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

2.1 Participants

CG patients were invited to participate in this study by their treating physician. Participating patients were studied in the galactosemia expertise outpatient clinic of the Amsterdam UMC.

CG patients with two known pathogenic variations in the GALT gene and/or an erythrocyte GALT activity <15% of the reference mean were eligible for participation in this study. This cohort comprises patients with varying geno- and phenotypes such as patients with classical phenotypes (two pathogenic GALT mutations and absent or barely detectable erythrocyte GALT activity), NBS detected variant patients (detected since 2007 with previously unknown geno- and phenotypes8) and patients with the homo- and heterozygous p.Ser135Leu mutation.18 The control group consisted of parents of paediatric patients with a confirmed heterozygous mutation in the GALT gene. Patients with swallowing difficulties and/or patients unable to follow breath test instructions were excluded.

2.2 Clinical outcomes

In order to investigate the association between galactose oxidation capacity and clinical outcomes, patients were divided into subgroups based on their intellectual and neurological outcome. Intellectual outcome was defined as poor (IQ < 85) or normal (IQ ≥ 85) and neurological outcome was based on the presence or absence of MDs.

2.3 Biochemical outcomes

The most recent Gal-1-P level measured by gas chromatography mass spectrometry (GC-MS) was used in this study (usual range in diet adherent patients 0.05–0.82 μmol/g Hb). Patients with self-reported dietary incompliance at the most recent Gal-1-P measurement were excluded from the Gal-1-P analysis.

2.4 ¹³-C breath test

The 1-¹³C₁ labelled galactose was produced by Cambridge Isotope Laboratories, Inc.©, Massachusetts, United States. Analysis in our laboratory showed a singly labelled stable isotope with a purity of 99%, as expected.

To ensure a steady baseline of ¹³C abundance in breath CO₂ over the study period, participants were instructed to eliminate ¹³C enriched products from their diet 2 days prior to the start of the ¹³C breath test19 and to keep a galactose restricted diet as specified in the international guideline20 for at least 24 hours prior to the start of the study. This prevented a decreased enrichment of supplemented 1-¹³C label with unlabelled galactose from the diet. A higher precursor enrichment (1-¹³C galactose) will lead to higher ¹³CO₂ enrichment in breath. Participants were fasted for a minimum of 2 hours before start of the test and were only allowed to drink water during the test. During the test, participants maintained a resting state. All participants received a 7 mg/kg oral dose of 1-¹³C labelled galactose dissolved in water. Two baseline breath samples were collected before galactose ingestion. Patients were instructed to take a deep breath, to hold their breath for 3 seconds and to blow out into glass Vacutainer tube through a straw. Exactly 60, 90, and 120 minutes after the galactose ingestion, two breath samples were collected. Breath samples were stored at room temperature until analysis.

2.5 Measurement of ¹³CO₂ in breath samples

In each breath sample, the enrichment of ¹³C in expired CO₂ was measured by automated gas-isotope-ratio mass spectrometry on a GC-IRMS Delta XL plus system (Thermo Fisher, Bremen, Germany) using a PoraBOND Q column (25 m × 0.32 mm; Agilent Technologies, the Netherlands).21, 22 All collected samples were measured in triplicate. Standards and control samples were measured with each sequence of analyses. Results were expressed as the δ‰ vs the international reference standard, PeeDee Belemnite. Subsequently, CO₂ production was calculated with the use of the Schofield equation, based on gender, age, height, and weight of participants and the Weir equation using a fasting respiratory quotient (RQ) of 0.80.23 The CO₂ production and the isotopic enrichment values were used to calculate the percentage dose recovered per participant at each time point and with these values the cumulative percentage dose of ¹³C recovered from the labelled galactose (CUMPCD) as ¹³CO₂ in exhaled air was calculated.24 The analytic variation was calculated for all participants.

2.6 Statistical analysis

SPSS version 25 (SPSS Inc. Chicago, Illinois) was used to perform all statistical analyses. Descriptive analyses included median and ranges because of a non-normal distribution. To determine if there were statistically significant differences in CUMPCDT120 between patients with poor and normal clinical outcomes the Mann-Whitney U test was used. The Spearman's rank coefficient test was used to test for correlations and in case of a significant correlation, linear regression was performed. P values below .05 were considered statistically significant.

3.1 Demographics

The data of 41 patients, 19 males, and 22 females with a median age of 18.0 years (5–47) and four controls, one male and three females with a median age of 44 years (38–49) are reported (Table 1). Our cohort consisted of 34 patients with a classical phenotype, hereafter classical patients, four NBS detected variant patients, two patients with the homozygous p.Ser135Leu genotype and one heterozygous p.Ser135Leu/p.*380Argext*50 patient. The erythrocyte GALT enzyme activity was below the limit of quantitation of the enzyme assay (<3.3%; <1.1 umol/h.g Hb) in 32/38 patients and between 3.6% and 8.7% in 6/38 patients.

3.2 CUMPCDT120

The CUMPCD levels of the participants are summarised in Table 2 and illustrated in Figure 1. The CUMPCD at 120 minutes (CUMPCDT120) of classical patients was significantly lower than of the NBS variants and of the homozygous p.Ser135Leu patients (P < .019). The CUMPCDT120 was significantly higher in paediatric patients when compared to adult patients (P = .001), which was also demonstrated in the classical paediatric patients (P = .019). The CUMPCDT120 did not differ between males and females.

