Homozygous HLA‐DQB1*06:02 combined with T‐cell receptor alpha polymorphism results in narcolepsy onset – A familial case report

Narcolepsy is a life‐long neurological disorder with well‐established genetic risk factors. Human leukocyte antigen‐DQB1*06:02 remains the strongest genetic predeterminant; however, polymorphisms in genes encoding the T‐cell receptor alpha chain are also strongly linked. This case report shows the inheritance pathway of these genetic markers contributing to narcolepsy onset in a 17‐year‐old female.

of narcolepsy type 2 are consistent with type 1 but are less extreme; individuals do not suffer with cataplexy and may have 'normal' levels of hypocretin in the cerebrospinal fluid.
Currently, the most impactful genetic risk factor for susceptibility to narcolepsy is conferred by the human leukocyte antigen (HLA) DQB1*06:02 encoded with the major histocompatibility complex (MHC), which is present in >95% of confirmed narcolepsy cases.
However, despite this strong association with the presence of a single copy of HLA-DQB1*06:02, only 0.001% of individuals with this HLA allele will develop narcolepsy, rendering the diagnostic benefit of HLA-DQB1 genotyping as debatable (Kornum et al., 2017).In addition to HLA-DQB1*06:02 other immune system related SNPs identified using genome-wide association studies (GWAS) have been implicated in the risk of developing narcolepsy.The most promising polymorphisms outside the MHC are sited within the genes encoding the T-cell receptor alpha (TRA).Hallmayer et al. (2009) first identified three SNPS on chromosome 14 (rs1154155, rs12587781 and rs1263646) where expression of the mutant variant was increased in narcolepsy patients (Hallmayer et al., 2009).Of the three SNPs identified, rs1154155 showed the greatest association and has since been confirmed in numerous narcolepsy GWAS studies (Han et al., 2013;Ollila et al., 2023;Tafti et al., 2014).Additionally, local frequency data (unpublished) showed that the minor rs1154155 allele was significantly associated with narcolepsy patients when compared against patients referred for genetic testing without a confirmed diagnosis (GG/GT vs. TT -OR 9.33 and p = .017).Both rs12587781 (GG/GT vs. TT, p = .33)and rs1263646 (GG/GT vs. TT and p = .74)were not significant.

Case report
Ethical committee approval was not required for this case and is presented with the consent of the patient's family.In May 2013, the index case, a 17-year-old female, presented with excessive daytime sleepiness and loss of muscle tone in the neck and upper body when experiencing strong emotion.Prior to this, the patient reports progressive onset of increased daytime sleepiness, which started coincidentally with the onset of puberty.Over the next 6-12 months, symptoms became more extensive, with paralysis extending down the spinal column during cataplexy episodes.The patient had not received the H1N1 influenza vaccine, Pandemrix, which has been associated with an increase in narcolepsy cases in numerous populations (Bardage et al., 2011;Dauvilliers et al., 2013;Heier et al., 2013;Nohynek et al., 2012;O'Flanagan et al., 2014).She had, however, contracted a streptococcal infection in June 2012, which is also been implicated as a risk factor associated with the onset of narcolepsy (Ambati et al., 2015;Aran et al., 2009).The patient continues to exhibit marked symptoms of narcolepsy while other family members report no narcolepsy associated symptoms.
To support a diagnosis of narcolepsy, HLA genotyping was conducted on the patient with additional testing of the mother, father and brother to confirm the presence or absence of risk factors within the immediate family.Additional genotyping of the SNP rs1154155 was also performed for all four family members using an end-point genotyping assay, confirmed by Sanger sequencing, to determine their status of the mutant TCR α variant.

