The transsulfuration pathway is a hepatic pathway that facilitates the conversion of homocysteine into cysteine to detoxify excess homocysteine, and contributes to the synthesis of glutathione, taurine and coenzyme A, which are important as antioxidants, for bile acid metabolism and for energy production (18). The transsulfuration pathway starts with the CBS enzyme, which catalyzes the conversion of homocysteine and serine into cystathionine, using pyridoxal 5′-phosphate (PLP), the active form of pyridoxine, as a cofactor. Afterwards, cystathionine γ-lyase (CGL) breaks down cystathionine into cysteine, α-ketobutyrate and ammonia. Absolute deficiency or malfunction of CBS disrupts this pathway, leading to elevated blood and urine levels of homocysteine and methionine, and reduced cysteine levels (18).

CBS mutations show genotype-phenotype variability, influencing the severity of the disease and the responsiveness to pyridoxine therapy. High-dose B6 supplementation may be beneficial in the case of partial CBS activity to enhance residual enzyme function, while total enzyme deficiency requires a methionine-restricted diet, cysteine-supplemented diets and betaine, folate and B12 supplementation to promote alternate remethylation pathways (19,20).

The remethylation pathway works in parallel with the transsulfuration pathway to normalize plasma homocysteine levels, especially in the brain where the transsulfuration pathway is inactive. In this reaction, homocysteine is remethylated to methionine, which is necessary for protein synthesis, through catalysis by MS, which requires B12 as a cofactor and employs 5-methyltetrahydrofolate produced by MTHFR as the methyl group donor (21).

Another hepatic remethylation route involves the enzyme betaine-homocysteine methyltransferase (BHMT), which uses betaine as a methyl donor to remethylate homocysteine into methionine. The BHMT pathway provides an alternative route for homocysteine metabolism in cases of folate or B12 deficiency or genetic disorders that affect the primary route (22).

These interrelated transsulfuration and remethylation pathways are summarized in Fig. 1, which illustrates the metabolic conversion of homocysteine and the key cofactors involved.

HCU usually presents with a wide range of clinical symptoms that affect multiple organs. Ocular complications, including ectopia lentis, myopia, astigmatism, glaucoma and blindness, are the earliest and most common manifestations, affecting 90% of poorly managed individuals (13–17).

In the early childhood growth phase, skeletal deformities are common, including marfanoid body habitus, long limbs, scoliosis, pectus deformities, genu valgum and severe osteoporosis, which predispose patients to recurrent bone fractures (14). Neurological deficits and developmental delays commonly occur in the early years of life, are noticed as intellectual disabilities and may progress to repeated seizures (17).

Thromboembolic events, such as DVT, PE and stroke, are the most serious complications that may affect both adolescents and adults. The severity of clinical presentation is markedly dependent on the degree of enzyme deficiency and therapeutic responsiveness (11,12).

Decreased homocysteine metabolism leads to a buildup of methionine, with levels ranging from 60 to 100 µmol/l. This concurrent elevation of tHcy and methionine can distinguish classical HCU from hyperhomocysteinemia caused by other remethylation pathway disorders, such as MTHFR deficiency or cobalamin metabolism malfunction, which lead to elevated tHcy levels while maintaining normal methionine levels. In patients on methionine-restricted diets, the normalization of plasma methionine and tHcy levels indicates effective metabolic control. The B6 trial also identifies B6-responsive individuals who show partial improvement in methionine levels (17).

Measurement of urinary homocysteine levels provides evidence for the diagnosis of classical HCU, particularly in limited-resource settings where plasma tHcy testing is not available. In CBS deficiency, elevated plasma homocysteine levels exceed the renal reabsorption capacity, leading to increased renal excretion of both free and tHcy. Although the urinary homocysteine level is less precise than the plasma tHcy test, it is used as an initial screening test, especially in symptomatic children. However, elevated urinary homocysteine levels can also be detected in other homocysteine metabolism disorders, and may vary with hydration status and renal function (31).

Being cofactors in the remethylation pathway that converts homocysteine to methionine, deficiencies in vitamin B12 or folate can lead to elevated plasma tHcy levels but are not indicative of CBS deficiency. Thus, measuring serum B12 and folate levels rules out nutritional or acquired causes of hyperhomocysteinemia (32,33).

