Unveiling Novel and Rare Variants in the α-1,4-Galactosyltransferase Gene Leading to Rare p Phenotype in Indian Patients

Abstract

Introduction

The rare p phenotype, characterized by the absence of P, P1, and P^k antigens, produces anti-PP1P^k that can cause severe hemolytic reactions and recurrent miscarriages. This phenotype is rare globally but shows notable prevalence in the Swedish population. This study focuses on the molecular characterization of 6 Indian patients to provide further insight into the genetic basis of the p phenotype.

Methods

A targeted next-generation sequencing assay covering 51 genes associated with 41 blood group systems was utilized to investigate the molecular basis of the A4GALT gene in 6 patients with a serologically confirmed p phenotype. The results were analyzed and annotated using bioinformatics tools, including the Integrative Genomics Viewer, with novel variants confirmed by Sanger sequencing. Family members were also screened to identify potential rare donors.

Results

Genomic analysis revealed novel and rare variants in the A4GALT gene, all confirming the p phenotype. Five frameshift variants (c.72dupC, c.218delG, c.592delC, c.972_997del, and c.547_548del) and one nonsense variant (c.C392G) were detected, resulting in truncated, non-functional proteins. The p phenotype was confirmed in a subset of family members, identifying three new donors for rare blood transfusion requirements.

Conclusion

This study presents the molecular characterization of the p phenotype in Indian patients, identifying novel variants not yet registered in the ISBT database. These findings enhance understanding of the p phenotype and highlight the significance of family screening for identifying rare blood donors. The study underscores the critical need for rare donor registries, especially for patients at high risk of severe hemolytic reactions during transfusion.

Introduction

The P1PK blood group system (ISBT 003) comprises three antigens: P1, P^k, and NOR [1]. The α-1,4-galactosyltransferase (A4GALT) gene is located on the long arm of chromosome 22 and consists of three exons. Among these, exon 3 contains the coding region responsible for producing α-1,4-galactosyltransferase, an enzyme essential for the synthesis of P1 and P^k antigens [2]. Various combinations or absence of P1 and P^k antigens define the five phenotypes of the P blood group system: P₁, P₂, P₁^k, P₂^k, and p [2]. The frequency of the P1 antigen differs significantly across ethnic groups, ranging from around 90% in people of African descent [3] to 20% in Asian populations [3]. The rare p phenotype is characterized by the absence of P, P1, and P^k antigens [2]. The reported frequency is approximately 5.8 per million Europeans [4], but is more common in regions such as Northern Sweden [5], Japan [5], and among the Amish community [5].

The anti-PP1P^k has been naturally produced in the absence of P, P1, and P^k red cell antigens [2]. These antibodies are of IgM or IgG type, or a mixture of both and are responsible for severe hemolytic transfusion reactions [6] and hemolytic disease of newborn [7]. These antibodies are previously reported to cause recurrent miscarriages. Identification of alloantibodies against high frequency antigens (HFAs) and provision of compatible blood units is often challenging, as HFA-negative donor units are extremely rare worldwide.

Single nucleotide variants (SNVs) [2, 5, 8], insertions/deletions [9], and large deletion [10] have been reported in the coding region of the A4GALT gene. This study was based on the molecular characterization of the p phenotype in 6 Indian patients. We report four novel variants in the A4GALT gene, resulting in the rare p phenotype.

Materials and Methods

Patients Sample

In 6 patients showing panagglutination reaction during antibody screening and identification testing, advanced serological and molecular analysis was performed simultaneously. These patients included one antenatal woman, two women with bad obstetric history, and three individuals who required blood transfusions but experienced difficulty in cross-matching. EDTA-anticoagulated whole blood samples were collected and processed for molecular analysis to identify specificity of antibody.

Molecular Analysis

Molecular analysis was performed using targeted next-generation sequencing (tNGS). Genomic DNA was extracted using whole blood (ReliaPrep Blood gDNA Miniprep System; Promega, Fitchburg, WI, USA) and quantified (Qubit 3, Invitrogen Thermofisher Scientific, USA). A customized blood group gene panel was employed to prepare the DNA library, targeting 51 genes across 41 blood group systems, including transcription factors (online suppl. Table S1; for all online suppl. material, see https://doi.org/10.1159/000548316). All steps adhered to the manufacturer’s guidelines (TWIST Biosciences, San Francisco, CA, USA). The DNA libraries were pooled and sequenced (NextSeq platform, paired-end, 2x150 bp; Illumina, San Diego, CA, USA).

