Autoimmune anti-D in an RhD-positive young infant: Learning from a rare case.

BACKGROUND AND OBJECTIVES: Anti-D is usually immune in nature and is formed in individuals lacking D antigen or having variants/altered D phenotypes. In the Indian population, 93.8% are RhD positive, and R1 R1 is the commonest Rh phenotype. Here we report a rare and interesting case of autoimmune anti-D in an RhD-positive 3-month-old infant leading to warm autoimmune haemolytic anaemia. STUDY DESIGN AND METHODS: Auto-anti-D was detected serologically by immunohaematological techniques such as direct antiglobulin test, antibody detection and identification, dithiothreitol, enzyme treatment, antibody titration and elution. Molecular studies were performed to rule out genetic variants of RhD. RESULTS: Anti-D was confirmed in eluate and blood group post elution was B RhD positive. On genotyping using the Indian-specific RHD genotyping assay, the sample was found to be negative for the RHD*01W.150 (most common RhD variant in Indians) but positive for RHD exon 5 and RHD exon 10 along with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The sample was further sequenced for RHD exons 1-10 by Sanger sequencing and found to be a wild type, thus, ruling out the presence of an RhD variant. CONCLUSION: This case is of interest because of the rare occurrence of autoimmune anti-D in an RhD-positive patient of such a young age (3 months). To the best of our knowledge, only two case reports have been published on autoimmune anti-D in infancy (in 1961 and 1964).

Molecular characterization of rare D--/D-- variants in individuals of Indian origin.

BACKGROUND: Rh antigens are critical in haemolytic disease of the foetus and newborn (HDFN). The D-- phenotype is a rare blood group characterised by the lack of expression of C, c, E and e antigens at the surface of red blood cells because of mutations in both RHCE alleles inactivating the expression of a "normal" protein. The aim of the study was to determine the molecular basis of D-- individuals of Indian origin. MATERIALS AND METHODS: Ten Rh D-positive postnatal women who had produced antibodies against all Rh antigens, except D, leading to HDFN and foetal loss, were investigated. Extensive serological and molecular (polymerase chain reaction [PCR] using sequence-specific primers), quantitative multiplex PCR of short fluorescent fragments (QMPSF), and Sanger sequencing analyses were carried out. RESULTS: Serological testing with anti-C, anti-c, anti-E, and anti-e reagents showed absence of the four antigens in all ten index cases, as well as in three siblings. Flow cytometry indicated absence of these antigens with a typical exalted expression of the D antigen, thus confirming the rare D-- phenotype. Molecular analysis by QMPSF suggested homozygous CE-D hybrid alleles causing the D-- phenotype: RHCE-D(3-9)-CE (n = 11), RHCE-D(3-8)-CE (n=1), and RHCE-D(2-6)-CE (n=1). DISCUSSION: For the first time, we report the molecular basis of the D-- phenotype in the Indian population. Identification and characterisation of RHCE-null variants by molecular methods can help resolve transfusion-related problems in these individuals. Family studies of index cases helped to identify rare blood donors and offer counselling to females of child-bearing age on the complications involved in such pregnancies.

Algorithm development and diagnostic accuracy testing for non-invasive foetal RHD genotyping: an Indian experience.

BACKGROUND: The discovery of the cell-free foetal DNA (cffDNA) circulating in the maternal plasma enabled prediction of foetal RHD thus eliminating the risks associated with invasive procedures. Non-invasive foetal RHD genotyping has now become the standard approach in developed countries for management of alloimmunised women and is also used for targeted antenatal prophylaxis in non-alloimmunised women. MATERIALS AND METHODS: cffDNA was extracted from the plasma of 217 RhD negative pregnant women at a gestational age of 10-34 weeks. The foetal RHD genotype was determined by real-time polymerase chain reaction (real-time PCR) amplification of exons 4, 5 and 10 in duplicates. After an initial 54 samples, foetal typing was carried out with RHD exons 5 and 10 for the remaining samples. CCR5, SRY and RASSF1A genes were used as controls. Results were compared with cord blood serological typing at birth. RESULTS: Out of the 217 women, 193 were non-immunised and 24 were alloimmunised. A conclusive diagnosis was obtained in 203 samples. Diagnosis was inconclusive in 14 samples; of these, foetal RHD genotype could be resolved in six samples after maternal and paternal RHD genotyping. A 100% diagnostic accuracy, sensitivity and specificity were demonstrated in 209 women who had had a conclusive result. When the inconclusive samples were included, diagnostic accuracy and sensitivity were more than 95% and specificity was 78.95%. DISCUSSION: Anti-D is still the leading cause of haemolytic disease of the foetus and the newborn in India. There is, therefore, a need to establish and develop an algorithm for antenatal RhD negative women in India. The positive results of non-invasive foetal RHD genotyping, from the start of the 10th week of gestation using two RHD exons giving 100% diagnostic accuracy, show promise for routine diagnostic use to the benefit of the antenatal RhD negative Indian population.

RHD-Positive Alleles among D- C/E+ Individuals from India.

