Glucose-6-Phosphate Dehydrogenase Deficiency: A Worldwide Potential Cause of Severe Neonatal Hyperbilirubinemia

Abstract

After completing this article, readers should be able to:Glucose-6-phosphate dehydrogenase (G6PD) deficiency, a commonly occurring X-linked genetic enzyme defect, is notorious for its association with acute hemolytic crises occurring in response to a frequently identifiable trigger (favism). Another potentially devastating danger of this condition is severe neonatal hyperbilirubinemia with its accompanying bilirubin encephalopathy, kernicterus, and death. Far from being a condition limited to historical time epochs and developing countries, G6PD deficiency-associated kernicterus still is seen in modern times. Indeed, among 80 infants from 21 states in the United States documented in a pilot Kernicterus Registry between 1984 and 1998, 18 (22.5%) were reported to have G6PD deficiency. Furthermore, Maisels recently listed G6PD deficiency among the 10 factors that are associated most commonly with an increased risk of nonhemolytic jaundice.Because of the association with favism, G6PD-associated hyperbilirubinemia traditionally has been regarded as hemolytic in origin. However, recent research has shown that although acute hemolysis does play a role, its contribution in many cases may be smaller than previously thought. The emphasis now has been placed on decreased bilirubin conjugation, with promoter polymorphism for the gene for the bilirubin conjugating enzyme, UDP glucuronoslytransferase 1A1 (UGT1A1), being a major factor in production of the icterus. In this review we highlight key aspects of the pathogenesis of this type of hyperbilirubinemia, explain the relevance of the condition, and describe a particularly problematic group that has a recently recognized high incidence of neonatal hyperbilirubinemia—the female heterozygote. Finally, we discuss the prevention and treatment program that we have developed at the Shaare Zedek Medical Center that has decreased the need for exchange transfusion.A major function of G6PD is its role in protecting cells against oxidative damage. Reduced glutathione plays a major part in this defense system by becoming oxidized, thereby neutralizing the damaging oxidative process. To play its detoxifying role, glutathione must be maintained in its reduced form and must be regenerated continuously from its oxidized form. The regeneration mechanism uses hydrogen ions obtained from nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH) (Fig. 1 ), which is formed from nicotinamide adenine dinucleotide phosphate (NADP) by G6PD catalyzing the first step in the hexose monophosphate pathway. The red blood cell (RBC) is at especially high risk for oxidative damage because the hexose monophosphate pathway is the only source of NADPH in these cells and because of the high concentrations of oxygen physiologically present in RBCs. In the absence of G6PD, NADPH will not be available for regenerating reduced glutathione, the oxidative process will not be counteracted, and RBC membrane damage and hemolysis will result.G6PD deficiency is the most common enzyme deficiency known and is estimated to affect hundreds of millions of people throughout the world (Fig. 2 ). Diverse population groups are affected, including Africans (G6PD A- mutation), southern Europeans and Middle Eastern populations (G6PD Mediterranean), and Asians (G6PD Canton). Many other mutations exist. With ease of travel and migration of large segments of many population groups, G6PD deficiency no longer is localized to those geographic areas to which the condition was indigenous; a newborn who has hyperbilirubinemia due to G6PD deficiency may be encountered in virtually any part of the world today.The apparently low overall frequency of G6PD deficiency in North America reflected in Figure 2 is deceptive because the frequency may be high within certain population subgroups. African-Americans comprise the largest affected population group in America; the incidence of G6PD deficiency in this population is as high as 11% to 13%. Neonates of this group traditionally have not been regarded as being at high risk for G6PD deficiency-associated hyperbilirubinemia, but more recent evidence has shown that these newborns may be especially endangered by the condition. We are aware of 13 North American cases of kernicterus in African-American G6PD-deficient neonates