- Open Access
- Open Peer Review
Genotyping of human neutrophil antigens (HNA) from whole genome sequencing data
© Chu et al.; licensee BioMed Central Ltd. 2013
- Received: 1 October 2012
- Accepted: 29 August 2013
- Published: 12 September 2013
Neutrophil antigens are involved in a variety of clinical conditions including transfusion-related acute lung injury (TRALI) and other transfusion-related diseases. Recently, there are five characterized groups of human neutrophil antigen (HNA) systems, the HNA1 to 5. Characterization of all neutrophil antigens from whole genome sequencing (WGS) data may be accomplished for revealing complete genotyping formats of neutrophil antigens collectively at genome level with molecular variations which may respectively be revealed with available genotyping techniques for neutrophil antigens conventionally.
We developed a computing method for the genotyping of human neutrophil antigens. Six samples from two families, available from the 1000 Genomes projects, were used for a HNA typing test. There are 500 ~ 3000 reads per sample filtered from the adopted human WGS datasets in order for identifying single nucleotide polymorphisms (SNPs) of neutrophil antigens. The visualization of read alignment shows that the yield reads from WGS dataset are enough to cover all of the SNP loci for the antigen system: HNA1, HNA3, HNA4 and HNA5. Consequently, our implemented Bioinformatics tool successfully revealed HNA types on all of the six samples including sequence-based typing (SBT) as well as PCR sequence-specific oligonucleotide probes (SSOP), PCR sequence-specific primers (SSP) and PCR restriction fragment length polymorphism (RFLP) along with parentage possibility.
The next-generation sequencing technology strives to deliver affordable and non-biased sequencing results, hence the complete genotyping formats of HNA may be reported collectively from mining the output data of WGS. The study shows the feasibility of HNA genotyping through new WGS technologies. Our proposed algorithmic methodology is implemented in a HNATyping software package with user’s guide available to the public at http://sourceforge.net/projects/hnatyping/.
- Whole genome sequencing
Transfusion-related acute lung injury (TRALI) is a serious cause of transfusion-related morbidity and mortality [1, 2]. Published evidence strongly suggests that antibodies against neutrophil antigens, including the 7 antigens HNA-1a, HNA-1b, HNA-1c, HNA-2, HNA-3a, HNA-4a and HNA-5a, have all been implicated in cases of TRALI . These antibodies lead to the activation of neutrophil that induces endothelial and alveolar damage in the lungs [2, 4, 5]. These antigens have also been reported to cause alloimmune neonatal neutropenia (ANN) .
Current nomenclature for human neutrophil antigens and corresponding genes
Nucleotides (dbSNP ID)
With the invention of polymerase chain reaction (PCR), different PCR-based genotyping assays on HNA have been developed, including sequence-based typing (SBT)  as well as PCR-sequence-specific oligonucleotide probes (SSOP) , PCR-sequence-specific primers (SSP)  and PCR-restriction fragment length polymorphism (RFLP) . Sequence-specific oligonucleotide probes (SSOP) and sequence-specific primers (SSP) were designed to detect variable sequence motifs in PCR-amplified HNA genes, revealing an extensive level of previously detected alleles. Sequence-based typing (SBT) enables all nucleotides to be identified so that it provides a better method for the evaluation of new alleles on HNA genes. PCR-based methods continue to be used in routine antigen detection. However, the next-generation sequencing technology (NGS) enables rapid whole-genome sequencing for more detailed and precise investigation of total variants of genes . It opens the opportunity to redesign genotyping strategies for more effective genetic mapping and genome analysis .
In this paper, we developed a bioinformatics tool for typing the HNA antigens from personal human whole-genome sequencing data. The NGS technology was evaluated for its potential in high-resolution HNA typing. Our tool combined with NGS data can produce unambiguous results regardless any new identified variants of the antigen systems.
