Analysis of the molecular mechanism and pedigree investigation of para-Bombay phenotype caused by combined mutations at position h⁶⁴⁹ and h⁷⁶⁸ of FUT1 gene

Abstract

Background

The para-Bombay phenotype is a rare red blood cell phenotype characterised by the lack of ABH antigens on red blood cells, but ABH substances can be found in saliva. The aim of this research was to study the mechanism of mutation of FUT1 and FUT2 genes and the pedigree of a family with the para-Bombay phenotype.

Material and methods

The blood group was detected by a conventional serological method, H antigen adsorption-elution test, and testing saliva for A, B, and H antigens. We amplified and sequenced the ABO, FUT1, and FUT2 genes of the proband and her family using a polymerase chain reaction method, and performed TA cloning and sequencing on the amplified products of the FUT1 gene to determine its genotype.

Results

With the conventional serological method, it was found that the red blood cell phenotype of the proband and her sister lacked H antigen, while the adsorption-elution test of H antigen could detect weak H antigen. Through FUT1 cloning and sequencing, it was found that the proband had a compound heterozygous mutation of c.649G>T and c.768delC, and the genotype was FUT1*01W.24/FUT1*01N.20; the proband’s father and mother had heterozygous mutations of c.768delC and c.649G>T, and their genotypes were FUT1*01N.20/FUT1*01 and FUT1*01W.24/FUT1*01. The sister’s FUT1 mutation site and genotype were the same as the those of the proband. FUT2 gene sequencing revealed that the proband and sister had a synonymous mutation of c.357C>T, while their parents both had a synonymous mutation of c.357C>T and a missense mutation of c.385A>T. The Lewis blood types of the four samples all showed Le (a–b+), all of which were secretory.

Conclusion

Blood group serology and molecular diagnostic techniques showed that the compound heterozygous mutations of the proband and her sister were inherited from their father and mother.

INTRODUCTION

The para-Bombay phenotype is a rare red blood cell phenotype characterised by the lack of ABH antigens on red blood cells, but ABH substances can be found in saliva¹. Two α-1,2 fucosyltransferases synthesise H antigen. α2Fuc-T1 (coded by FUT1) determines the expression of H antigen on red blood cells. α2Fuc-T2 (coded by FUT2) is not expressed on red blood cells, but is expressed in salivary glands, gastrointestinal tissues and genitourinary tissues^2,3. The Bombay and para-Bombay types are, therefore, closely connected to FUT1 and FUT2 gene mutations.

MATERIAL AND METHODS

Subjects

A 52-year-old female from Nantong (Jiangsu, China) was treated in our hospital for thrombocytopenia on March 27, 2021. When her blood group was tested, it was found that the H antigen was negative, so we collected peripheral blood (5 mL) and saliva (5 mL) from both this female proband and three members of her family (parents and a sister).

The research protocol was approved by the medical ethics committee of Nantong University Affiliated Hospital (approval number: 2021-K009). Informed consent was obtained from all participants of the study.

Main reagents and instruments

The main reagents and instruments used in this study included ABO forward blood grouping and RhD typing reagents (column agglutination technique, CAT) and anti-human globulin cards (Bioxun Biotech [Jilin, China], lot numbers 20201211, 20201103); anti-A, anti-B (Ig-M) monoclonal, Panel Cells, Referencells A1, B, O (Shanghai Blood Bio [Shanghai, China], lot numbers 20201018, 20201018, 2020521, 20215320), anti-H, anti-Le^a, and anti-Le^b reagents (Sanquin [Amsterdam, The Netherlands], lot numbers 8000258203, 8000451442, 8000452122), whole blood genome DNA isolation kit (BioTeke, lot number B008006021), 2 × Hieff^TM PCR Master Mix (Yeasen Biotechnology, Shangai, China), dNTP, MgCl₂, 10 × PCR buffer, Taq DNA enzyme (Thermo Fisher, Waltham, MA, USA), SanPrep Column DNA gel extraction kit (Sangon Biotech, Shangai, China), primers (synthesised by Sangon Biotech), a PCR amplification instrument (Bioer Technology [Hangzhou, China], TC-XP), and a gel imaging analysis system (BIOTOP [Beijing, China], SC850).

