Characterization of gallbladder disease in metachromatic leukodystrophy across the lifespan

Abstract

Metachromatic leukodystrophy (MLD) is a progressive demyelinating disorder resulting from the toxic accumulation of sulfatides. The stereotyped neurodegeneration of MLD is well understood, and cases are categorized into subtypes by age at neurologic onset: late infantile (LI), juvenile (J), and adult. The systemic burden of disease, such as gallbladder involvement, however, is less well characterized. It is important to understand the longitudinal trajectory of gallbladder complications in MLD and its relationship with neurologic progression as this has the potential to identify cases of active disease before neurologic onset. Additionally, as newborn screening is established in MLD, it will inform clinical care during the presymptomatic period.

To address this knowledge gap, we leveraged a retrospective natural history study of MLD and published cases in the medical literature. Medical records from subjects consented to a natural history study were used to collect information of disease course, including gallbladder abnormality. Neurologic function was retrospectively assessed using the gross motor function classification scale (GMFC-MLD). Additionally, a comprehensive review identified published cases of MLD with subject-level information around gallbladder disease. Data was summarized using descriptive statistics, Fisher’s exact test for significance, and survival analysis with log rank test.

The natural history cohort includes 40 subjects with gallbladder reports (imaging or pathology). The first gallbladder evaluation occurred after neurologic onset in 35/40 cases. Gallbladder abnormalities were noted in 36 subjects, often within the initial evaluation (97.2 %). There was no difference in the time to first gallbladder abnormality (log rank: p = 0.4170) and risk of polyps or higher (log rank: p = 0.6414) between the LI- and non-LI subtypes. The level of gallbladder involvement does not correlate with GMFC-MLD score (Fisher’s exact: p = 0.321). A review of the literature identified 87 additional cases of MLD with mention of gallbladder status across 40 published studies. Gallbladder involvement was noted in 74 cases and occurred at similar rates across subtypes (X² = 4.68, p = 0.7925).

Overall, the study showed a high prevalence of gallbladder complications in MLD. Gallbladder abnormalities were commonly found at first evaluation, even in pre- or early symptomatic disease. Since gallbladder disease has the potential to progress to malignancy, this supports the integration of regular gallbladder monitoring as clinical care and its potential as a predictive biomarker supporting disease onset.

1. Introduction

Metachromatic leukodystrophy (MLD) is an autosomal lysosomal storage disorder caused by biallelic disease-causing variants in the arylsulfatase A gene (ARSA). Deficient enzyme activity results in the toxic accumulation of sulfatides throughout the peripheral and central nervous system leading to progressive demyelination of neurons [1–3]. Disease subtype is defined by the age at neurologic symptom onset: late infantile (LI-MLD, onset before 2.5 years), juvenile [early J-MLD (EJ): before 7 years; late J-MLD (LJ): before 16 years], and adult MLD (onset after 16 years). While all forms of MLD are progressive, there is a spectrum of neurologic changes ranging from the subacute loss of gross motor skills in LI- and EJ-MLD to a chronic neuropsychiatric change associated with LJ- and adult MLD [2,4–6]. Depending on age, in the presymptomatic and minimally symptomatic populations, gene therapy [7,8] and allogeneic hematopoietic stem cell transplantation [6,9] are potential therapeutic options.

Current evidence of gallbladder involvement in MLD is predominantly anecdotal in the form of case reports. Exceptions to this are two cross-sectional studies that report on gallbladder evaluation at a single time point on a predominantly non-LI-MLD cohort [10,11]. The prevalence of gallbladder disease in MLD, correlation with disease subtype and severity is under characterized [10–16]. In addition, there have been case reports of gallbladder involvement early in the disease course [10,11,17–24]. However, gallbladder complications are rare among children and adolescents in the general population [25–27]. Collectively, this supports that gallbladder imaging may be an important biomarker in presymptomatic screening for MLD [6,17,28,29]. In addition, gallbladder disease can be symptomatic, and in MLD, has a risk of progression to cancer [11,17,29]. Existing literature note dysplasia, hyperplasia, and metaplasia across all MLD subtypes [11,14,21,22,24,29,30]. Of note, early gallbladder disease can also be asymptomatic, underscoring the need for screening even in a presymptomatic population identified by newborn screening [6,22,28,31].

