The Metabolite Pathway Behind the MS Severity Variant

The 2019 reference map of multiple sclerosis (MS) genetic architecture gathered more than 200 autosomal regulatory single nucleotide polymorphisms associated with MS risk, and not one of them was associated with severity. Against that background, rs10191329 is the only variant so far shown to correlate with clinical severity, found in two large independent cohorts. Which gene it regulates is a separate question from whether it associates, and the original report answered it by prioritizing ZNF638 and DYSF, both highly expressed by neural cells and especially by oligodendrocytes, concluding that the variant sets the level of brain tissue resilience. Nataf and colleagues ran the prioritization again with a different strategy and arrived at the opposite answer: that rs10191329 essentially targets NAGK, an immunometabolic gene, and that the effect operates in immune cells rather than neural ones. Their study is a data mining analysis built entirely from public databases, repeated at least three times between August 2023 and February 2024.

What Gene Prioritization Weighs, and in What Order
The disagreement is methodological before it is biological. Gene prioritization refers to the methods geneticists use to decide which gene or genes a given SNP is most likely to regulate transcriptionally, and it rests on several parameters: gene proximity, the relevance of candidate target genes to the pathology in question, the expression profile of those candidates in relevant cells and tissues, and, the one the authors rank highest, the measured impact of the SNP on gene expression or protein levels in those cells. The original prioritization rested mainly on the first of these, the close vicinity of rs10191329 to the transcription start sites of ZNF638 and DYSF. The counter-argument the authors make is that target genes frequently sit as far as 2 Mb from their regulatory SNP, which makes proximity a weak discriminator, and that eQTL studies remain the gold standard. They also note that no experimental proof was offered that rs10191329 alleles associate with distinct expression levels of ZNF638 or DYSF in neural cells.

What 60 eQTL Datasets Say About the Variant
The Open Targets Genetics platform surveys 60 eQTL datasets generated by distinct consortia across 92 tissues and cell types, performing integrated statistical fine-mapping across expression and protein quantitative trait loci. Queried for rs10191329, it returns no association with any expression or protein quantitative trait previously described in the CNS, across 18 distinct CNS regions. What it does return sits elsewhere. In blood cells the variant associates with NAGK at 385,525 bp from the transcription start site (beta −0.316, p = 6.7×10⁻¹⁰¹), with ZNF638 at 173,308 bp (+0.166, 3.2×10⁻⁵¹), and more weakly with MCEE, CLEC4F and MPHOSPH10. In interferon-gamma-stimulated monocytes it associates again with NAGK (−0.0972, 1.9×10⁻⁶). DYSF appears only in testis and induced pluripotent stem cells. Across the whole table NAGK carries by far the largest effect size and the most significant p-value, and the single protein quantitative trait the variant associates with anywhere is plasma NAGK (beta −0.165, p = 2.5×10⁻⁷).

The Expression Pattern Points at Classical Monocytes
Single-cell expression data from the Human Protein Atlas separate the candidates cleanly. NAGK runs high in immune cells, with mean normalized transcripts per kilobase million of 119.6 in dendritic cells, 104.9 in macrophages, 85.4 in monocytes, 44.2 in B cells and 25.4 in T cells, against 25 in microglia, 22.3 in oligodendrocytes, 14.5 in inhibitory neurons, 14.2 in astrocytes and 11.1 in excitatory neurons. ZNF638 and DYSF run the other way, ZNF638 reaching 576.6 in oligodendrocytes against 48.7 to 61.9 across immune cells, and DYSF 58.4 in oligodendrocytes and 33.6 in excitatory neurons against 0.4 to 10 in immune cells. Among blood-circulating immune cells, classical monocytes carry the highest NAGK mRNA levels. In the EMBL single-cell atlas, myeloid cells, chiefly monocytes and dendritic cells, make up the great majority of cell types to which NAGK was assigned by clustering analysis. And in COEXPRESdb, the 100 genes most significantly co-expressed with NAGK across 25,362 samples and 1,324 studies are most strongly enriched for monocytes (adjusted p = 6.2×10⁻¹⁷), with the phagocyte-related term "lysosome" the top pathway hit at an adjusted p of 0.00004.

