A homozygous KLF1 gene mutation presenting as mild Thalassemia Intermedia unraveled by targeted Next Generation Sequencing

Abstract

The krupple-like factor 1 (KLF1) is a crucial transcription factor that is responsible for the proper maturation of the erythroid cells. Recent studies have demonstrated that mutations in KLF1 gene may lead to increased fetal hemoglobin (HbF) and reduced or borderline hemoglobin A2 (HbA2) levels. Increased HbF levels and concomitant α-thalassemia are two main modifiers that can ameliorate the clinical and hematological severity of β-thalassemia. Mutations in KLF1 have been found in association with β thalassemia. DNA was extracted with QIAmp DNA Blood kit and quantified spectrophotometrically. Gap PCR was used to screen common HPFH deletions and Sanger's sequencing was done to screen β-globin (HBB) mutations. Libraries were prepared using TruSight One sequencing panel and sequenced on MiSeq Sequencing System. MiSeq Reporter and Variant Studio were used for data analysis. A 56 years male presented with splenomegaly and unconjugated hyperbilirubinemia with normal hematological indices. Hemoglobin high performance liquid chromatography revealed 72.3% HbF, 0.5% HbA 2 and 25.2% HbA 0 . Patient was found to be clinically consistent with mild TI. No mutation/s in HBB was found by Sangers sequencing. Hereditary Persistence of Fetal Hemoglobin (HPFH) deletions [HPFH1, HPFH2, HPFH3, Chinese G -Indian inversiondeletion] were also found to be negative. Targeted resequencing revealed a novel homozygous probably causative mutation in KLF1 [c. 943C>T (p.Arg301Cys)]. This mutation was found to be probably damaging via PolyPhen2 and SIFT. The patient's son showed 5% HbF with heterozygous mutation. This is the first report from India where a homozygous mutation in KLF1 gene is implicated with high HbF in a patient with TI. Thus, mutations which affect the activity of KLF1 gene may lead to high level of fetal hemoglobin in patients presenting as TI with no HBB mutations.

Severe neonatal haemolytic anaemia caused by compound heterozygous KLF1 mutations: report of four families and literature review

“…Table IV shows a summary of 15 previously reported cases of biallelic KLF1 mutations that were identified from literature review. [26][27][28][29][30][31][32] Fourteen cases presented with severe haemolytic anaemia in the fetal or neonatal periods. One case with compound heterozygous null mutations presented with severe anaemia and kernicterus.…”

“…31 A case with a homozygous R301C mutation presented with mild anaemia and splenomegaly in the adult period. 32…”

“…31 On the other hand, a case with homozygous R301C mutation, which is a class 2 mutation, presents in adulthood with mild anaemia and splenomegaly. 32 Apart from the findings of severe haemolytic anaemia and abnormal red cell morphology, pathologically reduced pyruvate kinase (PK) levels are seen in patients with compound heterozygous class 2/class 3 KLF1 mutations. 26 The low PK levels in KLF1 mutations may result in a misdiagnosis of congenital PK deficiency.…”

“…26 After the initial report, there were further reports of seven cases with compound heterozygous KLF1 mutationsassociated haemolytic anaemia with clinical manifestation ranging from hydrops fetalis to mild non-transfusiondependent anaemia. [27][28][29][30][31][32] As the condition is rare and may mimic other more common hereditary haemolytic anaemias, knowledge of clinical manifestations and haematologic characteristics should be useful for understanding the KLF1 mutations and establishing a diagnosis of new cases. Herein, we described the genotypes and haematologic phenotypes in cases of compound heterozygous KLF1 mutations-associated haemolytic anaemia and their parents who were carriers.…”