Epigenetic regulation in amyloid precursor protein and the Lesch-Nyhan syndrome

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Abstract

Lesch-Nyhan syndrome (LNS) is a neurogenetic disorder of purine metabolism in which the enzyme, hypoxanthine–guanine phosphoribosyltransferase (HPRT) is defective. A major unsolved question is how the loss of HPRT enzyme function affects the brain to cause the neurobehavioural syndrome in LNS and its attenuated variants (LNVs). To address this issue, a search for a link between LNS and the amyloid precursor protein (APP) is developed. Here, I identified, for the first time in fibroblasts from normal subjects as well as from LNS and LNV patients: (a) several APP-mRNA isoforms encoding divers APP protein isoforms ranging from 120 to 770 amino acids (with or without mutations and/or deletions) accounted for epigenetic mechanisms in the regulation of alternative APP pre-mRNA splicing and (b) five novel independent polymorphisms in the APP promoter: −956A>G, −1023T>C, −1161A>G, −2224G>A, −2335C>T relative to the transcription start site. A role for epistasis between mutated HPRT and APP genes affecting the regulation of alternative APP pre-mRNA splicing in LNS is suggested. An accurate quantification of various APP isoforms in brain tissues for detection of initial pathological changes or pathology development is needed. My findings may provide new directions not only for investigating the role of APP in neuropathology associated with HPRT-deficiency in LNS but also for the research in neurodevelopmental and neurodegenerative disorders by which various APP isoforms involved in the pathogenesis of the diseases such as Alzheimer’s disease.

Introduction

Lesch-Nyhan syndrome (LNS) is a neurogenetic disorder of purine metabolism in which the enzyme, hypoxanthine–guanine phosphoribosyltransferase (HPRT, EC. 2.4.2.8; MIM 308000)), is defective [1], [2]. The etiology involves a mutation of the HPRT gene, which is on the long arm of the X chromosome (Xq26.1), and it contains 9 exons and 8 introns [3]. Because the HPRT gene is on the X-chromosome, males are affected and females in the families are at risk of being carriers of the mutation. Complete or severe deficiency of HPRT activity leads to LNS (MIM 300322) [1]. Classical features of LNS include hyperuricemia and its sequelae (gout, nephrolithiasis, and tophi), motor disability (dystonia, chorea, and spasticity), intellectual impairment, and self-injurious behaviour. Partial deficiency of HPRT activity (MIM 300323) is characterized by the consequences of overproduction of uric acid and a variable spectrum of neurological manifestations, without the manifestations of self-injurious behaviour. The patients with partial deficiencies have been described as Lesch-Nyhan variants (LNVs) [4]. A major unsolved question is how the loss of HPRT enzyme function affects the brain to cause the neurobehavioural syndrome in LNS and its attenuated variants. To address this issue, a link between LNS and the aberrant basal ganglia function, including the dysfunction of dopaminergic pathways, was reported [5], [6]. However, the mechanism by which features of LNS result from impaired purine metabolism is still not well understood. It was also documented that adhesion of HPRT-deficient neuroblastoma as well as fibroblasts from patients with LNS exhibited dramatically enhanced adhesion compared to control cells [7] and could have consequences for the maturation of the central nervous system, as seen in the smaller brain size of LNS and LNVs children [8], [9]. Furthermore, it was also reported that HPRT deficiency was accompanied by aberrations in a variety of pathways know to regulate neurogenesis or to be implicated in neurodegenerative disease, including the canonical Wnt/β-catenin and the Alzheimer’s disease/presenilin signaling pathways [10]. A role for the amyloid precursor protein (APP) relates to cell–cell or cell-substrate adhesion and is important for brain morphology and highly coordinated brain functions such as memory and learning has been suggested [11], [12]. Hence, the APP pathway is possibly implicated in the development of LNS.

In an attempt to search for a link between LNS and APP, I have examined the APP-mRNA profile, the genomic APP-DNA, as well as the APP 5′ regulatory region in fibroblasts from four normal subjects and in HPRT-deficient fibroblasts derived from nine patients with LNS and three patients with LNV.

Section snippets

Patients

This study includes four normal subjects, controls (samples # 1–4), nine LNS affected male patients (samples # 5–13), and three LNV affected male patients (samples # 14–16).

Isolations of genomic DNA, mRNA, amplifications, and cloning

RNA-free genomic DNA, and mRNA were separately isolated from intact cultured fibroblasts. For genomic DNA isolation, the Puregene®DNA Purification Kit (Gentra System, Minneapolis, Minnesota, U.S.A.) was used. For mRNA isolation, the FastTract®2.0 mRNA Isolation Kit (Invitrogen, Carlsbad, CA, U.S.A.) was used. The DNA and

Analysis of APP coding region

Isolation of mRNA from intact cultured fibroblasts followed by RT-PCR and cloning showed the presence of different isoforms of the entire coding sequence (CDS) of the APP-cDNA produced from a single APP gene by alternative pre-mRNA splicing and encode divers APP protein isoforms ranging from 120 to 770 amino acids with or without mutations and/or deletions in normal subject, control (sample # 1), in LNS (samples # 7,13), and in LNV (samples # 14,15) patients (Table 1).

Analysis of the genomic APP-DNA

In order to access the

Discussion

The human APP is a type I transmembrane glycoprotein with a long N-terminal extracellular region and a short C-terminal cytoplasmic tail [11], [12]. The human APP gene is localized to chromosome 21 (21q21.2–3), spans approximately 240 kb and contains 18 exons. APP is the best known as the precursor molecule whose proteolysis generates beta amyloid (Aβ), a 39–42 amino acid peptide whose amyloid fibrillar form is the primary component of amyloid plaques found in the brains of individuals with

Acknowledgments

This work was supported by grants from the Lesch-Nyhan Syndrome Children’s Research Foundation and the Harold A. and Madeline R. Jacobs Fund at The San Diego Foundation. I am grateful to the patients and theirs families for agreeing to participate in this study.

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