Multi-omics in classical galactosemia: Evidence for the involvement of multiple metabolic pathways.

Classical galactosemia (CG) is one of the more frequent inborn errors of metabolism affecting approximately 1:40.000 people. Despite a life-saving galactose-restricted diet, patients develop highly variable long-term complications including intellectual disability and movement disorders. The pathophysiology of these complications is still poorly understood and development of new therapies is hampered by a lack of valid prognostic biomarkers. Multi-omics approaches may discover new biomarkers and improve prediction of patient outcome. In the current study, (semi-)targeted mass-spectrometry based metabolomics and lipidomics were performed in erythrocytes of 40 patients with both classical and variant phenotypes and 39 controls. Lipidomics did not show any significant changes or deficiencies. The metabolomics analysis revealed that CG does not only compromise the Leloir pathway, but also involves other metabolic pathways including glycolysis, the pentose phosphate pathway, and nucleotide metabolism in the erythrocyte. Moreover, the energy status of the cell appears to be compromised, with significantly decreased levels of ATP and ADP. This possibly is the consequence of two different mechanisms: impaired formation of ATP from ADP possibly due to reduced flux though the glycolytic pathway and trapping of phosphate in galactose-1-phosphate (Gal-1P) which accumulates in CG. Our findings are in line with the current notion that the accumulation of Gal-1P plays a key role in the pathophysiology of CG not only by depletion of intracellular phosphate levels but also by decreasing metabolite abundance downstream in the glycolytic pathway and affecting other pathways. New therapeutic options for CG could be directed towards the restoration of intracellular phosphate homeostasis.

Gray and white matter are both affected in classical galactosemia: An explorative study on the association between neuroimaging and clinical outcome.

BACKGROUND: Classical Galactosemia (CG) is an inherited disorder of galactose metabolism caused by a deficiency of the galactose-1-phosphate uridylyltransferase (GALT) enzyme resulting in neurocognitive complications. As in many Inborn Errors of Metabolism, the metabolic pathway of CG is well-defined, but the pathophysiology and high variability in clinical outcome are poorly understood. The aim of this study was to investigate structural changes of the brain of CG patients on MRI and their association with clinical outcome. METHODS: In this prospective cohort study an MRI protocol was developed to evaluate gray matter (GM) and white matter (WM) volume of the cerebrum and cerebellum, WM hyperintensity volume, WM microstructure and myelin content with the use of conventional MRI techniques, diffusion tensor imaging (DTI) and quantitative T1 mapping. The association between several neuroimaging parameters and both neurological and intellectual outcome was investigated. RESULTS: Twenty-one patients with CG (median age 22 years, range 8-47) and 24 controls (median age 30, range 16-52) were included. Compared to controls, the WM of CG patients was lower in volume and the microstructure of WM was impaired both in the whole brain and corticospinal tract (CST) and the lower R1 values of WM, GM and the CST were indicative of less myelin. The volume of WM lesions were comparable between patients and controls. The 9/16 patients with a poor neurological outcome (defined as the presence of a tremor and/or dystonia), demonstrated a lower WM volume, an impaired WM microstructure and lower R1 values of the WM indicative of less myelin content compared to 7/16 patients without movement disorders. In 15/21 patients with a poor intellectual outcome (defined as an IQ < 85) both GM and WM were affected with a lower cerebral and cerebellar WM and GM volume compared to 6/21 patients with an IQ ≥ 85. Both the severity of the tremor (as indicated by the Tremor Rating Scale) and IQ (as continuous measure) were associated with several neuroimaging parameters such as GM volume, WM volume, CSF volume, WM microstructure parameters and R1 values of GM and WM. CONCLUSION: In this explorative study performed in patients with Classical Galactosemia, not only WM but also GM pathology was found, with more severe brain abnormalities on MRI in patients with a poor neurological and intellectual outcome. The finding that structural changes of the brain were associated with the severity of long-term complications indicates that quantitative MRI techniques could be of use to explain neurological and cognitive dysfunction as part of the disease spectrum. Based on the clinical outcome of patients, the absence of widespread WM lesions and the finding that both GM and WM are affected, CG could be primarily a GM disease with secondary damage to the WM as a result of neuronal degeneration. To investigate this further the course of GM and WM should be evaluated in longitudinal research, which could also clarify if CG is a neurodegenerative disease.

