Acquisition of Developmental Milestones in Hypomyelination With Atrophy of the Basal Ganglia and Cerebellum and Other TUBB4A-Related Leukoencephalopathy

Abstract

Mutations in TUBB4A are associated with a spectrum of neurologic disorders categorized as TUBB4A-related leukoencephalopathy. Affected children can present with global developmental delay or normal early development, followed by a variable loss of skills over time. Further research is needed to characterize the factors associated with the divergent developmental trajectories in this rare monogenic disorder because this phenotypic spectrum is not fully explained by genotype alone.

To characterize early psychomotor features, developmental milestones and age of disease onset were collected from medical records (n=54 individuals). Three subcohorts were identified: individuals with the common p.Asp249Asn variant vs all other genotypes with either early (<12 months of age) or late onset of presentation. Individuals with the p.Asp249Asn variant or those with non-p.Asp249Asn genotypes with later disease onset attained key milestones, including head control, sitting, and independent walking. Subjects with early-onset, non-p.Asp249Asn–associated disease were less likely to achieve developmental milestones. Next, we defined the developmental severity as the percentage of milestones attained by age 2 years. The mild form was defined as attaining at least 75% of key developmental milestones. Among cohort categorized as mild, individuals with p.Asp249Asn variant were more likely to lose acquired abilities when compared with non-p.Asp249Asn individuals.

Our results suggest multiple influences on developmental trajectory, including a strong contribution from genotype and age of onset. Further studies are needed to identify additional factors that influence overall outcomes to better counsel families and to design clinical trials with appropriate clinical endpoints.

Keywords

developmental delay genetics leukodystrophy neurodevelopment pediatric

Mutations in TUBB4A are associated with a spectrum of neurologic disorders, including hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC), isolated hypomyelination, and DYT4/whispering dysphonia.^1

-8 Collectively, these disorders are categorized as TUBB4A-related leukoencephalopathy.

Microtubules are cytoskeletal complexes composed of heterodimers of alpha and beta tubulins.^1,2 One beta tubulin, TUBB4A (MIM 602662), plays an integral role in neuronal migration and axonal projections and is highly expressed in neurons and oligodendrocytes within the basal ganglia and cerebellum.¹ Pathogenic changes in TUBB4A result in a spectrum of clinical disorders ranging from isolated dystonia to severe hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC).^{5

-8,10

-15,} The most common pathologic variant associated with H-ABC in TUBB4A is p.Asp249Asn.^2,17 The developmental course of TUBB4A-related leukoencephalopathy can be variable and is only partially explained by genotype alone. ^{1

-9,16} Children can demonstrate mild or profound global developmental delay, or normal early development, followed by a variable loss of skills over time.

In this study, a retrospective international cohort of 54 individuals affected by TUBB4A-related leukoencephalopathy was analyzed for age of onset and genotype, and a detailed developmental history was obtained. Using age of acquisition of developmental milestones, we were able to evaluate the influence of genotype and age of onset on developmental trajectories. Future studies are needed to characterize additional variables that influence overall clinical course.

Methods

Patient Ascertainment and Enrollment

Fifty-four individuals with clinical and molecular confirmation of pediatric-onset TUBB4A-related leukoencephalopathy (defined as disease onset occurred before 18 years of age) were recruited through the Myelin Disorders Bioregistry Project (institutional review board approved), an arm of the Global Leukodystrophy Initiative Clinical Trial Network (GLIA-CTN) at the Children’s Hospital of Philadelphia (Supplemental Table S1). Individuals without a known genetic diagnosis and those with insufficient developmental histories (available up to less than 24 months of age) were excluded.

Data Extraction and Collection

Demographic information was collected for all individuals (Table 1), including genotype. A report of neuroimaging was available for all the individuals (Supplemental Table S1), and presence of leukodystrophy/hypomyelination, cerebellar atrophy, and basal ganglia atrophy was documented.

Table 1.

Demographic Table.

