Thun, G. A. | Search bioRxiv

bioRxiv2023

Novel regulatory variant in ABO intronic RUNX1 binding site inducing A3 phenotype

Background and ObjectivesMixed-field agglutination in ABO phenotyping (A3, B3) has been linked to genetically different blood cell populations like in chimerism, or to rare variants in either ABO exon 7 or regulatory regions. Clarification of such cases is challenging and would greatly benefit from sequencing technologies that allow resolving full-gene haplotypes at high resolution. Materials and MethodsWe used long-read sequencing by Oxford Nanopore Technologies to sequence the entire ABO gene, amplified in two overlapping long-range PCR fragments, in a blood donor presented with A3B phenotype. Confirmation analyses were carried out by Sanger sequencing and included samples from other family members. ResultsOur data revealed a novel heterozygous g.10924C>A variant on the ABO*A-allele located in the transcription factor binding site for RUNX1 in intron 1 (+5.8 kb site). Inheritance was shown by the results of the donors mother, who shared the novel variant and the anti-A specific mixed-field agglutination. ConclusionWe discovered a regulatory variant in the 8-bp RUNX1 motif of ABO, which extends current knowledge of three other variants affecting the same motif and also leading to A3 or B3 phenotypes. Overall, long-range PCR combined with nanopore sequencing proved powerful and showed great potential as emerging strategy for resolving cases with cryptic ABO phenotypes.

genetics↗

bioRxiv2021

The ulcerative colitis associated gene FUT8 regulates the quantity and quality of secreted mucins

Fucosylation of mucins, the main macrocomponents of the mucus layer that protects the digestive tract from pathogens, increases their viscoelasticity and shear stress resistance. These properties are altered in patients with ulcerative colitis (UC), which is marked by a chronic inflammation of the distal part of the colon. Here we show that the levels of Fucosyltransferase 8 (FUT8) and specific mucins are increased in the distal inflamed colon of UC patients compared to normal individuals. Overexpressing FUT8, as observed in UC, in mucin-producing HT29-18N2 colonic cell line increases trafficking of MUC1 to plasma membrane and secretion of MUC2/MUC5AC. FUT8 depletion (FUT8 KD), instead, causes intracellular accumulation of MUC1 and alters the ratio of secreted MUC2 to MUC5AC. Mucins secreted by FUT8 overexpressing cells are more resistant to shear stress compared to mucins secreted by FUT8 KD cells. These data fit well with the Fut8-/- mice phenotype, which are protected against UC. Fut8-/- mice exhibit a thinner proximal colon mucus layer with an altered ratio of neutral to acidic mucins compared to Fut8+/+ mice. Together, these data reveal that FUT8 optimizes the viscoelastic properties of the extracellular mucous by controlling the quantities of mucins secreted. SIGNIFICANCE STATEMENTMucins, the major components of the mucous barrier that protects our body from pathogens, are heavily glycosylated proteins. Changes their amounts and properties will alter the viscosity of mucous. Here we show that FUT8, a glycosylation enzyme of the Golgi apparatus, can control the viscosity of secreted mucins. Mucin secreting cells of the distal colon express FUT8, but their levels are altered in Ulcerative colitis patients. As a result, mucous produced by these cells is easily washed away, which exposes them to pathogens. We suggest that a defective mucous production is the main cause of initial inflammation observed in disease. Our findings help in understanding how cells control the quality of mucins and provide a means to prevent Ulcerative colitis.

cell biology↗