3.3 Correlation with age

Since the CUMPCDT120 was significantly higher in paediatric patients than in adult patients, the correlation between age and CUMPCDT120 was investigated in the largest group of patients with the same genotype, the homozygous p.Gln188Arg patients. This group consists of 14 classical patients with a median age of 23 years (6–35). Linear regression indicated a negative correlation between age and CUMPCDT120: F(1,12)5.77, β-0.30(95%CI −0,057 – −0,003), P = .033.

3.4 Analytic variation

Since the galactose oxidation capacity of classical patients was found to be within a narrow range, the analytic variability of the galactose oxidation test was assessed. The effect of the analytic variation varied between patients with a low and high CUMPCDT120, and was ±0.06 in the patient with the lowest CUMPCDT120 of 0.08 and ±0.16 in the patient with the highest CUMPCDT120 of 18.59.

3.5 Intellectual outcome

In total, 8/20 paediatric patients had a poor and 12/20 had a normal intellectual outcome. Paediatric patients with a normal intellectual outcome demonstrated a significantly higher CUMPCDT120 than patients with a poor intellectual outcome (P = .031). The difference remained significant after the exclusion of the homozygous p.Ser135Leu patient and the p.Ser135Leu/p.*380Argext*50 patient. For the classical paediatric patients, there was no significant difference in CUMPCDT120 between patients with a poor and normal intellectual outcome.

In total, 16/21 adult patients had a poor and 5/21 had a normal intellectual outcome. There was no significant difference in CUMPCDT120 between adult patients with a poor and normal intellectual outcome. This result remained unchanged after the exclusion of the homozygous p.Ser135Leu patient.

In both paediatric and adult patients, there was no significant correlation between the IQ (as continuous measure) and CUMPCDT120.

3.6 Neurological outcome

An MD was found in 8/19 paediatric patients and in 5/10 adult patients. The CUMPCDT120 in paediatric patients without an MDs was significantly higher when compared to paediatric patients with an MD (P = .013). The difference remained significant after the exclusion of the homozygous p.Ser135Leu patient and the p.Ser135Leu/ p.*380Argext*50 patient. For the classical paediatric patients, there was no significant difference in CUMPCDT120 between patients with and without an MD. There was no significant difference in CUMPCDT120 between adult patients with and without an MD. These results remained unchanged after the exclusion of the homozygous p.Ser135Leu patient.

3.7 Siblings

Our cohort includes seven sibling pairs and their data is summarised in Table S1.

4.1 Limitations

This cohort includes 41 patients with varying ages and genotypes and the necessity to analyse results separately for adults and paediatric patients resulted in an even smaller sample size and hampered statistical power. To minimise the burdensome fasting and resting state in children in particular, and because previous research demonstrated a peak release of ¹³CO₂ into expired air at 120 minutes, we decided to measure the galactose oxidation capacity until 120 minutes.25 After analysing the results, a majority of the patients showed flattening in the area under the curve at 120 minutes, but not all patients had reached their maximum oxidation capacity at 120 minutes yet and this might have influenced the results.

The negative correlation we found between age and galactose oxidation capacity in patients with an identical genotype poses a limitation to the use of this test. Further studies are warranted to investigate whether this is truly due to a higher galactose oxidation capacity at a younger age.

4.2 Strengths

For the rarity of the disorder, we included a relatively large number of patients that represent the full genetic, biochemical, and clinical outcome spectrum of CG. The inclusion of multiple sibling pairs with a different intellectual outcome in some siblings, and the inclusion of twins provided more insight into whole body galactose oxidation.

To ensure NBS variant and p.Ser135Leu patients did not influence the results, analyses were performed both with and without these patients.

4.3 Future perspectives

In our cohort, the galactose oxidation capacity of the classical patients was within a narrow range and some differences between patients may be attributed to analytic variation. Also, sibling pairs with different clinical outcomes did not demonstrate significant differences in oxidation capacity. The limited number of patients may have affected the results or the 1-¹³C breath test may not be sensitive enough to discriminate between classical patients.

Even though differences were small, the galactose oxidation capacity did differ between classical patients with identical genotypes. The use of universally 6-¹³C labelled galactose might be able to differentiate within the group of classical patients by providing larger differences in ¹³CO₂ enrichment and thus enable better distinction between these patients.

In order to correctly interpret the results of the galactose oxidation breath test and to improve our understanding of galactose metabolism, the effects of age, body composition and genotype on galactose oxidation capacity should be further investigated in a larger cohort for which international cooperation will be necessary. Also, repeating the ¹³C galactose breath test in the same patient cohort might provide valuable information with regard to the course of the galactose oxidation capacity with increasing age.

We demonstrated that NBS detects a group of variant patients with erythrocyte GALT activity up to 10%, with significantly higher galactose oxidation capacity than the classical patients, and no long-term complications so far. The question remains whether these patients are at risk for long-term complications or might even benefit from a less strict diet. Future studies addressing the galactose tolerance of these individuals are warranted to prevent overtreatment.