	Cohort	D249N	Non-D249N
Number	54	13 (24.07)	41 (75.92)
Average age at onset, mo (range)	20.33 (0-180)	25.77 (8.04-54.48)	18.61 (0-180)
Sex (n = 52)
Male	32 (61.5)	9 (69.2)	23 (59)
Female	20 (38.5)	4 (30.8)	16 (41)
Average age at last follow-up, mo (range)	139.44 (30-552.84)	149.76 (54.96-261)	136.17 (30-552.84)

We identified 17 skills from the Denver Developmental Screening Test II (DDST-II) to assess acquisition of developmental milestones in the TUBB4A-related leukoencephalopathy population (Supplemental Table S2), as previously applied in the leukodystrophy population.¹⁸ The age at acquisition and loss of milestones was obtained from historical medical records. Information was extracted from medical records synchronous with the time of acquisition and loss of a specific milestone when feasible to minimize recall bias. Ages were confirmed by telephone parental interview conducted by a child neurologist. In order to reduce the bias from missing data related to “delayed” early milestones, if a particular milestone was described as “delayed acquisition,” the date of acquisition was defined as the 50th percentile in typically developing individuals + 3 SD (based on DDST-II). We compared the average percentage of milestones achieved at 24 months of age between 3 clinically relevant thresholds for age of onset (6, 9, and 12 months). Age of disease onset was defined as the youngest age in which a disease-specific manifestation was noted. Neuroradiologic reports and disease-specific medical histories were collected for the cohort.

Statistical Approach

Analyses were performed using Stata 16, SAS 9.4, and PRISM graphical software. In order to determine the ideal age of onset cut off that best described discrete cohorts, we fit a linear regression model of the percentage of milestones achieved at 24 months. The presence or absences of the common genotype p.Asp249Asn was further used to define cohorts. Kaplan-Meier curves and the Mann-Whitney test were used to compare the percentage of milestones achieved by 24 months between the cohorts. Log rank testing was also used to compare subsequent loss of milestones between groups. We also estimated the 25th, 50th, 75th, 90th percentiles of the age of acquisition for each developmental milestone as the age at which the estimated Kaplan-Meier survival curve crossed 0.25, 0.50, 0.75, and 0.90, respectively. Two-sided tests of hypotheses were applied, with a P value <.05 as the criterion for statistical significance. We also performed a descriptive analysis of acquisition and loss of milestones by constructing a stacked bar chart that plotted the number of children in each of 3 categories (milestone not yet acquired, milestone acquired, milestone lost) by age in months.

Results

Demographics and Neuroimaging Findings

Fifty-four individuals with clinical and molecular confirmation of pediatric-onset TUBB4A-related leukoencephalopathy (defined as disease onset occurred before 18 years of age) were included in this analysis. Genotype in 13 individuals identified the common p.Asp249Asn pathogenic variant, whereas the remainder of the individuals has less common variants. A report of neuroimaging was available for all the individuals (Supplemental Table 1), obtained at variable ages relative to disease presentation. Of those, 53 had evidence of white matter changes consistent with leukodystrophy/hypomyelination (98.1% of the cohort), 41 with cerebellar atrophy (75.9% of the cohort), and 27 with basal ganglia atrophy, with predominant putaminal involvement (50% of the cohort). One subject (subject 40) met the overall study inclusion criteria but was noted to have basal ganglia atrophy on MRI, without evident white matter changes.

Determination of Age at Onset Threshold

Earlier age of onset is often associated with disease severity.¹⁹ To determine a more precise threshold for “early onset,” we compared the average percentage of milestones achieved at 24 months of age between 3 clinically relevant thresholds for age of onset (6, 9, and 12 months). Twenty-four months was selected because most early milestones are attained by this age. We fit a linear regression model of the percentage of milestones achieved at 24 months vs an indicator variable (having age of onset > the threshold). We estimated the effect-size for each threshold as the regression coefficient for the indicator variable divided by the root mean square error for the regression model. The 12-month age at onset threshold had the highest effect size (1.5) and R-squared value (0.4).

Determination of Cohorts

The enrolled cohort was divided by genotype: p.Asp249Asn variant vs all other genotypes. The remaining non-p.Asp249Asn variants included 25 discrete variants occurring between one and 6 individuals each. The low numbers of each non-p.Asp249Asn genotype precluded any additional genotype-phenotype correlations. We further divided individuals by age of onset: <12 vs ≥12 months. Most (11/13; 85%) children with p.Asp249Asn-related disease had an onset ≥12 months of age. The 2 children with early onset demonstrated a developmental trajectory similar to the other subjects with the p.Asp249Asn variant. We thus created 3 cohorts: p.Asp249Asn-related disease, early infantile (<12 months) onset non–p.Asp249Asn related disease, and late infantile (>12 months) onset non–p.Asp249Asn related disease. Cohort groups are visually presented in Supplemental Figure S1.

