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Immunohematology and Genetic Testing (red cells, leukocytes and platelets)

(P-IG-44) Value of Specialized Molecular Techniques at the Immunohematology Reference Laboratory in the Investigation of Two Fortuitous Cases of Apparent Paternity Exclusion

Sunday, October 20, 2024
12:00 PM - 1:00 PM  

    Presenting Author(s)

  • Thierry Peyrard, PharmD, PhD, EurSpLM

    Head of Department
    Etablissement Français du Sang Île-de-France, UMR_S1134 BIGR Université Paris Cité & Université des Antilles, Paris, France
    Paris, Ile-de-France, France

Background/Case Studies: RBC antigens are dominantly inherited so that a child obligatorily carries one allele passed by the father and one by the mother. However, failure of allele amplification sometimes occurs, leading heterozygous mutations to appear homozygous. One cause of an apparently homozygous status is that one of the 2 alleles is silent. We describe here 2 cases of apparent paternity exclusion due to RH hybrid alleles.

Study

Design/Methods: RBC genotyping was performed with HEA, wRHD, wRHCE kits (Werfen/USA). Genomic DNA was amplified by RHD/RHCE exon-specific primers and sequenced. QMPSF (quantitative multiplex polymerase chain reaction of short fluorescent fragments) was carried out with RHD/RHCE exon copy number calling (Fichou Y et al. Transfusion 2013).

Results/Findings: Case 1. Samples from a 44 y.o. male patient were referred for standard RBC genotyping. As he showed a homozygous c.733C >G change in RHCE, a full RHCE study was performed, which revealed a RHCE*ceAR allele in the homozygous state (Hr– rare type). RHD study showed an unexpected RHD*DAR/RHD*DAU0 genotype, as RHCE*ceAR has never been reported to be in cis with RHD*DAU0. A family study was initiated. One child was D+C–E+c+e– (R2R2 or R2r’’), which denoted a genetic inconsistency for a RoRo father expected to obligatorily pass a Ro haplotype. QMPSF showed in the propositus and child a hybrid RHCE*CE-D(3-9)-CE silent allele, in heterozygous state, likely in cis with RHD*DAU0. The propositus genotype was then RHCE*ceAR/RHCE*CE-D(3-9)-CE. RHCE*ceAR falsely appeared in the homozygous state with standard sequencing because all mutations in this allele occur in exons 5 and 6 (no RHCE exons 5 and 6 in RHCE*CE-D(3-9)-CE).
Case 2. Samples from a 31 y.o. pregnant woman were referred because of a weak e phenotype. RHCE genotyping showed a RHCE*ceMO allele (c.667G >T), in the homozygous state, typically found in people of African origin. This was an unexpected result, as the patient's father was of European origin. A family study was carried out, which revealed her mother to be heterozygous for RHCE*ceMO, and her father to be surprisingly D+C+E–c–e+, which indicated a genetic inconsistency as he was expected to obligatorily pass a R1 or r’ haplotype. QMPSF and RHCE exon 2 sequencing showed in the propositus and her father a hybrid RHCE*CE-D(3-7)-CE silent allele, in the heterozygous state. The propositus genotype was then RHCE*ceMO/RHCE*CE-D(3-7)-CE. RHCE*ceMO falsely appeared to be in the homozygous state with standard sequencing because the c.667T mutation occurs in exon 5 (no RHCE exon 5 in RHCE*CE-D(3-7)-CE).


Conclusions: Routine molecular investigation may fortuitously lead to genetic inconsistencies and apparent parenthood exclusion. In such cases, the presence of a silent allele should be suspected first. QMPSF, able to easily detect RH hybrid alleles and more accessible than long read NGS, is a helpful tool in solving those complex and intriguing cases.