P. Enzyme Replacement with PEGylated Cystathionine β-Synthase Ameliorates Homocystinuria in Murine Model. J. Clin. Investig. 2016, 126, 2372–2384. [Google Scholar] [CrossRef] [Scilit]
  9. Majtan, T.; Pey, A.L.; Gimenez-Mascarell, P.; Martínez-Cruz, L.A.; Szabo, C.; Kožich, V.; Kraus, J.P. Potential Pharmacological Chaperones for Cystathionine Beta-Synthase-Deficient Homocystinuria. In Targeting Trafficking in Drug Development; Ulloa-Aguirre, A., Tao, Y.-X., Eds.; Handbook of Experimental Pharmacology; Springer International Publishing: Cham, Switzerland, 2017; Volume 245, pp. 345–383. [Google Scholar]
  10. Park, I.; Bublil, E.M.; Glavin, F.; Majtan, T. Interplay of Enzyme Therapy and Dietary Management of Murine Homocystinuria. Nutrients 2020, 12, 2895. [Google Scholar] [CrossRef] [Scilit]
  11. Lee, H.-O.; Salami, C.O.; Sondhi, D.; Kaminsky, S.M.; Crystal, R.G.; Kruger, W.D. Long-Term Functional Correction of Cystathionine β-Synthase Deficiency in Mice by Adeno-Associated Viral Gene Therapy. J. Inherit. Metab. Dis. 2021, 44, 1382–1392. [Google Scholar] [CrossRef] [Scilit]
  12. Gus, P.I.; Donis, K.C.; Marinho, D.; Martins, T.F.; de Souza, C.F.M.; Carloto, R.B.; Leivas, G.; Schwartz, I.V.D. Ocular Manifestations in Classic Homocystinuria. Ophthalmic Genet. 2021, 42, 71–74. [Google Scholar] [CrossRef] [Scilit]
  13. Kalil, M.A.B.; Donis, K.C.; Poswar, F.d.O.; dos Santos, B.B.; Santos, Â.B.S.; Schwartz, I.V.D. Cardiovascular Findings in Classic Homocystinuria. Mol. Genet. Metab. Rep. 2020, 25, 100693. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  14. Magner, M.; Krupková, L.; Honzík, T.; Zeman, J.; Hyánek, J.; Kožich, V. Vascular Presentation of Cystathionine Beta-Synthase Deficiency in Adulthood. J. Inherit. Metab. Dis. 2011, 34, 33–37. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  15. Poloni, S.; Leistner-Segal, S.; Bandeira, I.C.; D’Almeida, V.; de Souza, C.F.M.; Spritzer, P.M.; Castro, K.; Tonon, T.; Nalin, T.; Imbard, A.; et al. Body Composition in Patients with Classical Homocystinuria: Body Mass Relates to Homocysteine and Choline Metabolism. Gene 2014, 546, 443–447. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  16. Kim, C.E.; Gallagher, P.M.; Guttormsen, A.B.; Refsum, H.; Ueland, P.M.; Ose, L.; Følling, I.; Whitehead, A.S.; Tsai, M.Y.; Kruger, W.D. Functional Modeling of Vitamin Responsiveness in Yeast: A Common Pyridoxine-Responsive Cystathionine β-Synthase Mutation in Homocystinuria. Hum. Mol. Genet. 1997, 6, 2213–2221. [Google Scholar] [CrossRef] [Scilit]
  17. Al-Sadeq, D.W.; Thanassoulas, A.; Islam, Z.; Kolatkar, P.; Al-Dewik, N.; Safieh-Garabedian, B.; Nasrallah, G.K.; Nomikos, M. Pyridoxine Non-Responsive p.R336C Mutation Alters the Molecular Properties of Cystathionine Beta-Synthase Leading to Severe Homocystinuria Phenotype. Biochim. Biophys. Acta (BBA)-Gen. Subj. 2022, 1866, 130148. [Google Scholar] [CrossRef] [Scilit]
  18. Collard, R.; Majtan, T. Genetic and Pharmacological Modulation of Cellular Proteostasis Leads to Partial Functional Rescue of Homocystinuria-Causing Cystathionine-Beta Synthase Variants. Mol. Cell. Biol. 2023, 43, 664–674. [Google Scholar] [CrossRef] [Scilit]
  19. Majtan, T.; Park, I.; Cox, A.; Branchford, B.R.; di Paola, J.; Bublil, E.M.; Kraus, J.P. Behavior, Body Composition, and Vascular Phenotype of Homocystinuric Mice on Methionine-Restricted Diet or Enzyme Replacement Therapy. FASEB J. 2019, 33, 12477–12486. [Google Scholar] [CrossRef] [Scilit]
  20. Petrosino, M.; Zuhra, K.; Mijatovic, E.; Philipp, T.M.; Bremer, O.; Ascenção, K.; Szabo, C.; Majtan, T. Givinostat Rescues Folding of Cystathionine Beta-Synthase and Ameliorates Murine Homocystinuria. Biochem. Pharmacol. 2025, 239, 117079, Correction in Biochem. Pharmacol. 2026, 248, 117842.. [Google Scholar] [CrossRef] [Scilit]
  21. Poloni, S.; Sperb-Ludwig, F.; Borsatto, T.; Hoss, G.W.; Doriqui, M.J.R.; Embiruçu, E.K.; Boa-Sorte, N.; Marques, C.; Kim, C.A.; de Souza, C.F.M.; et al. CBS Mutations Are Good Predictors for B6-Responsiveness: A Study Based on the Analysis of 35 Brazilian Classical Homocystinuria Patients. Mol. Genet. Genom. Med. 2018, 6, 160–170, Correction in Mol. Genet. Genom. Med. 2018, 6, 861.. [Google Scholar] [CrossRef] [Scilit]
  22. Grimm, D.; Lee, J.S.; Wang, L.; Desai, T.; Akache, B.; Storm, T.A.; Kay, M.A. In Vitro and In Vivo Gene Therapy Vector Evolution via Multispecies Interbreeding and Retargeting of Adeno-Associated Viruses. J. Virol. 2008, 82, 5887–5911. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  23. Tissue Expression of CBS—Summary—The Human Protein Atlas. Available online: https://www.proteinatlas.org/ENSG00000160200-CBS/tissue (accessed on 7 January 2026).