Bioinformatics Analysis

For each sample, the quality of raw FASTQ files was assessed using FastQC [11]. The sequence reads were aligned to the human reference genome (GRCh37/hg19) with the Burrow-Wheeler Aligner [12], producing SAM files. These files were converted into sorted BAM files using SAMtools [13]. Variant calling was performed with GATK HaplotypeCaller to generate compressed VCF files [14], which were subsequently annotated using wANNOVAR [15]. The annotated VCF files were carefully reviewed, and blood group alleles for each individual were identified based on the allele database of the ISBT [1]. Mapped reads were visualized using the Integrative Genomics Viewer [16].

Sanger Sequencing

Sanger sequencing was performed to validate any novel variants identified in the A4GALT gene. Specifically, the relevant regions of exon 3, based on patients findings, were amplified (online suppl. Table S2) using the following PCR protocol: initial denaturation at 95°C for 3 min; followed by 30 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 60 s; with a final extension step at 72°C for 8 min. The PCR amplicons were purified and sequenced using the ABI Prism 310 Genetic Analyzer (Applied Biosystems, CA, USA). Sequence data were analyzed with Chromas Software v2.6.6.

Family Screening

During the study, family members of the index cases were also screened for the p phenotype to identify additional rare blood donors. These donors could serve as an invaluable resource for future transfusions, especially in patients with complex immunohematological needs or those requiring matched rare blood types for safe and effective transfusion.

Results

Serological Analysis

Direct antiglobulin test was negative for all patients, suggesting absence of autoantibody. Antibody identification carried out at saline, enzyme, and IAT phase using in-house and commercially available red cell panels at different temp showed panaggutination reaction with negative autocontrol. Antibody to HFA was suspected. Extended phenotyping by serology showed absence of P1 antigen. These results suspected antibody against p phenotpe. To confirm antibody preserved ABO compatible p phenotype red cells were used and found to be compatible with patient’s serum.

Molecular Analysis

tNGS revealed novel and rare variants in exon 3 of the A4GALT gene confirming the presence of the p phenotype in all cases. A total of four novel variants were identified in the homozygous state, resulting in new alleles, as summarized in Table 1. Five frameshift variants were identified, one was a frameshift insertion c.72dupC, remaining four were frameshift deletions: c.218delG, c.592delC, c.972_997del, and c.547_548del. In 1 case, a nonsense variant c.C392G was identified (Fig. 1).

Table 1.

Novel and rare variants identified in the A4GALT gene

Case No.	Nucleotide change	Protein change	rs ID	Molecular consequence	Clinvar accsession No.	Additional polymorphic positions	Religion	Region
Case 1	NM_017436.4:c.72dupC	p.I25Hfs*30	​	Frameshift	SCV005328487	c.903C>G	Hindu	West
Case 2	NM_017436.4:c.218delG	p.G73Afs*41	​	Frameshift	SCV005328488	–	Muslim	West
Case 3	NM_017436.4:c.592delC	p.L198Sfs*7	​	Frameshift	SCV005328489	–	Hindu	West
Case 4	NM_017436.4:c.C392G	p.S131X	​	Nonsense	SCV005328490	–	Muslim	South
Case 5	NM_017436.4:c.972_997del	p.R325Afs*113	rs778387354	Frameshift	AY496234	–	Hindu	South
Case 6	NM_017436.4:c.547_548del	p.M183Vfs*99	rs771507737	Frameshift	HE818933	c.903C>G	Hindu	South

mRNA reference sequence: NM_017436.4 and protein reference sequence: NP_059132.1 or NP_001304967.1.

Fig. 1.

Schematic representation of novel and rare variants in the A4GALT gene. Exons are shown as white boxes, with the Open Reading Frame (ORF) in exon 3 highlighted in black, and exon sizes indicated below in base pairs (bp). Introns are represented by horizontal black lines. Frameshift and nonsense variants appear as vertical black lines, with frameshifts specifically indicated by dashed lines. The P1/P2-defining polymorphism in exon 2a is marked by a vertical gray line.

In 2 cases, additional silent variant c.C903G was identified in exon 3, which is not associated with the p phenotype [11]. In all cases, SNVs – chr22:43,114,551C/T (rs8138197) (Fig. 2) and chr22:43,113,793 G/T (rs5751348) – were present in the homozygous state. Furthermore, to validate the novel variants identified through tNGS, Sanger sequencing was performed (Fig. 3).

Fig. 2.

IGV analysis of novel and rare variant. IGV, Integrative Genomics Viewer.

Fig. 3.

Confirmation of novel variants using sanger sequencing.

