Akademik Veri Yönetim Sistemi
EHA-SWG Scientific Meeting on Red Cell and Iron Metabolism Defects: From Basic Science to Clinical Case Application, Budapest, Macaristan, 12 - 14 Ekim 2023
Background: Hereditary hemolytic anemias (HHA) are rare disorders with clinical and genetic heterogeneity. Erythrocyte membrane
defects, erythrocyte enzyme defects or ineffective erythropoiesis are the causes of HHA. Despite the expanded diagnostic panels that
have taken place in routine practice in recent years, it is possible to reach the diagnosis in rare diseases and to reveal new genes related
to the disease by whole exome sequencing (WES) in unresolved cases.
Aim: To establish genotype-phenotype relationship by WES in eleven index that could not be clinically classified by conventional
diagnostic approaches.
Methods: Eleven families whose molecular diagnosis could not be made with routine laboratory tests and whose written consent forms
were obtained, were included in the study. In DNA samples isolated from peripheral blood, index cases were studied by whole exome
sequencing. Raw data were aligned to the hg38 reference genome with BWA and SAMtools. Filtering was performed based on missense,
frameshift and splice variations and minor allele frequency (MAF<0.005). 1000Genomes, ExAC, gnomAD databases were used for
population data and in-silico prediction tools were used to determine the possible pathogenic effect of variations. The candidate genes
identified by the analysis were validated by performing familial segregation analyses with Sanger sequencing.
Results: The age distribution of the patients was between 29-183 months (mean 103 months ±59), and the follow-up period was 27-174
months (mean 84 months ±55). The hemoglobin level at the time of admission was 4-9 gr/dL (mean 6.95 ±1.89). Ten patients needed
regular transfusion. Splenectomy and colcystectomy were performed in one patient. The families had consanguineous marriage (n=5)
and family history suggestive of hemolytic anemia (n=6). Within the scope of our study, variations associated with hemolytic anemia and
compatible with the phenotype were determined in 63% (n=7) of the cases in which all exome sequencing was performed. Hereditary red
cell membrane defects in two families (SPTB c.5051G>A, R1684H, PIEZO1 c.7367G>A, p.R2456H), pyruvate kinase enzyme deficiency
with homozygous inheritance (c.1516G>A, p .V506I) in one family and compound heterozygous inheritance in the other (R486W and
I90T), one family (CDIN1 combined heterozygotes R101W and P248fs) with ineffective erythropoiesis and congenital dyserythropoetic
anemia (CDA1) phenotype, and one family (ALAS2, c.1435 C>T, R479W) sideroblastic anemia and a homozygous (RPS17, c.159T>G, Y53*)
variation associated with Diamond-Blackfan anemia in one family were detected. . In the families in which variation was detected,
confirmation was performed by segregation analyses in the mother, father and affected siblings, if any. New candidate genes associated
with the disease were identified in three families and functional studies are planned. Informatics analyses are ongoing in one case.
Conclusion: Diagnostic success with whole exome analysis in unresolved hemolytic anemia cases is only 60% for known hereditary anemia
genes. On the other hand, whole exome analyses are extremely valuable in finding new candidate genes/variants associated with the
disease, and in this study, 3 new candidate genes supporting clinical phenotypes were identified. Whole genome sequencing and/or
methylome investigations should be performed in cases where whole exome analyses remain unresolved.