either fully documented or alluded to in recent medical literature. Additional North American population subgroups that have a high frequency of G6PD deficiency include Greeks, Italians (especially of Sardinian ancestry), Asians, Sephardic Jews whose families emigrated from Asia Minor, and Southeast Asians.In some cases, the hyperbilirubinemia caused by G6PD deficiency may be the result of acute hemolytic crises, similar to favism, triggered by indentifiable chemical agents, such as naphthalene, triple dye, Chinese herb remedies, menthol-containing umbilical potions, vitamin K3 (not vitamin K1), and henna. We recently reported two cases of “favism by proxy” in breastfed G6PD-deficient neonates whose mothers had eaten fava beans. Cases of hydrops in utero due to maternal ingestion of fava beans also have been described. However, in contrast to favism, in which strict avoidance of contact with this agent will prevent fava-induced hemolysis, lack of contact with the previously mentioned compounds does not eliminate the neonatal hyperbilirubinemia associated with G6PD deficiency. Furthermore, in many instances, the hyperbilirubinemia is not associated with a fall in hemoglobin or hematocrit and an increase in reticulocyte count, as would be expected in acute hemolysis. Moreover, not all G6PD-deficient neonates develop severe hyperbilirubinemia, and studies in Greek infants have demonstrated a variable incidence of hyperbilirubinemia in geographically separated population subgroups. These findings suggest the existence of additional genetic or environmental factors that either dampen or exacerbate hyperbilirubinemia in specific individuals.Until recently there has been no unanimity of opinion regarding the pathogenesis of hyperbilirubinemia in G6PD-deficient neonates. Some have suggested that increased hemolysis is sufficient to explain the hyperbilirubinemia and that liver dysfunction or immaturity should not be implied as having a greater role than the physiologically decreased bilirubin conjugating ability seen in normal neonates. Others have proposed that hepatic handling of bilirubin is a more important factor than increased hemolysis.It is important to remember that serum total bilirubin levels at any point in time represent a balance between bilirubin production and excretion. An infant who has acute hemolysis resulting in a high bilirubin production rate, but excellent hepatic elimination ability, may not develop significant bilirubinemia. On the other hand, minimally increased bilirubin production in the face of poor bilirubin conjugation or excretion may result in clinically consequential icterus.Hematologic changes commonly associated with hemolysis have not been seen consistently in neonates who have G6PD deficiency. In some instances, such as in Nigerian neonates, decreased hemoglobin (Hb) and hematocrit (Hct) values have been associated with acute hyperbilirubinemia, with those who developed kernicterus having even lower values. In many instances, however, there have been only minimal or no changes in hematologic indices. Paradoxically, some Greek neonates who required exchange transfusion had higher Hb values than those who had less severe hyperbilirubinemia. Sardinian neonates consistently have shown no significant differences in hematologic indices between those who developed hyperbilirubinemia and those who remained only minimally icteric. However, there may be considerable overlap in hematologic values between hemolytic and nonhemolytic states in the newborn, making these inaccurate indicators of hemolysis in this age group.Carbon monoxide (CO), released in equimolar quantities for every molecule of biliverdin, and thence bilirubin, produced from the catabolism of heme, combines with Hb to form carboxyhemoglobin (COHb), which is transported to the lungs and excreted. Because most CO in the body is produced from the breakdown of heme, accurate measurement of blood COHb, with correction for inspired room air CO (COHbc), will reflect endogenous production of CO. This, in turn, provides an accurate index of heme catabolism and, therefore, bilirubin production.Several studies have used this technique to assess the role of hemolysis in G6PD deficiency. Some have shown a contribution of increased hemolysis to the pathogenesis of the hyperbilirubinemia, while others showed no such correlation. In Greek G6PD-deficient neonates sampled on