Elevated MMA levels suggest functional B12 deficiency or disorders of cobalamin metabolism, which may be associated with elevated tHcy levels. However, in classical HCU, MMA levels are typically normal, which helps to exclude remethylation disorders from the potential diagnosis and indicates that the elevated homocysteine level is due to transsulfuration pathway impairment rather than defective remethylation (34).

Genetic testing using next-generation sequencing that utilizes the CBS gene, or whole-exome sequencing in asymptomatic cases, is a decisive tool for confirming the diagnosis of classical HCU, uncovering the underlying CBS gene mutations responsible for CBS deficiency, as well as predicting B6 responsiveness and guiding treatment decisions (35,36).

Over 190 CBS gene mutations with clinically significant genotype-phenotype associations have been isolated. The common p.I278T mutation is often associated with partial or full responsiveness to B6 therapy, whereas p.R125Q, p.G307S and p.R266K mutations are associated with non-responsiveness and severe symptoms (37).

CBS activity assays performed on cultured skin fibroblasts or fresh liver biopsy samples evaluate the functional capacity of CBS, which is typically absent or markedly reduced in HCU. Enzymatic activity testing assists in distinguishing between the B6-responsive and non-responsive forms of CBS, as residual activity may be enhanced by the addition of PLP in vitro. The use of enzyme assays has lost its applicability in clinical practice as it requires an invasive tissue sampling technique, a long turnaround time and challenging interpretation in cases with residual activity (36,38).

A lifelong methionine-restricted diet remains the basic strategy for managing HCU due to CBS gene mutations. The diet aims to reduce methionine accumulation while ensuring adequate nutrition using methionine-free nutritional formulas. This approach requires careful follow-up of plasma amino acid levels to maintain metabolic control, prevent protein malnutrition and ensure the fulfillment of demands for normal growth, especially in pediatric patients (44).

Although a methionine-restricted diet is effective in lowering plasma tHcy levels, it poses a long-term compliance challenge because of the poor palatability of dietary formulas (45). In addition, dietary restrictions alone may not be sufficient to prevent disease complications and may require adjunctive therapies (44,45).

Numerous preclinical trials in animal models have attempted to overcome the challenges related to dietary restrictions. Using a mouse model, CDX-6512, an engineered orally-stable methionine γ-lyase (MGL), was administered following a high-protein meal and led to a dose-dependent ability to locally degrade methionine in the gastrointestinal tract (GIT), suppressing plasma methionine and homocysteine (46). Another experimental model by Perreault et al (47) used a bolus dose of an engineered probiotic E. coli Nissle strain to degrade methionine in the GIT. The resulting SYNB1353 strain metabolized methionine in mice, non-human primates and humans, resulting in lower plasma methionine levels.

Betaine, also known as trimethylglycine, is a commonly used adjunctive therapy for B6-unresponsive patients with HCU that acts as a methyl donor in the hepatic BHMT remethylation pathway (48). In a study enrolling patients with B6-unresponsive HCU, betaine administration resulted in individual mean reductions in plasma tHcy levels ranging from 47.4 to 105.0 µmol/l (49). Another study involving healthy subjects showed that betaine supplementation of 6 g/day for 3 weeks significantly reduced homocysteine levels (P=0.030) (50). A study by Lu et al (51) reported a 10% reduction in the plasma homocysteine concentration when using a combination of betaine and low-dose B vitamins: 400 µg folic acid, 8 mg vitamin B6, 6.4 µg vitamin B12 and 1 g betaine.

However, prolonged betaine therapy of >100 mg/kg/day in patients with inadequate dietary methionine restriction led to notable hypermethioninemia-related adverse events (52), while another study reported marked elevations in total cholesterol levels with betaine supplementation, necessitating close monitoring in patients at risk of cardiovascular disease (53).

Folate and B12 are important drugs in HCU management protocols, particularly in patients with MTHFR, MTR and MTRR mutations. These vitamins serve important roles in the remethylation of homocysteine to methionine (54). The active form of folate, 5-methyltetrahydrofolate, donates a methyl group to homocysteine, converting it to methionine. This reaction is catalyzed by MS with methylcobalamin, the active form of vitamin B12, as a cofactor. Insufficient folate and B12 supplies impair this pathway, leading to elevated tHcy levels (55). A randomized clinical trial by Kok et al (56) demonstrated marked reductions in tHcy levels in elderly participants consuming 400 g folic acid and 500 g vitamin B12 daily over 2 years.