Deep phenotyping classical galactosemia: clinical outcomes and biochemical markers.

Early diagnosis and dietary treatment do not prevent long-term complications, which mostly affect the central nervous system in classical galactosemia patients. The clinical outcome of patients is highly variable, and there is an urgent need for prognostic biomarkers. The aim of this study was first to increase knowledge on the natural history of classical galactosemia by studying a cohort of patients with varying geno- and phenotypes and second to study the association between clinical outcomes and two possible prognostic biomarkers. In addition, the association between abnormalities on brain MRI and clinical outcomes was investigated. Classical galactosemia patients visiting the galactosemia expertise outpatient clinic of the Amsterdam University Medical Centre were evaluated according to the International Classical Galactosemia guideline with the addition of an examination by a neurologist, serum immunoglobulin G N-glycan profiling and a brain MRI. The biomarkers of interest were galactose-1-phosphate levels and N-glycan profiles, and the clinical outcomes studied were intellectual outcome and the presence or absence of movement disorders and/or primary ovarian insufficiency. Data of 56 classical galactosemia patients are reported. The intellectual outcome ranged from 45 to 103 (mean 77 ± 14) and was <85 in 62%. Movement disorders were found in 17 (47%) of the 36 tested patients. In females aged 12 years and older, primary ovarian insufficiency was diagnosed in 12 (71%) of the 17 patients. Significant differences in N-glycan peaks were found between controls and patients. However, no significant differences in either N-glycans or galactose-1-phosphate levels were found between patients with a poor (intellectual outcome < 85) and normal intellectual outcome (intellectual outcome ≥ 85), and with or without movement disorders or primary ovarian insufficiency. The variant patients detected by newborn screening, with previously unknown geno- and phenotypes and currently no long-term complications, demonstrated significantly lower galactose-1-phospate levels than classical patients (P < 0.0005). Qualitative analysis of the MRI's demonstrated brain abnormalities in 18 of the 21 patients, more severely in patients with a lower intellectual outcome and/or with movement disorders. This study demonstrates a large variability in clinical outcome, which varies from a below average intelligence, movement disorders and in females primary ovarian insufficiency to a normal clinical outcome. In our cohort of classical galactosemia patients, galactose-1-phosphate levels and N-glycan variations were not associated with clinical outcomes, but galactose-1-phosphate levels did differentiate between classical and variant patients detected by newborn screening. The correlation between brain abnormalities and clinical outcome should be further investigated by quantitative analysis of the MR images. The variability in clinical outcome necessitates individual and standardized evaluation of all classical galactosemia patients.

Correction to: Classical galactosemia: neuropsychological and psychosocial functioning beyond intellectual abilities.

An amendment to this paper has been published and can be accessed via the original article.

Classical galactosemia: neuropsychological and psychosocial functioning beyond intellectual abilities.

