Red Blood Cell Enzymopathies

Introduction

G6PD deficiency was the first described and is the most common and best-studied RBC enzyme deficiency. G6PD deficiency is more common where Plasmodium falciparum malaria is or has been endemic. It was discovered in the 1950s as a result of investigations into a self-limited hemolysis that occurred after administration of the antimalarial drug primaquine, most commonly in individuals of African or Mediterranean ethnic origin. These early studies also determined that G6PD deficiency is X-linked. Subsequent studies in carrier females led to the discovery of X-inactivation, a phenomenon that has been exploited to study the hierarchy of hematopoiesis and the clonality of malignant neoplasms.

Epidemiology

G6PD deficiency is the most prevalent human enzyme deficiency in the world, affecting about 500 million people, although the vast majority of affected individuals never become symptomatic. Although most prevalent in individuals of African, Mediterranean, and southeast Asian ethnic origins, it has been found in almost every population. The highest prevalence is in the tropical belt of sub-Saharan Africa (>32%) and the Arabian Peninsula. In other populations, its prevalence ranges from less than 1 in 1000 among northern European populations to 50% of Kurdish Jews. The distribution across Asia is heterogeneous. Nearly 20% of males in Thailand are affected by one of the five prevalent variants. It is common in southern China (~5% in Hong Kong) and rare in other parts of China, while in India the prevalence varies from 0% to 27% in different regions, caste, ethnic, and linguistic groups. In the United States, G6PD deficiency affects about 10% of African American males, a lower proportion of individuals of Italian, Greek, Spanish, Corsican, and Sardinian ancestries and a variable proportion of Middle-Eastern and recent Asian immigrants. G6PD deficiency is virtually nonexistent among indigenous peoples of the Americas and Asian highlanders. The variable geographic distribution of G6PD deficiency implies that it confers a selective advantage and, as it coincides with the geographic distribution of endemic malaria, suggests protection from lethal malaria, although the exact mechanism has not been fully elucidated.

The wild-type enzyme is designated G6PD B. The most common G6PD low activity allelic variants in the United States are G6PD A− and G6PD Mediterranean; however, Asian variants are being encountered in the US population with increasing frequency. G6PD A− accounts for approximately 90% of G6PD deficient variants in Africa but is also prevalent in North and South America, the West Indies, Italy, the Canary Islands, Spain, Portugal, and the Middle East. The G6PD A− mutation (G202A; c.202 C>T) arose on a G6PD A + chromosome (A376G, c. C376C>T ); these two missense changes are in cis orientation. G6PD A+ has no obvious hematologic phenotype and has a gene frequency similar to that of G6PD A− among African Americans. G6PD Mediterranean is found in highest frequencies in the southern part of Italy, Greece, Spain, Sardinia and Corsica, as well as the Middle East, Iran, and the Arabian Peninsula, India, and Indonesia. G6PD Mediterranean is not homogeneous, but is composed of several distinct mutations, which G6PD Mediterranean (c.C563T) predominates. Several G6PD variants are pandemic in Asia. There are more than 100 different mutations in various Asian populations.

The high frequency of the most common G6PD variants and the diversity of the variants suggest selection of the variants, presumably because of protection from malaria, given the geographical distribution of these variants in areas of the world where malaria is or has been endemic. However, data on which genotype confers protection from malaria and the mechanism of the protection has been conflicting. The conflicting epidemiological data may be in part due to the large number of variants and phenotypical heterogeneity. The most common African variant G6PD A− (c.202 C>T) and possibly other variants associated with more severe G6PD deficiency is associated with a decreased risk of cerebral malaria in male hemizygotes and female heterozygotes, but an increased risk of severe malarial anemia in male hemizygotes and female homozygotes. The mechanism of malarial protection remains to be established. However, deficient cells infested with malaria parasites appear to be phagocytized more efficiently than normal cells. The host RBCs’ impaired ability to restore intracellular NADPH to maintain a high GSH/GSSG ratio may also mean that malarial parasites in G6PD-deficient RBCs are more vulnerable to ROS.