the third day of life, values greater than 1% (percentage of total Hb) were found in almost all of those who exhibited hyperbilirubinemia, contrasting to values of less than 1% for most of the G6PD-normal, nonhyperbilirubinemic infants in the control group. Three infants who developed kernicterus had COHb values ranging from 2.24% to 8.36%. In Nigerian neonates sampled on the fourth day of life, significantly higher COHb values were found in G6PD-deficient infants (2.3% [1.3% to 4.2%], median [interquartile range]) compared with controls (1.2% [1.0% to 1.7%]). Values correlated with the severity of the hyperbilirubinemia and were significantly higher among those who developed kernicterus (3.72% [2.13% to 5.93%]) than in those who did not (1.3% [0.43% to 5.44%]). In Sephardic Jewish neonates, COHbc levels sampled on the third day of life were higher in those who had G6PD deficiency than in normal controls (0.75±0.17% versus 0.62±0.19%, P<0.001). Whereas COHbc values correlated with the development of hyperbilirubinemia in the control group, there was no such correlation in the G6PD-deficient group. Possibly the most fascinating aspect of this study was the fact that COHbc values were increased to a similar extent in both G6PD-deficient neonates who developed serum total bilirubin values greater than 255 mcmol/L (15 mg/dL) and in those who remained only moderately jaundiced (Fig. 3 ). Therefore, although increased hemolysis clearly is a hallmark of G6PD-deficient neonates, it cannot be construed as the primary factor in the pathogenesis of the associated hyperbilirubinemia. Other factors must be working to a greater extent than increased bilirubin production.Physiologically, a small amount of conjugated bilirubin refluxes from the hepatocyte to the serum. Therefore, accurate assessment of the serum conjugated bilirubin fractions can be used to reflect intrahepatocytic bilirubin conjugation. Clinical laboratory methods of direct bilirubin measurement are not sufficiently accurate to accomplish this purpose, but a high degree of accuracy can be attained by using alkaline methanolysis followed by reverse-phase high-performance liquid chromatography. Kaplan et al used this method to compare serum conjugated bilirubin fractions in 29 G6PD-deficient neonates who had serum total bilirubin values of at least 255 mcmol/L (15 mg/dL) at a mean age of 76±22 hours with 35 G6PD-normal controls who had similar serum bilirubin values at a mean age of 80±16 hours. The serum diconjugated bilirubin fraction was significantly lower in the G6PD-deficient neonates than in the controls (median, 0.06 mcmol/L [range, 0.00 to 1.84 mcmol/L] and 0.21 mcmol/L [range, 0.00 to 1.02 mcmol/L], respectively) (P=0.006). Diglucuronide was unmeasurable in 38.9% of the G6PD-deficient neonates compared with 8.6% of controls (P=0.015). This pattern of conjugated bilirubin fractions was similar to that seen in adults who have Gilbert syndrome and was interpreted to reflect hepatic bilirubin conjugating ability, which was even more immature than that normally encountered in term neonates.In a second study, the same authors sampled serum for conjugated bilirubin fractions from G6PD-deficient neonates while serum bilirubin levels were rising and were in the range of 170 to 253 mcmol/L (10 to 14.9 mg/dL). By a process of natural selection, the infants separated into a nonhyperbilirubinemic group, in whom serum total bilirubin values did not exceed 253 mcmol/L (14.9 mg/dL) during the first week of life, and a hyperbilirubinemic group, whose serum bilirubin values subsequently rose to 255 mcmol/L (15.0 mg/dL) or more. At the time of sampling, the groups had similar serum total and unconjugated bilirubin values. However, conjugated bilirubin fractions, including total, monoconjugated, and diconjugated fractions, were significantly lower in infants who subsequently became hyperbilirubinemic compared with those who remained nonhyperbilirubinemic. These results imply that hepatic conjugation of bilirubin was significantly less efficient in those who became hyperbilirubinemic than in those who remained only moderately jaundiced. Decreased bilirubin conjugation capacity was, therefore, a crucial factor in the development of hyperbilirubinemia in G6PD-deficient neonates. A further investigation finetuned the genetic predisposition of those at risk for developing hyperbilirubinemia.Approximately 5% of most