Background
Narcolepsy is a serious chronic neurological sleep disorder affecting between 0.02–0.05% of the general Caucasian population [
1]. It is characterized by excessive daytime sleepiness, cataplexy, hypnagogic hallucination, sleep paralysis, and nocturnal fragmented/disorganized sleep [
2,
3]. Narcolepsy causes cognitive dysfunction, low academic performance and interpersonal problems [
4]. Cataplexy is a pathognomonic clinical symptom required for the diagnosis of narcolepsy, according to the International Classification of Sleep Disorders [
5]. It is a symptom that occurs when the muscle tension, in various areas of the body, is suddenly decreased involuntarily and lasts from few seconds to several minutes. Cataplexy is generally induced by laughter, excitement anger, and other emotional changes [
5]. Mutations in genes of the hypocretin (orexin) neurotransmitter system cause narcoleptic symptoms in animal models [
6]. Although most patients with narcolepsy-cataplexy have a reduction of hypocretin concentration in the cerebrospinal fluid [
7], mutations or polymorphisms in hypocretin-related genes are extremely rare [
8]. A potential autoimmune mechanism has been suggested, supported by the finding that in most narcolepsy patients, 80–90% of hypocretin cells in the hypothalamus were destroyed [
9]. The lost of hypocretin neurons [
10] has also been shown in a few post-mortem cases. In addition, the transcription of preprohypocretin mRNA was significantly decreased in the brain of these patients [
11]. This wake-promoting neuropeptide is involved in sleep regulation, energy homeostasis, reward-seeking, learning, and memory; and it is also involved in the body temperature regulation, endocrine function, and the cardiovascular system, among other systems [
12]. Recent studies also indicate that hypocretin/orexin neurons can alter their intrinsic electrical activity according to fluctuations in the levels of nutrients and appetite-regulating hormones [
13].
Narcolepsy transmission is polygenic, environmentally influenced and genetic factors play an important role in its expression. Family studies indicate the presence of a 20–40 times increased risk of disease expression in first-degree relatives, and monozygotic twin studies showed that concordance is partial (25–31%) [
1]. Early onset French and Chinese patients suffering from narcolepsy have shown a positive family history as compared with late-onset patients [
14,
15], suggesting that the disease may be more likely due to genetic factors in this subgroup of patients.
Based on the strong HLA association, family study segregation [
16,
17] and the finding of reduction of hypocretin-1 levels in the cerebrospinal fluid of
DQB1*0602 positive patients [
18,
19], an autoimmune mediated destruction of hypothalamic neurons secreting hypocretin has been suggested as the cause of the disease. However, serum autoantibody markers have not been detected and no immunological abnormalities have been found in patients with narcolepsy [
20].
The disease is strongly associated in Caucasians and Japanese with the
DRB1*1501-
DQA1*0102-
DQB1*0602 haplotype. In African Americans, it is associated with
DQB1*0602 haplotypes bearing different
DRB1 alleles (*1101 and *1503) suggesting that
DQA1 and
DQB1 play a primary role in susceptibility [
17].
DQB1*0602 rather than
DRB1*1501 has been found increased in the patients, indicating that the disease susceptibility allele or locus is within, or, in the vicinity of the DQ region [
21]. Haplotype analysis and contiguous genomic sequencing across the region have identified no other candidate gene [
22]. Two-five fold increased risk in
DQB1*0602 homozygous vs. heterozygote patients has been demonstrated in different ethnic groups [
23].
DQB1*0301/*0602 carriers are also at an increased risk, whereas
DQB1*0602/*0601 and
DQB1*0602/*0501 heterozygotes have a lower disease risk [
23‐
25].
Several authors reported that 85 to 95% of patients with narcolepsy carry
DQB1*0602 when cataplexy is clinically typical or severe. However, only 40–60% of the patients are
DQB1*0602 when mild, atypical or no cataplexy exists [
25,
26]. TNF alpha region has also been claimed to be involved in susceptibility independently of class II loci [
27].
Genetic factors in other chromosomes have also been implicated [
28]. A gender dimorphism and a strong effect of the catechol-O-methyltransferase (
COMT) genes seem to influence symptoms.
COMT genotype distribution between male and female patients was associated with the response to modafinil in Caucasians, since the optimal dose of modafinil was approximately 100 mg lower in females with narcolepsy, suggesting that females are better responders to the drug [
29].
The HLA genetic profile of Mexican and other Central American patients has not been published; therefore the aim of this study was to investigate the class II-DRB1/DQB1 allele distribution in a group of sporadic Mexican Mestizo patients with narcolepsy and to explore if the HLA association is gender related.