Despite being the basis of HCU management, conventional therapies have non-negligible limitations, including variability in patient responsiveness to B6. Adherence to low-methionine diets is often difficult, affecting metabolic control, even with strict diet control and vitamin supplements. Numerous patients fail to meet the desired tHcy levels, leaving them at risk of complications. Furthermore, the long-term outcomes of current interventions are inconsistent and there is limited capacity to reverse established complications (17,43–45).

Mechanistic and structural studies on CBS have provided the basis for emerging therapeutic concepts (57–62). Building on these and on prior reviews, such as that by Majtan et al (63), this section emphasizes treatment developments since 2023 and practical translational considerations across enzyme replacement, gene- and proteostasis-directed strategies.

Fig. 2 provides an integrated overview of these molecular pathways with annotated therapeutic targets and intervention points.

Human CBS is a complex multidomain enzyme that requires PLP for its activity and binds to a heme group that serves a regulatory role in its enzyme activity. The CBS protein is composed of four identical subunits comprising 551 amino acids. Each subunit consists of three domains, with the N-terminal domain containing the heme-binding site. In this region, the heme is linked by cysteine at position 52 and histidine at position 65 (57).

ERT is being developed as a mutation-agnostic approach for CBS deficiency that delivers functional enzymes directly, bypassing reliance on residual endogenous activity. While ERT can normalize biochemistry quickly, the need for chronic administration and the typical costs and immunogenicity risks of biologics remain practical challenges for widespread adoption (58).

i) Pegtibatinase. Pegtibatinase, also known as OT-58 or TVT-058, is a recombinant human CBS catalytic core (amino acids 1–413) that has been modified with the addition of polyethylene glycol (PEG) and lacks the CBS autoinhibitory regulatory domain, yielding a constitutively active, predominantly dimeric enzyme. This modification stimulates the catalytic activity of the enzyme, rendering it predominantly in a dimeric state (58). To boost pegtibatinase stability and minimize unwanted protein aggregation, a cysteine-to-serine substitution at position 15 is introduced to prevent the formation of interdimer disulfide bonds. The enzyme is also chemically modified by PEGylation, in which five PEG chains of 20 kDa each are attached to each CBS subunit. This extends the circulation half-life by 10-fold, making the PEGylated form more suitable for therapeutic use in HCU than the unmodified enzyme (59).

In the phase 1/2 COMPOSE study, pegtibatinase demonstrated promising tolerability and a safety profile with only two incidents of mild injection site urticaria that resolved upon temporary dose interruption and subsequent dose titration. Using a subcutaneous dose of 1.5 mg/kg twice weekly of pegtibatinase achieved a 67.1% mean relative reduction in tHcy levels from baseline (97 to 32 µM) at follow-up intervals of 6, 8, 10 and 12 weeks, maintaining tHcy levels below the clinically meaningful threshold of ≥100 µM (60). A preclinical study in CBS-deficient animal models has demonstrated that pegtibatinase ameliorated related complications such as ocular and skeletal manifestations, decreased facial alopecia, and enhanced liver metabolism of glucose and lipids (61).

ii) Pegtarviliase. Pegtarviliase, also known as AGLE-177, is an engineered CGL variant (CGL-ILMDRGVS) with markedly increased affinity for homocysteine compared with wild-type CGL. It contains eight missense mutations, referred to as the CGL-ILMDRGVS construct, and exhibits a 60-fold increased affinity for homocysteine compared with the wild-type human CGL enzyme (62). Furthermore, pegtarviliase shows an enhanced capability for degrading multiple forms of homocysteine compared with wild-type CGL, along with improved stability and circulation time, potentially resulting in a more comprehensive reduction in tHcy levels (62).

A preclinical study of pegtarviliase in mice using subcutaneous doses of 1, 3 and 10 mg/kg twice weekly between postnatal days 10 and 70 reported a marked improvement in 30-day survival rates, at >75 vs. <20% in the treated and control groups, respectively. The treated mice also showed resolution of liver steatosis and alopecia. Furthermore, a dose of 10 mg/kg led to an ~43% reduction in plasma tHcy levels and an 87% reduction in brain tHcy levels (63).