populations have Gilbert syndrome, a mild and benign form of bilirubinemia that is due to decreased activity of the bilirubin conjugating enzyme UGT1A1. The genetic basis for the condition has been identified as a polymorphism in the promoter for this gene in which the TATAA box contains seven (TA) repeats [(TA)7TAA] instead of the usual six [(TA)6TAA]. Patients who have Gilbert syndrome are homozygous for the abnormal promoter. The extra nucleotides decrease the expression of the UGT1A1 gene, leading to reduced enzyme activity and subsequent decreased bilirubin conjugation.It seemed logical to presume that neonates who had the homozygous form of (TA)7TAA would be at increased risk for hyperbilirubinemia, but an association between the Gilbert syndrome genotype and acute neonatal hyperbilirubinemia in healthy term infants has not been demonstrated. In contrast, in Sephardic Jewish G6PD-deficient neonates, the incidence of hyperbilirubinemia was significantly increased, in a stepwise, allele dose-dependent fashion, with the addition of one or two UGT1A1 genes with the variant promoter (Fig. 4 ). Neither the presence of G6PD deficiency in the absence of the variant UGT1A1 promoter nor the variant promoter in the absence of G6PD deficiency resulted in an increased incidence of neonatal hyperbilirubinemia. Crucial to the development of significant jaundice was the combination of G6PD deficiency and the presence of the UGT1A1 gene with the variant promoter. Because neither of these two factors alone acted as an icterogenic risk factor, the effect of the two genes in combination implies a gene interaction rather than a simple additive effect.Because G6PD deficiency is an X-linked condition, females are in the unique position of having three genotypes: normal homozygotes, deficient homozygotes, or heterozygotes. Because of X chromosome inactivation, heterozygotes have two erythrocyte populations: one G6PD-normal and one G6PD-deficient. In most cases, this division is approximately midway and such individuals will have intermediate enzyme activity. However, nonrandom X chromosome inactivation may result in as many as 10% of heterozygotes having a normal phenotype and a similar number having low enzymatic activity. Because of this apparent discrepancy, heterozyogotes cannot be distinguished on the basis of biochemical tests. As a result, females rarely have been included in studies of G6PD deficiency. Furthermore, the World Health Organization has suggested that heterozygotes have sufficient enzymatic activity to protect them from the dangers of G6PD deficiency, including neonatal hyperbilirubinemia.Modern molecular techniques have made it possible to identify the G6PD genotype. Because nonrandom X chromosome inactivation does not alter the mutant nucleotide sequence of the gene, heterozygotes now can be identified by DNA analysis. Using these techniques, Kaplan et al recently assessed the risk of developing significant hyperbilirubinemia (serum total bilirubin, ≥255 mcmol/L [15 mg/dL]) in a cohort of Sephardic Jewish female neonates at high risk for G6PD deficiency. Contrary to expectations, the incidence of hyperbilirubinemia followed a progression through the various G6PD genotypes, with both heterozygotes (22%) and deficient homozygotes (26.3%) having a significantly higher incidence of hyperbilirubinemia than the normal homozygotes (9.8%). Rather than being midway between the homozygote-normal and homozygote-deficient population, the incidence of hyperbilirubinemia in the heterozygotes was closer to that of the deficient homozygotes. However, COHbc values were unequivocally higher in only the G6PD-deficient homozygotes (0.74±0.14%, P=0.02), not in the heterozygotes (0.69±0.19%), when compared with the normal homozygotes (0.63±0.19%). Furthermore, high COHbc values were not a prerequisite for the development of hyperbilirubinemia in any of the G6PD genotypes. Increased hemolysis, therefore, could not be suggested as the primary factor in the etiology of the hyperbilirubinemia. A significantly greater incidence of hyperbilirubinemia was found among the G6PD-deficient heterozygotes who also had the UGT1A1 gene with the variant promoter in both its heterozygous and homozygous forms compared with those who had the normal UGT1A1 gene. This effect was not seen in those who were G6PD-normal homozygotes. Thus, even in the presence of a single G6PD-deficient