REPORT OF NEW ALLELES OR ANTIGENS New RHD variant alleles Felix Garcia,1 Miguel-Angel Rodriguez,1 Mindy Goldman,2 Maria-Nerea Azcarate,3 Maria-Inmaculada Rodriguez,4 Eduardo Muniz-Diaz,5 Fernando Puente,6 Hana Alshatti,7 Katri Haimila,8 Araitz Molano,9 Adirane Garaizar,10 and Gorka Ochoa-Garay10 SUMMARY STATEMENT This report describes 22 new RHD variants whose identification was prompted by discrepancies between the results of targeted genotyping and serology.

BACKGROUND The spread of targeted genotyping as a means to confirm serology data or to resolve hematologic discrepancies is accelerating the discovery of new blood group variants. Listing of blood group variants in public databases in association with their phenotype, haplotype, and racial background may facilitate decision making by the transfusion specialist.

MS201/MS26 (R2), Gammaclone 401/F8D8 (R3), human polyclonal serum (R4), and Novaclone 175-2/D415 1E4 (R5) from Immucor; Bioclone MAD2 (R6) and Bioclone D7B8/H1121G6/LORIFA (R7) from Ortho; TH-28 monoclonal (R8) and ID-Card human polyclonal serum (R9) from Bio-Rad; ESD-1 monoclonal (R10) from Diagnostics Scotland; Albaclone Partial D panel (R11) and Albaclone Weak D kit (R12) from Alba Bioscience; D-Screen 9-monoclonal panel (R13) from Diagast; Albaclone 6-monoclonal panel (R14), Albaclone 12-monoclonal panel (R15), and human polyclonal serum (R16) from DiaMed. DNA sequencing of RHD Exons 1 to 10 and flanking intron regions was performed by the Sanger dideoxy method, and testing for exon presence or absence by allele-specific polymerase chain reaction. Sequences of all primers used are available upon request.

BRIEF METHODS Targeted genotyping was performed on BloodChip Reference v4.0, an oligonucleotide microarray that interrogates 69 polymorphic positions in the RHD gene (Progenika Biopharma, Derio, Spain). Phenotyping was performed by tube, microplate, gel column, solid-phase, and adsorption-elution methods using the following anti-D reagents (see Table 1): Immuclone RUM-1 (R1), Series 4

From the 1Centro de Transfusion de Madrid, Madrid, Spain; 2 Canadian Blood Services, Ottawa, Ontario, Canada; 3Centro Vasco de Transfusion y Tejidos Humanos, Galdakao, Spain; 4 Centro de Transfusion de Galicia, Santiago de Compostela, Spain; 5Banc de Sang i Teixits, Barcelona, Spain; 6Banco de Sangre y Tejidos de Aragon, Zaragoza, Spain; 7Kuwait Central Blood Bank, Jabriya, Kuwait; 8Finnish Red Cross Bood Service, Helsinki, Finland; 9Progenika Biopharma, Derio, Spain; and 10 Progenika, Inc., Medford, Massachusetts. Address correspondence to: Gorka Ochoa-Garay, Progenika, Inc., 200 Rivers Edge Drive, Medford, MA 02155; e-mail: [email protected]. Received for publication March 3, 2014; revision received June 30, 2014, and accepted July 7, 2014. doi: 10.1111/trf.12828 © 2014 AABB TRANSFUSION **;**:**-**.

RESULTS Table 1 shows information associated with the molecular work (variant positions identified by sequencing, SNP ID when listed in dbSNP,1 SNP minor allele frequency when available, SNP minor allele count when available, predicted effect of the variant on the gene product, description of the novel RHD allele, its assigned GenBank ID, RH haplotype of which the RHD allele is or can be a part) and information associated with the serology (phenotype, number of samples [cases] identified, number of times each sample was tested, whether or not RBCs were analyzed by the adsorption-elution test, whether or not anti-D reactivity was found in the serum). For variant alleles found in heterozygous RHCE*C/c or RHCE*E/e samples, both possible RH haplotypes are listed. Although zygosity testing was not performed, most variant alleles are likely present in hemizygosity, given their low frequency relative to RHD*deletion (data not shown) and given that most are found in a heterozygous RH background. The assignment of partial D status is based on the development of anti-D (Sample 2.4) or on differential reactivity with a panel of monoclonals (Sample 3.1). In the absence of either type of evidence, and whether or not testing with a panel of monoclonals or testing for anti-D was performed, the sample has been assigned a weak D phenotype. The alleles span the spectrum of altered Volume **, ** **