BACKGROUND: Despite early diagnosis and treatment, Classical Galactosemia (CG) patients frequently develop long-term complications, such as cognitive impairment. Available literature primarily reports on general intellectual abilities and shows a substantially lower Full Scale Intelligence Quotient (FSIQ) in CG patients than in the general population. Both problems in social functioning as well as internalizing problems are often reported in CG patients. The combination of intelligence, cognitive functioning, behavior and social functioning has not been studied systematically in CG patients. METHODS: To determine if CG patients demonstrate a specific neuropsychological and psychosocial profile, we investigated intelligence, functioning on multiple cognitive domains, behavior and social functioning with a comprehensive neuropsychological test battery and questionnaires (self- and proxy-reported). RESULTS: The data of 48 patients, aged 4-47 years are reported. FSIQ ranged from 45 to 103 (mean 77 ± 14). A negative correlation between age and FSIQ was demonstrated (p = 0.037) which resulted directly from the inclusion of four young 'milder' patients detected by newborn screening (NBS) with an expected better clinical outcome. Compared to normative data, patients had significantly lower but highly variable scores on all cognitive domains, especially on tests requiring mental speed. In the context of the FSIQ, 43% of the cognitive test results exceeded IQ based expectations. Overall, the patients' scores on social functioning were in the normal range but internalizing problems were frequently reported. In our cohort, an early initiation of dietary treatment due to NBS or family screening did not result in a more favorable neuropsychological outcome. CONCLUSIONS: In this study, we demonstrated that as a cohort, CG patients have a below average intelligence and impaired cognitive functioning without a distinctive neuropsychological profile. The effect of age on neurocognitive functioning should be assessed in longitudinal studies. Social functioning was not impaired, but patients may be at risk for internalizing problems. Considering the large variability in cognitive, behavioral and social functioning and the finding that cognitive outcomes may exceed IQ based expectations, an individual evaluation and follow-up is warranted in all CG patients to ensure timely support if needed.

The Galactose Index measured in fibroblasts of GALT deficient patients distinguishes variant patients detected by newborn screening from patients with classical phenotypes.

BACKGROUND: The high variability in clinical outcome of patients with Classical Galactosemia (CG) is poorly understood and underlines the importance of prognostic biomarkers, which are currently lacking. The aim of this study was to investigate if residual galactose metabolism capacity is associated with clinical and biochemical outcomes in CG patients with varying geno- and phenotypes. METHODS: Galactose Metabolite Profiling (GMP) was used to determine residual galactose metabolism in fibroblasts of CG patients. The association between the galactose index (GI) defined as the ratio of the measured metabolites [U13C]Gal-1-P/ [13C6]UDP-galactose, and both intellectual and neurological outcome and galactose-1-phosphate (Gal-1-P) levels was investigated. RESULTS: GMP was performed in fibroblasts of 28 patients and 3 control subjects. The GI of the classical phenotype patients (n = 22) was significantly higher than the GI of four variant patients detected by newborn screening (NBS) (p = .002), two homozygous p.Ser135Leu patients (p = .022) and three controls (p = .006). In the classical phenotype patients, 13/18 (72%) had a poor intellectual outcome (IQ < 85) and 6/12 (50%) had a movement disorder. All the NBS detected variant patients (n = 4) had a normal intellectual outcome (IQ ≥ 85) and none of them has a movement disorder. In the classical phenotype patients, there was no significant difference in GI between patients with a poor and normal clinical outcome. The NBS detected variant patients had significantly lower GI levels and thus higher residual galactose metabolism than patients with classical phenotypes. There was a clear correlation between Gal-1-P levels in erythrocytes and the GI (p = .001). CONCLUSIONS: The GI was able to distinguish CG patients with varying geno- and phenotypes and correlated with Gal-1-P. The data of the NBS detected variant patients demonstrated that a higher residual galactose metabolism may result in a more favourable clinical outcome. Further research is needed to enable individual prognostication and treatment in all CG patients.

The 1-13 C galactose breath test in GALT deficient patients distinguishes NBS detected variant patients but does not predict outcome in classical phenotypes.

Classical galactosemia (CG) patients frequently develop long-term complications despite early dietary treatment. The highly variable clinical outcome is poorly understood and a lack of prognostic biomarkers hampers individual prognostication and treatment. The aim of this study was to investigate the association between residual galactose oxidation capacity and clinical and biochemical outcomes in CG patients with varying geno- and phenotypes. The noninvasive 1-13 C galactose breath test was used to assess whole body galactose oxidation capacity. Participants received a 7 mg/kg oral dose of 1-13 C labelled galactose. The galactose oxidation capacity was determined by calculating the cumulative percentage dose of the administered galactose (CUMPCD) recovered as 13 CO2 in exhaled air. Forty-one CG patients (5-47 years) and four adult controls were included. The median galactose oxidation capacity after 120 minutes (CUMPCDT120) of 34 classical patients (0.29; 0.08-7.51) was significantly lower when compared to two homozygous p.Ser135Leu patients (9.44; 8.66-10.22), one heterozygous p.Ser135Leu patient 18.59, four NBS detected variant patients (13.79; 12.73-14.87) and four controls (9.29; 8.94-10.02). There was a clear correlation between Gal-1-P levels and CUMPCDT120 (P < .0005). In the classical patients, the differences in CUMPCDT120 were small and did not distinguish between patients with poor and normal clinical outcomes. The galactose breath test distinguished classical patients from homo- and heterozygous p.Ser135Leu and NBS detected variant patients, but was not able to predict clinical outcomes in classical patients. Future studies are warranted to enable individualised prognostication and treatment, especially in NBS variants with galactose oxidation capacities in the control range.