In MLD, gallbladder abnormalities, including evidence of contraction, sludge, polyps, wall thickening and gallstones, can be found by imaging at the time of presymptomatic treatment [11], suggesting that gallbladder abnormality may precede neurological decline. There is a lack of information regarding the longitudinal progression of gallbladder manifestations in MLD. This study, by combining natural history cohort and published cases, aims to define the frequency and evolution of gallbladder disease in MLD in a larger cohort representing the MLD subtypes, as well as its relationship with neurologic severity.

2. Methods and materials

2.1. Natural history study

Subjects were accrued as part of a retrospective natural history study under the Myelin Disorders Biorepository Project (MDBP) within the Global Leukodystrophy Initiative Clinical Trial Network (GLIA-CTN) approved by the Children’s Hospital of Philadelphia (CHOP) Institutional Review Board (IRB approval number: 14–011236).

2.1.1. Cohort identification

The study included subjects with (i) a confirmed diagnosis of MLD based on identification of disease-causing ARSA variant and presence of both low blood arylsulfatase A activity and increased urinary sulfatide excretion; and (ii) have records indicating gallbladder evaluation during the span of the disease. All clinical encounters/medical records, irrespective of subject’s treatment status were reviewed and extracted for abnormal gallbladder evaluations. We excluded 2 subjects with confirmed secondary diagnoses of an additional neurogenetic disorder with phenotypic overlap.

2.1.2. Data collection and extraction

The data was extracted from existing medical records by trained study personnel into the study’s REDCap database hosted at CHOP. REDCap (Research Electronic Data Capture) is a secure, web-based software platform designed to support data capture for research studies, providing 1) an intuitive interface for validated data capture; 2) audit trails for tracking data manipulation and export procedures; 3) automated export procedures for seamless data downloads to common statistical packages; and 4) procedures for data integration and interoperability with external sources [32,33].

Baseline information on the subject’s sex, race, and age at diagnosis were collected. We abstracted data on the age at onset of neurologic symptoms attributable to MLD based on the first recorded note of delayed milestones, abnormal movement, cognitive difficulties, behavioral changes, or regression of previously attained skills [22].

For each enrolled subject, all available medical records (as of February 16th, 2024) were reviewed to collect data on the age at gallbladder evaluation, type of diagnostic technique, type of gallbladder abnormality, and if available pathological findings. The following search terms were used to identify gallbladder abnormality: “gallbladder”, “cholecystectomy,” “cholecystitis,” “sludge”, and “polyps.” Individual gallbladder events were extracted according to reported findings from diagnostic tools, including ultrasonogram, magnetic resonance imagining (MRI), computed tomography (CT), and other types of imaging, pathology, and autopsy evaluations. Whenever more than one study was obtained within a week, priority was given to abdominal ultrasound findings. If the original report was not available, the first noted clinical note of the status of the gallbladder was used to identify gallbladder event. Surgical pathology reports were also collected for subjects that underwent cholecystectomy.

Finally, to assess the relationship between the gallbladder disease and neurologic severity, clinical encounters within +/− 30 days of the gallbladder evaluation were identified [34] and gross motor function was assessed retrospectively by trained child neurologists using the Gross Motor Function Classification Metachromatic Leukodystrophy (GMFC-MLD) scale. The GMFC-MLD is a disease specific tool that enables the mapping of gross motor function in prospective and retrospective applications [35]. This assessment is scored in children above the age of 18 months according to an age-independent seven-item categorical scale from normal ambulation (M0) to no mobility or head control (M6). In this study, we used the GMFC-MLD scale as a proxy to assess neurological function. This cohort includes 4 subjects with pre- and post-treatment encounters, and 3 with post-treatment encounters only.

2.2. Review of published literature

In addition, we performed a comprehensive review of the literature using PubMed to identify cases of gallbladder abnormalities associated with MLD ever published in literature. The following search terms were used: “metachromatic leukodystrophy” and “gallbladder.” The search was limited to human studies and English language articles. Additionally, we manually reviewed the reference section within each identified publication to identify additional cases. Each patient described in the publication was assigned a unique subject ID. Corresponding data on the age of symptom onset, ages at the time of gallbladder evaluation and gallbladder abnormality were collected.

