First report of novel biallelic KLF1 variants causing prenatal onset severe transfusion-dependent non-spherocytic hemolytic anemia in North America Free

Kruppel-like factor 1 (KLF1) is an essential erythroid transcription factor that regulates globin gene switching and red blood cells (RBC) maturation. While heterozygous KLF1 variants are often associated with benign red cell traits, biallelic pathogenic variants can cause severe congenital non-spherocytic hemolytic anemia, with phenotypes overlapping congenital dyserythropoietic anemia, RBC enzymopathies, RBC membranopathies and thalassemia. Reported cases have largely originated from families in Southeast Asia. We report the first North American family with biallelic KLF1 loss of function variants, identified prenatally in two siblings who presented with fetal anemia requiring intrauterine transfusions.

Two fetuses in consecutive pregnancies were diagnosed with fetal anemia on second-trimester ultrasound. Amniocentesis was performed, and trio-based whole exome sequencing (WES) in the first pregnancy and targeted familial variant analysis in the subsequent pregnancy led to the prenatal diagnosis of a congenital hemolytic anemia due to compound heterozygous KLF1 variants. Intrauterine transfusions were administered in both pregnancies, with one transfusion completed in the first pregnancy, and five serial transfusions completed in the current pregnancy. Postnatally, the proband remains transfusion-dependent with persistent hemolytic anemia. A similar postnatal course is expected for the current pregnancy.

Novel compound heterozygous KLF1 variants were identified in both fetuses: a missense variant c.1081C>T (p.His361Tyr), a class 2 KLF1 variant, and a frameshift variant c.512del (p.Pro171Argfs*66), a class 3 KLF1 variant. Segregation analysis confirmed each parent carried one variant. Both parents were asymptomatic with normal hemoglobin concentration, red blood cell indices, and hemoglobin F levels, but exhibited borderline elevated hemoglobin A₂ levels (3.2-3.4%), suggestive of subtle erythroid regulatory perturbation. No additional disease-causing variants were found in other genes associated with congenital anemia. After birth, the proband displayed chronic hemolysis with marked reticulocytosis, indirect hyperbilirubinemia, and splenomegaly. RBC pyruvate kinase enzyme level was low with persistence of embryonic hemoglobin (Hb Portland and Gower-2) on postnatal hemoglobin electrophoresis. Peripheral smears showed anisopoikilocytosis without spherocytes.

We describe the first North American report of biallelic KLF1-related congenital hemolytic anemia, diagnosed prenatally through whole exome sequencing following detection of fetal anemia. This case highlights the expanding geographic distribution of KLF1 deficiency, including its potential to cause severe fetal anemia requiring intrauterine intervention. These findings underscore the utility of prenatal genomic diagnostics in guiding perinatal management and provide further evidence that KLF1 mutations should be considered in the differential diagnosis of unexplained fetal anemia or hydrops. The identification of subtle hematologic findings in asymptomatic carriers also raises the need for awareness of carrier phenotypes in KLF1-related disorders.