HLA genotyping
DNA was extracted from EDTA-anticoagulated peripheral blood from each family member using a Qiagen Ez-1 xl and stored at −

rs1154155 Sanger sequencing
The TRA gene on chromosome 14 at position 2253376, encoding the rs1154155 SNP was subject to Sanger sequencing in all four family members.Primers 5′-CCG TGA GTC GAT TTT CAC CTT G-3′ (forward) and 5′-CTA GCT TTT CTT GGA TTG AGC GTC-3′ (reverse) were designed using Primer Express version 3.0 (Thermo Fisher Scientific) to amplify a 250 bp fragment encompassing the rs1154155 SNP.
Sequencing was performed using Thermo Fisher BigDye terminator chemistry 3.1 and processed using the ABI 3500XL (Applied Biosystems).Results were analysed using CLC Sequence Viewer 7 (Qiagen).
Genotyping of the sequences identified the presence of the wild-type TT alleles in the father and the brother (Figure 2a), whereas both the mother and daughter genotyped as heterozygous GT at rs1154155 (Figure 2b).Thus, the patient had inherited the maternally-derived mutant variant TRA.

DISCUSSION
The TRA locus encodes the alpha chain of the TCR alpha-beta het- with multiple functional SNPs affecting the J segments TRAJ24 and TRAJ28 (Ollila et al., 2023).
rs1154155 is one of four SNPs (rs1154155, rs1483979, rs3764159 and rs3764160) within a 18 kb region in strong linkage disequilibrium.rs1154155 is encoded within the genes for TRA J regions and mediates its action through the TRA J24 and J28 repertoire (Ollila et al., 2023).Patients with narcolepsy have a significantly lower expression of TRA J28 when compared to healthy controls (Ollila et al., 2023).In addition, the presence of the mutant form rs1154155G results in increased expression of TRA J24 and as such increases the binding affinity of the TCR.This combination, mediated by the mutant rs1154155G allele, suggests it to be a strong risk factor for narcolepsy onset, displaying preferential binding capabilities of the autoantigen.
This familial study outlines the inheritance route of the minor allelic variant, rs1154155G in an individual with narcolepsy type 1.The mutant rs1154155G allele was inherited from the mother who also carries HLA-DQB1*06:02:01 but does not suffer from narcolepsy.This may be due to lack of exposure to an associated environmental trigger, examples of which have been cited to be vaccination against H1N1 (Pandemrix) (Bardage et al., 2011;Dauvilliers et al., 2013;Heier et al., 2013;Nohynek et al., 2012;O'Flanagan et al., 2014) or streptococcal infection (Ambati et al., 2015;Aran et al., 2009).Alternatively, the mother's heterozygosity at HLA-DQB1 may have a significant protective effect.mRNA concentration studies have shown that narcolepsy onset is impacted by HLA-DQB1*06:02 allelic dosage, whereby disease status and DQB1*06:02 revealed a 1.65-fold higher expression in homozygotes versus heterozygotes.This is translated to the level of protein expression on the surface of B cells to indicate that DQB1*06:02 homozygotes have a 1.59-fold higher level of expression than DQB1*06:02 heterozygotes (Weiner Lachmi et al., 2012).
The effect of homozygosity of HLA-DQB1*06:02 has been observed in African Americans, white Americans and Japanese populations.In the Japanese population, the increased risk of developing narcolepsy is five times greater in HLA-DQB1*06:02 homozygotes compared with heterozygotes (Mignot et al., 2001).The TRA recognises antigens presented by HLA class II glycoproteins, including DQB1*06:02.
Increased surface density of HLA-DQB1*06:02 on antigen-presenting cells may increase the likelihood of the autoantigen presentation to T cells, resulting in the destruction of hypocretin producing neurons.
The increased dosage effect of HLA homozygosity is consistent with other well established autoimmune conditions that have a strong HLA association, including coeliac disease (Megiorni et al., 2009) and type I diabetes (Aydemir et al., 2019).for HLA-DQB1*06:02, at increased risk.To our knowledge, despite evidence from the original GWAS results (Hallmayer et al., 2009) later confirmed by subsequent GWAS studies (Han et al., 2013;Ollila et al., 2023;Tafti et al., 2014), testing for additional loci is yet to be included in routine genetic screening to support a narcolepsy diagnosis.Although implementation would be subject to a prospective study, the GWAS results combined with our local findings suggest this may be of value in differentiating between narcolepsy and other sleep disorders.