  24. Ventura, P.; Rosa, M.C.; Abbati, G.; Marchini, S.; Grandone, E.; Vergura, P.; Tremosini, S.; Zeneroli, M.L. Hyperhomocysteinaemia in Chronic Liver Diseases: Role of Disease Stage, Vitamin Status and Methylenetetrahydrofolate Reductase Genetics. Liver Int. 2005, 25, 49–56. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  25. Look, M.P.; Riezler, R.; Reichel, C.; Brensing, K.A.; Rockstroh, J.K.; Stabler, S.P.; Spengler, U.; Berthold, H.K.; Sauerbruch, T. Is the Increase in Serum Cystathionine Levels in Patients with Liver Cirrhosis a Consequence of Impaired Homocysteine Transsulfuration at the Level of Gamma-Cystathionase? Scand. J. Gastroenterol. 2000, 35, 866–872. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  26. Nakladal, D.; Lambooy, S.P.H.; Mišúth, S.; Čepcová, D.; Joschko, C.P.; van Buiten, A.; Goris, M.; Hoogstra-Berends, F.; Kloosterhuis, N.J.; Huijkman, N.; et al. Homozygous Whole Body Cbs Knockout in Adult Mice Features Minimal Pathology during Ageing despite Severe Homocysteinemia. FASEB J. 2022, 36, e22260. [Google Scholar] [CrossRef] [Scilit]
  27. Hayashi, Y.; Utsuyama, M.; Kurashima, C.; Hirokawa, K. Spontaneous Development of Organ-Specific Autoimmune Lesions in Aged C57BL/6 Mice. Clin. Exp. Immunol. 1989, 78, 120–126. [Google Scholar]
  28. Nakai, H.; Montini, E.; Fuess, S.; Storm, T.A.; Grompe, M.; Kay, M.A. AAV Serotype 2 Vectors Preferentially Integrate into Active Genes in Mice. Nat. Genet. 2003, 34, 297–302. [Google Scholar] [CrossRef] [Scilit]
  29. Fausto, N.; Laird, A.D.; Webber, E.M. Liver Regeneration. 2. Role of Growth Factors and Cytokines in Hepatic Regeneration. FASEB J. 1995, 9, 1527–1536. [Google Scholar] [CrossRef] [Scilit]
  30. Wang, J.; Lozier, J.; Johnson, G.; Kirshner, S.; Verthelyi, D.; Pariser, A.; Shores, E.; Rosenberg, A. Neutralizing Antibodies to Therapeutic Enzymes: Considerations for Testing, Prevention and Treatment. Nat. Biotechnol. 2008, 26, 901–908. [Google Scholar] [CrossRef] [Scilit]
  31. Jiskoot, W.; Kijanka, G.; Randolph, T.W.; Carpenter, J.F.; Koulov, A.; Mahler, H.-C.; Joubert, M.K.; Jawa, V.; Narhi, L.O. Mouse Models for Assessing Protein Immunogenicity: Lessons and Challenges. J. Pharm. Sci. 2016, 105, 1567–1575. [Google Scholar] [CrossRef] [Scilit]
  32. Harding, F.A.; Stickler, M.M.; Razo, J.; DuBridge, R. The Immunogenicity of Humanized and Fully Human Antibodies: Residual Immunogenicity Resides in the CDR Regions. mAbs 2010, 2, 256–265. [Google Scholar] [CrossRef] [Scilit]
  33. Arjomandnejad, M.; Dasgupta, I.; Flotte, T.R.; Keeler, A.M. Immunogenicity of Recombinant Adeno-Associated Virus (AAV) Vectors for Gene Transfer. BioDrugs 2023, 37, 311–329. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  34. Costa Verdera, H.; Kuranda, K.; Mingozzi, F. AAV Vector Immunogenicity in Humans: A Long Journey to Successful Gene Transfer. Mol. Ther. 2020, 28, 723–746. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  35. Rudenko, A.Y.; Mariasina, S.S.; Sergiev, P.V.; Polshakov, V.I. Analogs of S-Adenosyl-L-Methionine in Studies of Methyltransferases. Mol. Biol. 2022, 56, 229–250. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  36. Zhang, C.; Sultan, S.A.; Rehka, T.; Chen, X. Biotechnological Applications of S-Adenosyl-Methionine-Dependent Methyltransferases for Natural Products Biosynthesis and Diversification. Bioresour. Bioprocess. 2021, 8, 72. [Google Scholar] [CrossRef] [Scilit]
  37. Menke, M.J.; Schneider, P.; Badenhorst, C.P.S.; Kunzendorf, A.; Heinz, F.; Dörr, M.; Hayes, M.A.; Bornscheuer, U.T. A Universal, Continuous Assay for SAM-dependent Methyltransferases. Angew. Chem. Int. Ed. 2023, 62, e202313912. [Google Scholar] [CrossRef] [Scilit]