In a phase 1/2 dose-escalation trial aiming to assess the safety, tolerability, pharmacokinetics and efficacy of pegtarviliase in patients with classical HCU, three cohorts of participants received weekly subcutaneous injections of pegtarviliase at doses of 0.15, 0.45 and 1.35 mg/kg, respectively over the course of 4 weeks. A 3-day post-treatment dose-dependent reduction in tHcy levels of 26.3 and 33.0% was observed in cohorts 1 and 2, respectively. However, some participants in cohort 3 experienced injection site reactions and increased tHcy levels, which can be explained by the development of anti-drug antibodies. The study highlighted the promising tolerability and efficacy of pegtarviliase at low doses, with a clear need for modified clinical strategies to resolve the immunogenicity issues (64). As of 2023, Aeglea BioTherapeutics, Inc., announced the exploration of strategic alternatives for the pegtarviliase program following interim phase 1/2 results (65), which ultimately led to the discontinuation of its clinical development in 2024.

iii) CDX-6512. CDX-6512 is an investigational modified MGL enzyme that is specifically engineered to maintain stability and activity in the GIT. In 2022, the U.S. Food and Drug Administration granted CDX-6512 the ‘orphan drug designation’ for the treatment of HCU. Using artificial intelligence and machine learning, 12 iterative rounds of enzyme evolution were performed, and >27,000 variants were screened for activity under simulated gastric and intestinal conditions. The resulting enzyme exhibited high resistance to deactivation by gastric pH and other enzymes (46).

In a preclinical study using the Tg-I278T CBS⁻/⁻mouse model of HCU, an oral dose of 148 mg/kg CDX-6512 administered after a high-protein meal led to an almost 50% reduction in plasma tHcy levels after 4 h. Plasma methionine levels also declined in a non-significant dose-dependent manner, with up to a one-third decrease at the highest dose (66).

A study in healthy non-human primates that received a high-protein meal followed by an oral dose of 370 mg/kg CDX-6512 treatment led to a statistically significant, dose-dependent reduction in plasma methionine levels, demonstrating the effectiveness of the enzyme in breaking down dietary methionine in a physiologically-similar model to humans (46). In the same preclinical program using Tg-I278T CBS⁻/⁻ mice, daily oral administration of CDX-6512 with a high-protein meal over 2 weeks effectively maintained baseline plasma tHcy levels. By contrast, untreated controls showed a 39% increase in homocysteine levels, suggesting that homocysteine levels can be sustained with long-term CDX-6512 treatment (46). In 2024, the program was acquired and rebranded as SYNT-202 by Syntis Bio, with continued preclinical optimization focused on gut-restricted methionine degradation; no human data under the new designation have been disclosed at present (67).

Although ERT offers mutation-independent metabolic control, injectable agents such as pegtibatinase face lifetime adherence and immunogenicity hurdles, and oral agents such as SYNT-202 still require durable efficacy and safety data; cost-of-goods and access considerations further suggest that ERT will likely complement, rather than fully replace, diet and vitamin-based care (58–61).

Gene therapy offers a potential one-time treatment for CBS deficiency by restoring intrinsic CBS function at the genetic level. The strategy delivers a functional CBS gene to the liver, via viral or non-viral vectors, to achieve sustained metabolic correction.

AAVrh.10-CBS uses an AAVrh.10 vector to deliver a functional human CBS gene under a cytomegalovirus early enhancer/chicken β-actin promoter, targeting hepatocytes involved in homocysteine metabolism. In the CBS-deficient mouse model I278T, a single intravenous dose reduced plasma tHcy levels by ~97% within 1 week and sustained an ~81% reduction at 1 year from administration, with observed reversal of alopecia, skeletal defects and abnormal fat distribution (68). Pre-existing neutralizing antibodies to AAVrh.10 appear to be less prevalent than those to AAV2 or AAV8, supporting the suitability of AAVrh.10 for liver-directed applications (69). Furthermore, the durability of liver-directed AAV expression observed in a human hemophilia B gene therapy trial (70) suggests the potential for long-term transgene persistence in clinical applications.