The identification of DNA variants for the HNA systems
DNA variants in human genome for the HNA antigens
Oligonucleotide (21mer) on the SNP
The filtering of HNA reads from WGS datasets
To filter the reads, we build a hash table of short keys (default is 14 bp) from the DNA templates stated in the previous section. We examined the non-overlapped occurrences of the keys on each read and the reads with more than two occurrences of keys are stored for the typing. The filtering process is used to efficiently eliminate most of the unrelated reads to speed up the following alignment process. Only very few reads remain in the output of this step.
The typing of HNA reads by the alignment of the filtered reads
Genotype calling would then proceed by counting the number of times each allele is observed and using a fixed cut off value. For example, a heterozygous genotype is called if the proportion of the non-reference allele is between 5% and 95%; otherwise, a homozygous genotype would be called. But if the coverage at the SNP site is less than 2, the “unknown” genotype would be called. The WgsHnaTyping program displays the results of HNA types in a user friendly graphical user interface and also outputs the following two files - (1) an ACE file which recorded the alignment of reads. The ace file can be displayed with the visualization tool UGENE (http://ugene.unipro.ru/), and (2) a TXT file to record the coverage at each allele.
Whole-genome sequencing samples
Whole genome sequencing samples
SRA ID of dataseta,b
Total reads in dataset
Filtered reads for HNA typing
ERX000172 ~ 3
ERX000174 ~ 5
ERX000170 ~ 1
Typing result of the HNA1 system
In the results, all of the six samples have HNA-1a antigens and only one African was without HNA-1b. Besides, two African samples have HNA-1c which was a rare type for other populations around the world. Figure 4A depicts the consensus of the allele:FCGR3B*141. All of the aligned nucleotides are Guanine and no Cytosine, thus only the HNA-1a type is positive and both the HNA1b and HNA1c types are negative for the sample NA18507. On the contrary, the same location in Figure 4B shows a different situation that both Guanine and Cytosine coexist for the sample NA18506. Combined with the allele (FCGR3B*266) in Figure 4C, it shows that the HNA-1a, HNA-1b and HNA-1c types are all positive.
The likely coexistence of 3 alleles (especially FCGR3B*266) for the sample NA18506 may need verification studies at aspects including the specific DNA structure for sequencing base preferences , the additional HNA loci on the same chromosome due to the unequal crossing-over events  and due to the gene duplication events , and the heterogeneous neutrophil specimen of mixed clonal lineages. Albeit, the detected coexistence of 3 alleles maybe beneficial for avoiding clinical crisis caused by the false negative results.
Typing result of the HNA3, HNA4 and HNA5 systems
In the typing of HNA-3, -4, -5 systems, there were four homozygous cases of HNA-3aa and two heterozygous cases of HNA-3ab. Similarly, there were three homozygous cases of HNA-5bb and three heterozygous cases of HNA-5ab as well. Besides, all of the six samples were homozygous in the typing of HNA-4. The results showed that the usefulness of genotyping from whole-genome sequencing. It provided an unambiguous analysis for the zygosity of alleles. Figure 4D illustrates a heterozygous case of HNA-5ab. There were sufficient reads aligned which contribute the nucleotides at the SNP 2296C/G. The SNP confirmed existence of both HNA-5a and HNA-5b antigens in the sample NA18507.
Requirement of sequencing coverage for HNA-Typing
Where n is the number of times a base is read, and C stands for coverage. The coverage of WGS dataset was defined as the total bases from the sequences reads divided by the size of human genome. (The estimated diploid genome sizes for human female and male genomes are 6.406G and 6.294G, respectively .)
Counts for the critical alleles of HNA antigens
Probabilities for the event which each haploid at a particular locus is sequence at least twice
P(X1 > 1∩ X2 > 1)
Our study provides a new approach of genotyping to the HNA systems. The conventional samples, analysed by PCR-based methods, cannot be easily used for testing new variants. However, once there are any new variant such as HNA-1d of the antigen systems discovered and confirmed , our bioinformatics tool can be easily modified to explore the WGS datasets again to find the updated and/or undiscovered variants. Our tool may adequately exploit the genotyping advantage of next generation sequencing data either for the HNA systems or for the extended systems such as human leukocyte antigen (HLA) systems.