gene, the pathogenesis of the hyperbilirubinemia seems to be mediated through its interaction with the variant UGT1A1 gene promoter.Several screening tests are available, some of which are qualitative and others of which are semiquantitative. Luzzatto recommends that an ideal screening test should not give false-negative results, but can be allowed to give a few false-positive results. In other words, G6PD-deficient neonates should not be misclassified as normal, but it is permissable for a few G6PD-normal individuals to be designated as G6PD-deficient. Ideally, an abnormal screening test result should be confirmed by a quantitative enzyme assay. The International Committee for Standardization in Hematology has recommended the fluorescent spot test as the preferred screening test for G6PD deficiency. The screening tests are usually accurate in males, but for the reasons stated previously, identification of female G6PD-deficient heterozygotes by simple biochemical tests is difficult. In the previously mentioned study, in which a commercial color reduction screening method was used, only 20% of the heterozygotes were identified correctly. Another recent comparison of three screening tests (the blue formazan spot test, the methemoglobin reduction test, and the fluorescent spot test) found all to be equally accurate in detecting male G6PD-deficient individuals, but none allowed the detection of 100% of heterozygotes. In light of the high incidence of hyperbilirubinemia now demonstrated in female heterozygotes, the poor performance of screening tests in this subgroup of the population is disturbing. Therefore, clinicians must maintain a high index of suspicion for G6PD deficiency when managing a jaundiced female infant who is a member of a high-risk ethnic group.Both classic hemolytic disease of the newborn due to Rh alloimmunization and G6PD deficiency are associated with increased hemolysis and the potential of kernicterus if untreated. Increased cord blood bilirubin levels, predictive of subsequent serum bilirubin concentrations, and decreased hemoglobin values are typical of Rh hemolytic disease. Kaplan et al determined whether hemolysis, anemia, and bilirubinemia of G6PD deficiency also commence in utero, which could be used to predict subsequent development of hyperbilirubinemia. Term male Sephardic Jewish neonates at risk for G6PD deficiency were studied by sampling serum total bilirubin, total Hb (tHb), and COHbc within 3 hours of delivery to reflect in utero status. A mandatory serum bilirubin determination was obtained again on the third day of life, with additional serum bilirubin determinations obtained as required by clinical indications. Hyperbilirubinemia (serum total bilirubin, ≥255 mcmol/L [15 mg/dL]) was present in 14/41 (34%) of the G6PD-deficient neonates compared with 6/111 (5.4%) of the controls (P<0.0001). Serum bilirubin values at the time of the first sampling were significantly higher (49±12 mcmol/L [2.9±0.7 mg/dL] versus 44±10 mcmol/L [2.6±0.6 mg/dL], P=0.02), tHb values significantly lower (11.47±2.42 mmol/L [185±39 g/dL] versus 12.4±1.43 mmol/L [200±23 g/dL], P=0.003), and COHbc values significantly higher (1.06±0.26% versus 0.81±0.22%, P<0.0001) in the G6PD-deficient neonates. On the third day of life, serum total bilirubin values still were significantly higher in the G6PD-deficient neonates than in controls (173±49 mcmol/L [10.2±2.9 mg/dL] versus 151±48 mcmol/L [8.9±2.8 mg/dL], P=0.01). COHbc values drawn within the first 3 hours did not correlate with simultaneously drawn bilirubin values, but first bilirubin values did correlate significantly with third-day serum bilirubin values (r=0.61, P<0.0001 and r=0.40, P<0.0001 for the G6PD-deficient and control infants, respectively).The authors concluded that hemolysis and bilirubinemia were greater in utero in the G6PD-deficient neonates than in controls and that these bilirubin values were predictive of subsequent serum bilirubin values. However, the degree of hemolysis and anemia contrasted strikingly to the severity noted in Rh hemolytic disease. Although increased COHbc values and diminished tHb concentrations clearly indicated ongoing hemolysis in utero, the degree of hemolysis did not correlate with serum bilirubin values. Therefore, hemolysis must have contributed to the in utero bilirubinemia, but it could not have been the sole or major factor in the pathogenesis. Other