Genetic testing has become clinically available and is now used for the confirmatory diagnosis of Mendelian disorders. However, when confirmatory genetic testing for an initial diagnosis is negative, the potential disease is diversified into various disorders showing similar clinical and laboratory features. In that situation, time and effort are required to make an accurate diagnosis. High-throughput sequencing, previously performed on a research basis, is very useful to genetically differentiate clinically similar diseases.1, 2
In this study, we present two sibling patients who initially exhibited congenital dyserythropoietic anemia (CDA)-associated features but were finally identified as having two PKLR null mutations using whole exome sequencing (WES). The patients were a Korean boy and his younger sister. The boy presented with neonatal anemia, a complete blood count (CBC) with a leukocyte count of 7.7 × 109/L, hemoglobin (Hb) of 7.9 g/dL and platelet count of 470 × 109/L. The RBC indices included a mean corpuscular volume (MCV) of 88.6 fL, a mean corpuscular Hb (MCH) of 30.1 pg and a MCH concentration (MCHC) of 34.0%. The values of the indices were characteristic of normocytic normochromic anemia. The total and indirect bilirubin levels were 2.92 and 2.61 mg/dL, respectively. The lactate dehydrogenase (LD) level was 1166 IU/L (reference range 250–450 IU/L), and the haptoglobin level was lower than the detection limit (reference range 30–200 mg/dL). A peripheral blood (PB) smear revealed anisocytosis, and a paroxysmal nocturnal hemoglobinuria investigation by flow cytometry was normal. The bone marrow (BM) revealed CDA-associated features and a normal karyotype. The patient was examined regularly and received blood transfusions and an iron chelating agent, deferasirox. A sister was born when the male patient was 3 years and 10 months old. She also developed neonatal anemia and required blood transfusions. When she was 7 months old, she was admitted for evaluation of a possible hematologic disorder similar to her brother's. She exhibited a leukocyte count of 3.3 × 109/L, Hb of 10.8 g/dL and platelet count of 202 × 109/L. Her RBC indices were as follows: MCV 86.4 fL, MCH 29.1 pg and MCHC 33.6%, indicating normocytic normochromic anemia. Her total and indirect bilirubin levels were 2.69 and 2.22 mg/dL, respectively. Her LD level was 894 IU/L, and haptoglobin was not detected. A PB smear revealed normal morphology, and the BM revealed 100% cellularity and marked erythrocytosis with dysplasia, similar to her brother. She attended regular follow-up visits with regular blood transfusions.
Because the siblings suffered from the same disease, Sanger sequencing for CDAN1, C15ORF41 and SEC23B was performed to identify CDA-causing mutations.3 All examinations were negative. Next, we performed WES using probands and parental samples to detect disease-causing mutations after obtaining written informed consent from all subjects. The patients’ exome DNA was captured using a SureSelect All Exon V4+UTRs kit (Agilent Technologies, Santa Clara, CA, USA) and sequenced as paired-end 150 bp reads on the HiSeq 2500 platform (Illumina Inc., San Diego, CA, USA). The obtained sequence reads were aligned to the human reference genome sequence 19 (hg19) using Novoalign v3.01.01 (Petaling Jaya selangor, Malaysia). After excluding the known common variations reported in public sequence databases (including dbSNP137, the 1000 Genome Project and HAPMAP) the remaining variants in both probands that can lead to pathogenic effects, such as nonsense, frameshift or splicing mutations, were further selected and finally sorted as those which showed compound heterozygous or homozygous status in the patients and heterozygous status only in the parents, but were not present in the healthy control. The compound heterozygous mutations in the PKLR gene that could be implicated in the patient phenotype were identified as c.1270-3C>A and c.1618G>T. The c.1270-3C>A mutation in intron 8 is predicted to produce ‘alteration of the WT acceptor site, most likely affecting splicing’ by in silico analysis using Human Splicing Finder Matrices (Variation: WT site broken-10.95%) and MaxEnt Scores (Variation: 3′ Motif-59.32%). The c.1618G>T on exon 10 induces stop codon formation p.(Gly540*) leading to a premature termination codon. The parents were each heterozygous for one of the mutations; c.1270-3C>A was inherited from their father, and c.1618G>T was inherited from their mother (Figure 1). Sanger sequencing validated the cosegregation of the novel PKLR mutations in the probands and their parents (RefSeq ID: NM_000298.5). Among two mutations, c.1618G>T of PKLR was reported only in the ExAC database at an extremely low frequency of 0.00000824 (0.0001156 in East Asia only). In addition, these mutations were absent from public sequence databases including dbSNP, 1000 genomes and EVS as well as the locus-specific database HGMD. The mutations were not detected in the analysis of an additional 100 normal Korean controls, the Korean reference genome database (KRGDB) and the TIARA genome database. PKLR mutations cause a pyruvate kinase (PK) deficiency in RBCs and induce hemolysis. They are the most common cause of congenital non-spherocytic hemolytic anemia, and over 200 mutations have been described.4, 5 The clinical features are highly variable, and some share features of other congenital anemias such as CDA.6 The worldwide spectrum of PKLR mutations shows that ~70% of PK-deficient alleles carry a missense mutation followed by splicing and stop codon mutations (13 and 5%, respectively; www.pklrmutationdatabase.com).7 It has been reported that a severe syndrome is commonly associated with disruptive mutations.8 Patients with homozygous ‘null’ mutations display intrauterine growth retardation, severe anemia at birth, blood transfusion dependence and, in