2.3. Cohort categorization

The published definitions of MLD subtypes, based on age at disease onset, was applied to published case reports-LI-MLD (age at onset <2.5 years), Juvenile (between 2.5 and 16 years) and adult (16 years and older). The subtypes were categorized also as LI versus non-LI. The gallbladder abnormalities were categorized by common language in the radiographic reports or pathologic evaluation reports from autopsy or post-cholecystectomy specimen: (0) normal, (1) stone and/or sludge, (2) contracted, evidence of inflammation or wall thickening, (3) presence of polyps, (4) concern for dysplasia or cancer, and (5) unspecified. In addition, the gallbladder abnormalities were clustered by severity as less severe (levels 1 & 2) and severe (levels 3 & 4).

2.4. Statistical analysis

Data was summarized using counts and proportions for categorical variables, and mean, standard deviation, median and ranges for continuous variables. Chi-square or Fisher’s exact tests, as appropriate, were performed to determine the associations between two categorical variables. Survival curves were created to illustrate time to certain gallbladder events and the log rank test was used to test for statistical significance. Longitudinal gallbladder disease evolution was depicted using the Swimlane plot. Data analysis was preformed using R and illustrations were developed on R and Prism.

3. Results

3.1. GLIA-MLD natural history cohort

3.1.1. Cohort characteristics

A total of 40 subjects (55 % female, 87.5 % white) met inclusion criteria (Table 1). This cohort was comprised of 25 subjects with LI-MLD and 15 non-LI MLD subjects (13 Juvenile). By MLD subtype, the median age at onset of neurological symptoms was 1.2 years (IQR: [1.0, 1.5]) and 6.5 years (IQR: [5.5, 8.8]) in the LI-MLD and non-LI-MLD cohorts, respectively. Overall, there was a gap between neurologic onset and diagnosis of MLD. The median age at diagnosis was 2.2 years in the LI-MLD (IQR: [2.0, 2.6]) and 8.3 years in the non-LI-MLD cohorts (IQR: [6.3, 9.4]). Commonly, the first gallbladder evaluation occurred after the diagnosis of MLD. Overall, the median time at first gallbladder evaluation was 3.3 years [95 % CI: 2.2, 7.2], and by MLD subtype 2.6 years (IQR: 2.0–3.1) in LI-MLD and 8.5 years (IQR: 6.6–10.7) among non-LI subjects (Table 1). Repeated gallbladder evaluations were available in 24 subjects. In this longitudinal sub-cohort, the frequency of evaluation was variable between subjects (median = 0.7 years, [Q1, Q3] = [0.5, 1.8], range = [0.0, 2.4]).

Table 1.

Cohort description overall and by the MLD subtype.

	Overall (N = 40)	Late Infantile (N = 25)	Non-Late Infantile (N = 15)	P-value
Sex, Female N (%)	22 (55.0 %)	13 (52.0 %)	9 (60.0 %)	0.7470
Race/Ethnicity
Non-Hispanic White N (%)	35 (87.5 %)	21 (84.0 %)	14 (93.3 %)	0.6330
Age at neurologic onset, (years)				<0.0001
Median [IQR]	1.5 [1.2, 5.5]	1.2 (1.0, 1.5)	6.5 (5.5, 8.8)
[Min, Max]	[0.7, 20.5]	[0.7, 2.1]	[3.0, 20.5]
Age at diagnosis (years)				<0.0001
Median (IQR)	2.8 [2.1, 6.5]	2.2 [2.0, 2.5]	8.6 [6.1, 9.5]
[Min, Max]	[0.1, 27.4]	[0.1, 3.8]	[4.2, 27.4]
Missing	2 (5.0 %)	1 (4.0 %)	1 (6.7 %)
Time to first gallbladder evaluation, (years)
Median (95 % CI)	3.3 [2.2, 7.2]	2.6 [2.0, 3.1]	8.5 [6.6, 10.7]	<0.0001
Age at first abnormal gallbladder report, (years)				<0.0001
Median [IQR]	3.9 [2.2, 7.1]	2.8 [2.1, 3.3]	7.9 [6.4, 9.6]
[Min, Max]	[1.0, 26.8]	[1.0, 9.4]	[5.6, 26.8]
Not applicable	4 (10.0 %)	3 (12.0 %)	1 (6.7 %)

Abbreviations: IQR- Interquartile range; CI- Confidence interval.

Four subjects (4/40; 10 %) were found to have no report of abnormalities in the earliest available gallbladder evaluation. Of these, record of second evaluations were available for only three subjects and continued to report normal gallbladder features.