Serological methods

Plasma and red cell tests for ABO and Rh blood groups of the proband and her family members were tested simultaneously with ABO.RhD Blood Grouping Tests Cards (CAT) and a tube test, which was used for the identification of H antigen. The H antigen adsorption-elution test was performed on samples from the proband and her sister. Anti-A, anti-B and anti-H monoclonal reagents were mixed with red blood cells for overnight absorption at 4°C, and after 10 min of heat elution at 56°C, the eluate was then evaluated for the presence of anti-A and anti-B by testing against A1 or B reagent red cells⁴. The presence or absence of ABH antigens in saliva was determined by haemagglutination inhibition with standard serological techniques. We indirectly inferred the secretor status through the Lewis phenotype. We used anti-human globulin cards for antibody detection, and the tube test in the Lewis blood group test.

DNA extraction

Genomic DNA was isolated from the samples using the Whole Blood Genome DNA Isolation Kit (Sangon Biotech) according to the manufacturer’s instructions.

Amplification and sequencing of ABO

PCR amplification of exons 6 and 7 of ABO with the most polymorphic sites in the ABO blood group gene was carried out and the purified amplification products were sequenced. The PCR amplification conditions, amplification primers and sequencing primer sequences used were those described by Chen et al.⁵.

Amplification and sequencing of FUT1 and FUT2

The PCR amplification reaction system for FUT1 and FUT2 gene amplification was as follows: template DNA 1 μL, forward primer 1 μL, reverse primer 1 μL, 2 × Hieff^TM PCR Master Mix 10 μL, double distilled H₂O 7 μL, for a total volume of 20 μL at 95°C; initial denaturation for 5 min, 95°C for 30 s, 58°C for 30 s and 72°C for 30 s, for 35 cycles, and finally 72°C for 10 min. Related primer sequences and sequencing primer sequences are described in the Online Supplementary Content (Table SI)⁶. After the PCR reaction was over, we collected 5 μL of the PCR products and observed them using 2% TBE agarose gel electrophoresis.

The PCR bands were cut and purified, following the instructions provided with the SanPrep Column DNA gel extraction kit (Sangon Biotech). Sangon Biotech was commissioned to sequence the purified amplified products. We used Chromas software (Technelysium Pty, South Brisbane, Australia) to read the sequencing chromatogram, and the results were analysed with BioEdit software (UNT Computing for Arts & Sciences, Denton, TX, USA). Finally, we compared the sequences with those in GenBank.

TA cloning sequencing

Amplified FUT1 fragments from the proband and her sister were cloned using the TOPO TA Cloning Kit (Yeasen Biotechnology) according to the manufacturer’s instructions. We sampled multiple positive colonies and extracted plasmid DNA for sequencing and identification to determine the haplotype of the specimens.

RESULTS

ABO blood group serology results

The results of the ABO blood group serology for the proband and her sister are shown in Table I. The ABO blood groups of the proband’s father and mother were A and B, respectively. According to the serological pattern, the proband and her sister were initially judged to be para-Bombay type.

Table I.

Results of ABO blood grouping

Sample	Reaction condition	Red cell grouping				Serum grouping				Other tests		Antigens in saliva
		Anti-A	Anti-B	Anti-D	Anti-A1	A1 cells	B cells	O cells	AC	Anti-A, B	Anti-H	A	B	H	Saline (Dilution Control)
Proband	Room temperature	2+	1+mf	4+	0	1+w	1+w	1+w	0	3+	0	+	+	+	0
	37°C	2+	2+mf	−	0	1+w	1+w	1+w	0	−	−	−	−	−	−
	4°C	3+	3+mf	−	0	1+s	1+s	1+s	0	−	−	−	−	−	−
Proband’s sister	Room temperature	0	0	4+	0	4+	4+	0	0	−	0	0	0	+	0

−: not tested; 0: no agglutination; 1+~4+: designation of agglutination reactions, AC: autocontrol; mf: mixed-field agglutination; +w: weakened expression of antigen; +s: strengthened expression of antigen.

Testing saliva for A, B, and H antigens, antibody detection and Lewis blood group testing

A, B and H substances were detected in the saliva of the proband; H substance was detected in her sister’s saliva, but A and B substances were not detected (Table I).

According to the Lewis blood group test, the proband and her family members were all Le (a–b+). Antibodies were detected in the proband, but not in any of the other family members. The proband’s anti-H antibody titre was 2 and it was found that the anti-H antibody activity decreased at 37°C, and that the classical anti-human globulin test was negative (Table II and III).

Table II.