rare cases, intrauterine death or death during the first few days of life.9, 10 The unusual manifestations in our patients including marked dyserythropoiesis are often found in patients with severe hemolysis sufficient to be considered CDA, which might be caused by two disruptive mutations (stop codon and splicing).11 A standard treatment protocol for PK deficiency has not been established because it is rare and presents variable manifestations. Maintaining an adequate Hb through transfusion and avoiding excessive iron overload are general strategies used for conservative treatment. In severe cases, a total splenectomy has proven to be an effective therapy for eliminating or decreasing transfusion dependence and raising the baseline Hb level.12 In the present cases, splenic infarction developed when the boy was 7 years old, and a splenectomy was performed. Marked congestion with erythrophagocytosis and extramedullary hematopoiesis were observed in the enlarged spleen and accessary spleen (Figures 2a and c). Reticulocyte and platelet counts increased to 51.46% and 1451 × 109/L after the splenectomy, respectively. However, the Hb level did not increase (7.5 g/dL), and the indirect bilirubin level did not decrease (up to 5.73 mg/dL). Although it is known that some patients may require intermittent transfusions after a splenectomy, the boy required continuous transfusion, and a spinal compression fracture occurred due to osteoporosis. The ferritin level was high, even with iron chelation therapy (up to 2000 ng/mL). These findings could be caused by continuing ineffective erythropoiesis in the BM and hemolysis even after the splenectomy.13 Hematopoietic stem cell transplantation (HSCT) is a possible candidate treatment modality in patients with PK deficiency. To date, one successful report in humans has been published.14 Therefore, HSCT was considered for life-long control of the boy. Because his sister (and only sibling) had the same disease, a sibling donor could not be considered. He underwent PBSCT from an unrelated donor with HLA-A, B, DR, DQ matches and one HLA-C allele mismatch at high resolution molecular typing at 11 years of age. The conditioning regimen consisted of fludarabine (30 mg/m2/day) and cyclophosphamide (50 mg/kg/day) from day −5 to day −2. GvHD prophylaxis consisted of anti-thymocyte globulin (Thymoglobulin; Genzyme. 2.5 mg/kg/day) from day −3 to day −1, IV cyclosporine (3 mg/kg/day IV initially, followed by 5 mg/kg/day oral) starting from day −1 and four infusions of mini-dose methotrexate (5 mg/m2) at days 1, 3, 6 and 11. The Hb level, indirect bilirubin level, and reticulocyte and platelet counts were gradually normalized and maintained for 3 years after HSCT. His sister also required regular transfusion and preferentially received HSCT from an unrelated donor at 8 years of age based on previous experience. The pretransplantation preparation and GvHD prophylaxis were the same as those used for her brother, except for a total dose of fludarabine of 200 mg/m2. However, pancytopenia developed and was maintained. Mixed (36–51%) donor chimerism was apparent. Although her indirect bilirubin level was maintained below 1.04 mg/dL, her LD level increased to 1111 IU/mL. Splenic infarction occurred 6 months after HSCT, and a splenectomy was performed. Severe congestion, hemosiderosis and extramedullary erythropoiesis were observed (Figures 2b and d). The post-splenectomy CBC (Hb 13.5 g/dL, leukocytes 8.3 × 109/L, platelets 391 × 109/L), indirect bilirubin and LD values were normalized (0.35 mg/dL and 391 IU/mL, respectively). Mixed chimerism transitioned into full donor chimerism. Hence, the mixed chimerism before the splenectomy might have originated from the coexistence of recipient cells produced in the spleen and transplanted donor cells. A splenectomy before HSCT may be required due to its potential to remove the source of extramedullary PK-deficient erythropoiesis and prevent sequestration of infused HSCs. The boy received a splenectomy before HSCT, whereas his sister received HSCT but ultimately underwent spleen removal. Therefore, a reciprocal sequence of the therapeutic approach shows that the combination of HSCT and splenectomy could be an advantageous therapeutic strategy for patients with severe transfusion-dependent PK deficiency. Recently, nonmyeloablative transplantation has been attempted with different degrees of preparative regimen intensity in patients with severe congenital hemolytic anemia.15 We successfully performed HSCT with reduced intensity conditioning for two children with PKLR mutations. They have remained in good condition while being treated with interim iron chelating therapy (phlebotomy) and have shown complete chimerism without GvHD, indicating that PK deficiency is an applicable condition for nonmyeloablative transplantation.
In this study, we efficiently diagnosed patients with null PKLR mutations using WES and successfully treated them by HSCT combined with a splenectomy. This trial provides a basis to establish a diagnostic and tailored treatment strategy for patients with deleterious PKLR mutations in the genetic era.
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We are grateful to the patients and their parents, and The Catholic Genetic Laboratory Center for assisting us in carrying out this study and compiling this report. This study was supported by a grant of the Korea Health technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A120175 and HI14C3417).
The authors declare no conflict of interest.
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Kim, M., Park, J., Lee, J. et al. Hemolytic anemia with null PKLR mutations identified using whole exome sequencing and cured by hematopoietic stem cell transplantation combined with splenectomy. Bone Marrow Transplant 51, 1605–1608 (2016) doi:10.1038/bmt.2016.218
Blood Research (2017)