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Caucasian (Spain) Caucasian (Morocco) Caucasian (Spain) Caucasian (Spain) Native American (Colombia) Caucasian (Ukraine) Caucasian (Spain) Arab Caucasian Black N/Av N/Av N/Av

N/Av N/Av N/Av Caucasian

Caucasian (Spain) Caucasian (Spain) Caucasian (Spain) Arab (Kuwait)

Caucasian (Finland)

1.1

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3.1 3.2 3.3 4.1

4.2

7.1

Donor

Donor

Donor

Donor

Donor

Donor Donor Donor Donor

Donor Prenatal Prenatal Patient Patient Donor

Prenatal

Donor

Donor

Donor

Donor

Donor

Donor

Case

c.201G>A c.203G>A c.1136C>T c.1145T>C

IVS9-1G>A

c.1010T>G

c.1154G>A IVS1+1G>T c.993C>G c.1187C>G c.1060G>A c.712G>A c.809T>G IVS4-2A>C c.1065C>T c.1248insG c.916G>A c.932A>G c.1154G>C c.1157T>A

c.67G>C

c.1179G>A

c.915delC

c.667T>G c.819G>A IVS6+1G>A

c.8C>G c.178A>C c.278T>G

Variant

Silent Ser68Asn Thr379Met Leu382Pro

Splice site

Leu337Arg

Gly385Asp Splice site Asn331Lys Pro396Arg Ala354Thr Val238Met Val270Gly Splice site Silent 416fs>476Stp Val306Ile Tyr311Cys Gly385Ala Leu386Stp

Ala23Pro

Trp393Stp

305fs>357Stp

Phe223Val Silent Splice site

Ser3Cys Ile60Leu Leu93Arg

Effect

rs41302032 rs62621068 rs199998930 N/Av

N/Av

N/Av

N/Av N/Av N/Av N/Av N/Av rs1053360 rs121912763 N/Av N/Av N/Av N/Av N/Av rs71652374 N/Av

