O N NE E PWA AG LE L RE EL PE OS RA TN DO AF N NT EI GWE NA SL L–ESLHEOS R OT RR EA PN OT RI GT ES N S A new Rhnull allele in francophone Quebecers Maryse St-Louis,1 Carole Éthier,2 Josée Perreault,1 and Josée Lavoie1 BACKGROUND AND CASE REPORT Rhnull phenotype is very rare. The first Rhnull phenotype was reported in 1961 where no Rh antigens could be detected on red blood cells (RBCs) of an Australian aboriginal woman.1,2 Two types of Rhnull have been observed so far. The extremely rare amorph type results from homozygosity for silent genes at RHD and RHCE.1,2 The more common regulator type is the result of RHAG inactivation. The reason why changes to the RhAG protein prevent Rh antigens’ expression is unclear. A rare intermediate type, called Rhmod, has also been described. It is caused by some RHAG mutations leading to a low level of Rh antigens expression.1 In this study, we report a new Rhnull allele (RHAG*01N.13 ISBT designation) of the regulator type found in a consanguineous French-speaking Quebecers’ family.
METHODS AND RESULTS The family was first studied in the late 1960s. A family pedigree is depicted in Fig. 1. The probands’ parents were first cousins (II-1 and II-2). Blood samples were collected in 1968 for serology workup using anti-D (Canadian Red Cross Lot 232), anti-C (Canadian Red Cross Lot 81), anti-c (Hyland Laboratories Lot 0337D004A1), anti-E (Canadian Red Cross Lot 62), and anti-e (Ortho Lot RSET-59). Other antigens were also tested: ABO, Lea, Leb, M, N, S, s, U, P1, K, k, Fya, Fyb, Jka, and Jkb (no data available concerning the reagents used).
From 1Research and Development and 2Immunohematology Reference Laboratory, Héma-Québec, Quebec City, Quebec, Canada. Address reprint requests to: Maryse St-Louis, PhD, Recherche et Développement, Héma-Québec, 1070, Avenue des Sciences-de-la-Vie, Québec, QC G1V 5C3, Canada; e-mail: [email protected]. GenBank Accession Number KJ543706, Received for publication May 28, 2014; revision received August 18, 2014, and accepted August 24, 2014. doi: 10.1111/trf.12887 © 2014 AABB TRANSFUSION **;**:**-**. 2015;55:1580–1581 TRANSFUSION
1580 TRANSFUSION Volume 55, June 2015
Rh phenotype was repeated on blood samples from III-5 in 1982 and 2006. Reagents used are unknown. Blood samples from III-3 were phenotyped in 1990 and again in 1991 and 1999 as a blood donor. Her Rh phenotype was confirmed by approved serology techniques in 2013 using anti-D blend (DBL Novaclone [IgM clone D175-2 + IgG Clone D415 1E4], anti-C Clone MS24 [Ortho Diagnostics BioClone reagents, Markham, Ontario, Canada]), anti-c Clone MS42 (Ortho Diagnostics BioClone), anti-E Clone C2 (Ortho Diagnostics BioClone), and anti-e clone MS16 (Ortho Diagnostics BioClone). No adsorption-elution was performed. According to the results available from the late 1960s, family members I-2, II-1, and II-2 presented weaker reactions with anti-D, anti-C, anti-c, and anti-e compared to controls. They were all negative with anti-E (R1r). The maternal grandparents were not tested (I-3 and I-4). Three of the five siblings (III-2, III-3, and III-5) were found to be Rhnull (D− C− c− E− e−). They also presented weaker reactions with anti-N (III-5) and anti-s (III-3, III-5) and were found to be U− (III-2, III-3, and III-5). Some individuals in the fourth generation (not shown in Fig. 1) showed weaker Rh antigen expression. Phenotype performed in the 2000s on the sisters’ blood samples (III-3 and III-5) confirmed the Rhnull status. DNA was extracted from III-3 and III-5 whole blood collected on EDTA using a genomic DNA purification kit (QIAamp DNA Blood Mini Kit, Qiagen, Mississauga, Ontario, Canada) according to the manufacturer’s instructions. RHD, RHCE*C/RHCE*c, RHCE*E, RHCE*e assays were performed as previously published.3 Results indicated a predicted phenotype of D+ C+ c+ E− e+ (R1r) for both probands. RNA was isolated from whole blood preserved in RNA stabilization solution (RNAlater, Thermo Fisher Scientific, Burlington, Ontario, Canada) with a RNA purification kit (RiboPure, Thermo Fisher Scientific) following the manufacturer’s instructions. RHAG was subjected to reverse transcription-polymerase chain reaction and sequenced. RHAG sequences showed a homozygous polymorphism in Exon 7 at Position c.1003G>A (p.Gly335Ser, RHAG*01N.13). This result was confirmed by genomic DNA sequencing. High consanguinity level has been observed in the vast majority of Rhnull cases reported.1,2 In the case described here, consanguinity was noted for two family branches. Two brothers married two sisters that happened Volume **, ** **
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A new Rhnull allele in francophone Quebecers
ST-LOUIS ET AL.
