A mutation in the signal sequence ofLRP5in a family with an osteoporosis-pseudoglioma syndrome (OPPG)-like phenotype indicates a novel disease mechanism for trinucleotide repeats

Abstract

We extend the spectrum of phenotypes caused by mutations in the Wnt/Norrin coreceptor low-density lipoprotein receptor-related protein 5 (LRP5) by identifying two novel types of mutation in related individuals whose presenting features were profound muscle hypotonia, mild mental retardation, blindness, and growth retardation. One mutation removes 6 out of 9 consecutive leucine residues in the LRP5 signal peptide (c.43_60del or p.Leu15_Leu20del), which impairs polypeptide entry into the endoplasmic reticulum (ER), trafficking to the cell membrane, and signal transduction. The second mutation resulted from nonhomologous recombination between Alu repeat sequences, which deleted exons 14-16 and would produce a nonfunctional, truncated, and frameshifted polypeptide, if expressed [chr11:g.(13871447_1387511)_(13879636_13879700)del (NW_925106.1) or p.Pro1010GlnfsX38]. We confirmed that the length of the LRP5 signal peptide poly-leucine repeat is polymorphic in the general population, and, importantly, we were able to demonstrate in independent in vitro assays that different allele sizes affect receptor processing and signal transduction. Consequently, this polymorphism may have physiologic effects in vivo. This latter finding is relevant since through a genomewide search we identified nearly 400 human proteins that contain poly-leucine repeats within their signal peptide. We chose 18 of these proteins and genotyped the underlying trinucleotide repeat in healthy Caucasian individuals. More than one length allele was observed in one-half of the proteins. We therefore propose that natural variation in poly-leucine-stretches within signal peptides constitutes a currently unrecognized source of variability in protein translation and expression.

Novel mutations affecting LRP5 splicing in patients with osteoporosis-pseudoglioma syndrome (OPPG)

“…The dual luciferase reporter assay for Wnt/Norrin signal transduction, secretion assay and western blot analyses were performed as described previously. 1,25 Western blot analyses of conditioned medium and of cell lysates from transfected cells were carried out using an anti-myc antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for immunodetection of LRP5. An anti-b-actin antibody (Sigma, St. Louis, MS, USA) to stain b-actin served as a loading control for equal amounts of cell lysates.…”

“…24 These plasmids have been described previously. 1,25 The identified p.E528_V529ins21 mutation was amplified by OneStep RT-PCR (Qiagen) from patient RNA (forward primer: 5¢-GTGTGCAGCTGCAG GACAAC-3¢ and reverse primer: 5¢-GGTAGTCAAGGCCAAACTCC-3¢) and introduced as a 1.1-kb PshAI (NEB) restriction fragment into full-length LRP5-WT 9L and LRP5N-WT 9L , respectively. The dual luciferase reporter assay for Wnt/Norrin signal transduction, secretion assay and western blot analyses were performed as described previously.…”

“…The generation of the following constructs has been described previously: 1,25 full-length human LRP5 (LRP5-WT 9L ) and a truncated form of human LRP5 (LRP5N-WT 9L ); mutant LRP5 with a deletion of six out of nine consecutive leucine residues in the signal peptide caused by the c.43_60del mutation (LRP5-Mut 3L ) and its truncated form (LRP5N-Mut 3L ). All these constructs have a myc epitope tag at the carboxy terminus.…”

“…26 Cytosolic and membrane fractions were analyzed by western blotting following reducing SDS-PAGE. 25 Endogenous b-actin and calnexin, which were detected with an anti-b-actin antibody (SIGMA) and an anti-calnexin antibody (BD Biosciences, Franklin Lakes, NJ, USA), served as positive controls for proper separation of cytosolic and membrane proteins. The truncated constructs (LRP5N-WT 9L , LRP5N-p.E528_V529ins21, LRP5N-Mut 3L ) were only used in the secretion assay.…”

Variants in the 3′UTR of SNCA do not affect miRNA-433 binding and alpha-synuclein expression

“…Both allele possibilities were generated by site-directed mutagenesis. The dual-luciferase reporter assay for both allele variants within the SNCA 3 0 UTR was performed following the protocol described by Chung et al, 13 with the exception that experiments were normalized using the topflash vector containing the luciferase with its own 3 0 UTR. To activate the wingless pathway, Neuro2A cells were transiently transfected with LRP5, Norrin, FZD4, Topflash and Renilla.…”