N/Av

N/Av

N/Av

rs1053356 rs150606530 N/Av

rs144969459 rs1053341 N/Av

SNP ID

N/Av N/Av N/Av N/Av

N/Av

N/Av

N/Av N/Av N/Av N/Av N/Av 0.045 0.000 N/Av N/Av N/Av N/Av N/Av N/Av N/Av

N/Av

N/Av

N/Av

0.066 N/Av N/Av

N/Av N/Av N/Av

MAF

N/Av N/Av N/Av N/Av

N/Av

N/Av

N/Av N/Av N/Av N/Av N/Av 99 1 N/Av N/Av N/Av N/Av N/Av N/Av N/Av

N/Av

N/Av

N/Av

143 N/Av N/Av

N/Av N/Av N/Av

MAC

RHD*1145C

RHD*201A,203A,1136T

RHD*IVS9-1A

RHD*1010G

RHD*1157A

RHD*IVS4-2C RHD*1065T RHD*1248insG RHD*916A,932G,1154C

RHD*1154A RHD*IVS1+1T RHD*993G RHD*1187G RHD*1060A RHD*712A,809G

RHD*67C

RHD*1179A

RHD*915delC

RHD*IVS6+1A

RHD*667G,819A

RHD*278G

RHD*8G,178C

Allele

KJ145907

KF861938

KF861924

KF861923

KF861929

KF861936 KF861937 KJ145906 KF861928

KJ145905 KF861925 KF861926 KF861927 KJ145901 KJ145902

KJ145904

KF861935

KF861933

KF861934

KF861932

KF861931

KF861930

GenBank

DCe or Dce

Dce

DCe or Dce

DCe or Dce

DCe or Dce

DcE or Dce DCe or Dce DCe or Dce DcE or Dce

DcE or Dce DcE or Dce DCe or Dce DCe or Dce DCe or Dce DCe or Dce

DCe or Dce

DCe or Dce

DCe or Dce

DCe or Dce

Dce

DCe or Dce

DCe or Dce

Haplotype

Weak D

D+ or partial D

D–

D– or Del

D–

Partial D Weak D Del Weak D

D– Del Weak D Weak D Partial D Weak D

Weak D

D–

D–

D–

Weak D

Del

Weak D

Phenotype

MAC = minor allele count; MAF = minor allele frequency as defined in dbSNP (NCBI); N/Ap = not applicable; N/Av = information not available; N/T = not tested.

6.1

5.2

5.1

1.8 2.1 2.2 2.3 2.4 2.5

1.7

1.6

1.5

1.4

1.3

1.2

Race/ethnicity

ID

TABLE 1. New RHD variants

R5, R4, R5, R5, R5, R5,

R16 R5, R6 R6, R12 R6 R6 R6, R12

R8, R9, R10, R11

R2, R7

R1, R5

R1, R5

R13, R15, R16

R1, R5, R7, R14, R15 R1, R5, R7 R1, R5, R7, R16 R15

R1, R3, R3, R3, R3, R3,

R1, R5

R1, R5, R16

R1, R5, R16

R1, R5, R16

R1, R5

R1, R5, R16

R1, R5

Reagents

1

1

1

1

1

1 1 1 1

1 2 1 1 1 2

1

1

1

1

1

1

2

Samples

2

10

12

2

1

1-4 1-3 2 1

2 2 N/Av N/Av N/Av 2

2

2

2

2

2

2

2

Tests

N/Ap

N/Ap

No

No

Yes

N/Ap N/Ap Yes N/Ap

Yes Yes N/Ap N/Ap N/Ap N/Ap

N/Ap

Yes

Yes

Yes

N/Ap

Yes

N/Ap

Adsorptionelution

Anti-D

No

N/T

N/T

N/T

Yes

N/T N/T N/T N/T

N/T N/T No No Yes No

No

N/T

N/T

N/T

N/T

N/T

N/T

GARCIA ET AL.

NEW RHD VARIANT ALLELES

RhD phenotypes: two associate with partial D (Samples 2.4 and 3.1), three with Del (Samples 1.2, 2.1, and 3.3), six with D– (Samples 1.4, 1.5, 1.6, 1.8, 4.2, and 5.2), and nine with weak D (Samples 1.1, 1.3, 1.7, 2.2, 2.3, 2.5, 3.2, 4.1, and 7.1) although some of these (Samples 1.1, 1.3, 3.2, and 4.1) were not tested for reactivity with a panel of anti-D monoclonals or for the presence of anti-D in serum. The protein encoded by RHD*667G,819A (Sample 1.3) and by the partial D/D+ variant RHD*DFV2 may be identical, since c.819G>A is a silent SNP. The weak D phenotype associated with allele RHD*1065T (Sample 3.2) is possibly due to the formation of a new splice site, consistent with the fact that c.1065C>T is a silent change. Sample 5.1 was not tested by adsorption-elution, so a Del phenotype has not been excluded for allele RHD*1010G. Allele RHD*201A,203A,1136T (Sample 6.1) includes c.1136C>T, a nucleotide substitution shared by RHD*DAU variants.

This sample was tested with an anti-D monoclonal antibody blend, so a partial D phenotype has not been excluded. CONFLICT OF INTEREST AG and GOG are employees of Progenika, Inc. The other authors have disclosed no conflicts of interest.

REFERENCES 1. National Center for Biotechnology Information. Database of single nucleotide polymorphisms (dbSNP). [cited 2014 Aug 16]. Available from: http://www.ncbi.nlm.nih.gov/snp 2. Noizat-Pirenne F, Lee K, Pennec PY, et al. Rare RHCE phenotypes in black individuals of Afro-Caribbean origin: identification and transfusion safety. Blood 2002;100:4223-31.

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New RHD variant alleles.

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