A Glycolytic Network and the Muramyl Dipeptide Trigger
Protein interaction data from BioGrid, restricted to experimentally demonstrated human interactions, put NAGK in company that fits a metabolic reading. Pathway enrichment across its known protein partners returned "glycogenesis and neoglucogenesis" as the most significant term (adjusted p = 0.002), comprising lactate dehydrogenase A, pyruvate kinase M1/M2, hexokinase 2, GOT1 and lactate dehydrogenase A like 6B. The authors then pushed the network a layer further: of the 45 human genes annotated with that pathway, they took the top 20% by expression in classical monocytes, nine genes, and found that each of their protein partner lists was itself highly enriched for the same pathway, giving a dense interconnected network of NAGK with PGK1, PKM, ALDOA, GAPDH, LDHB, ENO1, PGAM1, TPI1 and MDH1. The functional hook comes from published work showing that muramyl dipeptide, the main NOD2 ligand and a gut-derived postbiotic, must be phosphorylated by NAGK before it can activate NOD2. In the only transcriptomic study of muramyl dipeptide-stimulated classical monocytes, NAGK and eight of the nine glycolysis genes were significantly up-regulated, with GAPDH at 2.4-fold, PGK1 at 1.86, ALDOA at 1.70, ENO1 at 1.68 and NAGK itself at 1.53, while in non-classical patrolling monocytes the response was either different in direction or weaker.

The Glycosylation Arm
The other main function assigned to NAGK is recycling the GlcNAc molecules produced when lysosomes degrade glycosylated proteins. NAGK converts GlcNAc into GlcNAc-6-phosphate, from which the enzymes PGM3 and UAP1 generate UDP-GlcNAc, and since UDP-GlcNAc is a major glycosyl donor for both O- and N-glycosylation, NAGK sits at the entry of what is called the UDP-GlcNAc salvage pathway. Roughly half of all amino sugars from endocytosed glycans are recycled this way, with the parallel supply route being de novo synthesis from glucose and glutamine through the hexosamine biosynthesis pathway. Querying N-GlycositeAtlas for human blood-derived macrophages, the only immune cell type the database covers, the 121 glycosylated proteins identified include several molecules already tied to MS: complement component C3, the complement C3 receptor integrin alpha-M, the immune checkpoint molecule PD-L1, and the leucocyte antigen HLA-DR15. Supporting the same thread, serum GlcNAc levels, the main NAGK substrate in this pathway, have been shown to correlate inversely with clinical severity in MS patients, and oral GalNAc has been reported to treat EAE and to exert measurable anti-inflammatory effects in MS patients.

Three Pathways, and the Case Against the Tissue Resilience Reading
Their model predicts that carriers of the rs10191329A allele show decreased NAGK expression in immune cells, above all classical monocytes, with three possible routes from there to severity. The first is the NAGK–muramyl dipeptide–NOD2 axis, where NOD2 operates as an immunoregulatory receptor. Loss-of-function mutations cause the autoinflammatory disorder Blau syndrome and raise Crohn's disease risk more than 40-fold in homozygotes, and in EAE systemic muramyl dipeptide promotes immune tolerance through NOD2 in myeloid cells, partly by converting classical monocytes into non-classical patrolling ones, the classical subset being the one reported to exert pathogenic functions in EAE and MS. The second is the glycolytic reprogramming that accompanies that conversion, and the third the glycosylation changes above. On the original "cognitive reserve" reading the authors raise three specific objections, each drawn from published data rather than from new analysis. The association of rs10191329 with the trait "intelligence" has been reported in a single study, at a beta of −0.018 and a p-value of 1.92×10⁻⁶. In the original paper's own data supplements, adjusting for income dropped the link between years of schooling and MS severity from p = 2.09×10⁻¹³ and 4.61×10⁻¹⁶ in the two cohorts to 0.011 and 0.03, with separate work tying treatment escalation to socio-economic status as a whole. And the higher count of brainstem and cortical lesions reported in homozygous carriers does not by itself establish lower tissue resilience, which would require assessing demyelination, remyelination and axonal loss in patients carrying comparable levels of inflammation. The authors are direct that at this stage it is extremely difficult to tell whether carriers display lower CNS tissue resilience or an increased immune aggressiveness of their lesions, and their own conclusion is that the only SNP associating with MS severity appears to set the intensity of neuroinflammation rather than tissue resilience.

Disclaimer: This blog post is based on the cited study and is intended for informational purposes only. It is not intended to provide medical advice. Please consult with a healthcare professional for any health concerns.

Reference:
Nataf, S., Guillen, M., & Pays, L. (2024). The immunometabolic gene N-acetylglucosamine kinase is uniquely involved in the heritability of multiple sclerosis severity. International Journal of Molecular Sciences, 25(7), 3803. https://doi.org/10.3390/ijms25073803