Detailed information on patients in the two groups is shown in Table 1. Among the c.482G > A group (118 males, 77 females), a total of 125 (64.1%) affected individuals were identified with MS/MS-based newborn screening (NBS). One hundred and seventeen cases remained asymptomatic at the time of this study with a median age of 3 years and 3 months (range from 11 months to 8 years old). While waiting for the results of NBS, seven cases developed symptoms, respectively, at the age ranged from 1 to 22 days, and then treatment was administered immediately. Their symptoms included vomiting, feeding difficulty, prolonged neonatal jaundice, and lethargy. At the last follow-up, these seven patients were aged 2–6 years old, with normal physical and psychomotor development. Unfortunately, among the 125 cases detected by NBS, one patient was found with developmental delay at the age of 17 months, due to the refusal of confirmative testing and pre-symptomatic treatment.

Sixty patients in the c.482G > A group were diagnosed because of disease onset at the median age of 7 years and 6 months (ranging from 1 day to 33 years). Ten cases were diagnosed because of sibling MMA diagnosis. Among these 10 individuals, nine were asymptomatic, while the other one developed lower limb weakness and poor exercise tolerance, and a decline in learning and memory at 9 years of age for unknown reasons. The patient was diagnosed and treated until 4 years later when the younger sibling was diagnosed with MMA through NBS. Fortunately, this patient is currently 16 years old and has no obvious abnormality in motor and language development after reasonable treatment.

In the non-c.482G > A group, which included 107 males and 93 females, a total of 90 individuals were identified through NBS by MS/MS and received timely pre-symptomatic treatment. Of these, 41 cases remained asymptomatic with a median age of 3 years and 2 months (ranging from 2 to 10 years), while 49 cases continued to exhibit a range of symptoms. This included patients who developed symptoms very early, prior to receiving NBS results, and those who became symptomatic despite receiving adequate and timely treatment. The median age of symptom onset was 24 days (ranging from 1 day to 4 years and 9 months). There were 110 patients diagnosed because of the onset of the disease with a median age of onset of 40 days (ranging from 1 hour to 8 years). In general, the proportion of symptomatic patients in the c.482G > A group was significantly lower than that in the non-c.482G > A group (P < 0.01), even in patients who were diagnosed with NBS and started treatment on time.

The clinical characteristics of all symptomatic patients in different groups are summarized in Table 2. More patients presented with later onset in the c.482G > A group than those in the non-c.482G > A group. More than half of patients with a c.482G > A variation developed their first symptom after the age of 4 years. Seven of them showed symptomatic presentation during adulthood (the median age was 22 years, ranging from 19 to 33 years). In the non-c.482G > A group, 80.5% of patients developed their first symptom before the age of 1 year. The difference was statistically significant (P < 0.01).

In 69 symptomatic patients with c.482G > A variant, the most common clinical symptoms at onset were developmental delay (59.4%), lower limb weakness and poor exercise tolerance (50.7%), decline in cognitive, learning, and memory (37.7%), gait instability and abnormal posture (36.2%), seizures (26.1%), and psychiatric and behavioral disturbances (24.6%). Among the seven patients who had disease onset during adulthood, the initial clinical manifestations were psychiatric and behavioral disturbance. On the other hand, in the 159 symptomatic patients without the c.482G > A variant, the most common clinical symptoms at onset were developmental delay (81.8%), lethargy and feeding difficulty (62.9%), lower limb weakness and poor exercise tolerance (54.7%), prolonged neonatal jaundice (51.6%), vomiting (47.2%), and seizures (32.7%). For clinical manifestations that were less common, we observed that ocular problems and hydrocephalus mainly presented in the non-c.482G > A group while incontinence was found in the c.482G > A group only. Pulmonary hypertension was a rare symptom in both groups. Furthermore, compared with patients diagnosed due to disease onset, the proportion of clinical symptoms in individuals identified by NBS displayed no significant differences, with the exception of developmental delay, feeding difficulty, and prolonged neonatal jaundice in the c.482G > A group. In contrast, for the non-c.482G > A group, only the proportion of patients experiencing vomiting exhibited a significant difference between those diagnosed due to disease onset and those identified through NBS.

