, Introduction
Citrin deficiency, a rare urea cycle disorder discovered by Keiko Kobayashi et al., is caused by a mutation in the SLC25A13 gene located on chromosome 7q21.3, which is responsible for producing the urea cycle enzyme citrin [1]. The prevalence of citrin deficiency was found to be higher in East and Southeast Asian countries, with a prevalence of 1/10,000-1/38,000.2) According to the age of onset and phenotype, citrin deficiency is classified into 3 types: (1) neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD, OMIM#605814), (2) failure to thrive and dyslipidemia caused by citrin deficiency, and (3) adult-onset citrullinemia type II (CTLN-II, OMIM#603471) [1].
In adults with CLTN-II, abnormal neurological and psychiatric symptoms, including emotional and behavioral disturbances such as aggression, irritability, restlessness, and consciousness disorders, have been documented [2]. In addition to individuals with CLTN-II, individuals with other late-onset urea cycle disorders have also been shown to exhibit neurocognitive and behavioral impairment, particularly in aspects related to attention and executive functioning [3]. These symptoms are believed to be related to the effects of recurrent hyperammonemia on the brain, a phenomenon also seen in some individuals with citrin deficiency [4]. Unlike those with other urea cycle disorders, patients with NICCD typically exhibit a return to normal liver marker levels during childhood and adolescence. Growth retardation, underweight, and emaciation have been reported in children with citrin deficiency, with 25% experiencing developmental delay [5]. However, long-term neurophysiological outcomes, such as low intelligence quotient (IQ) scores and the development of attention deficit/ hyperactivity disorders (ADHD), in individuals with citrin deficiency remain unclear. It is unknown whether transient metabolic disturbances during early childhood in individuals with citrin deficiency may have a lasting impact on neurocognitive functions [6].
Newborn screening for citrullinemia using tandem mass spectrometry at the National Taiwan University Hospital Newborn Screening Center began in 2002 [7]. Early diagnosis and management of citrin deficiency through diet therapy and treatment for cholestasis have been shown to delay the onset of CLTN-II and improve prognosis [8,9]. However, no study has documented the impact of newborn screening on neuropsychological function via early intervention. We believe it is important to understand the physiological and psychosocial conditions of children and adolescents with citrin deficiency to obtain a more comprehensive understanding of the disorder.
Methods
1. Patients
This was a case-control study conducted from 2020 to 2023 that included a total of 30 patients diagnosed with citrin deficiency before the age of one year. The diagnosis was confirmed through genetic analysis of the SLC25A13 gene. The growth, development, liver function, metabolic profiles and dietary conditions of all patients were consistently monitored through regular follow-up either at the Department of Medical Genetics or the Department of Pediatric Gastroenterology at National Taiwan University Hospital. Patients between the ages of 3 and 25 years were invited to participate in this study for neuropsychological evaluation (Fig. 1). We collected data from historical medical records, including physical examination, biochemical, plasma amino acid analysis, tandem mass analysis, and nutritional evaluation data, for the assessment of markers at disease onset in infancy that correlated with long-term neuropsychological outcomes. All patients experienced a prompt recovery, with normal liver function and metabolic profiles following diagnosis and regular follow- up. Among the 30 patients, 10 were diagnosed with citrin deficiency based on abnormal findings in newborn screening (NBS group), while 20, who did not receive the newborn screening, were diagnosed clinically with neonatal cholestasis (clinical diagnosis group; patients with NICCD). All subjects gave their informed consent for inclusion before they participated in the study. This study was approved by NTUH-REC No: 202006156RINA.
2. Neuropsychiatric evaluation
All individuals underwent neuropsychological assessments, including the Wechsler Intelligence Scale for IQ evaluation [10], Conners Continuous Performance Test (CPT) [11], Conners Kiddie Continuous Performance Test, a semistructured diagnostic interview (Kiddie Schedule for Affective Disorders and Schizophrenia-Epidemiological version [K-SADS-E]) [12], and parent-reported Swanson, Nolan, and Pelham-Version IV (SNAP-IV) [13,14] for attention evaluation. The formal ADHD diagnosis for all patients was established through comprehensive clinical evaluations conducted by experienced pediatric psychiatrists (JCC and SSG), based on the diagnostic criteria outlined in the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5) [15]. The control group for this study was composed of completely healthy individuals, matched by age, gender, and overall IQ with cases of citrin deficiency collected from the Department of Psychiatry. These individuals had no history of citrin deficiency or any other significant medical conditions. The general characteristics and neuropsychiatric evaluation results of the control group are provided in Supplementary Table 1.