  38. Maas, M.N.; Hintzen, J.C.J.; Porzberg, M.R.B.; Mecinović, J. Trimethyllysine: From Carnitine Biosynthesis to Epigenetics. Int. J. Mol. Sci. 2020, 21, 9451. [Google Scholar] [CrossRef] [Scilit]
  39. Bremer, J. Carnitine--Metabolism and Functions. Physiol. Rev. 1983, 63, 1420–1480. [Google Scholar] [CrossRef] [Scilit]
  40. Vance, D.E. Phospholipid Methylation in Mammals: From Biochemistry to Physiological Function. Biochim. Biophys. Acta (BBA)-Biomembr. 2014, 1838, 1477–1487. [Google Scholar] [CrossRef] [Scilit]
  41. Li, J.; Xin, Y.; Li, J.; Chen, H.; Li, H. Phosphatidylethanolamine N-Methyltransferase: From Functions to Diseases. Aging Dis. 2023, 14, 879–891. [Google Scholar] [CrossRef] [Scilit]
  42. Lambooy, S.; Heida, A.; Joschko, C.; Nakladal, D.; Van Buiten, A.; Kloosterhuis, N.; Huijkman, N.; Gerding, A.; Van De Sluis, B.; Henning, R.; et al. Selective Hepatic Cbs Knockout Aggravates Liver Damage, Endothelial Dysfunction and ROS Stress in Mice Fed a Western Diet. Int. J. Mol. Sci. 2023, 24, 7019. [Google Scholar] [CrossRef] [Scilit]
  43. Zhang, N.; Geng, T.; Wang, Z.; Zhang, R.; Cao, T.; Camporez, J.P.; Cai, S.-Y.; Liu, Y.; Dandolo, L.; Shulman, G.I.; et al. Elevated Hepatic Expression of H19 Long Noncoding RNA Contributes to Diabetic Hyperglycemia. JCI Insight 2018, 3, e120304. [Google Scholar] [CrossRef] [Scilit]
  44. Ancey, P.-B.; Ecsedi, S.; Lambert, M.-P.; Talukdar, F.R.; Cros, M.-P.; Glaise, D.; Narvaez, D.M.; Chauvet, V.; Herceg, Z.; Corlu, A.; et al. TET-Catalyzed 5-Hydroxymethylation Precedes HNF4A Promoter Choice during Differentiation of Bipotent Liver Progenitors. Stem Cell Rep. 2017, 9, 264–278. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  45. Hatziapostolou, M.; Koutsioumpa, M.; Zaitoun, A.M.; Polytarchou, C.; Edderkaoui, M.; Mahurkar-Joshi, S.; Vadakekolathu, J.; D’Andrea, D.; Lay, A.R.; Christodoulou, N.; et al. Promoter Methylation Leads to Hepatocyte Nuclear Factor 4A Loss and Pancreatic Cancer Aggressiveness. Gastro Hep Adv. 2024, 3, 687–702. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  46. Lu, S.C.; Alvarez, L.; Huang, Z.-Z.; Chen, L.; An, W.; Corrales, F.J.; Avila, M.A.; Kanel, G.; Mato, J.M. Methionine Adenosyltransferase 1A Knockout Mice Are Predisposed to Liver Injury and Exhibit Increased Expression of Genes Involved in Proliferation. Proc. Natl. Acad. Sci. USA 2001, 98, 5560–5565. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  47. Ramani, K.; Lu, S.C. Methionine Adenosyltransferases in Liver Health and Diseases. Liver Res. 2017, 1, 103–111. [Google Scholar] [CrossRef] [Scilit]
  48. Capelo-Diz, A.; Lachiondo-Ortega, S.; Fernández-Ramos, D.; Cañas-Martín, J.; Goikoetxea-Usandizaga, N.; Serrano-Maciá, M.; González-Rellan, M.J.; Mosca, L.; Blazquez-Vicens, J.; Tinahones-Ruano, A.; et al. Hepatic Levels of S-Adenosylmethionine Regulate the Adaptive Response to Fasting. Cell Metab. 2023, 35, 1373–1389.e8. [Google Scholar] [CrossRef] [Scilit]
  49. Martínez-Uña, M.; Varela-Rey, M.; Mestre, D.; Fernández-Ares, L.; Fresnedo, O.; Fernandez-Ramos, D.; Juan, V.G.; Martin-Guerrero, I.; García-Orad, A.; Luka, Z.; et al. S-Adenosylmethionine Increases Circulating Very-Low Density Lipoprotein Clearance in Non-Alcoholic Fatty Liver Disease. J. Hepatol. 2015, 62, 673–681. [Google Scholar] [CrossRef] [Scilit]
  50. Martínez-Chantar, M.L.; Vázquez-Chantada, M.; Ariz, U.; Martínez, N.; Varela, M.; Luka, Z.; Capdevila, A.; Rodríguez, J.; Aransay, A.M.; Matthiesen, R.; et al. Loss of the Glycine N-methyltransferase Gene Leads to Steatosis and Hepatocellular Carcinoma in Mice†. Hepatology 2008, 47, 1191–1199. [Google Scholar] [CrossRef] [Scilit]