Antibody detection and Lewis grouping

Sample	O I cells	OII cells	OIII cells	O cord red blood cells	Anti-Lea	Anti-Leb
Proband	1+	1+	1+	1+	0	4+
Proband’s father	0	0	0	−	0	3+
Proband’s mother	0	0	0	−	0	4+
Proband’s sister	0	0	0	−	0	3+

−: not tested; 0: no agglutination; 1+~4+: designation of agglutination reactions.

Table III.

Antibody identification results of proband

Tube test	Antibody identification panels
	1	2	3	4	5	6	7	8	9	10	11	AC
Room temperature	1+	1+	1+	1+	1+	1+	1+	1+	1+	1+	1+	0
37°C	±/0	0	±/0	±/0	±/0	0	0	0	0	0	0	0
37°C AHG	0	0	0	0	0	0	0	0	0	0	0	0

0: no agglutination; ±~1+: designation of agglutination reactions; AC: autocontrol; AHG: antihuman globulin.

ABO, FUT1, FUT2 gene sequencing and cloning analysis

The ABO gene sequencing results of the proband, and her father, mother, and sister were ABO*A1.02/ABO*B.01, ABO*A1.02/ABO*O.01.02, ABO*B.01/ABO*O.01.01 and ABO*O.01.01/ABO*O.01.02, respectively. The sequencing results of FUT1 and FUT2 genes are shown in Table IV. The cloning results of FUT1 revealed that the two mutation sites of the proband and her sister were located on different haplotypes, and all their genotypes were FUT1*01W.24/FUT1*01N.20. (Figure 1 and Online Supplementary Content, Figure S1 and S2).

Table IV.

Gene sequencing results of FUT1 and FUT2

	FUT1			FUT2
	Nucleotide change	Genotype	Phenotype	Nucleotide change	Genotype	Phenotype
Proband	c.768delC	FUT1*01N.20	H+weak	c.357C>T	Se/Se2	Se (Secretor)
	c.649G>T	FUT1*01W.24		/
Proband’s father	c.768delC	FUT1*01N.20	H+	c.357C>T homozygous mutation	Sej/Se2	Se (Secretor)
	/	FUT1*01		c.385A>T
Proband’s mother	c.649G>T	FUT1*01W.24	H+	c.357C>T	Se/Sej	Se (Secretor)
	/	FUT1*01		c.385A>T
Proband’s sister	c.768delC	FUT1*01N.20	H+weak	c.357C>T	Se/Se2	Se (Secretor)
	c.649G>T	FUT1*01W.24		/

Figure 1.

DNA sequencing of FUT1 gene

I: Proband; II: Proband’s father; III: Proband’s mother; IV: Proband’s sister. I and IV (A, B: direct DNA sequencing; C, D: cloning sequencing of mutated FUT1 alleles) II and III (A: direct DNA sequencing; B: cloning sequencing of mutated FUT1 alleles; C: cloning sequencing of another normal FUT1 allele).

Pedigree investigation

The FUT1 gene of the proband and her sister both had compound heterozygous mutations with c.649G>T and c.768delC, and their genotype was FUT1*01W.24/FUT1*01N.20. The father’s c.768delC had a heterozygous mutation, and the mother’s c.649G>T also had a heterozygous mutation. The two rare mutations in the proband and her sister had been inherited from their parents (Online Supplementary Content, Figure S3)

DISCUSSION

Individuals with the para-Bombay phenotype are H-deficient secretors⁷. At present, this phenotype has been found in populations including Indians, Europeans, Japanese, Chinese, and Southeast Asians^4,8. It is a rare red blood cell type, and there are regional differences in its frequency. Compared to individuals with the Bombay type with mutations in both FUT1 (H gene) and FUT2 (Se gene), who have no enzyme activity, in subjects with para-Bombay, the activity of the α-2-L-fucosyltransferase encoded by the mutated FUT1 gene is greatly reduced. Therefore, small amounts of A, B, and H antigens are produced. When the silent FUT1 gene and the active FUT2 gene are inherited, the α-2-L-fucosyltransferase associated with the FUT2 gene (α2FucT2) produces A, B, and H antigens in secretions.