3. Statistics
The statistical analyses were performed using IBM SPSS Statistics ver. 25.0 (IBM Co., Armonk, NY, USA). Variables related to citrin deficiency and the control group were analyzed using the Mann-Whitney U test for comparison. This included the prevalence of ADHD between the patient group and the general population, as well as the basic characteristics and neurocognitive function of both the patient and control groups. Additionally, comparisons were made between the basic characteristics and neurocognitive function of the NBS group and the clinically diagnosed group, as well as between the ADHD and non- ADHD groups. A post hoc analysis for p value correction was also conducted for the multiple groups analysis: NBS group, clinical diagnosis group, and control group. A P value <0.05 was considered to indicate statistical significance. We also utilized logistic regression to investigate the relationship between the assessment results and laboratory test values.
Results
1. General characteristics and follow-up
Table 1 shows the demographic characteristics of the 30 patients with citrin deficiency. Among the 30 citrin deficiency patients, 15 were male, and 15 were female. All patients showed a preference for high-protein, high-fat, and low-carbohydrate diets for their daily nutrient intake, and 18 patients (60%) with liver function abnormalities were switched to medium-chain triglyceride (MCT) formula milk at the time of diagnosis. Peak plasma amino acid analysis before the age of one year showed an approximately 6-fold increase in the citrulline level and a 4-fold increase in the methionine level compared to the upper limit of normal values (Table 2). The peak liver function (aspartate aminotransferase [AST], alanine transaminase [ALT], direct bilirubin [D-Bil], total bilirubin [T-Bil], gamma-glutamyl transferase [γ-GT], and alkaline phospatase), ammonia, and bile acid levels before the age of one year were all greater than the reference ranges, while the albumin and glucose levels were lower than the reference ranges. The frequency of ammonia level measurements before the age of 1 varied depending on each patient's initial lab results. If the initial ammonia level was normal, no further testing was conducted. However, if elevated ammonia levels were detected, we monitored the levels until they returned to normal, typically requiring only 2–3 follow-up tests. Due to the challenges of drawing blood from infants, ammonia levels were not frequently monitored unless the patient presented symptoms. The patients’ liver function and ammonia levels returned to normal after 1 year of age. All patients underwent genetic testing, with the c.851del4 and c.615+5G>A variants accounting for 44.6% and 23.2% of the total sample, respectively. These 2 variants had the highest frequencies among the gene mutations investigated in this study.
2. Neuropsychiatric evaluation results
All patients underwent neuropsychiatric evaluations, including the full-scale IQ (FSIQ), CPT, SNAP-IV, and K-SADS-E. The mean FSIQ score in citrin deficiency patients was 96.32±15.49 (range, 70–135). Among the 30 citrin deficiency patients, 5 (16.7%) had a score of 70–79, 5 (16.7%) had a score of 80–89, 7 (23.3%) had a score of 90–99, 8 (26.7%) had a score of 100–109, 3 (10.0%) had a score of 110–119, 1 (3.3%) had a score of 120–129, and 1 (3.3%) had a score of 130 or above. The results of the Shapiro-Wilk normality test indicated that the FSIQ score in citrin deficiency patients followed a normal distribution. Although the proportion of individuals with FSIQ scores ranging from 70–79 was greater than that in the general pediatric population, the difference was not statistically significant (P=0.084 by Mann-Whitney U test) (Fig. 2).
After the detailed neuropsychiatric evaluation, we found that patients with citrin deficiency had a greater likelihood of being diagnosed with ADHD. We compared neurocognitive function between citrin deficiency patients and control individuals by age, sex, and IQ score. The parent-reported SNAP-IV results revealed that citrin deficiency patients had a higher tendency towards ADHD, with significant differences found in hyperactivity symptoms (P=0.020 by Mann-Whitney U test) (Fig. 3A). Further assessments based on DSM-5 diagnostic criteria, conducted by experienced pediatric psychiatrists (JCC and SSG), identified 14 cases (Supplementary Table 2), although 20 cases were initially reported by parent-reported SNAP-IV results. Supplementary Table 2 displays the results for citrin deficiency patients who were diagnosed with ADHD through a physician assessment. Among the 30 citrin deficiency patients, 14 were diagnosed with ADHD (6 with the combined type, 6 with the inattentive type, and 2 with the hyperactive-impulsive type). The proportion of citrin deficiency patients with ADHD was 47% (14 of 30). The average age of diagnosis with ADHD is 6.6±2.6 years (range, 3–11). Among the 14 patients with ADHD, 7 (50%) needed to take medicine to control their symptoms during daily school activities.
