Treatment Outcomes for Maple Syrup Urine Disease Detected by Newborn Screening Free

This multicenter, observational study of patients with confirmed MSUD born between January 1, 1998 and June 30, 2021 is a national extension of the regional NBS outcome study in southwest Germany.¹⁷  The study was first approved by the ethics committee of the coordinating site (Heidelberg University Hospital, #S104/2005; Trial ID: DRKS00013329) and subsequently, by the other sites. This study includes individuals with cMSUD and vMSUD but excludes individuals with thiamine-responsive MSUD and deficiency of the subunit E3. The inclusion criteria were (1) identification by NBS or false negative NBS result with later diagnosis of MSUD, (2) confirmed diagnosis through the detection of allo-isoleucine²¹  and/or pathogenic bi-allelic variants in BCKDHA, BCKDHB, or DBT, and (3) written informed consent from the patient and/or caregivers.

The German NBS program recommends a sampling at the age of 36 to 72 hours of life, a sample transport within 48 hours, and analysis in the NBS laboratory within 24 hours. Consequently, the NBS report is available on day of life 5 or 6 (median).¹⁷  The primary markers for MSUD in tandem mass spectrometry-based NBS are combined leucine and isoleucine levels (XLE) and their ratio to other amino acids. Massively elevated XLE above the cutoff results in a strong suspicion of MSUD and an immediate notification of the sender and the family, short-term clinical evaluation and confirmatory diagnostics (amino acid profile in plasma and genetic testing), and the initiation of specific treatment in a specialized metabolic center. For minor XLE elevations, a second dried blood sample is requested to exclude false positives.

We evaluated biochemical parameters of the NBS and confirmatory diagnostics, clinical follow-up parameters, and medical history at defined ages, as described previously.¹⁷  Cognitive functions were assessed by using age-adapted standardized tests, such as the Wechsler Preschool and Primary Scale of Intelligence, fourth edition, the Wechsler Intelligence Scale for Children, fifth edition, and the Snijders-Oomen Nonverbal Intelligence Test. In addition, the Denver Developmental Screening Test, the Bayley Scales of Infant Development, third edition, the Parent Report of Children’s Abilities-Revised, and the “Entwicklungstest für Kinder von 6 Monaten bis 6 Jahren” revision were used.

A quantitative analysis of BCAA in plasma was performed in different local metabolic laboratories by using either ion exchange chromatography or liquid chromatography (tandem) mass spectrometry. However, according to German national regulations, all laboratories participated in the same ring tests for benchmarking, so the quantitative results for BCAA are comparable, and therefore, all available BCAA values during the observation period were extracted from the patients’ medical records. Values less than the limit of quantification were graded as “0.” To approximate long-term metabolic control, we estimated the area under the curve (AUC) of leucine values. To standardize the AUC, it was divided by the individual observational period (AUC ratio).

The initial metabolic treatment after the NBS report was classified as (1) basic if a low-protein diet with BCAA-free supplements with or without additional IV glucose therapy was used, or (2) intensified if extracorporeal detoxification with hemodialysis was needed. The clinical presentation at the time of the first NBS report was divided into 3 groups: (1) asymptomatic, (2) characteristic clinical presentation of MSUD without, or (3) with severely impaired consciousness (ie, coma) and/or respiratory failure.

To enable the severity-adjusted evaluation of the benefit of NBS, we defined cMSUD and vMSUD according to previous studies.^1,2  For pre-symptomatically identified MSUD patients, the disease severity was estimated by using the residual BCKDH activity, known pathogenic gene variants, clinical data from symptomatic siblings (if available), and biochemical markers. Finally, cMSUD was distinguished from vMSUD by the presence of 1 or more of the following criteria: (1) residual enzyme activity of BCKDH <2%, (2) pathogenic variants known to result in cMSUD, (3) the phenotype of a previously diagnosed and treated sibling with cMSUD, and (4) initial peak pLeu >1000 µmol/L (this threshold was chosen because all neonatal symptomatic individuals in our cohort presented with an initial pLeu ≥1000 µmol/L).

