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Case Reports
. 2015 Feb 5;96(2):258-65.
doi: 10.1016/j.ajhg.2014.12.020. Epub 2015 Jan 15.

CLPB variants associated with autosomal-recessive mitochondrial disorder with cataract, neutropenia, epilepsy, and methylglutaconic aciduria

Carol Saunders  1 , Laurie Smith  2 , Flemming Wibrand  3 , Kirstine Ravn  3 , Peter Bross  4 , Isabelle Thiffault  5 , Mette Christensen  3 , Andrea Atherton  6 , Emily Farrow  2 , Neil Miller  5 , Stephen F Kingsmore  7 , Elsebet Ostergaard  8
Affiliations
Case Reports

CLPB variants associated with autosomal-recessive mitochondrial disorder with cataract, neutropenia, epilepsy, and methylglutaconic aciduria

Carol Saunders et al. Am J Hum Genet. .

Abstract

3-methylglutaconic aciduria (3-MGA-uria) is a nonspecific finding associated with mitochondrial dysfunction, including defects of oxidative phosphorylation. 3-MGA-uria is classified into five groups, of which one, type IV, is genetically heterogeneous. Here we report five children with a form of type IV 3-MGA-uria characterized by cataracts, severe psychomotor regression during febrile episodes, epilepsy, neutropenia with frequent infections, and death in early childhood. Four of the individuals were of Greenlandic descent, and one was North American, of Northern European and Asian descent. Through a combination of homozygosity mapping in the Greenlandic individuals and exome sequencing in the North American, we identified biallelic variants in the caseinolytic peptidase B homolog (CLPB). The causative variants included one missense variant, c.803C>T (p.Thr268Met), and two nonsense variants, c.961A>T (p.Lys321*) and c.1249C>T (p.Arg417*). The level of CLPB protein was markedly decreased in fibroblasts and liver of affected individuals. CLPB is proposed to function as a mitochondrial chaperone involved in disaggregation of misfolded proteins, resulting from stress such as heat denaturation.

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Figures

Figure 1
Figure 1
Identification of CLPB Variants in Five Individuals (A) DNA sequence analysis of CLPB shows the position of the homozygous c.803C>T variant in the subject compared to the control. (B) Analysis of cDNA encompassing the c.803C>T variant shows a band of normal size in individual 1. To assess the functional effect of the c.803C>T variant on splicing, RNA was extracted from fibroblasts and reverse transcribed to cDNA with the SuperScript II Reverse Transcriptase kit (Invitrogen), and PCR of a 381 bp cDNA fragment encompassing exons 4–8 was performed. To assess the carrier frequency for the c.803C>T variant, a TaqMan assay was developed (Applied Biosystems). The PCR conditions were: 10 μl Universal PCR Master Mix, 0.5 μl 40 × assay mix, and 20–100 ng DNA in a total volume of 20 μl. The PCR program was 95°C for 10 min, and 50 cycles at 92°C for 15 s and 60°C for 1 min. The samples were run on an ABI Prism 7000 and analyzed with ABI SDS software. (C) The alignment of the amino acid sequences of CLPB homologs in different vertebrate species shows the conservation of the mutated threonine at position 268. (D) A schematic representation of human CLPB (not to scale) shows the predicted domains and the position of the p.Thr268Met substitution. The following abbreviations are used: ANK, ankyrin repeat; CC, coiled-coil domain; AAA+, AAA+ ATPase; and D2-small, ClpB-D2-small. (E) DNA sequence analysis of CLPB DNA shows the position of the heterozygous c.961A>T variant in subject 5 compared to the control (left) and of the heterozygous c.1249C>T (right).
Figure 2
Figure 2
Analysis of CLPB Protein Levels and In-Gel Activity of Complexes I and IV (A) In-gel enzyme activity in liver from individual 5 shows normal activity of complexes I and IV. (B) Analysis by SDS-PAGE of CLPB protein in different human tissues showing ubiquitous localization of CLPB. (C and D) Immunoblot analysis of CLPB in fibroblasts from individuals 3 and 4 and liver from subject 5 (D) shows absence of CLPB protein. In brief, mitochondrial protein was isolated from fibroblasts and liver as previously reported. The samples (25–30 cμg protein/lane) were run on a 12% SDS polyacrylamide gel and transferred to a PVDF membrane. The membrane was probed with a polyclonal antibody against CLPB (Atlas Antibodies) at a 1:1,000 dilution and developed with a 1:1,000 dilution of goat anti-rabbit antibody (Dako). An antibody against porin (Proteintech) was used as a loading control at a 1:1,000 dilution, and SUCLA2 was used as a reference at a 1:1,000 dilution. The secondary antibody was goat anti-mouse at a 1:1,000 dilution (Dako). The bands were visualized with the Supersignal West Pico and Femto substrates (Thermo Fisher Scientific) and MicroChemi imaging (DNR Bioimaging Systems).
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