  51. Rome, F.I.; Hughey, C.C. Disrupted Liver Oxidative Metabolism in Glycine N-Methyltransferase-Deficient Mice Is Mitigated by Dietary Methionine Restriction. Mol. Metab. 2022, 58, 101452. [Google Scholar] [CrossRef] [Scilit]
  52. Hughey, C.C.; Trefts, E.; Bracy, D.P.; James, F.D.; Donahue, E.P.; Wasserman, D.H. Glycine N-Methyltransferase Deletion in Mice Diverts Carbon Flux from Gluconeogenesis to Pathways That Utilize Excess Methionine Cycle Intermediates. J. Biol. Chem. 2018, 293, 11944–11954. [Google Scholar] [CrossRef] [Scilit]
  53. Vizán, P.; Di Croce, L.; Aranda, S. Functional and Pathological Roles of AHCY. Front. Cell Dev. Biol. 2021, 9, 654344. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  54. Watanabe, M.; Osada, J.; Aratani, Y.; Kluckman, K.; Reddick, R.; Malinow, M.R.; Maeda, N. Mice Deficient in Cystathionine Beta-Synthase: Animal Models for Mild and Severe Homocyst(e)Inemia. Proc. Natl. Acad. Sci. USA 1995, 92, 1585–1589. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  55. Stover, P.J. One-Carbon Metabolism–Genome Interactions in Folate-Associated Pathologies. J. Nutr. 2009, 139, 2402–2405. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  56. Wilkinson, A.L.; Menelaou, K.; Rakoczy, J.; Tan, X.S.; Watson, E.D. Disruption of Folate Metabolism Causes Poor Alignment and Spacing of Mouse Conceptuses for Multiple Generations. Front. Cell Dev. Biol. 2021, 9, 723978. [Google Scholar] [CrossRef] [Scilit]
  57. Beaudin, A.E.; Abarinov, E.V.; Noden, D.M.; Perry, C.A.; Chu, S.; Stabler, S.P.; Allen, R.H.; Stover, P.J. Shmt1 and de Novo Thymidylate Biosynthesis Underlie Folate-Responsive Neural Tube Defects in Mice. Am. J. Clin. Nutr. 2011, 93, 789–798. [Google Scholar] [CrossRef] [Scilit]
  58. Mazzaccara, C.; Labruna, G.; Cito, G.; Scarfò, M.; De Felice, M.; Pastore, L.; Sacchetti, L. Age-Related Reference Intervals of the Main Biochemical and Hematological Parameters in C57BL/6J, 129SV/EV and C3H/HeJ Mouse Strains. PLoS ONE 2008, 3, e3772. [Google Scholar] [CrossRef] [Scilit]
  59. Hay, A.M.; Howie, H.L.; Gorham, J.D.; D’Alessandro, A.; Spitalnik, S.L.; Hudson, K.E.; Zimring, J.C. Mouse Background Genetics in Biomedical Research: The Devil’s in the Details. Transfusion 2021, 61, 3017–3025. [Google Scholar] [CrossRef] [Scilit]
  60. Zhao, P.; Qian, C.; Chen, Y.-J.; Sheng, Y.; Ke, Y.; Qian, Z.-M. Cystathionine β-Synthase (CBS) Deficiency Suppresses Erythropoiesis by Disrupting Expression of Heme Biosynthetic Enzymes and Transporter. Cell Death Dis. 2019, 10, 708. [Google Scholar] [CrossRef] [Scilit]
  61. Morrow, G.; Barness, L.A. Combined Vitamin Responsiveness in Homocystinuria. J. Pediatr. 1972, 81, 946–954. [Google Scholar] [CrossRef] [Scilit]
  62. Bhardwaj, P.; Sharma, R.; Sharma, M. Homocystinuria: A Rare Condition Presenting as Stroke and Megaloblastic Anemia. J. Pediatr. Neurosci. 2010, 5, 129–131. [Google Scholar] [CrossRef] [Scilit]
  63. Tehlivets, O.; Malanovic, N.; Visram, M.; Pavkov-Keller, T.; Keller, W. S-Adenosyl-L-Homocysteine Hydrolase and Methylation Disorders: Yeast as a Model System. Biochim. Biophys. Acta (BBA)-Mol. Basis Dis. 2013, 1832, 204–215. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  64. van der Veen, J.N.; Kennelly, J.P.; Wan, S.; Vance, J.E.; Vance, D.E.; Jacobs, R.L. The Critical Role of Phosphatidylcholine and Phosphatidylethanolamine Metabolism in Health and Disease. Biochim. et Biophys. Acta (BBA)-Biomembr. 2017, 1859, 1558–1572. