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Case Report

Case Report: Paroxysmal nocturnal hemoglobinuria in a woman heterozygous for G6PD A-

[version 1; peer review: 2 approved with reservations]
PUBLISHED 13 Aug 2014
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Abstract

We describe a case of paroxysmal nocturnal hemoglobinuria (PNH) in a woman who is heterozygous for the glucose-6-phosphate dehydrogenase A-   (G6PDA-) allele. PNH is associated with one or more clones of cells that lack complement inhibition due to loss of function somatic mutations in the PIGA gene.  PIGA encodes the enzyme phosphatidylinositol glycan anchor biosynthesis, class A, which catalyses the first step of glycosylphosphatidylinisotol (GPI)  anchor synthesis. Two GPI anchored red cell surface antigens regulate complement lysis. G6PD catalyses the first step of the pentose phosphate pathway and enzyme variants, frequent in some populations have been because they confer resistance to malaria, are associated with hemolysis in the presence of oxidizing agents including several drugs. The patient had suffered a hemolytic attack after taking Bactrim, a drug that precipitates hemolysis in G6PD deficient individuals. Since both G6PD and PIGA are X-linked we hypothesized that the PIGA mutation was on the X-chromosome carrying the G6PDA- allele. Investigations showed that in fact the PIGA mutation was on the X-chromosome carrying the normal G6PD B allele. We speculate that complement activation on G6PD A- red cells exposed to Bactrim might have triggered complement activation inducing the lysis of G6PD B PNH Type II red blood cells or that the patient may have had a PNH clone expressing G6PDA- at the time of the hemolytic episode.

Introduction

In paroxysmal nocturnal hemoglobinuria (PNH) one or more clones of blood cells develops from stem cells that have an acquired mutation in the X-linked PIGA gene1. The PIGA gene encodes phosphatidylinositol glycan complementation class A, an enzyme that catalyses an early and essential step in glycosylphosphatidylinositol (GPI) anchor synthesis. Thus cells are deficient in all GPI anchored proteins, including CD55 and CD59 which regulate complement activation. PNH usually develops in patients with aplastic anemia (AA) and it is thought that PNH cells have a growth or survival advantage over the AA cells although the mechanism is not known2. PNH cells can be completely deficient in GPI anchored proteins (Type III) or partially deficient due to residual activity of the PIGA protein (Type II), while PNH Type I cells express GPI-linked proteins normally.

Clinically, PNH is characterized by bone marrow failure, thrombosis and intravascular hemolysis. Recently the use of a complement inhibitor, eculizumab has greatly improved the quality of life of PNH patients as it causes a dramatic reduction in the hemolysis and thrombotic episodes, improvement in anemia, with a stabilization of the hemoglobin levels and reduced transfusion requirements3. Eculizumab leads to an increase in the number of circulating red blood cells that otherwise are subject to complement-mediated hemolysis4.

Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency is the most common red blood cell enzymopathy and is estimated to affect around 400 million people worldwide5. It is caused by mutations in the X-linked G6PD gene which usually lead to an unstable enzyme. G6PD is needed to maintain NADPH and consequently reduced glutathione levels in red blood cells. G6PD-deficient people, mainly males, can be asymptomatic but are subject to episodes of hemolysis when the red blood cells are subjected to oxidative stress caused by infections, certain drugs or in the case of favism, after eating fava beans6. Several polymorphic variants have been described with specific geographical distributions7. In the African population the most common deficient variant is the G6PD A- variant. Compared with normal G6PD, which is called G6PD B, G6PD A- has two amino acid substitutions Val68Met and Asn126Asp8. These are caused by mutations c.202 G->A and c.376A->G respectively. G6PD A- has a frequency of about 10% in Africans and African Americans. G6PD A differs from G6PD B only by the Asn126Asp change and is electrophoretically distinct but with no significant difference in activity. Though milder than other variants such as G6PD Mediterranean found in Italy, Greece and India, G6PD A- is associated with drug induced hemolysis and patients are advised against taking any substances from a list of those known to cause hemolysis. G6PD deficiency usually only affects hemizygous males and homozygous females but heterozygous females can be affected when, for example, biased X-inactivation has led to a predominance of red blood cells expressing the mutant protein9. Here we present a case of an African American woman who was heterozygous for G6PD deficiency and developed PNH, presenting an opportunity to observe the interaction of these two conditions.

Materials and methods

Peripheral blood from patient CHOP277.01 was obtained after obtaining written informed consent according to the declaration of Helsinki. The Internal Review Board of the Hospital of the University of Pennsylvania approved this study. DNA and RNA were extracted by using QIAamp DNA and RNA Blood mini Kits, respectively, according to manufacturers’ instructions. Blood samples for fluorescent cytometry and electrophoretic analyses were obtained from EDTA tubes and experiments were performed within 2 hours of blood withdrawal.