Long-term metabolic treatment was classified as conservative (dietary treatment, intermittent emergency treatment) or liver transplantation. The initial peak pLeu was defined as the highest pLeu detected during the first hospital admission. A metabolic decompensation was defined as a pLeu >500 µmol/L. Consecutive increases in pLeu over a 14-day period were rated as a single decompensation.

Cognitive functions were categorized as normal (IQ ≥85, normal result in a non-IQ-based test, or attendance of a regular school) or reduced (IQ <85, not age-appropriate result in a non-IQ-based test, or special educational needs). In the case of serial cognitive tests, the most recent test result was included.

Statistical analyses were performed by using the program R (https://www.R-project.org). Patients with missing data were excluded from the respective analysis. The number of patients for each analysis was given. Patients missed by NBS were included in the descriptive analysis but excluded from the comparative outcome analysis because of the small cohort. Independent variables used in the outcome analysis were age at first NBS report, initial peak pLeu, symptoms at initial presentation, initial metabolic treatment, the requirement of intensive care, number of decompensations, and sex. According to Hoffmann et al,⁸  we used hierarchical a cluster analysis with Canberra distance and the Ward fusion algorithm to cluster the annual median pLeu in the first 6 years of life, thereby identifying 2 groups with different leucine profiles. The median pLeu of these 2 groups were compared by using a linear mixed effect regression model (computed with R package “lme4”), with median pLeu as the dependent variable and predictor variables group derived from the cluster analysis, year of life (1–6 years), and random factor MSUD individual. In the case of serial IQ tests, the test result closest to the age of 6 years was selected for the analysis. For the outcome variables AUC ratio and IQ, a multiple regression analysis was performed with the independent variables described above. We used a stepwise forward and backward variable selection procedure based on Akaike Information Criteria to identify important independent variables in multiple regression models. The 2 groups were compared using the Wilcox–Mann–Whitney test.

A total of 35 individuals with confirmed MSUD were included. Thirty-three of these were identified through NBS (Table 1), whereas 2 patients were missed. The NBS cohort (N = 33) had a median age at last visit of 10.4 years (range 1.0–23.3; interquartile range [IQR] 3.6–15.9) and a cumulative follow-up time of 371 patient-years. Twenty-two patients in the NBS group had cMSUD, and 11 had vMSUD (Table 1). The diagnosis was confirmed biochemically in all patients, via enzymatic testing in 1, and genetically in 23 of them. Ten patients had bi-allelic pathogenic variants in BCKDHA, 11 patients in BCKDHB, and 2 patients in DBT (Table 1).

At the time of NBS report (median at 6 days of life [range 1–23; IQR 5–9]), 17 newborns (52%) were asymptomatic, whereas 15 (45%) already showed a characteristic clinical presentation of MSUD, 3 of them with severely impaired consciousness and/or respiratory failure (no data on neonatal presentation in 1 case). For cMSUD, the NBS report was communicated at an earlier point of time in newborns without clinical symptoms (N = 6; at median 2.5 days; range 1–5) than in those with symptoms at the time of NBS report (N = 15; at median 6 days; range 4–11; P = .009). When the high-risk family screening cases (N = 3) were excluded, this effect could not be reproduced.

In 4 newborns from known high-risk families (N = 3 cMSUD, N = 1 vMSUD), treatment was initiated within the first 2 days of life. In 6 individuals, treatment was started after the tenth day of life because of prolonged duration of transport (N = 3), technical difficulties at the beginning of the MSUD screening (N = 1), or the need for the analysis of a second dried blood sample before treatment because of minor abnormalities in the first sample (N = 2).

Fourteen patients (43%) required intensive care, and 8 of them required intensified treatment with hemodialysis (median initial pLeu 2063 µmol/L; range 1675–4142 µmol/L). A further 5 patients with an initial pLeu >1500 µmol/L (median 2269 µmol/L; range 1700–4037 µmol/L) were managed without hemodialysis.

Overall, the median initial peak pLeu (N = 32) was 1103 µmol/L (range 86–4142; IQR 483–1962). In the cMSUD group (N = 21), pLeu was markedly higher (median: 1712 µmol/L; range 307–4142; IQR 1180–2552; 59% >1500 µmol) than in vMSUD (median: 440 µmol/L; range 86–900; IQR 353–554) and increased linearly during the time interval required to report NBS results, whereas this period did not apparently affect the initial peak pLeu in vMSUD (N = 11; Fig 1).