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  65. Hoffman, D.R.; Haning, J.A.; Cornatzer, W.E. Microsomal Phosphatidylethanolamine Methyltransferase: Inhibition by S-Adenosylhomocysteine. Lipids 1981, 16, 561–567. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  66. Wan, S.; Kuipers, F.; Havinga, R.; Ando, H.; Vance, D.E.; Jacobs, R.L.; van der Veen, J.N. Impaired Hepatic Phosphatidylcholine Synthesis Leads to Cholestasis in Mice Challenged With a High-Fat Diet. Hepatol. Commun. 2019, 3, 262–276. [Google Scholar] [CrossRef] [Scilit]
  67. Nakatsuka, A.; Matsuyama, M.; Yamaguchi, S.; Katayama, A.; Eguchi, J.; Murakami, K.; Teshigawara, S.; Ogawa, D.; Wada, N.; Yasunaka, T.; et al. Insufficiency of Phosphatidylethanolamine N-Methyltransferase Is Risk for Lean Non-Alcoholic Steatohepatitis. Sci. Rep. 2016, 6, 21721. [Google Scholar] [CrossRef] [Scilit]
  68. Chen, L.; Vasoya, R.P.; Toke, N.H.; Parthasarathy, A.; Luo, S.; Chiles, E.; Flores, J.; Gao, N.; Bonder, E.M.; Su, X.; et al. HNF4 Regulates Fatty Acid Oxidation and Is Required for Renewal of Intestinal Stem Cells in Mice. Gastroenterology 2020, 158, 985–999.e9. [Google Scholar] [CrossRef] [Scilit]
  69. Huck, I.; Morris, E.M.; Thyfault, J.; Apte, U. Hepatocyte-Specific Hepatocyte Nuclear Factor 4 Alpha (HNF4α) Deletion Decreases Resting Energy Expenditure by Disrupting Lipid and Carbohydrate Homeostasis. Gene Expr. 2021, 20, 157–168. [Google Scholar] [CrossRef] [Scilit]
  70. Fontana, L.; Cummings, N.E.; Arriola Apelo, S.I.; Neuman, J.C.; Kasza, I.; Schmidt, B.A.; Cava, E.; Spelta, F.; Tosti, V.; Syed, F.A.; et al. Decreased Consumption of Branched Chain Amino Acids Improves Metabolic Health. Cell Rep. 2016, 16, 520–530. [Google Scholar] [CrossRef] [Scilit]
  71. Kruger, W.D. Cystathionine β-Synthase Deficiency: Of Mice and Men. Mol. Genet. Metab. 2017, 121, 199–205. [Google Scholar] [CrossRef] [Scilit]
  72. Gupta, S.; Kruger, W.D. Cystathionine Beta-Synthase Deficiency Causes Fat Loss in Mice. PLoS ONE 2011, 6, e27598. [Google Scholar] [CrossRef] [Scilit]
  73. Wang, L.; Chen, X.; Tang, B.; Hua, X.; Klein-Szanto, A.; Kruger, W.D. Expression of Mutant Human Cystathionine β-Synthase Rescues Neonatal Lethality but Not Homocystinuria in a Mouse Model. Hum. Mol. Genet. 2005, 14, 2201–2208. [Google Scholar] [CrossRef] [Scilit]
  74. Dai, H.; Wang, W.; Tang, X.; Chen, R.; Chen, Z.; Lu, Y.; Yuan, H. Association between Homocysteine and Non-Alcoholic Fatty Liver Disease in Chinese Adults: A Cross-Sectional Study. Nutr. J. 2016, 15, 102. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  75. Hu, Y.; Liu, J.; Dong, X.; Xu, Y.; Leng, S.; Wang, G. Clinical Study of Serum Homocysteine and Non-Alcoholic Fatty Liver Disease in Euglycemic Patients. Med. Sci. Monit. 2016, 22, 4146–4151. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  76. Yan, Y.; Wu, X.; Wang, P.; Zhang, S.; Sun, L.; Zhao, Y.; Zeng, G.; Liu, B.; Xu, G.; Liu, H.; et al. Homocysteine Promotes Hepatic Steatosis by Activating the Adipocyte Lipolysis in a HIF1α-ERO1α-Dependent Oxidative Stress Manner. Redox Biol. 2020, 37, 101742. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  77. Åberg, F.; Jula, A.; Lundqvist, A.; Männistö, V. Hyperhomocysteinemia Predicts Liver-Related Clinical Outcomes in the General Population. J. Hepatol. 2023, 78, e172–e174. [Google Scholar] [CrossRef] [Scilit]
  78. Paganelli, F.; Mottola, G.; Fromonot, J.; Marlinge, M.; Deharo, P.; Guieu, R.; Ruf, J. Hyperhomocysteinemia and Cardiovascular Disease: Is the Adenosinergic System the Missing Link? Int. J. Mol. Sci. 2021, 22, 1690. [Google Scholar] [CrossRef] [Scilit]