Among those in whom gallbladder involvement was reported, the mean age at first abnormal gallbladder report in the overall cohort was 3.8 years (IQR: 2.2, 7.2). The most common gallbladder abnormalities reported at first evaluation were wall thickening (n = 15) and sludge (n = 10). Among those with serial evaluations, sludge and stones were reported in 16, wall thickening, radiographic evidence of inflammation, or contraction in 18, polyps in 7, and pathological evidence of dysplasia or cancer in 3 individuals.

3.1.2. Comparison of gallbladder involvement by disease subtype

Despite a delay in initial imaging (Table 1), the median time to first documentation of a gallbladder abnormality was 2.8 years (95 % CI: 2.1, 3.5) in LI-MLD and 8.5 years (95 % CI: 7.0, 18.4) in the non-LI-MLD cohort. There was a significant difference between the median age at neurologic symptom onset and first imaging with gallbladder abnormality in the LI-MLD cohort (Fig. 1A, log rank test: p < 0.0001). The median time to first abnormal imaging was not significant in the non-LI-MLD cohort (Fig. 1B, log rank test: p = 0.149). In the non-LI cohort, the median time to occurrence of polyp or greater was 26.8 years (95 % CI: [8.80, NA]). Next, the time to gallbladder abnormality was compared between LI- and non-LI cohorts (Fig. 1C). The time to any identified gallbladder abnormality from neurologic onset and to first polyp or greater did not differ between the LI-MLD and the non-LI-MLD subtypes (log-rank test, p = 0.4170 and p = 0.6414, respectively).

Fig. 1.

Kaplan-Meier Curve of gallbladder abnormalities. A) Time to MLD disease onset and first gallbladder abnormality in LI-MLD. B) Time to MLD disease onset and first gallbladder abnormality in non-LI MLD. C) Comparison of time to occurrence of polys, dysplasia or cancer, from neurologic disease onset between Li- and non-LI MLD cohorts. Of note, one subject had gallbladder abnormality identified before neurologic disease onset and hence was censored at time 0.

3.1.3. Comparison of gallbladder with neurologic burden in MLD

Next, clinical encounters concurrent with gallbladder assessment were identified, and neurologic function was assessed by GMFC-MLD (n = 57 encounters from 33 subjects). Severe gallbladder abnormalities were noted in 8 encounters across 6 subjects with limited neurologic disease (GMFC-MLD M0-M2), compared to 6 encounters across 3 subjects with impaired neurologic function (GMFC-MLD M3-M6) (Fisher’s exact, p-value = 0.7772) (Fig. 2). Among the treated subjects, pre-treatment gallbladder evaluations were available for 7 individuals; sludge and/or wall thickening were noted for eight individuals and normal gallbladder imaging for 1 individual. Post-treatment data was available for 10 subjects and identified normal features in 1 and pathologies in 9 individuals-3 with sludge, 1 with wall thickening, 2 with polyps, 1 with stones, and 1 with cancer.

Fig. 2.

Comparison of frequency of gallbladder pathologies and neurological function at time of gallbladder evaluation. Gallbladder features were categorized according to described levels of increasing severity. Within each level, the distribution of neurologic function based on the GMFC-MLD score assigned within ±30 days of the gallbladder evaluation is plotted: high functioning M0-M2 (light blue), lower functioning M3-M4 (medium blue), and lowest function M5-M6 (dark blue).

3.1.4. Description of natural history subjects who developed dysplasia

Patient 1 (Untreated):

The patient first presented with MLD-related neurologic symptoms at approximately 12 months of age (Late infantile MLD) and was diagnosed at 3.8 years. Based on available medical records, the gallbladder was first evaluated at 3.4 years and was consistent with sludge. Concurrent GMFC-MLD (3.5 years) was M1. A repeat ultrasound performed at 3.8 years identified wall thickening in addition to sludge. At 4.7 years of age, the patient underwent surgery for gastrostomy tube placement with laparoscopic cholecystectomy. Pathological evaluation of the surgical specimen reported diffuse villous hyperplasia, epithelial hyperplasia of multiple cell lineages, low-grade dysplasia, and focal changes suspicious for high-grade dysplasia. Macrophage accumulation of sulfatides was also noted. The motor function at the time of cholecystectomy (4.6 years), was ambulatory with assistance (M2).

