Deciphering the Causal Diagnosis of Hydrops Fetalis or Unexplained Fetal Anemia Using Targeted Next Generation and Exome Sequencing

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.