Pathobiology

G6PD is a housekeeping enzyme that catalyzes the oxidation of glucose-6-phosphate to 6-phosphogluconate in the first reaction of a pentose shunt, which reduces NADP ⁺ to NADPH (see Figs. 45.1 and 45.2 ). In RBCs, the pentose shunt is the only source of NADPH, which is crucial to maintaining high cellular levels of GSH to protect the cell from oxidative, stress-induced damage. Within the RBCs, oxidant injury leads to the oxidation of sulfhydryl (SH) groups on the hemoglobin molecule. This results in the formation of disulfide bridges (-S-S-), which in turn leads to decreased hemoglobin solubility and ultimately the irreversible precipitation of oxidized hemoglobin. Under normal conditions these oxidized -S-S- groups of hemoglobin and other RBC proteins are reduced by GSH to SH-groups by glutathione peroxidase, which in turn is oxidized to GSSG that is restored back to GSH by glutathione reductase in a reaction requiring NADPH. NADPH levels are maintained by G6PD, meaning in G6PD-deficient RBCs, GSH is not restored to adequate levels under oxidative stress, leading to a buildup of free radicals and insoluble hemoglobin within the cell. Precipitated hemoglobin is disruptive to the structure and function of the RBC membrane and leads to increased membrane permeability, osmotic fragility, and cell rigidity. This compromised integrity of the RBC membrane in G6PD-deficient RBCs results in extravascular hemolysis from the rapid removal of these cells within the splenic pulp and also in varying degrees of intravascular hemolysis.

The G6PD gene localizes to Xq28, spans 18 kb, and contains 13 exons. The active G6PD enzyme exists as a tetramer or dimer. Stability of the multimeric structures is crucial for optimal G6PD activity. G6PD activity decreases significantly as RBCs age, with a half-life of about 60 days. Reticulocytes have five times higher enzyme activity than the oldest RBC subpopulation. An even greater decrease in G6PD activity with aging is present in some mutant variants and is particularly pronounced in the African G6PD A− mutant.

G6PD variants can be divided into three categories based on the type of hemolysis they cause: acute intermittent (most common), chronic (rare), or none. The World Health Organization also classifies the different G6PD variants according to the degree of enzyme deficiency and severity of hemolysis. Class I deficiencies are the most severe and cause chronic hemolysis. Less severe G6PD Mediterranean is a class II deficiency. The even less deficient G6PD A− is a class III deficiency. Classes IV and V do not cause hemolysis and are of no clinical significance.

Over 400 variants of the G6PD enzyme have been identified by biochemical methods. However, this is likely an overestimate, as some previously described different mutations were found to be caused by the same mutation through molecular genetic studies. Currently, 237 G6PD mutations have been characterized by DNA sequencing in the Human Gene Mutation Database (HGMD, http://www.hgmd.org ; accessed June 23, 2020); 212 of these cause disease. The majority are missense mutations but small deletions (13), splicing mutations (4), and small indels and gross deletions (4) also exist. Only one regulatory domain substitution has so far been identified. Of these mutations, at least 100 have the prevalence of a polymorphism (a rate of at least 1% in tested population). Mutations associated with chronic hemolysis tend to cluster in the vicinity of the NADP-binding domain of the G6PD gene and cause more severe deficiency, whereas those associated with acute intermittent hemolysis or no hemolysis are scattered throughout the gene. Unlike disease-causing mutations of other genes, total gene deletions and insertions causing frameshift and stop codon mutations are not observed. These events would be expected to be fatal, since G6PD is a housekeeping gene essential for basic cellular functions.

Clinical Manifestations

Laboratory Manifestations

Under normal conditions, most G6PD-deficient individuals are not anemic and have no laboratory evidence of hemolysis. In the setting of oxidative stress, laboratory findings indicative of acute hemolysis including anemia and reticulocytosis are seen. As the hemolysis is mainly extravascular with a variable intravascular component, variable degrees of hyperbilirubinemia, increased LDH, and decreased haptoglobin occur. Heinz bodies may be visible in the erythrocytes during an acute hemolytic episode, but not under normal circumstances. “Bite cells” have been described and purported to be indicative of G6PD deficiency, but they are not specific for G6PD deficiency and these cells are usually not present in acute hemolytic states of patients with common G6PD variants or in G6PD-deficient patients with chronic hemolysis.

Individuals with the chronic hemolytic G6PD variants have varying degrees of anemia and reticulocytosis.