Discussion
The results of this study are considered preliminary, due to the small sample size. However, it is important to mention that we are not "Hypothesis generating" but "hypothesis testing". As demonstrated by many statistician experts in the HLA field: "If an association is detected in the first case, it can be tested and confirmed in the latter without having to correct in multiple comparisons" [
33]. Nevertheless, future studies in Hispanic admixed populations are needed to confirm the presence of other HLA allele associations. It is worth to mention, that
DRB1/
DQB1 association has been tested and repeatedly demonstrated by many authors in different ethnic groups [
23‐
25,
34]; therefore, the associations shown here, are real. In this study, we report on the genetic profile in a sample of narcoleptic Mestizo patients.
DRB1*1501 (OR = 8.2; pc < 0.0001) and
DQB1*0602 (OR = 8.4; pc < 0.0001) were the strongest associated alleles found in narcoleptic Mexicans, similarly to African, White Americans and different Oriental groups [
23‐
25,
34‐
37]. Fifteen of the 27
DRB1 typed patients, were positive for
DRB1*1501 (allele frequency = 27.8 vs. 4.4% in the controls) and 21 of the 32
DQB1 typed patients were positive for
DQB1*0602 (allele frequency = 32.8% vs. 5.4% in the controls). In five
DQB1 typed patients,
DRB1 was not typed because of insufficient DNA, thus we were not able to assemble the
DRB1-
DQB1 combinations in them. Interestingly, the
DRB1*1501-
DQB1*0602 haplotype was present in most of the
DQB1*0602 positive patients but other DR2 allele combinations were also found. Indeed, two narcoleptic patients had
DRB1*1502-
DQB1*0602 and two had
DRB1*1503-
DQB1*0602. A
DRB1*1503 but not
DRB1*1502 association with
DQB1*0602 has been reported in patients from Martinique [
38] and in African Americans [
23]. These results show, beyond doubt, that
DQB1*0602, rather than
DRB1*1501 is the major narcolepsy susceptibility allele in Mestizos. Interestingly, none of the DR52 associated-
DQB1*0602 haplotypes were found in Mexican patients, but their frequency is low in the general healthy population. As an example, in 160 Mexican Mestizo healthy individuals typed in our laboratory, the haplotype frequency was 0.31% for each of the following combinations:
DRB1*1101-
DQB1*0602,
DRB1*1201-
DQB1*0602 and
DRB1*1301-
DQB1*0602 (unpublished data). Some of these haplotypes have been reported in narcoleptic patients in other populations, most notably in African Americans [
23].
No increase in
DQB1*0602 homozygosity was found, as previously reported in White Americans and in African American patients, in whom a two to four fold higher risk has been described, compared to heterozygotes [
23]. The same has been shown in Japanese patients [
24]. This fact cannot be explained only based on the different allele frequencies of
DQB1*0602 across ethnic groups, since the frequency shown in Japanese (AF = 6.4%) [
24] was similar to the one found in the control group of the present study (AF = 5.5%). The lack of homozygote patients may be due to the reduced number of cases. Thus, again, a larger number is needed to confirm these results.
As in other populations, other
DQB1 alleles, beside
DQB1*0602, influence narcolepsy susceptibility [
24,
39]. The analysis of
DQB1 distribution in
DQB1*0602 negative patients showed that
DQB1*0301 (OR = 2.7, p = 0.03) was significantly increased in this subgroup of patients.
DQB1*0301 had also the second strongest susceptibility effect, after
DQB1*0602 in Africans, Japanese and White Americans [
24]. In Mexican patients,
DQB1*0301 occurred in the context of several HLA haplotypes that included
DRB1*1101, *1303, *1304, *0806 and *1602; however the number of patients in this group was insufficient to perform additional comparisons and to explain the possible independent contribution of the mentioned
DRB1 alleles to susceptibility. None of these patients had the
DRB1*04-
DQB1*0301, found associated in White Americans [
24]. Genotype distribution in patients and controls showed that
DQB1*0602/
DQB1*0301 conferred the highest risk for susceptibility (OR = 11.4) compared to
DQB1*0602/X (non *0301) (OR = 9.4). The former combination was also described as the one with the highest risk for the development of narcolepsy, across three different ethnic groups [
24].
DRB1*0407, which is the most frequent allele in Mexican population, seemed to be linked to protection in the present study. This allele is in strong linkage disequilibrium with
DQB1*0302 in Mexicans and the haplotype frequency of
DRB1*0407-
DQB1*0302 is 13.1% [
31]. In Koreans
DRB1*0406-
DQB1*0302 was found protective in patients with narcolepsy [
39]. None of these studies confirmed a possible effect of
DRB1*04 in susceptibility as previously shown in Whites and Japanese [
24]. It may be claimed that the
DQB1 locus may also be involved in protection, since in Korean as well as in Mexican patients,
DQB1*0302 was decreased, although combined with *0406 in Koreans and with *0407 in Mexicans.