forces, such as decreased bilirubin conjugation in utero, altered placental bilirubin clearance, or deficient maternal bilirubin elimination, must have contributed to the in utero bilirubinemia as well.Because phototherapy frequently is introduced to prevent serum bilirubin values from reaching dangerous levels, serum bilirubin values may not reach their natural peak. It is, therefore, diffucult to compare the peak point of the hyperbilirubinemia with that of exaggerated physiologic jaundice or pathologic jaundice of causes other than G6PD deficiency. However, in the Sephardic Jewish female study, the jaundice did commence earlier in G6PD-deficient neonates compared with controls. Third-day serum bilirubin values were significantly higher in G6PD-deficient homozygotes and heterozygotes (204±51 mcmol/L [12.0±3.0 mg/dL] and 190±63 mcmol/L [11.2±3.7 mg/dL], respectively) than normal homozygotes (160±58 mcmol/L [9.4±3.4 mg/dL], P<0.01). By the third day, significantly more G6PD-deficient homozygotes (27.8%) had developed a serum bilirubin value of at least 255 mcmol/L (15.0 mg/dL) compared with normal homozygotes (4.9%, P<0.01). Once phototherapy had been started, Tan and Boey showed that the rate of decline of serum bilirubin was greater among those who had normal G6PD status. Significant rebound following discontinuation of phototherapy does not appear to occur commonly.Phototherapy is the mainstay of treatment for hyperbilirubinemic G6PD-deficient neonates. For several years we have been managing G6PD-deficient neonates according to the protocol outlined in the Table. The protocol involves frequent serum bilirubin determinations and earlier onset of phototherapy than many neonatologists institute. Except for occasional cases, such as infants of mothers who ate fava beans while nursing, this protocol has been successful in containing the rise of bilirubin. We have limited the number of infants requiring exchange transfusions to approximately 1 in 100 neonates who has a deficient-reading on the screening test that we encounter annually. Luzzatto is slightly more aggressive in his recommended management of severe hyperbilirubinemia, suggesting exchange transfusions if the serum bilirubin value exceeds 255 mcmol/L (15 mg/dL) during the first two days of life. The success of pre- or postnatal phenobarbital administration, a potent inducer of the bilirubin conjugating enzyme, which has been used in the prophylaxis of hyperbilirubinemia in Greek and Sardinian neonates, can be understood in the light of recent advances in our understanding of the pathogenesis of the jaundice.Because there is no correlation between bilirubin production and the development of hyperbilirubinemia in G6PD-deficient neonates, end tidal CO measurements, currently being evaluated for clinical use, will have a minimal role in the prediction of hyperbilirubinemia in these infants. However, timed serum bilirubin testing, with plotting of the value on a percentile nomogram adapted from that of Bhutani et al, successfully predicted hyperbilirubinemia not only in low-risk patients, but in G6PD-deficient neonates. In both groups, those whose serum bilirubin value was less than the 50th percentile for postnatal age had a very low risk of developing a serum bilirubin value of 255 mcmol/L (15 mg/dL) or greater; those who did develop hyperbilirubinemia had serum bilirubin values greater than the 50th percentile. Use of bilirubin screening should allow for a selective approach toward discharge. If adequate arrangements have been made for follow-up within 1 to 2 days, those G6PD-deficient neonates who are unlikely to develop hyperbilirubinemia can be discharged at 48 hours along with their G6PD-normal counterparts, and delayed discharge can be reserved for those who are at high risk for hyperbilirubinemia. (TableT1 ). G6PD deficiency is likely to cause severe hyperbilirubinemia and can be encountered by pediatricians or neonatologists practicing in all parts of the world. Major advances in the understanding of the pathophysiology of the condition demonstrate a striking contrast between this and other, more frequently encountered forms of neonatal hyperbilirubinemia. A high index of suspicion, liberal serum bilirubin testing as clinically indicated, early institution of phototherapy, and timely performance of exchange transfusion should be instrumental in preventing kernicterus in these patients.