PCR primers to detect mutations confirming the G6PD A- genotype were designed with Primer3 v4.0. (primers for c.202 G->A mutation: forward 5’- agaagaagatctaccccaccatct-3’ and reverse 5’- ctggtacagagggcagaaccag-3’; primers for c.376A->G: forward 5’-catctgtctgtgtgtctgtctgtc-3’ and reverse 5’- ctcatagagtggtgggaggac-3’). Sanger sequencing was done by the Nucleic Acids core facility at CHOP.

The HUMARA assay was performed as previously described10. Briefly, HhaI digested and non digested DNA was subjected to PCR amplification of the first exon of the HUMARA locus (containing a CAG repeat) using fluorochrome-coupled primers. Amplification products were then migrated on an ABI PRISM 3100 Automatic Genetic Analyzer (Applied Biosystems). Allele calling and the area under the curve (AUC) were determined using GeneMapper v.4.0 software (Applied Biosystems). The AUC was used to calculate the skewing from X chromosome inactivation (XCI) The XCI ratio of the digested fraction was corrected with that of the undigested fraction to allow for preferential amplification of the smallest allele (i.e., the allele containing less CAG repeats). Skewing is present when the percentage of the predominant allele exceeds 74%. A percentage of predominant allele between 90% and 100% is considered extreme skewing.

Measurements of oxidative stress ROS assay was performed as previously described11. Briefly, red blood cells were incubated with 20-70-dichlorofluorescein diacetate (DCF; Sigma) dissolved in methanol. After incubation at 37ºC for 15 minutes in a humidified atmosphere of 5% CO2 in air, the cells were washed, resuspended in PBS and analyzed by flow cytometry (FACSCalibur; Becton-Dickinson, Immunofluorometry Systems, Mountain View, CA, USA). The mean fluorescence channel (MFC) was calculated by FACSDiva software. The identity of the red cell population was verified by staining with an antibody anti to glycophorin-A. To determine the presence of GPI proteins, cells were labeled with a phycoerythrin-conjugated anti-CD55 antibody. For our experiment, cells from a non PNH- non G6PD individual served as control. The MFC of cells stained with DCF, is proportional to generation of ROS.

The electrophoretic mobility of the protein was performed in cellogel strips as previously described12. Hemolysates treated with and without acidified serum were run in order to assess differences in mobility of the G6PD enzyme.

Case report

The patient, a 23 year old African American woman, presented with left upper abdominal pain, vomiting and blurry vision. She had a four year history of episodes of hemolysis, abdominal pain and dark urine.

She was diagnosed with acute hemolytic exacerbation of PNH and was admitted. The patient was found to have anemia, leukocytosis and mild transaminitis. A Magnetic Resonance Venogram (MRV) of the abdomen and pelvis revealed several ill-defined low attenuation lesions of the posterior segment of the right hepatic lobe consistent with liver thrombosis so she was started on anticoagulation a concomitant urinary tract infection was treated with Trimethoprim-Sulfamethoxazole (Bactrim) and she was discharged.

After discharge, she followed up with a hematologist. On review of systems, the patient complained of a hemolytic attack with dark urine after taking Bactrim. On physical exam, she was found to have mildly icteric sclera. On further questioning of family history of other hematologic disorders, the patient said that one of her nephews has Glucose-6-Phosphate-Dehydrogenase deficiency. Because of her diagnosis of PNH and abdominal thrombosis, she was started on Eculizumab. The laboratory workup revealed a white blood cell count 4.6 k/uL, RBC count of 3.8 k/uL, hemoglobin of 11.8 g/dL, hematocrit 35%, MCV 91 fl, platelets 327 k/uL, reticulocytes 24.5%, PTT 27.8, INR 1.7, D dimer 2.5 ug/mL, bilirubin 1.3 mg/dL, bilirubin direct 0.2 mg/dL, bilirubin indirect 1.1 mg/dL, ANC 2.7 k/uL, ALC 1.3 k/uL, G6PD screen normal, LDH 720 U/L. The flow cytometry at this point revealed: 69% of the red blood cells were partially deficient for CD59 and 9.1% of the red blood cells were completely deficient for CD59; 86.3% of the granulocytes were partially deficient for CD59 and 6.4% were completely deficient for CD59.