The concentrations of blood propionylcarnitine (C3), propionylcarnitine/acetylcarnitine ratio (C3/C2) and total plasma HCY, and urinary methylmalonic acid values for each group of patients before and after medical intervention are summarized in Table 3. Finally, complete biochemical data both before and after treatment of 149 patients in the c.482G > A group and 150 patients in the non-c.482G > A group were available. In this study, the levels of blood C3, C3/C2 ratio and total plasma HCY, as well as urinary methylmalonic acid levels were above the cutoff values before medical intervention in all patients. Before treatment, levels of these metabolites were lower in patients in the c.482G < A group compared with those in the non-c.482G < A group. Between the two groups, differences in the levels of blood C3, C3/C2 ratio, and total plasma HCY were statistically significant (P < 0.05). However, urinary methylmalonic acid values showed no statistical differences (P > 0.05). After treatment, these metabolic markers showed a remarkable tendency to decrease, with a significant statistical difference in both groups (P < 0.05). On comparing the degree of decline in all the above indicators after treatment, both a significant difference in plasma total HCY values and urinary methylmalonic acid levels could be identified (P < 0.05), while there was only a slight difference in the reduction in blood C3 values and C3/C2 ratios (P > 0.05). For patients carrying a c.482G > A mutation in a homozygous state or in compound heterozygosity, there was no statistical difference in the degree of decline in all the above biochemical indicators (P > 0.05).

To better understand the genotype of the 195 patients carrying the c.482G > A variant in this study, we summarized their “another mutation” in another allele. Details are shown in Table 4. Biallelic mutations in the MMACHC gene were detected in 186/195 patients, while in the other nine cases, no mutations were detected in another allele except for the c.482G > A variant. Fifteen patients were homozygous for the c.482G > A variant and 171 were compound heterozygotes. As a result, 27 molecular variants in another allele of the MMACHC gene were identified, including nine frameshift mutations, eight nonsense mutations, five missense mutations, four deletion variations, and one splicing mutation. Among these variations, c.541G > T and c.435_436dup were novel. The five most prevalent variants were c.609G > A, c.658_660del, c.567dup, c.482G > A, and c.80A > G. The genotypes of all samples in the non-c.482G > A group are shown in Supplementary Table 1.

The clinical outcome evaluations of the biochemical results after treatment and the dosage of hydroxocobalamin at the last follow-up are shown in Table 3. The median dosage of hydroxocobalamin of patients in the c.482G > A group was significantly lower than that in non-c.482G > A group (P < 0.05). Furthermore, their metabolic conditions were better controlled after treatment, with much lower concentrations of blood C3, C3/C2 ratio, total plasma HCY, and urinary methylmalonic acid values. However, for patients carrying the c.482G > A mutation in a homozygous state or in compound heterozygosity, there was no statistical difference in the dosage of hydroxocobalamin (P > 0.05).

The clinical outcome evaluations of disease onset and basic motor function and language development are shown in Table 5. For individuals diagnosed through NBS or because of sibling MMA diagnosis, fewer cases developed clinical symptoms and more than 90% of cases remained with normal psychomotor and language development in the c.482G > A group. Compared with those in the non-c.482G > A group, the differences were statistically significant (P < 0.05). Similar results were found in patients diagnosed due to disease onset. In the c.482G > A group, only one neonatal-onset patient died at 4 months old because of refusal of treatment, 65.0% of patients had movement and/or speech impediments, while 33.3% of patients developed normally. In the non-c.482G > A group, four cases died of acute metabolic decompensation and/or encephalopathic crises induced by infection during follow-up, 85.5% patients had movement and/or speech development delay, while only 10.9% of cases developed normally. Compared with those in the non-c.482G > A group, individuals with a c.482G > A variation had significantly lower mortality and a higher proportion of patients with normal psychomotor and language development (P < 0.05). Compared with those diagnosed due to disease onset, individuals diagnosed with NBS in either group showed a better prognosis, with significantly lower mortality and a higher proportion of patients with normal psychomotor and language development (P < 0.05).

At the final follow-up, within the c.482G > A group, none of the 69 symptomatic patients experienced vomiting, feeding difficulty, lethargy, coma, mental abnormalities, jaundice, or urinary incontinence. Out of the 35 patients who initially presented with lower limb weakness and poor exercise tolerance, 10 recovered, while 25 continued to exhibit limb weakness. Among the 26 patients with cognitive, learning, and memory decline at onset, eight returned to normal, and 18 experienced slow response and memory decline. Of the 25 patients with gait instability and abnormal posture at onset, only five recovered. Out of the 18 patients who had seizures at onset, 16 did not experience further seizures, while two patients, classified as having “secondary epilepsy,”, remained seizure-free after oral antiepileptic drug treatment. Among the three patients with hydrocephalus at onset, one underwent surgery and recovered well, while two patients continued to have mild hydrocephalus without surgery. Two patients with visual impairment maintained impaired vision but did not undergo regular fundus and visual acuity examinations. Of the three patients with abnormal renal function at onset, two recovered, and one still exhibited abnormal renal function with increased proteinuria and blood creatinine levels. One patient with pulmonary hypertension returned to normal.

Additionally, our cohort included 15 patients with homozygous c.482G > A mutations. Ten were diagnosed through NBS, two were identified following sibling onset confirmation and presented no onset or abnormal symptoms, and three were diagnosed after onset (with ages of onset at 3 months, 6 months, and 26 years, respectively). Among these, two patients were developmentally delayed, displaying abnormal walking posture, gait instability, and cognitive regression.