3. Neurocognitive functional prognostic indicators
We then compared the peak laboratory values before the age of one year between ADHD patients and non-ADHD patients and found no statistically significant differences in either amino acid analysis or biochemical test results. In addition, logistic regression analysis revealed no significant associations between citrin deficiency blood test results before the age of one year, duration of cholestasis and ADHD. However, when ADHD was further divided into inattentive and hyperactivity-impulsivity symptoms, a one-unit increase in ammonia levels was associated with a 1.023-fold increase in the likelihood of developing future hyperactivity-impulsivity symptoms (P=0.038; 95% confidence interval [CI], 1.001–1.046), as assessed by both specialist evaluations and K-SADS-E assessments.
4. Other neurobehavioral disorders in citrin deficiency patients
In addition to being diagnosed with ADHD, some patients were diagnosed with other neurobehavioral disorders, including major depressive disorder (3.3%, n=1/30) [16], social anxiety disorder (3.3%, n=1/30) [17], Tourette's disorder (3.3%, n=1/30) [18], and Asperger syndrome (3.3%, n=1/30). Furthermore, based on the semistructured K-SADS-E inter view, some patients were found to have a history of neurobehavioral disorders, including tic disorders (10.0%, n=3/30) [18], sleep disorders (6.7%, n=2/30) [19], and separation anxiety disorders (6.7%, n=2/30) [20], which improved or did not affect their quality of life as they aged. The prevalence of all the aforementioned neurobehavioral disorders in the study population was similar to that of the general population.
5. Subgroup analysis according to patient source
Among the 30 patients with citrin deficiency, 10 were diagnosed through NBS, and 20 were diagnosed through clinical evaluation, with male-to-female ratios of 1.5:1 and 1:1.2, respectively. Among the 18 patients who were switched to MCT formula milk, 4 (22.2%) were in the NBS group, and 14 (77.8%) were in the clinical diagnosis group. There were no significant differences in growth, including height, weight, or body mass index, between the 2 groups (Table 1). Regarding the duration of cholestasis, the clinical diagnosis group had a longer duration (149±136 days) than did the NBS group (95±35 days), but the difference was not statistically significant (P=0.471). The peak laboratory values of the NBS and clinical diagnosis groups before one year of age are presented in Table 2. Compared to the NBS group, the clinical diagnosis group had significantly lower glutamine levels (P=0.034) and significantly higher AST (P≤0.001), ALT (P=0.017), D-Bil (P=0.014) and ornithine (P=0.040) levels. There were no significant differences in the ammonia or citrulline levels.
For the neuropsychiatric evaluation, when comparing the NBS, clinical diagnosis, and control groups, it was found that the clinical diagnosis group had significantly greater hyperactivity scores than did the control group (P=0.020 by post hoc analysis) (Fig. 3B). Of the 14 patients with ADHD, 11 (78.6%) were in the clinical diagnosis group, and 3 (21.4%) were in the NBS group. However, Fisher exact test showed no significant difference between the NBS and clinical diagnosis groups, indicating that the time of diagnosis was not related to the development of ADHD.
Discussion
In this study, we revealed a lasting impact of early metabolic disruptions on ADHD symptoms in citrin deficiency patients. Individuals diagnosed based on clinical symptoms were more prone to ADHD than those diagnosed through newborn screening. This emphasizes the urgent necessity for early detection and management strategies to address ADHD risk linked to citrin deficiency. Despite these findings, long-term follow-up of individuals with citrin deficiency demonstrated that the FSIQ scores were normally distributed, and the incidence of other neurobehavioral disorders, such as major depressive disorder, social anxiety disorder, and Tourette's disorder, in the study population was comparable to that in the general population. This suggests that, notwithstanding specific challenges, individuals with citrin deficiency can lead generally normal lives.