Results

The family history and the case history suggested that the patient may have both G6PD deficiency and PNH, since the G6PD deficiency might explain the hemolysis precipitated by Bactrim, a drug that is reported to cause hemolysis in G6PD patients. Sequencing of DNA from her granulocytes confirmed that she was heterozygous for G6PD A- having a G6PD B allele on one X-chromosome and a G6PD A- allele on the other. This finding raised the question as to whether the somatic PIGA mutation causing her PNH took place on the X-chromosome carrying the B or the A- G6PD gene. The flow cytometry data showed that the patient most likely had 2 PNH clones, a class I clone (partial deficiency) of about 86% and a class II clone (complete deficiency) of about 6%. The HUMARA assay, which measures X-inactivation, showed a single clone of about 90% (Figure 1), suggesting that in both clones the mutation had taken place on the same X-chromosome. We hypothesized that the mutations in PIGA would have taken place on the chromosome carrying the G6PD A- allele since this would help explain the patient’s reaction to Bactrim. Another factor was that treatment with eculizimab, by inhibiting lysis of PNH red cells may have led to a higher level of PNH (and concomitantly G6PD deficient) red cells than would be present in untreated patients. To determine which G6PD allele was expressed in the PNH clone we sequenced cDNA from the patient’s granulocytes. The sequencing trace showed that the vast majority of expressed G6PD cDNA contained the wild type (G6PD B) sequence at both nucleotides where it differs from G6PD A- (Figure 2), leaving us to conclude that the PIGA mutations had taken place on the X-chromosome containing the G6PD B allele. This was confirmed at the protein level since the red blood cells lysed by acidified serum (the PNH cells) contained most of the G6PD activity while the residual cells did not contain detectable G6PD activity. While developing our hypothesis, which turned out to be incorrect, we also considered whether PNH/G6PD A- cells might have high levels of oxidative stress since both G6PD deficiency and PNH have been shown to be associated with elevated levels of reactive oxygen species11,13. We found that the patient’s red blood cells contained ROS levels that were significantly higher than those from healthy controls, though surprisingly we did not detect any difference in ROS between PNH (CD55-) and normal (CD55+) cells (Figure 3).

b576501f-7b03-4b47-8d46-e413074a080f_figure1.gif

Figure 1. Clonal hematopoiesis in patient CHOP 277.01.

The panel at the bottom shows the migration of the 2 microsatellite alleles in patient CHOP277.01 revealed by PCR analysis of a region of the Androgen receptor gene on the X-chromosome. The amplified region contains both a polymorphic repeat and a site for the methylation sensitive restriction enzyme HhaI. This site is methylated on the inactive X-chromosome. The top panel shows the same sample digested with HhaI before the PCR so only the fragment on the inactive X-chromosome is amplified. An imbalance in the allelic ratio reflects an imbalance in X inactivation and therefore indicates clonality.

b576501f-7b03-4b47-8d46-e413074a080f_figure2.gif

Figure 2. Patient CHOP 277.01 is heterozygous for G6PD B/A- and the B allele is predominantly expressed.

A1 and A2 shows sequencing of genomic DNA around the 202 G->A and the 376 A->G mutation that lead to Val68Met and Asn126Asp changes in G6PD, respectively. Figures B1 and B2 show the cDNA sequence of the exact same mutations in the peripheral blood of the patient. The WT (B) allele is expressed in the majority of cells.

b576501f-7b03-4b47-8d46-e413074a080f_figure3.gif

Figure 3. Flow cytometry analysis of the oxidative status of RBCs in the patient and a healthy donor.

Peripheral blood mononuclear cells from the patient and a normal control were treated with the oxidation sensitive dye, CM-H2DCFDA, and the conversion to its oxidized fluorescent derivative assessed by flow cytometry. The fluorescence distribution histogram and the mean fluorescence channels (MFC) of each population derived from the normal control (orange) and the patient (blue for CD55 negative and red for CD55 positive cells) are shown.

Discussion

PNH is a rare condition, having an incidence of about 1 in a million, so the co-incidental finding of a female with PNH and heterozygous for G6PD deficiency was an opportunity to observe the interaction between these 2 conditions which both involve red blood cell hemolysis mediated by X-linked genes. Notably the first demonstration that PNH was a clonal disease took advantage of a female PNH patient who was heterozygous for the electrophoretic variant G6PD A14. In this patient both isozymes were present in a lysate of total red blood cells, but only one was present after acidified serum lysis, demonstrating clonality of the PNH cells.