DQB1*0601 has also been associated with protection in Koreans [
25,
39] and Japanese [
40], but the latter was not related with protection in Mexicans, perhaps due to its low frequency (AF = 0.5%) [
31]. Similar findings have been published recently in Koreans, where again,
DRB1*0406 was found negatively associated and
DQB1*0301 was described as a susceptibility allele. The authors claim based on their own work, and previous work, that a remarkable consistency of the HLA association pattern across multiple ethnic groups and cultures exists [
41]. Thus, even if our sample size is small and the results of protection and secondary association may be regarded as preliminary, our data are consistent with those published [
21,
23‐
26,
39,
41].
Interestingly, the overall positive rate for
DQB1*0602 in Mexican patients was 65.6%, while in Japanese, White Americans and African Americans; the rate is between 75–80% [
24]. To analyse if this difference was significant or not, we performed a statistical comparison between patients and controls from the present study and those from the Mignot et al. study [
24]. No significant difference, regarding
DQB1*0602 distribution, was found between Japanese and Mexican controls but we did find a significant deviation when comparing Mexican controls with White or African Americans healthy people (p = 0.001 and p = 0.00000001, respectively). These differences are due, undoubtedly, to the lower frequency of
DQB1*0602 existing in the general Mexican population [
42], which is similar to the one found in Orientals, but it is lower than the frequency in Caucasian and African Americans [
43]. The lower frequency of the
DQB1*0602 allele in Mexican patients compared with African (p = 0.0007), Caucasian (p = 0.003) and Japanese (p = 0.0009) patients, is due to our small number of cases.
Gender stratification showed a differential distribution of
DRB1 and
DQB1 alleles. The patients selection was unbiased since no significant difference was found when distribution of males Vs. females was compared (Z = 0.561, p = 0.29). To demonstrate if the gender selection among the controls was biased, we performed several analyses comparing the mean allele frequency in the overall control group with the frequency for HLA
DQB1*0602 among male and female patients. However, the distribution of
DQB1*0602 alleles among female and male controls was found in the limit of the significance (X
2Y = 3.841, p = 0.05; data not shown). Therefore, it must be mentioned that even if the p value was p = 0.05, the control selection may have been biased. When HLA distribution was compared matching for gender,
DRB1*1501 (OR = 15.8; pc < 0.0001) and
DQB1*0602 (OR = 19.8; p < 0.0001) showed a higher risk in female patients, although the same alleles were significantly increased in males, but with less intensity. This stronger female HLA association was described for the first time. However, it is important to confirm these data with a larger sample size and in different ethnic groups. Thus, the gender association reported here should be regarded as preliminary. Even if we have a stronger HLA
DRB1/
DQB1 association in females, this does not imply, by any means, that females are more or less affected than males; this only means that there is a stronger HLA genetic predisposition to narcolepsy in females than in males. A better response in affected women to certain drugs [
29] is not contradictory at all, with the HLA/female association.
The explanation for this gender specific association is not clear, but a sexual dimorphism and a strong effect of the COMT genotype on disease severity and response to modafinil have been shown [
44]. Females with narcolepsy with high COMT activity fell asleep twice as fast as those with low COMT activity during the multiple sleep latency test, while the opposite was true for men [
45]. A gender difference in body weight gain and leptin signaling in hypocretin/orexin deficient mouse models has been also claimed [
46]. Obesity was more prominent in females in both preprohypocretin knockout mice and orexin/ataxin-3 transgenic narcoleptic mice and was associated with higher serum leptin levels, suggesting a partial leptin resistance [
46].
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
CA Drafted the manuscript and supervised the laboratory work. LL Collaborated in drafting the manuscript and extracted the DNA at Stanford University. HF-A Carry out the statistical analysis and revised the drafted manuscript. MV Collaborated with the technical HLA DNA typing and revised the drafted manuscript. AM Collaborated with the technical HLA DNA typing and revised the drafted manuscript. EM Selected the clinical cases and wrote the criteria for all Centres and for the 13th International Histocompatibility Workshop and revised the drafted manuscript. RH Diagnosed and selected the cases from The Instituto Nacional de Neurologia and revised the drafted manuscript. HB Diagnosed and selected the cases from and revised the drafted manuscript. CG Coordinated all the work, revised the results and statistical analysis, revised the drafted manuscript thoroughly; revised and corrected the final version. All authors read and approved the final manuscript.