Patient 2 (Treated):

This was a juvenile MLD subject with age at neurologic symptom onset of 6.5 years, who was diagnosed at 6.7 years. Patient was treated at 7.1 years. The first gallbladder evaluation reported sludge and wall thickening. A repeat ultrasound performed post-treatment at 8.8 years because of worsening abdominal discomfort and vomiting. The repeat imaging noted sludge in addition to increased wall thickness with lobular margins and increased vascularity. This progression of gallbladder morphology prompted a cholecystectomy at 8.8 years (GMFC-MLD: M5). Pathological evaluation (Fig. 3) of the surgical specimen confirmed villous papilloma of fundus with focal dysplasia, hyperplasia of the adjacent mucosa, multiple foci of antral-like mucinous metaplasia, focus of epithelial dysplasia, and accumulation of metachromatic material in macrophages and epithelial cells. The abdominal discomfort and vomiting resolved post-cholecystectomy.

Fig. 3.

Pathological evaluation of gallbladder surgical specimen (Patient 2). A. Gross inspection of the gallbladder specimen showed the macroscopic view of the opened gallbladder showing a frond-like polypoid lesion located at the fundus. Lower (B) and higher (C) power view of gallbladder by H&E staining demonstrating clusters of foamy macrophages within the lamina propria of the papillary excrescences.

Patient 3 (Untreated):

This late-infantile MLD subject presented with neurological symptoms at 1.5 years and was diagnosed at 3.0 years. The first evaluation of the gallbladder occurred at the age of 3.1, revealing sludge. The neurologic function was non-mobile and no head control (M6) at the time. The subsequent two ultrasounds at age 3.6 and 4.3 years noted persistent sludge. The fourth ultrasound at 4.4 years showed wall thickening, and cholecystectomy was performed (4.6 years). Surgical pathology reported low grade intracholecystic papillary neoplasm (3.0 × 2.5 × 2.2 cm).

3.2. Published cohort characteristics

The literature review identified 54 publications between June 1952 and January 2023. Of these, 14 articles were excluded due to screen failure, yielding a total of 40 articles accounting for 87 published MLD-affected individuals for further review and extraction (Fig. 4) [10,11,13,14,16–22,24,29–31,36–60]. Among the 40 studies, 38 provided single case reports and 2 were larger cross-sectional studies [10,11]. Two from the total included reports on treated MLD cases [11,61]. This cohort comprised of 36 LI-MLD, 34 J-MLD, 14 Adult MLD, and 3 cases for whom disease onset was not reported.

Fig. 4.

Diagram of included published reports. In total, the review identified 40 published articles and 87 cases of MLD with mention of gallbladder status. This included 36 subjects with Li-MLD, 34 Juvenile MLD and 14 with Adult-onset MLD.

For 11/87 MLD-affected individuals, gallbladder involvement was noted on post-mortem examination, where all cases were noted to document wall thickening and/or polyps [46,62]. Diagnostic gallbladder imaging was performed in 74 cases accounting for a total of 99 events. There were heterogenous approaches to defining gallbladder abnormalities: ultrasound (N = 66 events), X-ray (6 events), CT (4 events), and endoscopic retrograde cholangiography (2 events). Sixty cases (80 %) were found to have abnormal gallbladder findings at the earliest reported diagnostic evaluation. Of these, 22 individuals showed early changes (level 1: 21 with sludge and 1 with gallstone), 40 with intermediate changes (level 2: 7 with contraction, 4 with radiographic evidence of inflammation, 29 with wall thickening), 32 with polyps (level 3), 1 with pathological evidence of dysplasia or cancer (level 4) [14], and 5 with unspecified abnormalities. Post-cholecystectomy pathology identified 2 additional cases with dysplasia or cancer (level 4) [11,61]. Fifteen reported cases had received disease-modifying interventions prior to gallbladder imaging with gallbladder findings ranging from unremarkable (n = 5), sludge (n = 1), thickening or inflammation (n = 3), and polyps (n = 6).

3.2.1. Evolution of gallbladder disease

In the natural history cohort (Fig. 5A), the first abnormal gallbladder finding was reported after the onset of neurological symptoms in most subjects (n = 35/40, 97.2 %). One subject without neurologic disease at the time of imaging was noted to have polyps. A range of features were noted at first evaluation: polyps (n = 7), sludge (n = 17), or wall thickening (n = 17). A change from sludge and wall thickening to the development of polyps, dysplasia or cancer was observed in 3 individuals. Cholecystectomy was performed in 10 subjects. The pathological evaluations reported: stones (n = 1), wall thickening/inflammation (n = 1), polyps (n = 3), and dysplasia/cancer (n = 3). The subjects with dysplasia or cancer are detailed below. For two individuals, the gallbladder pathology report was unavailable for review.