Family Screening

In an extensive family screening of 28 members of case 1, three more members – the mother, one brother and uncle – were identified as of rare p phenotype. Among this family, a heterozygous variant was identified in nine members, resulting in a normal phenotype, presenting a carrier status without phenotypic expression. The brother identified as p phenotype in the family of case 1 donated blood to case 2 twice for surgery.

None of the members tested in the other 5 families were of rare p phenotype. Only one sibling from the families of case 5 and case 6 was available for screening and was also found to have a normal phenotype (Fig. 4).

Fig. 4.

Pedigree analysis of the families with the p phenotype. The proband is indicated by an arrow. Circles represent females, and squares represent males. Affected individuals are shown with solid symbols, while carriers are indicated by half-filled symbols. NT, not tested.

Discussion

Molecular characterization of rare blood groups like Bombay phenotype [17], weak D [18], ABO blood weaker variants [19], D-- [20], and Rh null [21] has been studied in Indians, thus enhancing the understanding of blood group diversity. Although reports of the p phenotype have been documented across the country, only a three case studies have provided molecular confirmation identifying the following variants: c.972_997del, c.526G>A, c.995_1015 [6, 22, 23]. This study aimed to address this gap by using tNGS for molecular characterization and contributing to expand the existing knowledge on the p phenotype.

Globally, genetic polymorphisms in the A4GALT gene have been reported across diverse ethnic backgrounds, including populations from Japan, Sweden, France, Israel, Italy, and other regions [9, 10, 24–26]. The A4GALT protein comprises three domains: cytoplasmic (1-22 amino acid [aa]), transmembrane (23-43 aa), and luminal (44-353 aa). One of the novel variant (c.72dupC) identified in this study, an insertion of cytosine at position 72, results in a frameshift, disrupting translation in the transmembrane domain and preventing the formation of the catalytically active protein. This variant is similar to the previously reported 68_69insT found in the Israeli population [27].

Other variants identified in this study occurred within the luminal domain. We identified rare frameshift deletions in 2 cases: a 26 bp deletion (c.972_997del, rs778387354, global frequency 0.00002) and a 2 bp deletion (c.547_548del; rs771507737; global frequency 0.00001), previously classified by the ISBT as A4GALT*01N.19 and A4GALT*01N.34, respectively. Although A4GALT*01N.34 was earlier associated with additional silent variants (c.109A>G and c.367T>C), these were absent in our cases. Notably, we found only the c.547_548del variant in conjunction with c.C903G, suggesting a potential new allele [2]. Two more novel frameshift variants, c.218delG and c.592delC, were identified, both leading to a shift in the reading frame and resulting in protein truncation within the luminal domain. We also identified a novel nonsense variant (c.C392G), which prematurely terminates protein translation at position 131. Several studies have reported single nucleotide substitutions in the A4GALT gene that cause the rare p phenotype, [1]. In all cases, SNVs; rs8138197 and rs5751348 were present in the homozygous state are associated with the P1 blood group system. Specifically, these SNVs are located within the A4GALT gene, which encodes the enzyme responsible for producing the P1 antigen. The specific alleles at these positions influence the expression levels of the P1 antigen [28, 29].

Testing the family members of the index cases for the rare phenotype not only contributes to a better understanding of its inheritance patterns but also expands the inventory of rare blood donors. These individuals could be added to rare donor registries, allowing for timely and targeted blood donations in critical medical situations.

Conclusion

This study presents the molecular basis of the p phenotype among a cohort of Indian patients, and reports the several novel variants in the A4GALT gene that contribute to this rare blood type. The novel unreported variants in the ISBT database will enhance the understanding of the genetic basis of the P1PK blood group system. Importantly, the study underscores the value of family screening to locate additional rare donors and support the need for a national rare donor registry to improve transfusion for patients reliant on rare compatible blood.

Statement of Ethics

This study received approval from the Institutional Ethics Committee of ICMR-NIIH, Mumbai, India, with Approval No. NIIH/IES/04-2019. Written informed consent was obtained from each participant.

Conflict of Interest Statement

The authors have disclosed no conflicts of interest.

Funding Sources

This work was supported by funding through an extramural grant received from the Indian Council of Medical Research.

Author Contributions

S.K. and P.K. designed the research study and wrote the manuscript. S.K., N.B., S.B., and S.N. contributed to sample management. P.K. and G.R. carried out the experiments and data analysis. S.K. and M.M. revised and approved the manuscript.

Funding Statement

This work was supported by funding through an extramural grant received from the Indian Council of Medical Research.

Data Availability Statement

All data generated or analyzed during this study are included in this article and its online supplementary material files. Further inquiries can be directed to the corresponding author.