Deficiency for the ubiquitin ligase UBE3B in a blepharophimosis-ptosis-intellectual-disability syndrome

Abstract

Ubiquitination plays a crucial role in neurodevelopment as exemplified by Angelman syndrome, which is caused by genetic alterations of the ubiquitin ligase-encoding UBE3A gene. Although the function of UBE3A has been widely studied, little is known about its paralog UBE3B. By using exome and capillary sequencing, we here identify biallelic UBE3B mutations in four patients from three unrelated families presenting an autosomal-recessive blepharophimosis-ptosis-intellectual-disability syndrome characterized by developmental delay, growth retardation with a small head circumference, facial dysmorphisms, and low cholesterol levels. UBE3B encodes an uncharacterized E3 ubiquitin ligase. The identified UBE3B variants include one frameshift and two splice-site mutations as well as a missense substitution affecting the highly conserved HECT domain. Disruption of mouse Ube3b leads to reduced viability and recapitulates key aspects of the human disorder, such as reduced weight and brain size and a downregulation of cholesterol synthesis. We establish that the probable Caenorhabditis elegans ortholog of UBE3B, oxi-1, functions in the ubiquitin/proteasome system in vivo and is especially required under oxidative stress conditions. Our data reveal the pleiotropic effects of UBE3B deficiency and reinforce the physiological importance of ubiquitination in neuronal development and function in mammals.

Figure 1

A Blepharophimosis-Ptosis-Intellectual-Disability Syndrome (A–D) Individual 1 at the age of 1 year (A), individual 2 at the age of 7 years 7 months (B), individual 3 at the age of 3 years 1 month (C), and individual 4 at the age of 1 year 2 months (D). Note characteristic facial features with blepharophimosis and ptosis, sparse hair, sparse and arched eyebrows, long philtrum, anteverted nares, retrognathia, and low-set, posteriorly rotated ears. Individuals 2 and 3 also have upslanting palpebral fissures; this feature develops with age. (E) Cranial MRI scan of individual 1 (Ind. 1) at age 1 year 10 months shows cerebellar tonsillar ectopia consistent with Chiari 1 malformation, thin corpus callosum, and an overall small head. A cranial MRI scan of a healthy age-matched control (Co) is also shown.

Figure 2

UBE3B Mutations Cause the BPID Syndrome (A and B) Sequence chromatograms showing the homozygous (MUT/MUT) and heterozygous (WT/MUT) UBE3B splice donor mutation c.1741+2T>C identified in family 1 (A) that leads to skipping of exon 16 (119 bp; out-of-frame) and exons 16 and 17 (234 bp; in-frame) as shown by RT-PCR on RNA extracted from whole blood of individual 1 (B). Exon skipping was confirmed by sequence analysis of the respective RT-PCR products (not shown). (C and D) Individuals 2 and 3 are compound heterozygotes for c.2223_2224delAG and the splice acceptor mutation c.545−2A>G (C), the latter leading to out-of-frame skipping of exon 8 (86 bp) and exons 8 and 9 (169 bp) as shown by RT-PCR (D) and sequence analysis (not shown). (E) Protein diagram of human UBE3B depicting the N-terminal IQ domain (short calmodulin-binding motif containing conserved Ile [I] and Gln [Q] residues) and the C-terminal HECT (homologous to the E6-AP carboxyl terminus) domain. The positions of the three truncating mutations identified in individuals 1–3 are indicated by black arrows and the p.Gln727Pro missense alteration is shown in red. (F) Sequence chromatograms showing the homozygous (MUT/MUT) and heterozygous (WT/MUT) UBE3B missense alteration p.Gln727Pro (c.2180A>C) identified in individual 4 and multiple sequence alignment of UBE3B orthologs surrounding the Gln727 position in the human protein.

Figure 3

oxi-1, the Caenorhabditis elegans Ortholog of UBE3B, Is Involved in Ubiquitin/Proteasome-Mediated Protein Turnover In Vivo (A) Immunoblot of lysates derived from worms after control (ctrl) RNAi or RNAi against oxi-1 or hecd-1. (B) Immunoblot of lysates derived from worms after control or oxi-1 RNAi without (0) or with addition of 0.5 or 1 mM paraquat. The blot was performed with a GFP-specific antibody; tubulin serves as loading control. (C) Images of C. elegans that express the UbV-GFP reporter to measure protein turnover. Worms were treated as in (B). Top, transmitted light; bottom, GFP fluorescence. Scale bar represents 0.5 mm.

Figure 4

Expression Pattern of Ube3b in Mouse In situ hybridization via a Ube3b antisense probe on sagittal (A, B, D–F) and coronal (C) sections at embryonic (E) and postnatal (P) days as indicated. Ch, cortical hem; Cp, cortical plate; Cx, cerebral cortex; Dr, dorsal root ganglia; Eg, external granular layer; En, epithelium of the medial nasal process; Gl, granular layer; Hi, hippocampus; Ig, internal granular layer; Pn, preoptic neuroepithelium; Te, tectum; Vz, ventricular zone; Wh, whiskers.

Figure 5

Reduced Growth of Ube3b Mutant Mice (A) Homozygous Ube3b^−/− mice (Hom) show a significantly reduced growth rate (weight curve comparison wild-type [WT] versus Hom: p < 0.001) than WT or heterozygote (Het) controls. The data are presented relative to a reference range that encompasses 95% of the natural variation, shown as the green shaded area, constructed with 134 WT mice from a variety of genetic backgrounds. Error bars represent SD. (B) A representative Ube3b^−/− mouse is smaller and thinner than a WT control. (C and D) A significantly reduced brain section area (C) and a smaller hippocampus area (D) are also evident. ^∗p = 0.0151, ^∗∗p = 0.0049. Error bars represent SEM.

Figure 6

Neurosensory Abnormalities in Ube3b^−/− Mice (A and B) Ube3b^−/− mice show reduced grip strength (A; p < 0.0001 WT versus Hom), and low serum low density lipoprotein (LDL) levels were also observed (B; p < 0.0001 WT versus Hom). The data are presented relative to a reference range that encompasses 95% of the natural variation, shown as the green shaded area, constructed with 132 (A) and 93 (B) WT mice from a variety of genetic backgrounds. Data are shown both as individual data points and as a five point box plot summary that indicates the median, first and third quartiles (box), and minimum and maximum values (whiskers). (C and D) Auditory brainstem response (ABR) thresholds (±SEM) were significantly elevated in homozygotes (red) compared with heterozygotes (blue) and WT (green) littermates at 3 months (C) and at 6 months (D) (mean elevations cf. wild-types 15.0 ± 3.5 dB, Q = 4.427, p < 0.05 at 3 months; 22.8 ± 5.4 dB, Q = 4.671, p < 0.05 at 6 months).