At present, more than 50 FUT1 alleles related to H antigen deficiency on red blood cells have been found, including FUT1*01N.06 (c.551_552delAG), FUT1*01N.13 (c.881_882delTT) and FUT1*01W.09 (c.658C>T), which is the most common in the Chinese population⁹. The pedigree investigation showed that the FUT1 gene in the four samples did not belong to the most common genotype. The genotype of the proband is FUT1*01W.24/FUT1*01N.20, which is a rare compound heterozygous mutation with c.649G>T and c.768delC. The proband’s father and mother have single c.768delC and c.649G>T heterozygous point mutations and their genotypes are FUT1*01N.20/FUT1*01 and FUT1*01W.24/FUT1*01, respectively. The mutation site and genotype of the proband’s sister are the same as those of the proband. The single point mutations in the father and mother did not cause the loss of H antigen on red blood cells, so no abnormality was found in serology of their samples. However, the compound heterozygous mutation at two rare positions caused the proband’s H antigen expression to be extremely weak and here the H+weak phenotype could only be detected by an adsorption-elution test.

Su et al. sequenced and analysed the secretory genes of 41 random Chinese Han people, and found c.357C>T synonymous mutations and c.385A>T missense mutations¹⁰; the newly generated codon of FUT2 gene c.357C>T was synonymous with the codon before the mutation, which did not affect amino acid coding. The missense c.385A>T mutation led to the change of an amino acid (p. Ile129Phe). Henry et al. found that the enzyme encoded by the mutant gene may be more susceptible to hydrolysis¹¹, because the amount of mutated enzyme was less than the amount of wild-type enzyme, resulting in a weak secretory state. In the study, 70% of ten samples of the FUT2 gene with homozygous mutation of c.385A>T were non-secretory, due to instability of the enzyme encoded by the mutant gene. It was also reported that the Sew gene was associated with the Le(a+b+) phenotype, that is, Le(a+b+) was associated with a low level of expression of ABH substances in saliva¹². The proband and her sister in this pedigree had a c.357C>T synonymous mutation, the proband’s father had a c.357C>T homozygous mutation and a c.385A>T heterozygous mutation, and her mother had heterozygous mutations with c.357C>T and c.385A>T. Although the Lewis gene and secretory gene are inherited independently, the Lewis phenotype of red blood cells is affected by ABH secretory status, people who inherit the Le gene have the Le(a+b−) phenotype if they are non-secretory (sese) and have the Le(a–b+) phenotype if they are secretory (Sese or SeSe). The Lewis blood group in all our cases was Le(a–b+) since the c.385A>T heterozygous mutations did not cause changes in enzyme content and so they were all secreting type.

The anti-H in Bombay and para-Bombay phenotypes is clinically significant and associated with acute haemolytic transfusion reactions. The antibody is predominantly of IgM isotype and exhibits a broad thermal range (4 to 37°C) with all red cells except Oh red cells. As with anti-A and anti-B, alloanti-H is capable of activating complement and causing red cell haemolysis¹³. Consequently, patients with alloanti-H caused by a Bombay or para-Bombay phenotype must be transfused with H-negative (Oh) red blood cells. Because the para-Bombay phenotype has secretory type genes, the antibodies in the serum are weak, and there is often no response at 37°C. In this study, the proband had anti-H antibodies, and the reaction activity decreased at 37°C. The proband was admitted to hospital with thrombocytopenia (platelet count: 7×10⁹/L), and was diagnosed as having immune thrombocytopenic purpura. After treatment with carbazochrome sodium sulfonate, methylprednisolone hemisuccinate and other drugs, the platelet count gradually rose to normal (251×10⁹/L), and no blood was transfused during the whole treatment. However, if such patients need red blood cell transfusions, when there is no blood of the same phenotype, the red blood cells with the weakest response should be transfused or efforts should be made to find out whether there is another para-Bombay individual in the family who could donate blood.

The following strategies can be adopted for clinical blood transfusions for patients with Bombay and para-Bombay phenotypes: (i) establishment of a rare blood group bank, so that units can be selected directly from the bank when needed to prepare for emergency transfusion; (ii) encouragement of autotransfusions of pre-donated, stored autologous blood, if blood is needed; and (iii) search for compatible para-Bombay blood in the family, but irradiated red blood cells should be used to avoid the occurrence of blood transfusion-related graft-versus-host disease¹⁴.

CONCLUSIONS

In this article, we report the results of a thorough exploration of the molecular mechanisms underlying a para-Bombay Hweak phenotype caused by two rare mutations of FUT1, combining blood group serology and genetic methods, and investigated the proband’s pedigree, which is helpful to clarify the heterogeneity of the para-Bombay gene polymorphism and inheritance. Furthermore, it provides supporting data and a theoretical basis for the identification of para-Bombay blood group and safe transfusions.