  79. Clarke, R.; Daly, L.; Robinson, K.; Naughten, E.; Cahalane, S.; Fowler, B.; Graham, I. Hyperhomocysteinemia: An Independent Risk Factor for Vascular Disease. N. Engl. J. Med. 1991, 324, 1149–1155. [Google Scholar] [CrossRef] [Scilit]
  80. Bautista, L.E.; Arenas, I.A.; Peñuela, A.; Martínez, L.X. Total Plasma Homocysteine Level and Risk of Cardiovascular Disease. J. Clin. Epidemiol. 2002, 55, 882–887. [Google Scholar] [CrossRef] [Scilit]
  81. Kerins, D.M.; Koury, M.J.; Capdevila, A.; Rana, S.; Wagner, C. Plasma S-Adenosylhomocysteine Is a More Sensitive Indicator of Cardiovascular Disease than Plasma Homocysteine. Am. J. Clin. Nutr. 2001, 74, 723–729. [Google Scholar] [CrossRef] [Scilit]
  82. Tang, Y.; Chen, X.; Chen, Q.; Xiao, J.; Mi, J.; Liu, Q.; You, Y.; Chen, Y.; Ling, W. Association of Serum Methionine Metabolites with Non-Alcoholic Fatty Liver Disease: A Cross-Sectional Study. Nutr. Metab. 2022, 19, 21. [Google Scholar] [CrossRef] [Scilit]
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Figure 1. Liver-targeted AAV-DJ-hCBS corrects hyperhomocysteinemia and systemic phenotypes in Cbs-/- mice. (A) Longitudinal plasma homocysteine (Hcy) concentrations in mice following tamoxifen-induced Cbs deletion (Tx) and AAV-DJ administration. Lines show group means with 95% confidence interval. (B) Time course of body weight (percentage of baseline) represented as group mean ± SEM. (C–E) Body composition at 48 weeks: fat mass (C), lean mass (D), and fluid mass (E) as percentage of body weight. (F) Representative images, at 48 weeks, illustrating facial alopecia in Cbs-/- + GFP and its prevention by AAV-DJ-hCBS treatment. (G) Hepatic CBS expression normalized for beta-actin expression was similar to WT in Cbs-/- + hCBSlow, whereas Cbs-/- + hCBShigh showed approximately 5-fold higher CBS expression than WT; (H) hCBS vector genome copy number per 10 ng genomic DNA in liver, kidney, and spleen. Values are normalized to the median liver value of the Cbs-/- + hCBShigh group (set to 1). If not otherwise stated, data are represented as mean ± SEM. Circle = male mouse. Square = female mouse. Diamond = sex unknown. Data are from WT (n = 7; black), Cbs-/- + GFP (n = 8; red), Cbs-/- + hCBSlow (n = 9; blue), and Cbs-/- + hCBShigh (n = 10; beige) mice. * p-value < 0.05.
Figure 1. Liver-targeted AAV-DJ-hCBS corrects hyperhomocysteinemia and systemic phenotypes in Cbs-/- mice. (A) Longitudinal plasma homocysteine (Hcy) concentrations in mice following tamoxifen-induced Cbs deletion (Tx) and AAV-DJ administration. Lines show group means with 95% confidence interval. (B) Time course of body weight (percentage of baseline) represented as group mean ± SEM. (C–E) Body composition at 48 weeks: fat mass (C), lean mass (D), and fluid mass (E) as percentage of body weight. (F) Representative images, at 48 weeks, illustrating facial alopecia in Cbs-/- + GFP and its prevention by AAV-DJ-hCBS treatment. (G) Hepatic CBS expression normalized for beta-actin expression was similar to WT in Cbs-/- + hCBSlow, whereas Cbs-/- + hCBShigh showed approximately 5-fold higher CBS expression than WT; (H) hCBS vector genome copy number per 10 ng genomic DNA in liver, kidney, and spleen. Values are normalized to the median liver value of the Cbs-/- + hCBShigh group (set to 1). If not otherwise stated, data are represented as mean ± SEM. Circle = male mouse. Square = female mouse. Diamond = sex unknown. Data are from WT (n = 7; black), Cbs-/- + GFP (n = 8; red), Cbs-/- + hCBSlow (n = 9; blue), and Cbs-/- + hCBShigh (n = 10; beige) mice. * p-value < 0.05.