Bone mineral density is within normal range in most adult phenylketonuria patients.

Low bone mineral density (BMD) as a risk factor for fractures has been a long-standing concern in phenylketonuria (PKU). It is hypothesised that the disease itself or the dietary treatment might lead to a low BMD. Previous studies show conflicting results of BMD in PKU due to differences in age, techniques to assess BMD and criteria used. To assess the prevalence of low BMD and define possible risk factors in a large number of adult, early treated PKU (ETPKU) patients. European centres were invited for a survey, collecting retrospective data including results of dual-energy X-ray absorptiometry (DXA) scans of adult ETPKU patients. BMD of 183 adult ETPKU patients aged 18-46 (median age 28, all females premenopausal) years was lower than in the general population at most skeletal sites but the frequency of low BMD (Z-score <-2) was at maximum 5.5%. No risk factors for low BMD in PKU patients could be identified. Low BMD occurs only in a small subset of PKU patients. DXA scans should be considered for well controlled patients from age 35-40 years and up and on indication in those PKU patients considered to be at increased risk for fractures.

Reducing complexity: explaining inborn errors of metabolism and their treatment to children and adolescents.

BACKGROUND: Inborn errors of metabolism (IEM) are a group of rare, heterogeneous and complex genetic conditions. Clinically, IEM often affect the central nervous system and other organs. Some carry the risk of progression and / or potentially life-threatening crises. Many patients have to adhere to lifelong dietary or drug treatment. The complexity of IEM makes it difficult for patients and caregivers to understand their pathophysiology, inheritance and therapy rationale. Especially patients reaching adolescence may have only limited knowledge of their condition since medical care has often entirely been handled by their parents. Knowledge about disease and treatment, however, constitute pillars of self-responsible disease management. Not many standardized patient education materials on IEM are available and their comprehensibility has not been systematically investigated. METHODS: We developed and tested patient education materials for school-aged children and adolescents with IEM. Informative texts and illustrations in paper form and as videos were developed by an international network of metabolic care professionals together with a graphic artist and experts for easy-to-read language. The materials were presented in standardized single or group training sessions to 111 individuals; first, to 74 healthy children and adolescents (recruited via public schools) and consecutively to 37 paediatric patients with IEM (phenylketonuria, galactosemia, urea cycle defects, lysosomal storage disorders) from six metabolic centres. Knowledge-gain was assessed by pre- and post-testing. RESULTS: Knowledge-gain was significant in healthy children and adolescents as well as in patients (p < .001, r =. -77 /. -70). Effect sizes were large in both groups (r = -.77 / -.70). This result was independent from family language and teacher-rated concentration or cognitive capacity in healthy children. CONCLUSION: The newly developed patient education materials are a powerful tool to improve disease- and treatment-related knowledge. They facilitate communication between the medical team and children and adolescents with IEM and their caregivers.

Cognitive functioning in patients with classical galactosemia: a systematic review.

