Elsevier

Sleep Medicine

Volume 14, Issue 6, June 2013, Pages 482-487
Sleep Medicine

Original Article
Hypocretin (orexin) neuropeptide precursor gene, HCRT, polymorphisms in early-onset narcolepsy with cataplexy

https://doi.org/10.1016/j.sleep.2013.01.016Get rights and content

Abstract

Background

To test if the hypocretin (orexin) neuropeptide precursor (HCRT) gene, HCRT, mutations are implicated in the development of narcolepsy with cataplexy deficiency in young children.

Methods

The entire HCRT gene and ∼2000 bp promoter region was first sequenced in 181 patients and 153 controls, and rare polymorphisms including three nonsynonymous amino acid changes were identified. Next the 557 bp region of exon 2 harboring the three nonsynonymous changes was sequenced in an additional 298 early-onset subjects and in 148 control samples.

Results

A previously known common polymorphism (rs760282) and nine rare novel polymorphisms were identified in subjects and controls without significant differences. Two nonsynonymous exon 2 substitutions (+977 H54A, +979 G55R) were detected in two subjects with early onset at 7 and 6 years, respectively, but were not found in any controls. These substitutions are not likely to vastly change peptide binding to hypocretin receptors. One additional exon 2 substitution (+1019, K68R) was found in two patients and one control. Additional sequencing that focused on exon 2 showed additional subjects and controls with the +1019 K68R polymorphism and without significant differences between the subjects and the control. Segregation of two of these three nonsynonymous single nucleotide polymorphisms (SNPs) were observed from unaffected parents to offspring.

Conclusions

Sequencing of a large number of early-onset narcolepsy subjects revealed three novel nonsynonymous substitutions within the preprohypocretin protein, two of which were only found in patients with early-onset narcolepsy but are not likely to be functionally significant, especially in heterozygote subjects.

Introduction

Narcolepsy with cataplexy is a common sleep disorder closely associated with HLA-DQB1  06:02 and is caused by the loss of the hypocretin-producing cell in the hypothalamus. Onset of narcolepsy with cataplexy typically occurs around adolescence, and approximately 99% of cases with hypocretin deficiency are DQB1  06:02 positive, making narcolepsy-hypocretin deficiency one of the most highly known HLA-associated diseases. A probable cause for the hypocretin cell loss is autoimmunity, as evidenced through genome-wide association studies and infectious triggers for the condition. Systematic screening of mutations in the orexin system in US and European patients with narcolepsy with cataplexy has excluded hypocretin (orexin) receptor 1 and hypocretin (orexin) receptor 2 but not hypocretin (orexin) neuropeptide precursor gene, HCRT, to be major contributors of genetic predisposition to human narcolepsy [1], as HCRT gene mutation directly implicated in the development of narcolepsy has been evidenced [2].

Interestingly patients with early-onset narcolepsy (before puberty) have been shown to more frequently have a family history of narcolepsy [3], [4], [5], [6] suggesting that, similar to several other diseases early-onset subjects may be more adequate models to identify highly penetrating disease-causing mutations. In agreement with this concept, only one potential disease-causing mutation (rs104894574) has been identified in hypocretin system genes in a DQB1  06:02 negative patient with low cerebrospinal fluid (CSF) hypocretin 1 and an unusually early onset of narcolepsy at 6 months of age [2]. In this case, a single HCRT gene mutation substituting a leucine for a polar residue in the signal peptide area of the protein was observed and found to cause abnormal protein trafficking in vitro. Following this hypothesis, we systematically sequenced the HCRT gene region in Chinese patients, a population characterized by earlier age of onset than populations identified in other countries [3].

Section snippets

Subjects

Patients, guardians, and unrelated gender-matched controls (Table 1) were recruited from the Sleep Center at Beijing University People’s Hospital as previously described in Han et al. [3]. Subjects provided written consent and guardians (if the patient was a minor) consented for inclusion in this study. The institutional review board of Beijing University approved the study.

Patients all had cataplexy and were identified over a 10-year period (2001–2010) at the sleep laboratory of Beijing

First-stage sequencing of the extended HCRT gene region in Chinese subjects vs controls

Sequencing of the whole HCRT gene and of at least 2000 bp promoter identified one previously known common polymorphism (−909C/T, rs760282) [10] and nine rare polymorphisms in 181 subjects and 153 matched controls (Table 2). Previously reported −22C/T and −20C/A (rs4796777) polymorphisms [2], [10] were not identified in our sample. In regard to the common rs760282 polymorphism, subject genotypes were 47CC, 84CT, and 50TT in subjects, and 37CC, 75CT, and 41TT in controls in agreement with

Discussion

Our study extends on previous studies that have reported on rare and common polymorphisms in the HCRT gene region [1], [2], [10], [12], [13], [14]. In our study, we confirmed that common promoter area polymorphisms such as rs760282 are not associated with narcolepsy in China, similar to data found in white individuals [10]. Ethnic-specific HCRT gene polymorphisms have been reported in different populations, indicating the importance to study novel ethnic groups [1], [12], [13]. In addition we

Conclusion

We screened a large number of Chinese narcolepsy subjects with childhood onset for rare HCRT gene polyomorphisms but could not find any disease causing mutation; however, a large number of rare new SNPs were found including three novel nonsynonymous substitutions. Thus although defects in hypocretin neurotransmission underlie narcolepsy, this deficit is not due to loss of expression or function of the hypocretin system loci but rather to dramatic impairment or loss of the ∼70,000 cells,

Conflict of interest

The ICMJE Uniform Disclosure Form for Potential Conflicts of Interest associated with this article can be viewed by clicking on the following link: http://dx.doi.org/10.1016/j.sleep.2013.01.016.

. ICMJE Form for Disclosure of Potential Conflicts of Interest form.

Acknowledgments

This work was supported by research grants from the National Science Foundation of china (81070069 and 30890031), Sino-German Center for Research Promotion (GZ538).

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