Developmental Heat Maps and Developmental Function in Cohort Groups

We compared developmental achievements between cohorts by ranking percentiles estimated by the Kaplan-Meier curves with heat maps. These heat maps display the ages at which a given percentiles of the cohort achieved a milestone (p10 for 10%, p25 for 25% etc.).¹⁸ Additionally, the milestone status for each individual was divided into the categories of: not yet achieved, achieved, and lost. These milestone categories were assigned to each individual at 1-month intervals to tract the transition between each category.

The p.Asp249Asn-related disease was correlated with a higher percentage of developmental skills achievement by age 24 months (Mann-Whitney test, P value <.0001) (Figure 1), followed by late infantile (>12 months) onset non–p.Asp249Asn related disease. Early infantile (<12 months) onset non–p.Asp249Asn related disease was associated with a more severe developmental trajectory, for all assessed milestones (log-rank test, P value < .0001), except smiling responsively, which was attained universally across the cohorts (Figure 2). p.Asp249Asn-related disease was also associated with the earliest gain of milestones. Age of milestone acquisition was used to create plots of developmental skill acquisition by genotype and age of onset (Figure 3). Across all 3 cohorts, we characterized developmental outcomes as mild if individuals attained at least 75% of key developmental milestones. Individuals with the p.Asp249Asn-related disease, or those with late infantile onset non–p.Asp249Asn related disease were more likely to be characterized as mild. Subjects with early infantile non–p.Asp249Asn related disease were more likely to be characterized as severe.

The evolution of gain and loss of skills was tracked for each individual over time (Figure 4). In addition to having delayed acquisition of milestones, many individuals lost skills over time. The loss of previously acquired specific milestones (roll over, get to sitting, pull to stand, stand alone, and one word) was greater in individuals with p.Asp249Asn-related disease (log-rank test: roll over P < .05, get to sitting P < .005, pull to stand P < .005, stands alone P < .05, one word P < .05). Additionally, when the percentage of all milestones lost at last evaluation was compared between individuals having achieved at least 75% of all milestones (mildly affected), individuals with p.Asp249Asn variant were more likely to have lost milestones (Mann-Whitney test, P < .001) (Figure 5).

Discussion

TUBB4A-related leukoencephalopathy is a heterogeneous disorder caused by mutations in the beta-tubulin isotype encoded by TUBB4A. Although all children described to date with TUBB4A-related leukoencephalopathy demonstrate some degree of neurologic impairment, the magnitude of neurodevelopmental delay can be variable, as can the presence of regression of developmental skills.

Three distinct patterns within childhood-onset TUBB4A-associated leukoencephalopathy emerged: the p.Asp249Asn-related disease, characterized by late infantile onset, with acquisition of early milestones followed by neurologic regression; early infantile (<12 months) onset non–p.Asp249Asn related disease, characterized by minimal developmental gain; and late infantile (>12 months) onset non–p.Asp249Asn related disease, characterized by late infantile onset, with acquisition of early milestones with more moderate neurologic regression. Because of the heterogeneous psychomotor development observed in the late infantile (>12 months) onset non–p.Asp249Asn related disease cohort, we hypothesize that additional factors may influence neurologic outcome. It will be relevant to future clinical trial planning to better understand factors that may influence outcomes in the late infantile (>12 months) onset non–p.Asp249Asn related disease cohort. For example, the presence or absence of radiographic features such as cerebellar atrophy or basal ganglia atrophy, may impact long term developmental outcomes. However, in this study, imaging was obtained clinically based on the presence of developmental abnormalities. This ascertainment bias prevented the analysis of these features as predictive of developmental outcomes.

This study explores developmental outcomes in a group of children affected by TUBB4A-related leukoencephalopathy. Although this cohort is relatively small (n = 54), we anticipate that this international cohort is representative of pediatric-onset TUBB4A-related leukoencephalopathy. As the participants were enrolled through a pediatric leukodystrophy research network, it is important to note that there was an ascertainment bias in the identification of cases. This study did not include cases of adult-onset forms of TUBB4A-related leukoencephalopathy and may underrepresent cases without hypomyelination on imaging. All but one individual was noted to have MRI findings consistent with a leukodystrophy. Individuals with adult-onset disease or without hypomyelination are likely underdiagnosed in childhood. We hypothesize that these individuals could have normal or near-normal early development.

In this study, we characterize the association between age of onset, genotype, and phenotype in TUBB4A-related leukoencephalopathy. Future identification of variables that may affect long-term outcomes, in addition to age of onset and genotype, will be important in the design of future clinical trials and the selection of appropriate clinical endpoints.