BACKGROUND: Patients with the metabolic disorder classical galactosemia suffer from long-term complications despite a galactose-restricted diet, including a below average intelligence level. The aim of the current review was to investigate the incidence and profile of cognitive impairments in patients with classical galactosemia. METHOD: MEDLINE, EMBASE and PsychINFO were searched up to 23 October 2018 for studies examining information processing speed, attention, memory, language, visuospatial functioning, executive functioning and social cognition in patients with confirmed classical galactosemia utilizing standardized neuropsychological tests. Data synthesis followed a narrative approach, since the planned meta-analysis was not possible due to large variability between the neuropsychological assessments. RESULTS: Eleven studies were included, including case-studies. The quality of most studies was moderate to low. As a group, patients with classical galactosemia exhibit below average to low scores on all cognitive domains. A large proportion of the patients perform on an impaired level on attention, memory and vocabulary. Evidence for impairments in information processing speed, language, visuospatial functioning and aspects of executive functioning was limited due to the small number of studies investigating these cognitive functions. Social cognition was not examined at all. CONCLUSIONS: Given the moderate to low quality of the included studies and the limited evidence in many cognitive domains, the incidence of cognitive impairment in patients with classical galactosemia is not yet clear. Both clinicians and researchers encountering patients with classical galactosemia need to be aware of possible cognitive impairments. Future well-designed studies are needed to determine the cognitive profile of classical galactosemia. This can be the basis for the development of intervention strategies.

Normal MRI findings of the knee in patients with clinically active juvenile idiopathic arthritis.

OBJECTIVE: In a number of patients with clinically active juvenile idiopathic arthritis (JIA), contrast-enhanced MRI shows no signs of synovitis. The objective of this study was to assess the frequency and the patient characteristics in clinically active JIA patients in which MRI showed no signs of synovitis. METHODS: From our cohort of 313 patients in which contrast-enhanced MRI of the knee had been performed, we selected 72 JIA patients with clinically active disease involving the target joint. The validated Juvenile Arthritis MRI Scoring (JAMRIS) system was used to evaluate synovial thickening. Patients were divided into two groups based on MRI outcome: Group 1: thickened synovium on MRI (JAMRIS score ≥1) or Group 2: normal synovium on MRI (JAMRIS score 0). Patient characteristics and disease activity parameters were then compared. RESULTS: In 35% (25/72) of these patients, MRI results contrasted with the clinical assessment (Group 2). In comparison to Group 1, the patients with discrepant findings were significantly older at the date of examination and JIA had been diagnosed at later age (median age of 13.2 vs. 10.9 and median age 10.0 vs. 8.0 respectively). In Group 2 there were significantly more patients with RF-negative polyarticular disease. CONCLUSION: Patients with RF-negative polyarticular JIA who had been diagnosed at a later age and were older at the time of MRI were most likely to be considered clinically active while MRI showed no signs of synovitis. These particular JIA patients may benefit from monitoring of disease activity by MRI to prevent overtreatment.

Juvenile idiopathic arthritis: magnetic resonance imaging of the clinically unaffected knee.

BACKGROUND: Synovial thickening detected on magnetic resonance imaging (MRI) is present in a significant number of children with clinically inactive juvenile idiopathic arthritis (JIA). OBJECTIVE: To evaluate patient characteristics and disease activity parameters in a cohort of children with clinically inactive JIA, both with and without synovial thickening, in order to clarify the observed discrepancy between clinical and MRI assessments. MATERIALS AND METHODS: We prospectively enrolled 52 clinically inactive JIA patients (median age 13.3 years, 63.5% girls) who underwent MRI of the knee as major target joint in JIA. Children were divided into two groups based on MRI outcome: group 1, with synovial thickening on MRI; and group 2, with no synovial thickening on MRI. We used the Juvenile Arthritis MRI Scoring system to evaluate synovial thickness. We compared patient characteristics and disease activity parameters between the groups. RESULTS: Synovial thickening on MRI was present in 18 clinically inactive patients (group 1, 34.6%). The age was significantly lower for the patients in group 1 (median 10.7 versus 14.4, P=0.008). No significant differences were observed in any of the other patient characteristics nor the disease activity parameters tested. CONCLUSION: Synovial thickening on MRI was present in nearly 35% of the children with clinically inactive JIA. Children with synovial thickening on MRI were significantly younger than those without. This might indicate that younger patients are at risk of subclinical disease activity and under-treatment, although the exact clinical relevance of synovial thickening on MRI has not been determined.