Fig. 5.

Gallbladder disease in MLD by subtype. A) In the natural history cohort, gallbladder involvement is presented in swim lane plots in the late-infantile MLD (top) and non-late-infantile MLD (bottom) subtypes. The grey circles, by colour gradient, represent gallbladder features with white indicating normal gallbladder and darker shades indicating severe gallbladder features. Individual subjects are represented by a single lane with depiction of age at onset of neurological symptoms (broken line). Age at diagnosis is represented by red crosses and cholecystectomy by crossed squares. For subjects that underwent MLD therapy, yellow triangles mark the age at treatment. A crossed square without an overlapping solid circle is used when the gallbladder abnormality is unknown from the medical record. B) In the published cohort, the distribution of gallbladder involvement in the published cohort by subtypes is shown (87 individual cases, 112 evaluations. Gallbladder abnormality categories within each MLD disease subtype are plotted: LI MLD (triangle), juvenile MLD (circle), and adult (square). Longitudinal results were available for 20 subjects, often obtained serially. When no detailed age data was available, the encounters were separated by 0.1 years for visualization. C) Incidence of severe gallbladder abnormalities (polyp or greater) across MLD subtypes as shown by Kaplan-Meier curves, comparing the onset of severe gallbladder (polyps, dysplasia, cancer) abnormalities by MLD subtype. Comparison of risk of occurrence between LI-MLD (light blue), J-MLD, (dark blue) and Adult (grey) subjects across published and GLIA-CTN natural history cohort, anchored from age at neurological disease onset on the x-axis. Six subjects with evaluations prior to neurologic disease onset were censored at time 0; 5 subjects had severe gallbladder abnormalities pre-onset and for one did not report any subsequent evaluations.

Among published cases with a known MLD subtype (Fig. 5B), the occurrence of polyps was comparable across phenotypes, including the LI-MLD patients (14; 27.5 %) juvenile disease (15; 32.6 %), and adult-onset disease (9; 39.1 %). A total of four individuals were noted to have cancer or dysplasia (2 LI-MLD and 2 juvenile). The distribution of gallbladder abnormalities in the case reports was not significantly different between the MLD subtypes (Chi-square test of independence, X² = 4.68, p = 0.7925).

Within the combined natural history and published cohorts, the time from disease onset to severe gallbladder pathology (e.g., polyps, dysplasia or cancer) was not significantly different between MLD subtypes (log-rank test, p = 0.8586) (Fig. 5C). Overall, the median time to severe gallbladder abnormality across the case reports and natural history cohort combined was 8 years (95 % Confidence Interval (CI): 5, 12.5) after start of disease onset. Within each subtype, median time to severe gallbladder abnormality was: LI-MLD 7.2 (95 % CI: 3.7, NA) years, J-MLD: 11 years (95 % CI: 2.3, NA) years and Adult 6 years (95 % CI: 4, NA) years. In the combined cohort, 6 subjects were reported with gallbladder evaluations prior to disease onset and hence dropped from analysis-5 with severe gallbladder abnormalities. For one subject with pre-onset gallbladder evaluation, there was no report of subsequent gallbladder evaluations.

4. Discussion

MLD is a progressive disorder that results in profound clinical sequelae and early death. The approved therapy, atidarsagene autotemcel, has demonstrated maximal benefit in pre-symptomatic populations [6,8,28]. Because of the need for early intervention, there is a clinical challenge in identifying who would potentially benefit prior to disease onset as genotype alone only partially predicts subtype [3,6,63–68]. As such, there is an urgent need for early disease stratification and additional monitoring tools. Additionally, it is essential to understand the systemic burden of disease to support the optimal medical care for the symptomatic MLD population.

To characterize gallbladder disease in MLD, this study leverages a longitudinal natural history database for MLD in combination with published case reports. This combined cohort suggests a high prevalence of gallbladder abnormalities early in disease across all MLD subtypes. Our study is limited in its ability to map longitudinal change of gallbladder disease, as only 60 % of subjects had more than one available report of gallbladder evaluation, and by variable approaches to capturing gallbladder abnormalities. For example, a neoplasm was found in a patient with wall thickening by ultrasound, underscoring the importance of how the abnormality was defined. One challenge is inconsistencies with imaging modalities and practicality of obtaining histological evidence of disease. This limits our ability to directly and contemporaneously correlate neurologic disease with gallbladder pathology.

