CLPB mutations cause 3-methylglutaconic aciduria, progressive brain atrophy, intellectual disability, congenital neutropenia, cataracts, movement disorder

Abstract

We studied a group of individuals with elevated urinary excretion of 3-methylglutaconic acid, neutropenia that can develop into leukemia, a neurological phenotype ranging from nonprogressive intellectual disability to a prenatal encephalopathy with progressive brain atrophy, movement disorder, cataracts, and early death. Exome sequencing of two unrelated individuals and subsequent Sanger sequencing of 16 individuals with an overlapping phenotype identified a total of 14 rare, predicted deleterious alleles in CLPB in 14 individuals from 9 unrelated families. CLPB encodes caseinolytic peptidase B homolog ClpB, a member of the AAA+ protein family. To evaluate the relevance of CLPB in the pathogenesis of this syndrome, we developed a zebrafish model and an in vitro assay to measure ATPase activity. Suppression of clpb in zebrafish embryos induced a central nervous system phenotype that was consistent with cerebellar and cerebral atrophy that could be rescued by wild-type, but not mutant, human CLPB mRNA. Consistent with these data, the loss-of-function effect of one of the identified variants (c.1222A>G [p.Arg408Gly]) was supported further by in vitro evidence with the mutant peptides abolishing ATPase function. Additionally, we show that CLPB interacts biochemically with ATP2A2, known to be involved in apoptotic processes in severe congenital neutropenia (SCN) 3 (Kostmann disease [caused by HAX1 mutations]). Taken together, mutations in CLPB define a syndrome with intellectual disability, congenital neutropenia, progressive brain atrophy, movement disorder, cataracts, and 3-methylglutaconic aciduria.

Figure 1

Individual Photographs and MRI Findings (A) Individual #10 was born with increased muscle tone. (B and C) Study participants #11 (B) and #9 (C) with tented mouth, hypertelorism, and truncal hypotonia. (D) Individual #6 displayed no facial dysmorphisms and is able to stand freely. (E–H) Consecutive T2-weighted MR images of individual #8, axial view, at the age of 2.5 months (E), 16 months (F), 3.5 years (G), and 7 years (H), demonstrating progressive brain atrophy with both cortical and white matter volume decrease over time. Progressive, symmetrical basal ganglia atrophy was supported by abnormally increased T2 signal intensity in the caudate nucleus and putamen starting at the age of 16 months. (I) Isolated cerebellar atrophy was observed in study participant #6 as determined T1-weighted sagittal MRI.

Figure 2

Leucocyte Counts, Images, and Maturation Arrest (A) Leucocyte (reference range 4.5–10 g/l) and absolute neutrophil count (reference range at birth, 12–15 g/l; 2–12 months, >2 g/l; >12 months, >1.5 g/l) of individual #9 before and during successful treatment with G-CSF. (B) The total bone marrow composition at 20 months of life of the same individual: blasts 4%, promyelocytes 13%, myelocytes 1%, metamyelocytes 1%, bands and segmented cells 1%, neutrophils 2%, basophils 0%, eosinophils 8%, lymphocytes 32%, monocytes 18%, plasma-cells 0%, normoblasts 20% in comparison with a normal bone marrow. (C and D) Crista biopsy of individual #9 at 20 months of age shown in two distinct magnifications. The bone marrow contains many promyelocytes (^∗), but no mature neutrophils (maturation arrest at promyelocyte stage), many macrophages, hemophagocytosis, and atypical lymphocytes.

Figure 3

Schematic Representation of CLBP in Human, Fungi, and Bacteria (A) The positions of all mutations identified in human. Black boxes represent the two main functional domains: the ankyrin domain (ANK) consisting of a short 34 residue repeat implicated in a wide range of protein-protein interactions and the ATPase domain (AAA+). (B) Evolutionary changes in domain composition of CLPB proteins in human, fungi (mitochondrial HSP78 and cytosolic HSP104), and bacteria. All CLPB-family homologs possess at least a single AAA+ domain and the specific CLPB-D2 domain. In contrast to human CLPB, fungi and bacteria possess an “M domain,” a ClpB N domain, and an additional AAA+ domain. Ankyrin repeats are present only in the human homolog and are probably species specific.

Figure 4

The CLPB Zebrafish Model In vivo complementation of four CLPB variants (p.Arg408Gly, p.Met411Ile, p.Tyr617Cys, and p.Gly646Val) identified in evaluated study participants, in zebrafish. (A–G) Dorsal view of the developing zebrafish brain stained with acetylated tubulin at 3 days postfertilization (dpf). The cerebellum (CB) is highlighted by a white dashed rectangle in (A) showing a control embryo. (A–C) Control (A), morpholino injection (B), and rescue (C) by human WT mRNA. (D–G) Illustration of the effects of the tested alleles. (H) Graphic representation of the scoring for CB defects in control and injected embryos. Coinjection of MO with WT human CLPB mRNA results is a statistically significant reduction in the number of embryos with cerebellar defects (p < 0.0001). By contrast, coinjection of MO with each of the human CLPB mRNAs carrying the four mutations tested score as pathogenic being indistinguishable from the MO-injected embryos.

Figure 5

Protein Interaction Network for CLPB and HAX1 A database search resulted in 17 proteins with an established physical interaction with CLPB. The live-cell screen of CLPB against a library of 100 proteins by means of bioluminescence resonance energy transfer (BRET) identified 19 additional direct interactions (Tables S4 and S5). For HAX1, a total of 38 protein interactions were listed in the BioGRID database. Within the networks of first-order protein interactions for both proteins, a link between CLPB and HAX1 is established by mutual interaction with the sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SR Ca²⁺-ATPase 2 encoded by ATP2A2).