In the HnaTyping software package, both the programs WgsReadFilter and WgsHnaTyping were implemented in C# with the .NET Framework which can be run on 64-bit Windows/Linux. The HnaTyping software with a user manual is available at the Web site: http://sourceforge.net/projects/hnatyping/.
The authors would like to thank the National Science Council, Taiwan, R.O.C. for financially supporting this research under Contract No. NSC 102-2221-E-002-172.
- Muller MC, Porcelijn L, Vlaar AP: Prevention of Immune-mediated Transfusion-related Acute Lung Injury; from Bloodbank to patient. Curr Pharm Des. 2012, 18 (22): 3241-3248. 10.2174/1381612811209023241.View ArticlePubMedGoogle Scholar
- Vlaar AP: Transfusion-related acute lung injury: Current understanding and preventive strategies. Transfus Clin Biol. 2012, 19 (13): 117-124.View ArticlePubMedGoogle Scholar
- Moritz E, Norcia AM, Cardone JD, Kuwano ST, Chiba AK, Yamamoto M, Bordin JO: Human neutrophil alloantigens systems. An Acad Bras Cienc. 2009, 81 (3): 559-569. 10.1590/S0001-37652009000300019.View ArticlePubMedGoogle Scholar
- Bayat B, Sachs UJ: Transfusion-related acute lung injury: An overview. Curr Pharm Des. 2012, 18 (22): 3236-3240. 10.2174/1381612811209023236.View ArticlePubMedGoogle Scholar
- Caudrillier A, Looney MR: Platelet-neutrophil interactions as a target for prevention and treatment of transfusion- related acute lung injury. Curr Pharm Des. 2012, 18 (22): 3260-3266. 10.2174/1381612811209023260.View ArticlePubMedGoogle Scholar
- Black LV, Maheshwari A: Immune-mediated neutropenia in the neonate. NeoReviews. 2009, 10 (9): e446-e453. 10.1542/neo.10-9-e446.View ArticleGoogle Scholar
- Huizinga TW, Kleijer M, Tetteroo PA, Roos D, von dem Borne AE: Biallelic neutrophil Na-antigen system is associated with a polymorphism on the phospho-inositol-linked Fc gamma receptor III (CD16). Blood. 1990, 75 (1): 213-217.PubMedGoogle Scholar
- Moritz E, Chiba AK, Kimura EY, Albuquerque D, Guirao FP, Yamamoto M, Costa FF, Bordin JO: Molecular studies reveal that A134T, G156A and G1333A SNPs in the CD177 gene are associated with atypical expression of human neutrophil antigen-2. Vox Sang. 2010, 98 (2): 160-166. 10.1111/j.1423-0410.2009.01233.x.View ArticlePubMedGoogle Scholar
- Curtis BR, Cox NJ, Sullivan MJ, Konkashbaev A, Bowens K, Hansen K, Aster RH: The neutrophil alloantigen HNA-3a (5b) is located on choline transporter-like protein 2 and appears to be encoded by an R > Q154 amino acid substitution. Blood. 2010, 115 (10): 2073-2076. 10.1182/blood-2009-11-248336.View ArticlePubMedPubMed CentralGoogle Scholar
- Simsek S, van der Schoot CE, Daams M, Huiskes E, Clay M, McCullough J, van Dalen C, Stroncek D, von dem Borne AE: Molecular characterization of antigenic polymorphisms (Ond(a) and Mart(a)) of the beta 2 family recognized by human leukocyte alloantisera. Blood. 1996, 88 (4): 1350-1358.PubMedGoogle Scholar
- Huvard MJ, Schmid P, Stroncek DF, Flegel WA: Frequencies of SLC44A2 alleles encoding human neutrophil antigen-3 variants in the African American population. Transfusion. 2012, 52 (5): 1106-1111. 10.1111/j.1537-2995.2011.03396.x.View ArticlePubMedGoogle Scholar