REFERENCES I
1. Daniels G. Human blood groups. 3rd 1 AB, R1r (RHAG*01/ RHAG*01)#
2 B, R1r (RHAG*01/ RHAG*01N.13)#
3 Not tested (RHAG*01/ RHAG*01N.13)#†
ed. Oxford, UK: Wiley-Blackwell; 2013. 2. Klein HG, Anstee DJ. Mollison’s blood
4 Not tested (RHAG*01/ RHAG*01)#
transfusion in clinical medicine. 12th ed. Oxford, UK: Wiley Blackwell; 2014. 3. St-Louis M, Perreault J, Lemieux R. Extended blood grouping of blood
II
donors with automatable PCR-ELISA 1 AB, R1r (RHAG*01/ RHAG*01N.13)#
2 O, R1r (RHAG*01/ RHAG*01N.13)#
genotyping. Transfusion 2003;43:1126-32.
III 1 A, R1R1 RHAG not tested
2 B, Rhnull RHAG not tested
3 B, Rhnull RHAG*01N.13/ RHAG*01N.13 R1r on DNA
4 Not tested
5 A, Rhnull RHAG*01N.13/ RHAG*01N.13 R1r on DNA
6 B, rr RHAG not tested
Fig. 1. Family pedigree. The arrows indicate the two probands studied in the 2000s. The diagonal line indicates deceased individuals at the time of writing. #Results in parentheses are presumed. They are based on the results obtained for generation III. †The carrier state of I-3 is presumed, based on his brother’s results (I-2).
to be their first-degree cousins. The Rhnull phenotype caused by this new RHAG variant allele was found in one branch. CONFLICT OF INTEREST The authors have disclosed no conflicts of interest.
2
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METHODS AND RESULTS The family was first studied in the late 1960s. A family pedigree is depicted in Fig. 1. The probands’ parents were first cousins (II-1 and II-2). Blood samples were collected in 1968 for serology workup using anti-D (Canadian Red Cross Lot 232), anti-C (Canadian Red Cross Lot 81), anti-c (Hyland Laboratories Lot 0337D004A1), anti-E (Canadian Red Cross Lot 62), and anti-e (Ortho Lot RSET-59). Other antigens were also tested: ABO, Lea, Leb, M, N, S, s, U, P1, K, k, Fya, Fyb, Jka, and Jkb (no data available concerning the reagents used).