Minicircular DNA vectors, which lack bacterial backbone sequences, can more efficiently enhance transgene expression and reduce innate immune activation compared with plasmids. Foundational work on vector design and dosing has also highlighted safety principles to minimize hepatic genotoxicity in gene-delivery programs (71).

In CBS-deficient mice, a single liver-targeted injection of the minicircle CBS construct MC.P3-hCBS reduced plasma homocysteine levels by ~50% within 1 week, restored hepatic CBS activity ~34-fold and sustained metabolic correction for >6 months (72).

Gene therapy directly addresses the genetic cause of HCU, offering the possibility of durable metabolic correction. However, immune responses to viral vectors, the dilution of treatment effects with hepatic growth in children and complex manufacturing remain key challenges (68–72).

Pharmacological chaperones are small molecules that bind to misfolded CBS proteins, stabilizing their structure and enhancing catalytic activity. Examples include glycerol, trimethylamine-N-oxide and dimethyl sulfoxide, which have been shown to restore activity in certain CBS mutants in cell and animal models (73,74). Yeast expression systems for human CBS mutants have also demonstrated that chaperones can promote proper assembly and tetramerization, supporting mutation-specific therapeutic potential (75).

SAM, an allosteric activator of CBS, binds to the regulatory domain of the enzyme to boost activity (76,77). A study by Mendes et al (77) demonstrated that ~50% of tested patients with HCU showed defective SAM activation, often linked to specific CBS mutations.

CBS is a heme-dependent enzyme; some CBS gene mutations impair heme binding. Heme arginate can stabilize these variants and restore heme-binding activity in cell models (78). A study by Melenovská et al (78) showed that administration of heme arginate restored heme binding, improved tetramer formation and enhanced catalytic activity of the CBS enzyme in several B6-resistant mutants.

Chaperones may complement diet and gene therapy, but most remain unvalidated in clinical trials. Mutation-specific responsiveness suggests that genotype-guided therapy may be required (73–78).

Proteasome inhibition can stabilize partially active but misfolded CBS proteins. Bortezomib, an oncology drug, increased hepatic CBS activity >20-fold and reduced plasma tHcy levels by 97% in a p.R266K mouse model (79–81). Carfilzomib has shown similar biochemical benefits in preclinical HCU models, although its long-term safety remains yet to be fully elucidated (82).

Potential risks of PIs include systemic proteasome suppression. Liver-targeted delivery strategies and dose minimization are being explored to mitigate toxicity (80–82).

SYNB1353 is an engineered E. coli Nissle strain expressing methionine γ-lyase to degrade dietary methionine. In preclinical models, it reduces plasma homocysteine and methionine levels without adverse events (83). A phase 1 trial in healthy volunteers showed promising tolerability and dose-dependent methionine reduction (84). A phase 2 study in classical HCU (ClinicalTrials.gov identifier, NCT05651054) is ongoing, with efficacy data pending.

OLT has emerged as the only definitive treatment for severe pyridoxine-non-responsive HCU. Multiple case reports have demonstrated complete metabolic normalization following OLT. A case report by Lin et al (85) described a 24-year-old patient with HCU with notable complications, including cerebellar infarctions and hypertension, who underwent a liver transplant, after which their metabolic control normalized without dietary restrictions. Another case report described a patient with HCU who demonstrated post-transplant normalized homocysteine and methionine levels at the age of 4 years and remained stable for 6 years, indicating the resolution of HCU. The patient had no complications before the transplantation, highlighting the potential of early liver transplantation as a curative strategy for HCU (86).

A retrospective genetic analysis uncovered 18 CBS variants in 13 patients diagnosed with classic HCU between 10 days of age and 14 years of age, all of whom exhibited elevated methionine and tHcy levels. Three B6 non-responders underwent liver transplantation at the ages of 3, 8 and 8 years, and achieved normalized methionine and homocysteine levels within a week post-transplant (87).

Although liver transplantation offers a curative approach for B6 non-respondents, it carries notable challenges, including surgical risks, the need for prolonged immunosuppression and limited donor availability. Additionally, OLT does not reverse existing complications, and early diagnosis is important to optimize outcomes (85–87).