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Figure 2. Representative images of liver histology and CBS immunohistochemistry. Paraffin-embedded liver sections were stained with hematoxylin and eosin (H&E; left) and anti-CBS (DAB, brown; right). (A,B) WT control; (C,D) Cbs-/- + GFP; (E,F) Cbs-/- + hCBSlow; (G,H) Cbs-/- + hCBShigh. Large arrows indicate perivascular inflammation; small arrows indicate interstitial inflammation. Bars indicate 250 µm.
Figure 2. Representative images of liver histology and CBS immunohistochemistry. Paraffin-embedded liver sections were stained with hematoxylin and eosin (H&E; left) and anti-CBS (DAB, brown; right). (A,B) WT control; (C,D) Cbs-/- + GFP; (E,F) Cbs-/- + hCBSlow; (G,H) Cbs-/- + hCBShigh. Large arrows indicate perivascular inflammation; small arrows indicate interstitial inflammation. Bars indicate 250 µm.
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Figure 3. Liver protein signature. (A) PCA analysis of all detected proteins. (B) Heatmap of significant proteins (ANOVA; FDR < 0.05) with hierarchical clustering. (C) Assessing treatment effects by AAV-DJ-hCBS on protein expression in liver. Left: mapping log2FC of knockout affected compared to treatment effect in significantly altered proteins. Right: diagram of regulation of detected proteins in the methionine cycle (showing protein expression in Cbs-/- + GFP relative to WT; black = not significantly altered; strikethrough = protein was not detected.). (D) Top 21 enriched GO: biological processes organized by theme and mapped to their mean log2FC in Cbs-/- + GFP livers (calculated by taking the mean of all differentially expressed proteins in the specific pathway). (E) Top 10 KEGG enriched pathways. Enrichment was separately run on down and upregulated proteins. (F) Top 10 Reactome enriched pathways. Enrichment was separately run on down and upregulated proteins.
Figure 3. Liver protein signature. (A) PCA analysis of all detected proteins. (B) Heatmap of significant proteins (ANOVA; FDR < 0.05) with hierarchical clustering. (C) Assessing treatment effects by AAV-DJ-hCBS on protein expression in liver. Left: mapping log2FC of knockout affected compared to treatment effect in significantly altered proteins. Right: diagram of regulation of detected proteins in the methionine cycle (showing protein expression in Cbs-/- + GFP relative to WT; black = not significantly altered; strikethrough = protein was not detected.). (D) Top 21 enriched GO: biological processes organized by theme and mapped to their mean log2FC in Cbs-/- + GFP livers (calculated by taking the mean of all differentially expressed proteins in the specific pathway). (E) Top 10 KEGG enriched pathways. Enrichment was separately run on down and upregulated proteins. (F) Top 10 Reactome enriched pathways. Enrichment was separately run on down and upregulated proteins.
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Figure 4. Liver metabolome. (A) PCA analysis of all confidently detected metabolites in liver. (B) Heatmap of significant metabolites (ANOVA; FDR < 0.05) with hierarchical clustering. (C) Assessing treatment effects by AAV-DJ-hCBS on metabolite abundance in liver. Mapping log2FC of knockout effect compared to treatment effect in significantly altered metabolites in liver; (D) KEGG enrichment with MetaboAnalyst on significantly altered metabolites. (E) Detected metabolites in the cysteine and methionine metabolism KEGG pathway. (F) Detected metabolites in the glycerophospholipid metabolism KEGG pathway. Bars are represented as mean and standard error. Stars show significance tested by post hoc Tukey analysis.; * p < 0.05 vs. WT.
Figure 4. Liver metabolome. (A) PCA analysis of all confidently detected metabolites in liver. (B) Heatmap of significant metabolites (ANOVA; FDR < 0.05) with hierarchical clustering. (C) Assessing treatment effects by AAV-DJ-hCBS on metabolite abundance in liver. Mapping log2FC of knockout effect compared to treatment effect in significantly altered metabolites in liver; (D) KEGG enrichment with MetaboAnalyst on significantly altered metabolites. (E) Detected metabolites in the cysteine and methionine metabolism KEGG pathway. (F) Detected metabolites in the glycerophospholipid metabolism KEGG pathway. Bars are represented as mean and standard error. Stars show significance tested by post hoc Tukey analysis.; * p < 0.05 vs. WT.
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Figure 5. Plasma proteome and metabolome. (A) PCA analysis of all detected proteins in plasma. (B) PCA analysis of all confidently detected metabolites in plasma. (C) Heatmap of significantly altered metabolites (ANOVA; FDR < 0.05) with hierarchical clustering. (D) Assessing treatment effects by AAV-DJ-hCBS on metabolite abundance in plasma. Mapping log2FC of knockout effect compared to treatment effect in significantly altered metabolites in plasma. Hcn = L-Homocystine; SAH = S-Adenosylhomocysteine; Hcy = Homocysteine; NAGln = N-Acetylglutamine; GSSG = Oxidized glutathione.
