NM_000148.4(FUT1):c.[229C>T;302C>T] with 2 missense variations in the FUT1 gene associated with a para-Bombay phenotype

1 |. INTRODUCTION

The H antigen, a highly prevalent and only antigen of the H blood group system (ISBT 018), is encoded by the highly homologous FUT1 and FUT2 genes.¹ The genes are located 35 kbp apart on the long arm of chromosome 19 (Figure 1).¹ The FUT1 gene produces the α-1,2-fucosyl transferase 1 (α2FucT1) enzyme that form the H antigen in many tissues, including red cells and vascular endothelial cells.¹ The FUT2 gene produces the α-1,2-fucosyl transferase 2 (α2FucT2) enzyme in the secretory cells of the digestive and respiratory tracts that forms the H antigen in body fluids and intestinal mucosa.¹

FIGURE 1.

Schematic representation of the FUT1 and FUT2 genes. The FUT1 and FUT2 genes are closely linked, oriented in opposite directions, located at chromosome 19p13.33. The FUT1 and FUT2 genes consists of 5 and 2 exons of 365 and 343 amino acids, respectively. The coding sequences are found in only 1 exon of each gene (black box)

The complete lack of the H antigen (nonfunctional FUT1 and FUT2) on red cells, tissues, and body fluids leads to the Bombay phenotype, while the lack of the H antigen only on red cells (nonfunctional FUT1 and functional FUT2) leads to the para-Bombay phenotype.¹ The geographic and ethnic distribution of para-Bombay phenotype varies greatly among populations ranging from 1 in 8000 in Taiwan Chinese to less than 1 in 1,000,000 in Europeans.¹ The para-Bombay phenotype, albeit rare, is clinically significant, as incompatible blood transfusion can lead to acute hemolytic transfusion reactions.

A large number of variant FUT1 and FUT2 alleles are known that lead to weak or null expression of the H antigen on red cells or body fluids or both.^2,3 Many FUT1 alleles harboring single missense variants are known giving rise to variant H phenotypes, but only 1 allele (FUT1*01W.05.02) has been observed with 2 missense variants. We present a novel FUT1 allele harboring 2 missense single nucleotide variants (SNVs), c.229C>T and c.302C>T.

2 |. BRIEF METHODS

Red cell phenotype was determined using standard serologic techniques, including an adsorption-elution test. The patient’s saliva, plasma, and samples from family members were not available.

Genomic DNA was isolated (TIANamp Blood DNA kit, Tiangen Biotech) and the coding regions of the FUT1 (1439 bp) and FUT2 (1980 bp) were amplified and sequenced. The amplification primers for FUT1 were 5’-CTCAGGCTCTCCCTACCTC-3′ (fut1F, sense) and 5’-ACAGTTCCAGAAACGTCCC-3′ (fut1R1, antisense) and for FUT2 were 5′- ACACCTGAAGTAGAAGCACAC-3′ (fut2F, sense) and 5’-TTTCTTTTGCCTCCTATCGGGTC-3′ (fut2R, antisense). The amplicons were visualized by agarose gel electrophoresis and purified (AxyPrep PCR clean-up kit; Axygen Biosciences, Union City, CA, USA).

The FUT1 amplicon was sequenced using primers fut1F and fut1R1. The FUT2 amplicon was sequenced using primers fut2F and 5’-CTTTGTAGGGGTCCATGTT CG-3’ (futfs1, sense). Sequencing data were aligned to NCBI RefSeq NG_007510.2 (FUT1) and NG_007511.1 (FUT2) and analyzed with Bioedit (ver. 7.1.8). The FUT1 amplicon was cloned (TOPO TA Cloning kit, Life Technologies) and transformed in Escherichia coli DH5 competent cells. Plasmids were isolated (Wizard Plus Mini Prep DNA purification system; Promega, Madison, WI, USA) and the inserts sequenced.

The ABO gene exons 6 and 7 as well as their adjacent intronic regions were also amplified and sequenced.⁴

3 |. RESULTS

A 29-year-old Han Chinese female was identified during routine blood group typing for an abortion. She had 4 previous pregnancies (2 children, 1 ectopic, and 1 abortion) without history of transfusion. Anti-A, anti-B, anti-A,B, anti-A₁, and anti-H did not agglutinate the patient’s red cells. In reverse typing, the plasma was reactive with A₁ (4+), B (2+ weak) and O red cells (2+) at room temperature. The direct anti-globulin test was negative. However, the B antigen was detected on red cells by adsorption-elution, and we suspected a para-Bombay phenotype.

The patient was compound heterozygous for a novel FUT1 allele (c.229C>T; c.302C>T) and the FUT1*01N.06 allele (c.551_552delAG) (Table 1). According to gnomAD, p.Leu77Phe was not found among 41,470 African and African American alleles but occurred in 2 of 5190 East Asian alleles; p.Ala101Val was detected in 2 of 68,042 European alleles but no other population.

TABLE 1.

FUT1 alleles in the compound heterozygous patient with a para-Bombay phenotype

FUT1 allele			Nucleotide variant in the FUT1 genea
GenBank number	ISBT allele name	Status	n	Single nucleotide n variant (SNV)	dbSNP number	Amino acid substitution	Effect on proteinb	HGVS nomenclaturec	References
MN971576	FUT1*01W.30	Novel	2	c.229C>T	rs148719736	p.Leu77Phe	neutral	NM__000148.4:c.229C>T	This study
				c.302C>T	rs752107701	p.Ala101Val	deleterious	NM__000148.4:c.302C>T
None	FUT1*01N.06	Known	1	c.551_552delAG	rs573412368	p.Glu184Valfs*85	deleterious	NM_000148.4:c.551_552del	3

^aRelative to NCBI Reference Sequence NG_007510.2.

^bAccording to Provean.⁵

^cAccording to HGVS nomenclature recommendations⁶.

The patient was homozygous for the functional FUT2 allele, Se³⁵⁷ (c.357C>T) indicating a Secretor status. This homozygosity indicated that each of the 2 distinct FUT1 alleles was linked to a copy of the common Se³⁵⁷ FUT2 allele. The genomic sequencing of the ABO exons 6 and 7 indicated ABO*B.01 and ABO*O.01.01 alleles.

Almost all the serology could be explained assuming a nonfunctional FUT1 allele, which is however unlikely considering the type of the 2 missense variations. This limitation of the allele description can be overcome by future expression studies or an observation of the allele in conjunction with a nonfunctional FUT2 gene in cis.

The patient did not require blood transfusion and was discharged without further clinical follow up. Because alloanti-H is clinically significant, patients with a para-Bombay phenotype and alloanti-H should be transfused with H-negative, ABO compatible red cells. However, an anti-H titer is often lower in individuals with para-Bombay than Bombay phenotype. In an acute situation, careful and limited transfusion of ABO group-specific red cells may be possible in patients with a para-Bombay phenotype.

4 |. BRIEF SUMMARY

We report a Han Chinese individual who was compound heterozygous for 2 FUT1 alleles (FUT1*01W.30 + FUT1*01N.06). Due to the presence of B antigens on the red cells, our novel FUT1 allele, with the 2 missense variants c.229C>T and c.302C>T, is listed as a weak H phenotype.

Abbreviations:

α2FucT1

α-1,2-fucosyltransferase 1

α2FucT2

α-1,2-fucosyltransferase 2

SNVs

single nucleotide variants