Diagnosis

A biochemical diagnosis of G6PD deficiency can be made using quantitative spectrophotometric analysis to measure the generation of NADPH from NADP in RBC hemolysates. A more convenient rapid fluorescent screening test can be used to test at-risk populations. False-negative results are not unusual, however, especially if enzymatic analysis is performed during or shortly after resolution of acute hemolytic episodes, or in heterozygous females. After acute hemolysis, reticulocytes and young RBCs, which have much higher enzymatic activity, predominate. These false-negative test results are more likely to occur when a screening test rather than a quantitative spectrophotometric analysis of the enzyme activity is used. However, this obstacle can be overcome when hexokinase (HK) activity, another glycolytic enzyme with higher enzymatic activity in reticulocytes and young RBCs than in aged RBCs, is concomitantly measured; the HK/G6PD enzyme activity ratio can then be used to diagnose or rule out G6PD deficiency.

Females heterozygous for G6PD are particularly difficult to diagnose because of their mosaicism for this X-chromosome enzyme and may have total RBC enzymatic activity ranging anywhere from hemizygote to normal. However, these females have a variable mixture of deficient and nondeficient RBCs and their deficient RBCs are subject to same hemolytic destruction as those of males. Since the nucleotide substitutions of most G6PD-deficient isoenzymes have been identified, molecular diagnostic methods are more reliable for the accurate diagnosis of females who are heterozygous for G6PD deficiency and also in males with ongoing or recent acute hemolysis, or after transfusion.

Severe sporadic variants causing chronic hemolytic anemia may be considered for prenatal diagnosis in some circumstances.

Prognosis

Most patients with G6PD deficiency have normal life spans with no clinical sequelae. Neonatal icterus with resultant kernicterus that may lead to neurological deficits and mental retardation has the gravest consequences.

Therapy

Treatment of an acute hemolytic crisis includes withdrawal of any offending agent and supportive care, which in severe cases includes RBC transfusions. Folic acid supplementation is advocated for patients with chronic hemolysis, but even without folic acid supplementation, significant folate deficiency rarely occurs. Neonatal icterus associated with G6PD deficiency is treated in the same manner as neonatal icterus arising from other causes; G6PD deficiency should be considered in any neonate with hyperbilirubinemia, especially those of high-risk ethnic descent. The chronic hemolytic anemia in some exceedingly rare G6PD variants may be severe enough to require chronic transfusions and iron chelation.

Future Directions

In certain areas of the world where G6PD deficiency reaches epidemic proportions and ingestion of fava beans is staple, screening for endemic G6PD-deficient variants and avoiding fava beans in those that are G6PD deficient reduces hospitalizations and RBC transfusions. Screening of neonates in endemic areas can also reduce mortality (from kernicterus).

Introduction

PK deficiency is the most common enzyme deficiency causing hemolysis. Although this disorder is far less common than G6PD deficiency, the vast majority of patients with G6PD deficiency never suffer a hemolytic episode, while PK deficiency has a high penetrance, although also a highly variable phenotype.

Genetics

PK enzymes consist of several isoforms. They are products of two distinct genes, PKLR and PKM located on different chromosomes. PKLR (encoding L, liver, and R, RBC isoenzymes) is located on chromosome 1q21. The R isoform, unique to RBCs, is 33 amino acids larger than the L isoform, which is unique to hepatocytes. Expression in RBCs versus liver is due to differential use of tissue-specific promoters, which drive expression as well as tissue-specific exon usage (use of exon 1 but not exon 2 in RBCs and exon 2 but not exon 1 in liver). Regulatory elements in PKLR -gene expression are not fully defined, but one is a key erythroid transcription factor, Kruppel Like Factor 1 (KLF1).

The PKM gene encodes the M (muscle) enzymes. There are two isoforms, M1 and M2, which are different splicing products of the PKM -gene’s single transcript. The M1 isoform is expressed in muscle, heart, brain. The M2 isoform initially predominates in fetal erythropoiesis but is progressively replaced in RBCs with the R form during fetal development. In contrast, M2 persists in leukocytes and platelets. M2 is overexpressed in many tumors and is present in other cells such as lung, fat, retina, and pancreatic islets. PKM proteins have co-stimulatory activity with hypoxia-inducible transcription factor 1 (HIF-1). The PKM gene is located on chromosome 15q22.

Thus far, 290 PKLR mutations have been characterized by DNA sequencing (Human Gene Mutation Database [HGMD, http://www.hgmd.org ; accessed June 23, 2020]). Of these mutations, 276 are disease-causing, most of which are missense mutations.