From 1Research and Development and 2Immunohematology Reference Laboratory, Héma-Québec, Quebec City, Quebec, Canada. Address reprint requests to: Maryse St-Louis, PhD, Recherche et Développement, Héma-Québec, 1070, Avenue des Sciences-de-la-Vie, Québec, QC G1V 5C3, Canada; e-mail: [email protected]. GenBank Accession Number KJ543706, Received for publication May 28, 2014; revision received August 18, 2014, and accepted August 24, 2014. doi: 10.1111/trf.12887 © 2014 AABB TRANSFUSION **;**:**-**. 2015;55:1580–1581 TRANSFUSION
1580 TRANSFUSION Volume 55, June 2015
Rh phenotype was repeated on blood samples from III-5 in 1982 and 2006. Reagents used are unknown. Blood samples from III-3 were phenotyped in 1990 and again in 1991 and 1999 as a blood donor. Her Rh phenotype was confirmed by approved serology techniques in 2013 using anti-D blend (DBL Novaclone [IgM clone D175-2 + IgG Clone D415 1E4], anti-C Clone MS24 [Ortho Diagnostics BioClone reagents, Markham, Ontario, Canada]), anti-c Clone MS42 (Ortho Diagnostics BioClone), anti-E Clone C2 (Ortho Diagnostics BioClone), and anti-e clone MS16 (Ortho Diagnostics BioClone). No adsorption-elution was performed. According to the results available from the late 1960s, family members I-2, II-1, and II-2 presented weaker reactions with anti-D, anti-C, anti-c, and anti-e compared to controls. They were all negative with anti-E (R1r). The maternal grandparents were not tested (I-3 and I-4). Three of the five siblings (III-2, III-3, and III-5) were found to be Rhnull (D− C− c− E− e−). They also presented weaker reactions with anti-N (III-5) and anti-s (III-3, III-5) and were found to be U− (III-2, III-3, and III-5). Some individuals in the fourth generation (not shown in Fig. 1) showed weaker Rh antigen expression. Phenotype performed in the 2000s on the sisters’ blood samples (III-3 and III-5) confirmed the Rhnull status. DNA was extracted from III-3 and III-5 whole blood collected on EDTA using a genomic DNA purification kit (QIAamp DNA Blood Mini Kit, Qiagen, Mississauga, Ontario, Canada) according to the manufacturer’s instructions. RHD, RHCE*C/RHCE*c, RHCE*E, RHCE*e assays were performed as previously published.3 Results indicated a predicted phenotype of D+ C+ c+ E− e+ (R1r) for both probands. RNA was isolated from whole blood preserved in RNA stabilization solution (RNAlater, Thermo Fisher Scientific, Burlington, Ontario, Canada) with a RNA purification kit (RiboPure, Thermo Fisher Scientific) following the manufacturer’s instructions. RHAG was subjected to reverse transcription-polymerase chain reaction and sequenced. RHAG sequences showed a homozygous polymorphism in Exon 7 at Position c.1003G>A (p.Gly335Ser, RHAG*01N.13). This result was confirmed by genomic DNA sequencing. High consanguinity level has been observed in the vast majority of Rhnull cases reported.1,2 In the case described here, consanguinity was noted for two family branches. Two brothers married two sisters that happened Volume **, ** **
TRANSFUSION
1
A new Rhnull allele in francophone Quebecers
ST-LOUIS ET AL.
REFERENCES I
1. Daniels G. Human blood groups. 3rd 1 AB, R1r (RHAG*01/ RHAG*01)#
2 B, R1r (RHAG*01/ RHAG*01N.13)#
3 Not tested (RHAG*01/ RHAG*01N.13)#†
ed. Oxford, UK: Wiley-Blackwell; 2013. 2. Klein HG, Anstee DJ. Mollison’s blood
4 Not tested (RHAG*01/ RHAG*01)#
transfusion in clinical medicine. 12th ed. Oxford, UK: Wiley Blackwell; 2014. 3. St-Louis M, Perreault J, Lemieux R. Extended blood grouping of blood
II
donors with automatable PCR-ELISA 1 AB, R1r (RHAG*01/ RHAG*01N.13)#
2 O, R1r (RHAG*01/ RHAG*01N.13)#
genotyping. Transfusion 2003;43:1126-32.
III 1 A, R1R1 RHAG not tested
2 B, Rhnull RHAG not tested
3 B, Rhnull RHAG*01N.13/ RHAG*01N.13 R1r on DNA
4 Not tested
5 A, Rhnull RHAG*01N.13/ RHAG*01N.13 R1r on DNA
6 B, rr RHAG not tested
Fig. 1. Family pedigree. The arrows indicate the two probands studied in the 2000s. The diagonal line indicates deceased individuals at the time of writing. #Results in parentheses are presumed. They are based on the results obtained for generation III. †The carrier state of I-3 is presumed, based on his brother’s results (I-2).
to be their first-degree cousins. The Rhnull phenotype caused by this new RHAG variant allele was found in one branch. CONFLICT OF INTEREST The authors have disclosed no conflicts of interest.
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