Thirty-two participants in the NBS group (97%) survived. One patient died at the age of 16 years during a fatal metabolic decompensation precipitated by perforated appendicitis.

In 9457 BCAA analyses in plasma (target ranges: pLeu 75–200 µmol/L; isoleucine 100–400 µmol/L; valine 100–400 µmol/L⁹ ), MSUD patients achieved a median (range, IQR) concentration of pLeu (214 µmol/L [0–4152; 122–351]), isoleucine (164 µmol/L [0–4276; 104–250]), and valine (257 µmol/L [0–4888; 161–376]). The median (range, IQR) number of metabolic decompensations per patient was 11 (0–55; 2–27) and 0.9 per patient and year (0–3.5; 0.4–2.4). The frequency of metabolic decompensations was higher in individuals with cMSUD (median 1.9 per year; 0.3–3.5; 0.7–3.0) compared with those with vMSUD (0.4 per year; 0–1.4; 0–0.9; P < .001).

In 30 individuals, neurocognitive development was assessed at least once at a median age of 10.3 years (range 0.9–23.3; IQR 4.3–15.3), mostly through IQ-based tests (N = 23). In patients with serial neuropsychological testing (N = 11), we did not observe significant changes with age (P = .49). In 19 participants (58%), we observed normal cognitive functions, whereas 12 of them (36%) were below the normal range. Two patients at preschool age without cognitive testing could not be scaled.

Overall, the median (range, IQR) IQ at the last testing was at the lower end of the normal range (N = 23; 87 [55–141, 75–100.5]), with 57% of patients having an IQ of 85 or above. Individuals with cMSUD had a lower IQ (82.5 [55–101; 70.5–94.5]) than those with vMSUD (105 [83–141; 96.5–122.5; P = .001]). Overall, the global IQ correlated inversely with the initial peak pLeu (N = 22; P = .04; β = –0.0081, confidence interval [CI; 0.0004 to 0.0011]; R² = 0.1869; Fig 2A) and mean annual frequency of metabolic decompensations (N = 22; P = .02; β = –9.133; CI [–16.40 to –1.87]; R² = 0.2558; Fig 2B), and correlated positively with the age at first NBS report (N = 23; P = .03). However, this effect disappeared when the disease variant was included as an independent variable in multiple regression models. AUC ratio (N = 23), sex (N = 23), symptoms at diagnosis (N = 22), and the initial metabolic treatment (N = 23) had no apparent impact on IQ.

According to a previous study,⁸  we analyzed the median annual pLeu in the first 6 years of life of patients who received a low-leucine diet during this period (N = 21; 16 cMSUD, 5 vMSUD) to evaluate the impact of treatment control. Later, 3 individuals from this group underwent liver transplantation (2 cMSUD, 1 vMSUD; age 10–14 years). A cluster analysis of the median annual pLeu over the first 6 years of life was used to identify 2 groups (Fig 3A) that differ in their therapeutic achievement of target pLeu. Clinical phenotypes (cMSUD, vMSUD) were similarly distributed between the 2 groups (P = .70), and the groups did not differ in IQ (Fig 3B), age at last visit (P = .35), initial peak leucine level (P = .60), or number of decompensations per year (P = .68). In contrast, the long-term achievement of target pLeu had an impact on cognitive functions in cMSUD (N = 16; Fig 4A). Patients with lower long-term pLeu had higher IQs (median 95.5) compared with those with higher pLeu (median 80; P = .008; Fig 4B). It is of note that both groups did not differ in initial pLeu (P = .41) or the decompensation rate per year (P = .96), excluding alternative explanations for the discrepant IQ in these groups.