Epidemiology

PK deficiency is distributed worldwide but is more common among people of northern European extraction. PK deficiency is an autosomal recessive disease, and affected patients are typically double heterozygotes, or, less commonly, homozygous for the same mutation. Homozygous mutations are usually seen in groups with marked consanguinity, and homozygous PK deficiency has been well studied in the Amish populations of Pennsylvania and Ohio and also in an isolated fundamental Mormon settlement at the Utah/Arizona border. Common mutations have well-defined geographic associations. PKLR c.1529 G>A mutation is the most common mutation in the United States, northern and central Europe with a population prevalence in the US of ~50/million, while c.1456 C>T is the most common mutation in southern Europe, and c.1468 C>T in Asia.

PK deficiency does not localize to geographic areas of malarial endemicity. However, there is in vitro evidence that PK deficiency provides protection against infection and replication of Plasmodium falciparum in human RBCs, an effect possibly mediated by reduced ATP levels in PK-deficient RBCs. PK deficiency was also shown to be protective in a mouse model of infection with Plasmodium chabaudi .

Pathobiology

PK enzymes catalyze the final step in glycolysis: the irreversible transfer of phosphate from phosphoenolpyruvate to adenosine diphosphate (ADP), yielding one molecule of pyruvate and one molecule of ATP (see Fig. 45.1 ). PK is allosterically activated by the binding of fructose 1,6-diphosphate (FDP); that is, FDP binds to a site other than the active site for substrate binding, causing a conformational change in the PK enzyme. FDP is an intermediate in the glycolytic pathway, and higher concentrations of FDP increase PK activation (a process known as feedforward stimulation). PK deficiency results in decreased glycolytic ATP production and an accumulation of glycolytic intermediates, including 2,3-BPG.

PKLR mutations affecting the active catalytic site are associated with more severe hemolytic anemia. However, the phenotypical expression of identical mutations can be strikingly different, even within the same family. Clinical PK deficiency with hemolytic anemia is largely limited to mutations of the PKLR gene. However, a mutation in the transcription factor KLF1 that regulates PKLR transcription is a rare cause of PK deficiency (see below). Since PKM encoded proteins are present on leukocytes and platelets, using whole blood to analyze for PK deficiency may result in false-negative results.

PK-R exists as a heterotetramer. Since most PK-deficient patients are compound heterozygous for two different mutations, rather than homozygous for one, several different tetrameric forms of PK-R may be present, each with distinct structural and kinetic properties. This complicates genotype-to-phenotype correlations in these individuals, as it is difficult to infer which mutation is primarily responsible for deficient enzyme function and the clinical phenotype. There are even cases in which the activity of PK as measured in vitro is higher than normal, but a kinetically abnormal enzyme with low in vivo activity in erythrocytes is responsible for the hemolytic anemia. These mutations can only be detected if PK-enzyme activity is measured with and without FDP and at several concentrations of PK’s substrate phosphoenolpyruvate.

PK deficiency may be also caused by mutations not directly involving the PKLR gene. Mutations in the erythroid transcription factor KLF1 caused severe congenital hemolytic anemia because of a deficiency of PK. In this instance, the PKLR gene was intact and thus sequencing of the PKLR gene would miss the diagnosis of PK deficiency.

The mechanism of hemolysis in PK deficiency is not clear. The defect in ATP generation is unlikely to be the sole cause, as ATP deficiency is difficult to demonstrate in many patients and other disorders with more severe ATP deficiency are not associated with significant hemolysis. Increased apoptosis and ineffective erythropoiesis may be features of PK deficiency, although this has only been studied in splenic erythroid progenitors. Following splenectomy, despite decreased hemolysis and improved anemia, patients paradoxically have a higher number of reticulocytes, sometimes reaching over 50%. The reasons for this phenomenon is as yet unexplained.

Tolerance of Anemia

The anemia of PK deficiency is better tolerated than a comparable level of anemia seen in patients with hexokinase deficiency, since the block in glycolysis occurs after the Rapoport-Leubering shunt (see Fig. 45.1 ); (see section on 2,3-BPG deficiency). Levels of 2,3-BPG may be elevated up to two times normal in PK-deficient individuals, resulting in decreased hemoglobin oxygen affinity. This accumulation of 2,3-BPG shifts the oxyhemoglobin dissociation curve to the right, leading to better oxygen delivery to the tissue and improved tolerance of anemia than would be otherwise expected.

Clinical and Laboratory Manifestations