40 • C prior to use.The HLA types for all four family members were determined by next-generation sequencing at HLA-A, -B, -C, -DRB1/3/4/5, -DQA1, -DQB1, -DPA1 and -DPB1 using Illumina TruSight v2 according to the manufacturer's instructions.Following amplification, the product was cleaned, purified and normalised prior to library preparation, then tagmented, pooled and indexed followed by processing on an Illumina MiSeq.Results were analysed using Assign 2.1 Analysis suite.The father and both children were homozygous for the presence of HLA-DQB1*06:02:01, whereas the mother was heterozygous at this locus, expressing DQB1*06:02:01 and *03:19:01 (Figure 1).1.2.1 rs1154155 end point genotyping DNA from each family member was initially genotyped for the presence or absence of the rs1154155 minor allele using commercially synthesised primers supplied by Thermo Fisher Scientific combined with the TaqMan SNP genotyping kit.The assay was performed following the manufacturers recommended concentrations for all reagents and run on a Roche Lightcycler 480 II, the assay was then validated by Sanger sequencing.
expressed by T lymphocytes.The TCR expressed on CD4 + T helper cells interacts with HLA class II, in part encoded by HLA-DQB1 alleles.The TCR undergoes somatic hypermutation to increase the genetic variability, expanding the of recognition capability of the TCR when ligated with a corresponding HLA glycoprotein.This process occurs in the thymus, whereby naïve T lymphocytes undergo the process of negative and positive selection, ensuring the T cells generated bind self-HLA while remaining unresponsive towards self-antigen, maintaining immunological tolerance.TRA recombination occurs with one of the 46 functional variable (V) segments combined with one of the 49 joining (J) segments, producing a diverse TCR alpha and beta repertoire that is able to recognise presented antigens.Located 176 bp upstream to J10 (TRAJ10), rs1154155 has been associated F I G U R E 1 Familial human leukocyte antigen (HLA) haplotype inheritance.The index case has inherited the maternal haplotype A and paternal haplotype D, whereas the unaffected child has the paternal haplotype C with the maternal haplotype A. F I G U R E 2 (a) Sanger sequencing of rs1154155 for the father and unaffected brother.* indicates the presence of only a T at position 591 of the amplified fragment.(b) Sanger sequencing of rs1154155 for the mother and the affected child.* indicates the presence of both G and T at position 591 of the amplified fragment.
This case outlines the combined effects of familial inheritance of HLA alleles with additional SNPs outside the MHC, which are risk factors associated with narcolepsy.The mutant TRA variant, rs1154155G, combined with homozygosity for HLA-DQB1*06:02, is associated with a significant increase in the likelihood of narcolepsy onset in the presence of an environmental trigger.In this study, the patient reports experiencing daytime sleepiness prior to the initial diagnosis in May 2013.It is possible that, given her genetic background, an additional environmental trigger may have caused progression of her symptoms to include cataplexy, which led to her eventual diagnosis.This case has important implications for the potential of immunogenetic testing to support the diagnosis and classification of narcolepsy.In this family, the mother, who is heterozygous for HLA-DQB1*06:02 but also has the rs1154155G variant, did not develop cataplexy, further promoting the requirement for an environmental trigger and reinforcing an additional dosage effect associated with HLA-DQB1*06:02.The inclusion of genotyping for the presence of TRA rs1154155G as an additional diagnostic assay alongside genotyping for HLA-DQB1*06:02 to support the diagnosis of narcolepsy would be doubly beneficial.It could potentially identify individuals with excessive daytime sleepiness at risk of progressing to develop cataplexy.In addition, it may also increase the specificity of the immunogenetic testing to highlight individuals carrying both susceptibility alleles, in combination with homozygosity