ALBERT

Description: Fetal anemias are serious complications during pregnancies, which could lead to fetal death in case of hydrops fetalis (1/3000 pregnancies). Fetal anemia and hydrops fetalis are in most cases the result of fetal maternal alloimmunization, parvovirus B19 infection, fetal maternal hemorrhage, chromosomal abnormalities, congenital malformations, metabolic diseases, and in the context of hematological disorders, alpha-thalassemia. However, in one case out of 5, fetal anemia remains unexplained after an exhaustive first line of etiological evaluation. In order to identify the cause of the unexplained fetal anemia and to provide advice regarding prenatal diagnosis for next pregnancy, we have developed useful diagnostic tools on fetal blood based on erythrocyte and reticulocyte indices, examination of red cell morphology, flow cytometry (EMA test), osmotic gradient ektacytometry and molecular screening analysis. 43 fetal samples (30 probands) have been referred to our Hematology diagnostic lab in the time span between 2012-2018. In majority of the cases, various analyses were performed on fetal blood (23 out of 43) and in other instances during post-mortem examination following death (17 out of 43). Fetal blood purity was confirmed by microsatellite analysis on both parental and fetal DNAs. Informed consent was obtained from the mother in all cases. In 6 of 43 cases, prenatal diagnosis was performed after identification of the causal mutation responsible for the hydrops fetalis in the first fetus. Hydrops fetalis was suspected in 24 cases at the time of fetal sample collection. Red cell morphology and ektacytometry enabled the establishment of clinical diagnostic in two cases (congenital dyserythropoiesis type II (CDAII) and xerocytosis). Molecular screening analysis was performed by Sanger sequencing technique from 2012 to July 2016 and subsequently we designed a targeted Next Generation Sequencing (NGS) library including 74 genes involved in red cell disorders (n=9 fetuses) and exome sequencing (WES) was performed for 4 fetuses. Each identified allelic variation was confirmed by Sanger sequencing technique. Molecular Biology analysis (except the 6 prenatal diagnosis cases) was performed on 21 of 37 fetuses that enabled identification of the molecular defect in 10 fetuses. Rare red cell disorders were diagnosed in these cases including Diamond-Blackfan anemia (n=2), congenital dyserythropoiesis (n=6) and stomatocytosis (n=2) respectively. No putative pathogenous allelic variation following molecular screening could be identified in 4 fetuses. In the 6 cases which were screened for the molecular defect previously identified, none of the tested fetuses exhibited the allelic variation identified in the first fetus. In summary, targeted-NGS and WES are very valuable tools in the causal diagnosis of hydrops fetalis dues to unexplained anemia in addition to routine hematological tests (erythrocyte and reticulocyte indices, red cell morphology, flow cytometry, and ektacytometry) and after elimination of the most frequent causes of hydrops fetalis. This clinical problem is more frequent than has been previously surmised and needs more attention. Disclosures No relevant conflicts of interest to declare.

Description: Diamond-Blackfan anemia (DBA) is a rare congenital erythroblastopenia associated with haploinsufficiency of more than 15 ribosomal protein genes. We identified in the French registry a DBA patient exhibiting a large heterozygous deletion in ribosomal protein gene RPSA, making it a new candidate as a DBA-associated gene. The deletion was confirmed by qPCR using specific intron/exon primers that could discriminate the RPSA gene from pseudo-genes. Consistent loss of function of this allele, RPSA mRNAs were underrepresented in erythroid cultures and in lymphoblastoid cells (LCLs) derived from the patient. Additionally, an important decrease in erythroid proliferation was observed in erythroid progenitor and precursor cells derived from the patient's bone marrow CD34+ cells, together with a delay in erythroid differentiation, the activation of the p53 pathway, and a G0/G1 cell cycle arrest, which are regular features of DBA patient cells. Similar to other RP genes linked to DBA, interruption of RPSA expression in zebrafish embryos strongly affected development of the head and erythroid cell differentiation, supporting its relevance as a candidate DBA gene. Northern blot analysis also revealed impaired pre-ribosomal RNA (pre-rRNA) processing in patient LCLs compared to controls. But unexpectedly, the observed pre-rRNA profile was clearly different from that observed in HeLa cells upon RPSA knockdown and suggested a dysfunction of pre-rRNA processing upstream of that produced by a shortage of RPSA. We noticed that the RPSA gene hosts the intron-encoded H/ACA snoRNAs snoRA6 and snoRA62, the latter being included in the deletion. Q-RT-PCR analysis of patient cells showed decreased levels of snoRA62 when compared to controls and to other RP-mutated DBA patients. Strikingly, knocking out the snoRNA62 coding sequence in human haploid HAP1 cells with Crispr/Cas9 resulted in a pre-rRNA maturation defect similar to that observed in the RPSA+/mut DBA patient, thereby revealing a role for snoRA62 in human pre-rRNA processing. These data strongly suggest that, in addition to RPSA haploinsufficiency, the primum movens of DBA in this particular patient includes the deletion of the intronic snoRA62 hosted in the RPSA gene. To our knowledge, this is the first time that a snoRNA is linked to DBA. We will discuss this ongoing work in light of our most recent results in cells and zebrafish models. Disclosures No relevant conflicts of interest to declare.