- Stein EL, Santoso S, Behrens G, Mueller-Eckhardt C, Bux J: Genotyping of the granulocyte-specific NA antigens from small quantities of blood or serum. Tissue Antigens. 1995, 45 (1): 69-72. 10.1111/j.1399-0039.1995.tb02417.x.View ArticlePubMedGoogle Scholar
- Reil A, Wesche J, Greinacher A, Bux J: Geno- and phenotyping and immunogenicity of HNA-3. Transfusion. 2011, 51 (1): 18-24. 10.1111/j.1537-2995.2010.02751.x.View ArticlePubMedGoogle Scholar
- Cardone JD, Bordin JO, Chiba AK, Norcia AM, Vieira-Filho JP: Gene frequencies of the HNA-4a and -5a neutrophil antigens in Brazilian persons and a new polymerase chain reaction-restriction fragment length polymorphism method for HNA-5a genotyping. Transfusion. 2006, 46 (9): 1515-1520. 10.1111/j.1537-2995.2006.00943.x.View ArticlePubMedGoogle Scholar
- Zhang J, Chiodini R, Badr A, Zhang G: The impact of next-generation sequencing on genomics. J Genet Genomics. 2011, 38 (3): 95-109. 10.1016/j.jgg.2011.02.003.View ArticlePubMedPubMed CentralGoogle Scholar
- Huang X, Feng Q, Qian Q, Zhao Q, Wang L, Wang A, Guan J, Fan D, Weng Q, Huang T, et al: High-throughput genotyping by whole-genome resequencing. Genome Res. 2009, 19 (6): 1068-1076. 10.1101/gr.089516.108.View ArticlePubMedPubMed CentralGoogle Scholar
- Bux J: Human neutrophil alloantigens. Vox Sang. 2008, 94 (4): 277-285. 10.1111/j.1423-0410.2007.01031.x.View ArticlePubMedGoogle Scholar
- Xia W, Bayat B, Sachs U, Chen Y, Shao Y, Xu X, Deng J, Ding H, Fu Y, Ye X, et al: The frequencies of human neutrophil alloantigens in the Chinese Han population of Guangzhou. Transfusion. 2011, 51 (6): 1271-1277. 10.1111/j.1537-2995.2010.02979.x.View ArticlePubMedGoogle Scholar
- Durbin RM, Abecasis GR, Altshuler DL, Auton A, Brooks LD, Gibbs RA, Hurles ME, McVean GA: A map of human genome variation from population-scale sequencing. Nature. 2010, 467 (7319): 1061-1073. 10.1038/nature09534.View ArticlePubMedGoogle Scholar
- Rodriguez FA, Cai Y, Lin C, Tang Y, Kolbanovskiy A, Amin S, Patel DJ, Broyde S, Geacintov NE: Exocyclic amino groups of flanking guanines govern sequence-dependent adduct conformations and local structural distortions for minor groove-aligned benzo[a]pyrenyl-guanine lesions in a GG mutation hotspot context. Nucleic Acids Res. 2007, 35 (5): 1555-1568. 10.1093/nar/gkm022.View ArticlePubMedPubMed CentralGoogle Scholar
- Reil A, Sachs UJ, Siahanidou T, Flesch BK, Bux J: HNA-1d: a new human neutrophil antigen located on Fcgamma receptor IIIb associated with neonatal immune neutropenia. Transfusion. 2013, Advance online publication. doi:10.1111/trf.12086Google Scholar
- Kissel K, Hofmann C, Gittinger FS, Daniels G, Bux J: HNA-1a, HNA-1b, and HNA-1c (NA1, NA2, SH) frequencies in African and American Blacks and in Chinese. Tissue Antigens. 2000, 56 (2): 143-148. 10.1034/j.1399-0039.2000.560205.x.View ArticlePubMedGoogle Scholar
- Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, Devon K, Dewar K, Doyle M, FitzHugh W, et al: Initial sequencing and analysis of the human genome. Nature. 2001, 409 (6822): 860-921. 10.1038/35057062.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1755-8794/6/31/prepub
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