Six children received liver transplantation at a median age of 5.8 years (range 0.4–14.3; IQR 0.4–11.8). The median follow-up time after liver transplantation was 3.6 years (range 0.3–5.1; IQR 2.3–5.0). All patients received organs from deceased donors, and all organs from MSUD patients were passed on as domino transplants. Patient survival was 100%, and allograft survival was 86%, with 1 patient requiring re-transplantation on postoperative day 6 because of hepatic artery dissection. Two patients underwent post-transplant surgery for bleeding complications and secondary abdominal wall closure. Three patients with biliary complications required intervention, and 1 developed pancreatitis. Medical complications were cytomegalovirus- (N = 2) and Epstein-Barr virus-related (N = 3) diseases and acute rejection (N = 1; Supplemental Table 3). Increased protein intake after liver transplantation did not precipitate metabolic decompensations. A comparative analysis of conservative treatment versus liver transplantation was not feasible because of the small size of the transplantation group (Table 2).

Two siblings with vMSUD and intermittent normal leucine levels were not identified by NBS because of normal XLE concentrations in the NBS sample (269 µmol/L, 286 µmol/L [cutoff 300 µmol/L]). The younger sibling was diagnosed at the age of 44 months during an episode of impaired consciousness precipitated by a febrile infection (plasma concentrations: pLeu 3049 µmol/L; allo-isoleucine 130 µmol/L). Subsequently, his older sister was examined and diagnosed at the age of 60 months, with mildly elevated pLeu (245 µmol/L) and detection of allo-isoleucine. Their medical histories revealed previous transient episodes of drowsiness during infectious diseases but a normal development for both. Low-protein diets and intermittent emergency treatments were started. Their IQs were normal at 10.6 and 11.9 years (Table 1).

In this national, multicenter observational study, we followed 35 individuals with MSUD longitudinally to determine the impact of NBS, the type and quality of treatment, and therapy-independent variables on the health outcome. This study proves the overall benefit of NBS for MSUD patients but also uncovers opportunities for optimization.

NBS programs with a high sensitivity for cMSUD, but not for vMSUD, were implemented >20 years ago.^16,20,22,23  Previous studies have revealed that NBS and the early start of metabolic therapy reduce (neonatal) mortality.^18,20,24  In pre-NBS cohorts,^3,8,25,26  the cognitive outcome of surviving MSUD patients was commonly impaired (median IQ 77⁸ ; median 66³ ). Small NBS cohort studies revealed some improvement, but cognitive outcomes were still below the reference population (median IQ 93¹⁸  and 90¹⁷ ). In the current study, we confirmed that the IQs of screened individuals with MSUD (median IQ 87) are at the lower end of the normal range, and normal IQs are found in 58%. We show that the variation in IQ in this group is best explained by the time to diagnosis, intrinsic disease severity, and quality of therapy, highlighting opportunities for further optimization. Analogous to impaired IQ, previous studies have indicated impaired executive functions in MSUD patients.^27,28  This was not part of the neuropsychological testing in the current study but should be evaluated in future studies.

MSUD patients are traditionally grouped according to parameters that reflect disease severity, such as residual enzyme activity, age at disease onset, clinical endpoints, and dietary leucine tolerance.^2,29  However, because disease severity follows a continuous spectrum, the existing classifications are partially overlapping.^2,30  The most severe form, cMSUD, is biochemically characterized by a (near) loss of BCKDH activity and clinically characterized by a life-threatening neonatal disease onset and neurocognitive impairment in surviving individuals. Patients with vMSUD have retained some residual BCKDH activity, resulting in a more attenuated biochemical and clinical phenotype compared with cMSUD. Regardless of this, all MSUD patients carry a lifelong risk of a potentially fatal metabolic decompensation precipitated by catabolic stress.^1,2 

In NBS cohorts with mostly preclinical diagnosis, disease severity cannot be estimated early and reliably by using clinical parameters. To overcome these shortcomings, we established a set of robust criteria that allow reliable early prediction of disease severity in NBS patients. By using these criteria, we have shown that disease severity had a major impact on the individual risk of metabolic decompensations and cognitive outcome, highlighting the need for severity-adjusted evaluation of NBS programs for MSUD. Of note, these criteria might have limitations because of the continuous spectrum of disease severity and possible overlapping initial peak leucine levels for very early identified and treated cMSUD and vMSUD.

