Aicha Ait Soussan

SMIM1 underlies the Vel blood group and influences red blood cell traits

The blood group Vel was discovered 60 years ago, but the underlying gene is unknown. Individuals negative for the Vel antigen are rare and are required for the safe transfusion of patients with antibodies to Vel. To identify the responsible gene, we sequenced the exomes of five individuals negative for the Vel antigen and found that four were homozygous and one was heterozygous for a low-frequency 17-nucleotide frameshift deletion in the gene encoding the 78-amino-acid transmembrane protein SMIM1. A follow-up study showing that 59 of 64 Vel-negative individuals were homozygous for the same deletion and expression of the Vel antigen on SMIM1-transfected cells confirm SMIM1 as the gene underlying the Vel blood group. An expression quantitative trait locus (eQTL), the common SNP rs1175550 contributes to variable expression of the Vel antigen (P = 0.003) and influences the mean hemoglobin concentration of red blood cells (RBCs; P = 8.6 × 10−15). In vivo, zebrafish with smim1 knockdown showed a mild reduction in the number of RBCs, identifying SMIM1 as a new regulator of RBC formation. Our findings are of immediate relevance, as the homozygous presence of the deletion allows the unequivocal identification of Vel-negative blood donors.

Evaluation of prenatal RHD typing strategies on cell-free fetal DNA from maternal plasma

BACKGROUND: The discovery of cell‐free fetal DNA in maternal plasma led to the development of assays to predict the fetal D status with RHD‐specific sequences. Few assays are designed in such a way that the fetus can be typed in RHDψ mothers and that RHDψ fetuses are correctly typed. Owing to the limited knowledge about the mechanism responsible for the presence of fetal DNA in maternal plasma, precautions in developing prenatal genotyping strategies must be made.

Sensitivity of fetal RHD screening for safe guidance of targeted anti-D immunoglobulin prophylaxis: Prospective cohort study of a nationwide programme in the Netherlands

Objective To determine the accuracy of non-invasive fetal testing for the RHD gene in week 27 of pregnancy as part of an antenatal screening programme to restrict anti-D immunoglobulin use to women carrying a child positive for RHD. Design Prospectively monitoring of fetal RHD testing accuracy compared with serological cord blood typing on introduction of the test. Fetal RHD testing was performed with a duplex real time quantitative polymerase chain reaction, with cell-free fetal DNA isolated from 1 mL of maternal plasma The study period was between 4 July 2011 and 7 October 2012. The proportion of women participating in screening was determined. Setting Nationwide screening programme, the Netherlands. Tests are performed in a centralised setting. Participants 25 789 RhD negative pregnant women. Main outcome measures Sensitivity, specificity, false negative rate, and false positive rate of fetal RHD testing compared with serological cord blood typing; proportion of technical failures; and compliance to the screening programme. Results A fetal RHD test result and serological cord blood result were available for 25 789 pregnancies. Sensitivity for detection of fetal RHD was 99.94% (95% confidence interval 99.89% to 99.97%) and specificity was 97.74% (97.43% to 98.02%). Nine false negative results for fetal RHD testing were registered (0.03%, 95% confidence interval 0.01% to 0.06%). In two cases these were due to technical failures. False positive fetal RHD testing results were registered for 225 samples (0.87%, 0.76% to 0.99%). Weak RhD expression was shown in 22 of these cases, justifying anti-D immunoglobulin use. The negative and positive predictive values were 99.91% (95% confidence interval 99.82% to 99.95%) and 98.60% (98.40% to 98.77%), respectively. More than 98% of the women participated in the screening programme. Conclusions Fetal RHD testing in week 27 of pregnancy as part of a national antenatal screening programme is highly reliable and can be used to target both antenatal and postnatal anti-D immunoglobulin use.

Fetal DNA: strategies for optimal recovery.

