Severe Congenital Hemolytic Anemia Caused by a Novel Compound Heterozygous PKLR Gene Mutation in a Chinese Boy
Congenital hemolytic anemia (CHA) is a group of disorders characterized by the premature destruction of red blood cells (RBCs) due to defects in their structure, function, or metabolism. These defects can arise from abnormalities in hemoglobin, the RBC membrane, or metabolic enzymes. The clinical presentation of CHA typically includes normocytic or macrocytic anemia, reticulocytosis, and elevated levels of unconjugated bilirubin. In this case report, we describe a Chinese Han infant with severe transfusion-dependent CHA caused by novel compound heterozygous mutations in the PKLR gene, which encodes pyruvate kinase (PK), a critical enzyme in anaerobic glycolysis.
The patient was the first child of a non-consanguineous Chinese couple with no family history of jaundice or anemia. He was born via cesarean section at 36 weeks and 6 days of gestation due to suspected intrauterine distress. At birth, he exhibited pallor and shortness of breath, with an Apgar score of 8, 9, and 9 at 1, 5, and 10 minutes, respectively. His birth weight was 3150 g. Initial blood tests revealed severe anemia with a hemoglobin (Hb) level of 73 g/L, an RBC count of 1.64 × 10^12/L, and a reticulocyte percentage (Ret%) of 15.3%. His total bilirubin was significantly elevated at 307 µmol/L (reference range: 5–21 µmol/L), with indirect bilirubin at 280.8 µmol/L (reference range: <17 µmol/L). The patient’s blood type was O Rh D+, and his mother’s blood type was A Rh D+. He received phototherapy and whole blood replacement, which improved his Hb and RBC counts to 119 g/L and 3.88 × 10^12/L, respectively, before discharge.
Two months later, the patient was admitted to the emergency unit with pallor and reduced movement. Blood tests showed severe normocytic anemia (Hb 41 g/L, mean corpuscular volume [MCV] 85.8 fL, mean corpuscular hemoglobin [MCH] 29.1 pg) and reticulocytosis (Ret%: 7.47%, Ret count: 0.0956 × 10^12/L). An emergency RBC transfusion was administered, and he subsequently required monthly transfusions to maintain Hb levels between 65 and 75 g/L. At the age of 3 years, the patient was referred to our clinic, where he continued to suffer from life-threatening anemia requiring transfusions every 4 weeks.
Further investigations revealed elevated total and indirect bilirubin levels, with a haptoglobin level of 60 g/L. Direct antiglobulin and Ham tests were negative, and CD59 and CD55 expression on erythrocytes was normal. Bone marrow smear showed erythroid hyperplasia, and peripheral blood smear revealed mostly normal RBC morphology with occasional RBC fragments. Glucose-6-phosphate dehydrogenase (G6PD) and PK enzyme activities were within normal ranges, but these results were considered unreliable due to frequent transfusions. The patient’s parents were asymptomatic with normal blood test results and peripheral smears, although their PK enzyme activities were near the lower limit of the normal range.
Next-generation sequencing (NGS) of a panel of 600 genes associated with blood diseases was performed on the patient and his parents. The analysis ruled out α/β-thalassemia and Fanconi anemia and identified compound heterozygous mutations in both the PKLR and SPTA1 genes. The PKLR mutations included a previously described pathogenic non-synonymous mutation, c.941T>C (p.I314T), known as the Hong-Kong PK mutation, and a novel frameshift deletion, c.979delC (p.L327fs), which we named the Chengdu PK mutation. The SPTA1 mutations were also novel: c.3334G>T (p.D1112Y) and c.6359C>G (p.T2120S). Family analysis confirmed that the patient’s father was heterozygous for the c.941T>C PKLR mutation and the c.3334G>T SPTA1 mutation, while his mother was heterozygous for the c.979delC PKLR mutation and the c.6359C>G SPTA1 mutation.
Computer modeling of the protein variants revealed significant structural disruptions caused by the PKLR mutations. The Hong-Kong PK mutation (c.941T>C) resulted in the substitution of isoleucine with threonine at position 314, disrupting the hydrophobic core of PK’s conservative A domain. This mutation affected critical residues involved in acid-base catalysis and magnesium binding. The Chengdu PK mutation (c.979delC) caused a frameshift and premature stop codon at position 329, resulting in a truncated protein lacking more than 40% of its normal structure, including the entire C domain and part of the A domain. This truncated protein was unstable and nonfunctional.
In contrast, the SPTA1 mutations did not significantly affect the structure or function of α-spectrin, a major component of the RBC cytoskeleton. SDS-PAGE analysis of the patient’s RBC membrane proteins showed no decrease in α-spectrin levels compared to a normal control. Peripheral blood smear after splenectomy revealed larger RBCs with acanthocytes, target cells, and stomatocytes but no spherocytes or elliptocytes, which are typically associated with SPTA1 dysfunction. These findings suggested that the patient’s hemolytic anemia was primarily attributable to pyruvate kinase deficiency (PKD) rather than SPTA1 mutations.
At the age of 4 years, the patient underwent splenectomy to manage severe anemia, iron overload, and splenomegaly. Postoperatively, his Hb level increased to 103 g/L but subsequently dropped to around 70 g/L, necessitating another transfusion. Six months after splenectomy and three months after his latest transfusion, his Hb level stabilized between 80 and 100 g/L.
This case highlights the importance of genetic testing in diagnosing congenital hemolytic anemias, particularly in complex cases with multiple gene mutations. The identification of the novel Chengdu PK mutation expands the spectrum of PKD genotypes and underscores the need for further research into the molecular mechanisms underlying this disorder. The patient’s clinical course also demonstrates the challenges of managing severe PKD, including the need for frequent transfusions and the potential benefits of splenectomy.
In conclusion, this report describes a severe case of congenital hemolytic anemia caused by compound heterozygous mutations in the PKLR gene, including a novel frameshift mutation, c.979delC (p.L327fs). The findings contribute to the understanding of PKD and emphasize the role of genetic analysis in diagnosing and managing this condition.
doi.org/10.1097/CM9.0000000000000027
Was this helpful?
0 / 0