Figure 5. Plasma proteome and metabolome. (A) PCA analysis of all detected proteins in plasma. (B) PCA analysis of all confidently detected metabolites in plasma. (C) Heatmap of significantly altered metabolites (ANOVA; FDR < 0.05) with hierarchical clustering. (D) Assessing treatment effects by AAV-DJ-hCBS on metabolite abundance in plasma. Mapping log2FC of knockout effect compared to treatment effect in significantly altered metabolites in plasma. Hcn = L-Homocystine; SAH = S-Adenosylhomocysteine; Hcy = Homocysteine; NAGln = N-Acetylglutamine; GSSG = Oxidized glutathione.
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Table 1. Organ weights and blood parameters.
Table 1. Organ weights and blood parameters.
Wild TypeCbs-/- + GFPCbs-/- + hCBSlowCbs-/- + hCBShigh
Heart (mg)175.2 ± 13.5170.0 ± 47.1181.3 ± 34.2175.6 ± 29.7
Left kidney (mg)199.6 ± 21.5213.7 ± 34.5205.2 ± 17.9211.1 ± 25.3
Right kidney (mg)221.6 ± 18.1224.3 ± 23.4214.7 ± 30.9230.3 ± 27.7
Spleen (mg)97.6 ± 28.694.3 ± 29.488.3 ± 39.484.1 ± 17.7
Liver (mg)1682 ± 1661591 ± 1711579 ± 3211762 ± 408
Brain (mg)473.0 ± 18.1464.7 ± 17.8477.1 ± 24.7494.8 ± 36.5
Glucose (mM)13.4 ± 1.29.7 ± 1.4 *12.8 ± 3.313.2 ± 0.9
WBC (×109/L)6.3 ± 1.94.2 ± 2.36.0 ± 4.64.8 ± 1.7
RBC (×1012/L)12.4 ± 1.48.4 ± 3.3 *11.4 ± 1.412.1 ± 1.7
HGB (mmol/L)10.5 ± 1.18.9 ± 1.59.8 ± 1.110.4 ± 1.2
HCT (L/L)0.63 ± 0.070.51 ± 0.10 *0.58 ± 0.070.61 ± 0.08
MCV (fL)50.6 ± 1.051.9 ± 4.050.4 ± 0.650.5 ± 1.0
MCH (amol)852.2 ± 28.3914.4 ± 59.4 *863.1 ± 21.6864.5 ± 32.9
MCHC (mmol/L)16.8 ± 0.317.6 ± 0.617.1 ± 0.317.0 ± 0.6
PLT (×109/L)1273 ± 1861240 ± 6551001 ± 4791163 ± 372
WBC, white blood cell; RBC, red blood cell; HGB, hemoglobin; HCT, hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; PLT, platelet; represented as mean ± SEM; * p < 0.05 vs. WT.
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Joschko, C.P.; Wang, C.-C.; van Buiten, A.; Goris, M.; Hoogstra-Berends, F.; Wang, J.; Bacurio, J.H.; Chen, Y.; Jia, N.; Deng, J.; et al. Liver-Targeted AAV-DJ-hCBS Therapy Achieves Long-Term Correction of Metabolic Imbalance in CBS-Deficient Mice. Int. J. Mol. Sci. 2026, 27, 3338. https://doi.org/10.3390/ijms27073338

AMA Style

Joschko CP, Wang C-C, van Buiten A, Goris M, Hoogstra-Berends F, Wang J, Bacurio JH, Chen Y, Jia N, Deng J, et al. Liver-Targeted AAV-DJ-hCBS Therapy Achieves Long-Term Correction of Metabolic Imbalance in CBS-Deficient Mice. International Journal of Molecular Sciences. 2026; 27(7):3338. https://doi.org/10.3390/ijms27073338

Chicago/Turabian Style

Joschko, Christian P., Chih-Chieh Wang, Azuwerus van Buiten, Maaike Goris, Femke Hoogstra-Berends, Joy Wang, Jan Henric Bacurio, Yinxing Chen, Nannan Jia, June Deng, and et al. 2026. "Liver-Targeted AAV-DJ-hCBS Therapy Achieves Long-Term Correction of Metabolic Imbalance in CBS-Deficient Mice" International Journal of Molecular Sciences 27, no. 7: 3338. https://doi.org/10.3390/ijms27073338

APA Style

Joschko, C. P., Wang, C.-C., van Buiten, A., Goris, M., Hoogstra-Berends, F., Wang, J., Bacurio, J. H., Chen, Y., Jia, N., Deng, J., Hu, S., Nacht, M., Chiocco, M. J., Henning, R. H., & Deelman, L. E. (2026). Liver-Targeted AAV-DJ-hCBS Therapy Achieves Long-Term Correction of Metabolic Imbalance in CBS-Deficient Mice. International Journal of Molecular Sciences, 27(7), 3338. https://doi.org/10.3390/ijms27073338

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