For fetal DNA extraction, in principle each DNA extraction method can be used; however, because most methods have been optimized for genomic DNA from leucocytes, we describe here the methods that have been optimized for the extraction of fetal DNA from maternal plasma and validated for this purpose in our laboratories. The use of the QIAamp DSP Virus kit (QIAGEN), the QIAamp DNA Blood Mini kit (QIAGEN), and the Magna Pure LC (Roche) is based on the kit components provided by the respective companies. However, we noticed that the yield of fetal DNA from maternal plasma can be increased when higher volumes are processed or some slight modifications of the protocols provided by the manufacturer are followed. Here, we also describe an in-house method that allows the specific capture of target molecules in an extremely low volume by using magnetic beads and magnetic tips. This method can be either performed by hand, or it can be adapted to a commercially available pipetting workstation.

Impact of genetic variation in the SMIM1 gene on Vel expression levels

Serologic determination of the Vel– phenotype is challenging due to variable Vel expression levels. In this study we investigated the genetic basis for weak Vel expression levels and developed a high‐throughput genotyping assay to detect Vel– donors.

Novel alleles at the Kell blood group locus that lead to Kell variant phenotype in the Dutch population

Alloantibodies directed against antigens of the Kell blood group system are clinically significant. In the Netherlands, the KEL1 antigen is determined in all blood donors. In this study, after phenotyping of KEL:1‐positive donors, genotyping analysis was conducted in KEL:1,–2 donors to identify possible KEL*02 variant alleles.

Molecular analysis of immunized Jr(a–) or Lan– patients and validation of a high-throughput genotyping assay to screen blood donors for Jr(a–) and Lan– phenotypes

Individuals with anti‐Jra or anti‐Lan are ideally transfused with rare Jr(a–) or Lan– red blood cells. We characterized mutations in Dutch Jr(a–) and Lan– individuals and developed a high‐throughput genotyping assay to detect Jr(a–) and Lan– donors.

Fetal RHD genotyping after bone marrow transplantation

Fetal RHD genotyping allows targeted diagnostic testing, fetal surveillance, and eventually intrauterine treatment to D‐alloimmunized pregnant women who carry an RHD+ fetus. However, false‐positive and false‐negative results of noninvasive prenatal fetal RHD genotyping have been described due to a variety of causes. In this case report we present two cases where noninvasive fetal RHD typing was complicated by a previous bone marrow transplantation (BMT).

Sensitivity of Fetal RhD Screening for Safe Guidance of Targeted Anti-D Immunoglobulin Prophylaxis: Prospective Cohort Study of a Nationwide Programme in the Netherlands

The risk ofmaternal alloimmunization due to RhD incompatibility has decreased with use of antenatal and postnatal anti-D immunoglobulin prophylaxis. The discovery of cell-free fetal (cff) DNA in maternal plasma during pregnancy and the feasibility of fetal RhD testing using this source of DNA make it possible to determine fetal RhD type and to restrict the use of antenatal anti-D immunoglobulin to only those RhD-negative women carrying an RhD-positive fetus. Variable and low amount of fetal DNA present in maternal plasma and the genetic variation of RHD alleles in the fetus or mother complicate such noninvasive testing. This study was done to evaluate the efficacy of noninvasive fetal RHD testing in week 27 of pregnancy to restrict use of antenatal and postnatal anti-D immunoglobulin use to RhD-negative women carrying an RhD-positive fetus. Duplex real-time polymerase chain reaction analysis was carried out for RHD exon 5 and RHD exon 7 in triplicate on cellfreeDNA isolated frommaternal plasma. Cord blood serologywas used as the reference standard as it is the best test available for determination of neonatal RhD status. The sensitivity for detection of fetal RHD was 99.94% (95% confidence interval [CI], 99.89%-99.97%), and specificity was 97.74% (95% CI, 97.43%-98.02%). Cord blood serology showed 9 falsenegative fetal RHD test results (0.03%; 95% CI, 0.02%-0.07%) and 225 false-positive fetal RHD test results (0.87%; 95% CI, 0.76%-1.00%). The overall negative predictive value was 99.91%(95% CI, 99.82%-99.95%), and the positive predictive value was 98.60% (95% CI, 98.40%-98.77%). The results demonstrate that fetal RHD assay performed at week 27 of pregnancy as part of an antenatal screening program is reliable and effective and can be used to restrict the use of both antenatal and postnatal anti-D immunoglobulin in RhD-negative pregnant women