In the case discussed here a female African American patient with PNH suffered episodes of hemolysis, often following treatment with Trimethoprim-Sulfamethoxazole (Bactrim), one of the drugs that is known to cause hemolysis in G6PD patients15. When it emerged that she was heterozygous for G6PD A- we hypothesized that her expanded PNH clones may be expressing only the G6PD A- protein. The hypothesis proved incorrect and the clone expressed the wild type G6PD allele. A possible explanation is that complement activation on G6PD A- red cells exposed to Bactrim might have triggered complement activation inducing the lysis of G6PD B PNH Type II red blood cells16. Alternatively since PNH patients often have several clones17 which change in prevalence it is possible that earlier she did have a G6PD A- clone but this was replaced by wild type clones. Finally we can also speculate that the combination of G6PD and PIGA deficiency confers a serious growth disadvantage and PNH clones in this situation are more likely to be G6PD wild type. There is no clear mechanism for this however as G6PD A- nucleated cells have similar enzyme activity to WT cells18 – the deficiency becoming apparent in red blood cells, which do not synthesize new protein19.

Patient consent

Written informed consent for publication of their clinical details was obtained from the patient.

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Perdigones N, Morales M, Mason P and Bessler M. Case Report: Paroxysmal nocturnal hemoglobinuria in a woman heterozygous for G6PD A- [version 1; peer review: 2 approved with reservations]. F1000Research 2014, 3:194 (https://doi.org/10.12688/f1000research.4980.1)
NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.
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ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions
Version 1
VERSION 1
PUBLISHED 13 Aug 2014
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Reviewer Report 04 Sep 2014
ANASTASIOS KARADIMITRIS, Department of Haematology, Imperial College Healthcare NHS Trust and Imperial College, London, UK 
Approved with Reservations
VIEWS 28
Perdigones and colleagues describe the rare co-occurence in the same patient of two X-linked disorders, one acquired the other inherited, both causing intravascular haemolysis.
 
However how the interaction of the two disorders led to the clinical episode described in the case ... Continue reading
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HOW TO CITE THIS REPORT
KARADIMITRIS A. Reviewer Report For: Case Report: Paroxysmal nocturnal hemoglobinuria in a woman heterozygous for G6PD A- [version 1; peer review: 2 approved with reservations]. F1000Research 2014, 3:194 (https://doi.org/10.5256/f1000research.5317.r6022)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
  • Author Response (F1000Research Advisory Board Member) 15 Oct 2014
    Philip Mason, The Children's Hospital of Philadelphia, USA
    15 Oct 2014
    Author Response F1000Research Advisory Board Member
    It appears that the majority of the PNH clone (in both granulocytes and red cells) is type II, i.e., only partially deficient of GPI. One assumes that this picture was ... Continue reading
COMMENTS ON THIS REPORT
  • Author Response (F1000Research Advisory Board Member) 15 Oct 2014
    Philip Mason, The Children's Hospital of Philadelphia, USA
    15 Oct 2014
    Author Response F1000Research Advisory Board Member
    It appears that the majority of the PNH clone (in both granulocytes and red cells) is type II, i.e., only partially deficient of GPI. One assumes that this picture was ... Continue reading
Views
24
Cite
Reviewer Report 28 Aug 2014
Rosario Notaro, Laboratory of Genetics and Gene Transfer, Core Research Laboratory, Istituto Toscano Tumori, Florence, Italy 
Approved with Reservations
VIEWS 24
Perdigones and collaborators report an interesting clinical case about the association of PNH and G6PD deficiency. However, the timing of the clinical history is not clear and the report is scanty of some relevant clinical/laboratory data.

Major scientific points.
  1. The Authors should
... Continue reading
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CITE
HOW TO CITE THIS REPORT
Notaro R. Reviewer Report For: Case Report: Paroxysmal nocturnal hemoglobinuria in a woman heterozygous for G6PD A- [version 1; peer review: 2 approved with reservations]. F1000Research 2014, 3:194 (https://doi.org/10.5256/f1000research.5317.r5816)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
  • Author Response (F1000Research Advisory Board Member) 15 Oct 2014
    Philip Mason, The Children's Hospital of Philadelphia, USA
    15 Oct 2014
    Author Response F1000Research Advisory Board Member
    Perdigones and collaborators report an interesting clinical case about the association of PNH and G6PD deficiency. However, the timing of the clinical history is not clear and the report is ... Continue reading
COMMENTS ON THIS REPORT
  • Author Response (F1000Research Advisory Board Member) 15 Oct 2014
    Philip Mason, The Children's Hospital of Philadelphia, USA
    15 Oct 2014
    Author Response F1000Research Advisory Board Member
    Perdigones and collaborators report an interesting clinical case about the association of PNH and G6PD deficiency. However, the timing of the clinical history is not clear and the report is ... Continue reading

Comments on this article Comments (0)

Version 2
VERSION 2 PUBLISHED 13 Aug 2014
Comment
Alongside their report, reviewers assign a status to the article:
Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions
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