Current monitoring guidelines recommend regular gallbladder imaging as a potential early indicator of disease and consideration of cholecystectomy [6]. To minimize inclusion bias, we collected both published cases (which includes 2 case series [10,11]) to complement the natural history study, which allows for collection of medical records across institutions and does not require in person evaluations. This study suggests a high prevalence of gallbladder abnormalities in MLD, affecting all disease subtypes. This study, however, underscores a key gap in our understanding: when does the gallbladder abnormality begin across the cohorts? There was a similar time from neurologic onset to first gallbladder abnormality across MLD subtypes, although this was complicated by delayed imaging. At the time of first screening, most individuals were noted to have gallbladder involvement, including polyps in nearly a fifth of the cohort. When the imaging was obtained earlier in the disease course, abnormalities were noted even in the ambulatory cohort. Importantly, this study’s cohort captured a few cases in the presymptomatic period that were noted to be normal. This underscores the potential for this as a disease biomarker, suggesting that not all individuals have disease from birth. Gallbladder imaging potentially may serve as an early indicator of disease manifestation, especially in the context of newborn screening. Further studies on the longitudinal progression of gallbladder disease in MLD may help establish a phenotype or predict the onset of neurological disease. Also, given that gallbladder abnormalities may likely precede neurological symptoms, MLD should be considered an etiology for young individuals presenting with gallbladder disease [10,42,50,53].

Review of pathologic evaluations of post-cholecystectomy specimens reported accumulation of lipoid substances within the macrophages. We hypothesize that the accumulated lipoid materials represent sulfatides, based on published evidence that showed stored sulfatides within the macrophages of the MLD-affected gallbladder [41]. The accumulation of sulfatides in related disorders, such as saposin B deficiency [69] and multiple sulfatase deficiency (MSD) [70], supports gallbladder screening in these disorders as well.

This study also suggests a likely sequential change from sludge and radiographic evidence of inflammation to polyps and cancer. Within our cohort, precancerous lesions were identified in two subjects through pathological evaluation of gallbladder specimens post-cholecystectomy despite reassuring earlier ultrasound imaging. Similarly, the case with intracholecystic papillary neoplasm, the preceding ultrasound finding of wall thickening and sludge was noted in the absence of other abnormalities. These findings align with that of a cross-sectional study in a cohort of predominantly juvenile and adult MLD patients that recommends cholecystectomy in individuals with wall thickening when polyps cannot be excluded [11]. However, the findings are limited in the evaluation of gallbladder disease evolution in that certain features may mask other co-existing abnormalities. For example, sludge and inflammation may be masked by a polyp on ultrasound imaging. Also, identification of dysplasia or localized malignant lesions require pathological evaluation. In the current study, we included multiple modalities of gallbladder evaluation to reduce bias associated with this limitation. The standard recommendation for cholecystectomy when the polyp size reaches >5 mm may not be applicable to the MLD population as the disease evolution is less well understood. The transition from normal tissue to dysplasia to malignancy is well-understood in other cancer-predisposing genetic syndromes but has not yet been described in the MLD gallbladder [71,72]. Thus, the significance of early gallbladder dysplastic changes is unknown, but given incidence of early onset neoplasm in this population, the trend noted in this cohort is concerning [73]. The U.S. consensus guidelines for MLD management recommend annual ultrasound screening of the gallbladder for all ages and consideration of cholecystectomy at the time gastrostomy tube placement [74]. Refinement of timing and assessment modality will be needed once prospective data is collected in the context of newborn screening [6,28]. Indeed, we attempted to minimize these biases by establishing categorical levels for gallbladder disease and used original reports whenever feasible.

The recent approval of MLD gene therapy offers a new therapeutic option to improve outcomes and allow individuals with disease to live longer. However, it is unclear how interventions affect the evolution of gallbladder disease. This manuscript provides a comprehensive analysis of published cases in addition to a larger cohort within a natural history study to provide longitudinal analysis across all disease subtypes. Additionally, this work compares gallbladder disease to neurological severity. Gallbladder abnormalities can present asymptomatically in early stages and thus may be an important tool for presymptomatic monitoring. Therefore, given the increased risk of gallbladder abnormality, there is an urgent need to define the evolution of disease in MLD diagnosed individuals.

Data availability

The data that support the findings of the study not publicly available due to privacy and ethical concerns.