BACKGROUND: Molecular bases of blood group systems, including Rh blood group, have been poorly studied in the Indian population so far, while specificities of Europeans, East Asians and Africans have been well known for years. In order to gain insights into the molecular bases of this population, we sought to characterize the RHD allele in D- Indian donors expressing C and/or E antigen(s). METHODS: RHD gene was analyzed in 171 serologically D-, C/E+ samples by standard molecular methods such as quantitative, multiplex PCR of short fluorescent fragments (QMPSF) and direct sequencing when necessary. RESULTS: RHD whole gene deletion at the homozygous state was found to be the most common genotype associated with D- phenotype (118/171, 69.0%). Nonfunctional, negative hybrid genes with reported molecular backgrounds were observed in approximately one-third of the samples, while only four samples carry single-nucleotide variations, including one novel nonsense (RHD(Y243X)), one novel frameshift (RHD(c.701delG)), and two missense (RHD(T148R) and RHD(T148R, T195M)) alleles. CONCLUSION: Overall we report for the first time the molecular bases of D antigen negativity in the D-, C/E+ Indian population, which appears to be qualitatively similar to other populations, but with a population-specific, quantitative distribution of D-- alleles.

Molecular basis of weak D expression in the Indian population and report of a novel, predominant variant RHD allele.

BACKGROUND: The Rh blood group system is the most polymorphic system and is implicated in hemolytic transfusion reaction and hemolytic disease of the fetus and newborn. Molecular genetics of the RH genes have been extensively studied in Caucasians, Africans, and East Asians and the variant alleles giving rise to weak and partial D phenotypes have been reported. However, limited genetic studies have been carried out in the large Indian population, even though the variability of Rh expression has been documented. STUDY DESIGN AND METHODS: In this study we sought to characterize the molecular bases of weak D expression in Indians. RHD gene in samples presenting with a weak D phenotype by serologic analyses (n = 223) was genotyped by conventional molecular approaches. RESULTS: In addition to referenced and novel single-nucleotide variations, a novel approximately 12-kb duplication event, including Exon 3, was identified predominantly in variant D samples (130/223, 58.3%) and characterized at the nucleotide sequence level. Functional analyses suggested that this genetic variation quantitatively affects the expression of the normal transcript and then subsequently the expression of the normal RhD protein. CONCLUSION: We describe a major novel, variant RHD allele in Indians that can be easily identified routinely by implementing a simple genotyping assay. Although we may consider this variation as a weak partial D variant, further studies and observations are needed to confirm the same. These findings may contribute to improve significantly Rh blood group diagnostics in more than one billion Indians.

Screening for DEL phenotype in RhD negative Indians.

BACKGROUND: DEL phenotype represents a very weak form of D variant detected only by adsorption and elution technique. DEL phenotype individuals mistyped as RhD-negative can lead to alloimmunization after transfusion or pregnancy. Molecular techniques have now been used to identify DEL variants. They are commonly encountered in the East Asian population with RHD(K409K) being the most frequent allele. RHD(M295I) is the most common DEL allele in Caucasians. As there is a paucity of data on DEL phenotype in the Indian population, the study aims to screen RhD negative individuals for two most common DEL mutations. MATERIAL AND METHODS: EDTA blood was collected from 900 RhD negative individuals. Serological analysis included testing for the five major Rh antigens- C, c, D, E, and e by tube technique. Samples showing negative reaction for the presence of D antigen by Indirect Antiglobulin test were further tested for DEL phenotype by adsorption and elution technique. Molecular analysis involved DNA extraction and testing by PCR-SSP for RHD(K409K) and RHD(M295I) DEL alleles. RESULTS: Rh phenotyping showed 153 Rh negative individuals with r'r, ten with r''r and 737 with rr phenotype. All the samples tested negative for RhD antigen by adsorption and elution method. The two common DEL mutations RHD(K409K) and RHD(M295I) were also not detected in the study population. CONCLUSION: The study population showed the absence of the two common DEL alleles, concluding the variant to be rare. A comprehensive study with a larger sample size to look for other DEL mutations should be performed.

First report of Rhnull individuals in the Indian population and characterization of the underlying molecular mechanisms.

BACKGROUND: Rhnull phenotype is an extremely rare condition characterized by no expression of Rh antigens at the surface of red blood cells. Although rare, genetic bases of this phenotype are well known and include mutations within either the RH (RHD and RHCE) genes or the RHAG gene. So far Rhnull has been reported in individuals of Caucasian, African, and Asian origin. Here, we report individuals from two families of Indian origin representing such a rare phenotype. STUDY DESIGN AND METHODS: Serologic analysis was carried out by testing with anti-D, -C, -c, -E, and -e in Rhnull individuals and their family members. RH genes were analyzed by standard molecular approaches, including Sanger sequencing and quantitative multiplex polymerase chain reaction (PCR) of short fluorescent fragments. RHAG gene was investigated by exon-specific PCR amplification and Sanger sequencing. RESULTS: In one family, RHAG gene was found to be deleted at the homozygous state in the propositus, suggesting Rhnull of the regulator type. In the other family, a novel splice site variant in RHCE in cis with whole RHD gene deletion was identified at the homozygous state. Further functional analysis by minigene splicing assay showed that this variation, that is, c.801 + 1G>A, completely impairs normal splicing, then inactivating the expression of RhCE protein. Contrary to the former case, these data suggest Rhnull of the amorph type. CONCLUSION: Overall, we report for the first time the molecular mechanisms responsible for Rhnull phenotype in individuals of Indian origin. This study contributes to extend the molecular spectrum of variations in Rhnull individuals.