Supplemental Material

Supplemental Material, sj-jpg-1-jcn-10.1177_0883073821000977 - Acquisition of Developmental Milestones in Hypomyelination With Atrophy of the Basal Ganglia and Cerebellum and Other TUBB4A-Related Leukoencephalopathy

Supplemental Material, sj-jpg-1-jcn-10.1177_0883073821000977 for Acquisition of Developmental Milestones in Hypomyelination With Atrophy of the Basal Ganglia and Cerebellum and Other TUBB4A-Related Leukoencephalopathy by Francesco Gavazzi, Brittany A. Charsar, Catherine Williams, Justine Shults, Cesar A. Alves, Laura Adang and Adeline Vanderver in Journal of Child Neurology

Supplemental Material

Supplemental Material, sj-pdf-1-jcn-10.1177_0883073821000977 - Acquisition of Developmental Milestones in Hypomyelination With Atrophy of the Basal Ganglia and Cerebellum and Other TUBB4A-Related Leukoencephalopathy

Supplemental Material, sj-pdf-1-jcn-10.1177_0883073821000977 for Acquisition of Developmental Milestones in Hypomyelination With Atrophy of the Basal Ganglia and Cerebellum and Other TUBB4A-Related Leukoencephalopathy by Francesco Gavazzi, Brittany A. Charsar, Catherine Williams, Justine Shults, Cesar A. Alves, Laura Adang and Adeline Vanderver in Journal of Child Neurology

Supplemental Material

Supplemental Material, sj-pdf-2-jcn-10.1177_0883073821000977 - Acquisition of Developmental Milestones in Hypomyelination With Atrophy of the Basal Ganglia and Cerebellum and Other TUBB4A-Related Leukoencephalopathy

Supplemental Material, sj-pdf-2-jcn-10.1177_0883073821000977 for Acquisition of Developmental Milestones in Hypomyelination With Atrophy of the Basal Ganglia and Cerebellum and Other TUBB4A-Related Leukoencephalopathy by Francesco Gavazzi, Brittany A. Charsar, Catherine Williams, Justine Shults, Cesar A. Alves, Laura Adang and Adeline Vanderver in Journal of Child Neurology

Footnotes

Author Note

Information on data in the Myelin Disorders Bioregistry Project can be requested by emailing the corresponding author.

Laura Adang: consultant to Orchard Therapeutics and Takeda Pharmaceuticals. Research reported in this publication was supported by the National Institute of Neurological Disorders And Stroke of the National Institutes of Health under Award Number K23NS114113 and U54TR002823.

Adeline Vanderver: receives research support from Gilead Sciences Inc., Orchard Therapeutics, Eli Lily and Company, Biogen, Homology, Takeda, Ionis and Illumina Inc. Research reported in this publication was supported by the National Institutes of Health under Award Number U54TR002823.

Justine Shults: receives research support from Eli Lily and Company, Homology, Takeda. Research reported in this publication was supported by the National Institutes of Health under Award Number U54TR002823.

Author Contributions

FG, BC, JS, CA, LA, AV, CW, made a substantial contribution to the concept or design of the work; or acquisition, analysis or interpretation of data.

FFG, BC, JS, CA, LA, AV, CW, drafted the article or revised it critically for important intellectual content.

FG, BC, JS, CA, LA, AV, CW, approved the version to be published.

FG, BC, JS, CA, LA, AV, CW, each author should have participated sufficiently in the work to take public responsibility for appropriate portions of the content.

Declaration of Conflicting Interests

The authors declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: FG: no conflicts of interest to disclose. BAC: no conflicts of interest to disclose. LA: consultant to Orchard Therapeutics and Takeda Pharmaceuticals. AV: research support from Gilead Sciences Inc, Orchard Therapeutics, Eli Lily and Company, Biogen, Homology, Takeda, Ionis and Illumina Inc. Justine Shults: research support from Eli Lily and Company, Homology, and Takeda. CAA: no conflicts of interest to disclose.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: research reported in this publication was supported by the National Institute of Neurological Disorders and Stroke of the National Institutes of Health under award numbers K23NS114113 (to LA) and U54TR002823 (to LA, AV, and JS).

ORCID iD

Francesco Gavazzi, MD

Supplemental Material

Supplemental material for this article is available online.

Ethical Approval

The Myelin Disorders Bioregistry Project was approved by the Institutional Review Board at the Children’s Hospital of Philadelphia.

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