Citrin deficiency presents distinct clinical manifestations characterized by metabolic abnormalities, including unique dietary patterns and specific onset periods. Given the prevalence of psychiatric disorders, such as depression, anxiety, irritability, and impulsive behavior [21,22], observed in individuals with other metabolic diseases, we conducted a neuropsychiatric evaluation of patients with citrin deficiency. Our findings revealed that 46.7% of the individuals with citrin deficiency were diagnosed with ADHD, which was significantly greater than the prevalence of ADHD in the Taiwanese community (7.5%) [23] and the global prevalence of approximately 1.7 to 16% [24,25]. Additionally, the prevalence of ADHD-related problems was also greater in patients with citrin deficiency than in patients with other urea cycle disorders, which were reported to affect 20% to 30% of patients [26,27]. We also noted that the ammonia level before the age of one year may be a predictor of hyperactivity-impulsivity symptoms. Compared with adult brains, newborn brains are more vulnerable. When enzymes involved in the urea cycle are deficient, the gradual accumulation of ammonia may occur, resulting in hyperammonemia. Ammonia can cross the blood-brain barrier through diffusion and affect the glutamate-glutamine cycle in the brain, which is responsible for maintaining the balance of excitatory and inhibitory neurotransmitters [28]. During acute decompensation, ammonia enters the brain and combines with glutamate in astrocytes, which leads to the formation of glutamine. The accumulation of glutamine in the brain causes increased osmotic pressure in astrocytes, cell swelling, toxic edema, intracranial hypertension, and impaired blood perfusion. In addition, the activation of glutamate/N-methyl-D-aspartic acid (NMDA) receptors result in excitotoxicity, energy deficiency, and cell death in the brain. The immature central nervous system in newborns and infants is particularly sensitive to ammonia, which can lead to irreversible brain damage.29) In addition, Hasan et al. indicated that patients with ADHD have levels of ammonia and lactate that are significantly higher than the reference values [30].
Newborn screening is typically conducted 2 days after birth, allowing for early diagnosis and treatment before noticeable symptoms appear. In contrast, clinically diagnosed patients often present with evident symptoms and are diagnosed at a later stage, leading to more severe liver dysfunction and impaired metabolic functions. Previous studies have shown that patients who are clinically diagnosed often experience poorer argininosuccinate synthesis, gluconeogenesis, ketogenesis, fatty acid oxidation, liver function impairment, and bile retention than patients diagnosed through newborn screening [31]. In this study, we observed that the clinical diagnosis group exhibited a longer duration of bile retention, greater levels of abnormal biochemical markers (AST, ALT, and D-Bil) and more pronounced ADHD symptoms according to the SNAP-IV score than did the control group. As mentioned previously, some studies have reported that an imbalance in the metabolism of glutamine and glutamate leads to microstructural changes in the brain in ADHD patients [32]. The ratio of glutamine to glutamate in ADHD patients is lower than that in the general population [32-34]. In the comparison between the NBS group and the clinical diagnosis group, the clinical diagnosis group had significantly lower levels of glutamine and a lower ratio of glutamine to glutamate. Therefore, it was inferred that the clinical diagnosis group had a greater likelihood of developing ADHD than the NBS group. However, due to the small sample size, significant differences could not be demonstrated. These findings support the hypothesis that patients diagnosed clinically are more susceptible to neurocognitive impairments. Besides, we also analyzed the association between the duration of cholestasis, AST/ALT levels, bilirubin, and coagulopathy with ADHD in these patients. However, we did not find a significant correlation or increased risk. The relevant data are provided in Supplementary Table 3. While no increased risk was found between the duration of cholestasis and liver function abnormalities and ADHD, early diagnosis can prevent recurrent episodes of hyperammonemia. Given that hyperammonemia has been linked to an increased risk of ADHD in these patients, we believe that early detection through NBS can help identify affected individuals at an early stage, particularly by preventing hyperammonemia caused by liver dysfunction. This could, in turn, potentially reduce the future risk of ADHD development. Compared with clinically diagnosed patients, patients diagnosed through newborn screening show faster recovery from cholestasis and liver function abnormalities, highlighting the importance of newborn screening.
While our study has some limitations, including the small number of patients and the possibility that individuals with citrin deficiency who already exhibited ADHD symptoms may have been more inclined to participate, it is worth noting that we included the largest cohort of citrin deficiency patients who underwent neurocognitive assessments during long-term follow-up. Patients with citrin deficiency are relatively rare in Taiwan, which poses challenges to recruitment. Thus, the prevalence of ADHD in citrin deficiency patients may have increased the likelihood of overestimating the prevalence of ADHD. It is worth considering further inclusion of more patients in future research to explore the relationship between citrin deficiency and ADHD.
In conclusion, we found that citrin deficiency patients may have a greater risk of developing ADHD than the general population. In the future, we aim to track the neurocognitive development of citrin deficiency patients diagnosed with the disease in infancy during childhood and adolescence and to conduct statistical analysis to gain insights into the long-term course of the disease. It is believed that this study can contribute to improving the quality of life of patients with citrin deficiency.
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