The authors of previous clinical studies have suggested that the age at diagnosis,^25,31  the postnatal time interval with pLeu >1000 µmol/L,^3,25  and the quality of long-term metabolic control affect cognitive functions.^3,8,25,32  In this study, we demonstrate that pLeu remain stable in the neonatal period in vMSUD, whereas they increase linearly with age in cMSUD. Notably, in 59% of cMSUD patients, pLeu increased to >1500 µmol/L before the start of treatment, often necessitating hemodialysis. Although not all infants in our cohort could be detected pre-symptomatically, all neonates were identified through NBS before the diagnosis was made and, especially, before specific treatment was started. This underlines the importance of NBS for the early treatment of MSUD. These results support the notion of a short span of time to identify individuals with cMSUD in the preclinical stage, similar to other intoxication-type inherited metabolic diseases, such as galactosemia, long-chain 3-hydroxyacyl-coenzyme A dehydrogenase deficiency, and isovaleric acidemia.^17,33  These early decompensating diseases require an optimized NBS process and continuous efforts to avoid transport delays.

To elucidate the impact of therapy-independent variables on cognitive outcome, we investigated treatment adherence and long-term achievement of target pLeu. In line with a previous study,⁸  a cluster analysis identified 2 groups with different long-term pLeu. Overall, individuals with cMSUD with lower pLeu had higher IQ values compared with those with higher pLeu, highlighting the impact of the therapeutic quality on the outcome.

There is increasing evidence that conservative management does not reliably protect against recurrent metabolic decompensations, particularly in individuals with cMSUD.^17,20,34  This notion is supported by a patient with MSUD who died during a severe metabolic decompensation at the age of 16 years. Although the risk of mortality and severe morbidity can be reduced by adherence to treatment regimens, daily adherence to strict dietary protocols is often a significant burden for patients and families.^35,36  In recent years, liver transplantation has emerged as an alternative treatment option for individuals with cMSUD because post-transplant survival is good,^12,34  and sufficient BCKDH activity usually enables an unrestricted diet after transplantation.^10,11,37  However, liver transplantation does not reliably protect against hyperleucinemic encephalopathy during pronounced catabolic stress,³⁸  highlighting that this intervention does not cure but attenuates the disease. Because BCKDH is expressed mainly in skeletal muscle, but also in the liver, brain, and kidneys,³⁹  domino liver transplantation using MSUD liver grafts for other recipients is an effective and safe option.^12,40 

So far, little is known about the long-term impact of liver transplantation on cognitive outcomes using standardized neuropsychological tests. It has been suggested that early liver transplantation may be beneficial, particularly in individuals without irreversible brain damage and cerebral dysfunction.^10,41  The inverse association between long-term leucine concentrations and IQ revealed by this study supports this assumption. On the other hand, a high rate of transplant-related complications was reported. More structured data from the follow-up of transplanted MSUD cohorts are required to evaluate the benefits and harms of conservative treatment versus liver transplantation.

This study is limited by the small cohort size and the fact that the test results (IQ) might be influenced by variable patient cooperation, particularly at a young age. The influence of the age at diagnosis on the cognitive outcome might be affected by the disease severity.

NBS, which allows an early start of treatment, enables neonatal survival and a favorable long-term cognitive outcome in individuals with MSUD. The beneficial effect of NBS is largely dependent on the time to diagnosis, treatment quality, and disease severity. Because disease severity is an important modifier of outcome, it should be included in any evaluation of NBS programs for MSUD to avoid overestimating its benefit. The long-term achievement of good metabolic control can partially overrule the impact of disease severity on cognitive outcome, highlighting the importance of high-quality therapy.

We thank all patients and their families for participating and trusting us. In addition, we thank Annette Hess, Heidelberg, Catharina Enzingmüller, Hamburg, Peter Freisinger, Reutlingen, Frauke Lang, Mainz, Ulrike Och, Münster, and Natalie Weinhold, Berlin, for helping us to recruit patients and to collect data. We thank Elena Boyd, Heidelberg, for critically proofreading the manuscript.

AUC

area under the curve

BCAA

branched-chain amino acids

BCKDH

branched-chain α-ketoacid dehydrogenase

CI

confidence interval

cMSUD

classic maple syrup urine disease

IQR

interquartile range

MSUD

maple syrup urine disease

NBS

newborn screening

pLeu

plasma leucine concentration

vMSUD

variant maple syrup urine disease

XLE

combined leucine and isoleucine levels