Prevention is better than Treatment
Glucose-6-Phosphate Dehydrogenase Neonatal Screening : Preliminary evidence that a high percentage of partially deficient female neonates is missed during routine screening.
CHRISTINE J. HATZIDAKIS 1
and KLEOPATRA Η. SCHULPIS 2
1 R&D DIAGNOSTICS Ltd, R&D department, 41 El.Venizelou Street, 15561 Holargos, Greece and
2 INSTITUTE OF CHILD HEALTH, "Aghia Sophia" Children's Hospital, 11527 Athens, Greece.
Running Title : Deficient G-6-PD female neonates probably missed during routine screening.
Key Words : G-6-PD Deficiency, Female G-6-PD Heterozygotes
Glucose- 6-Phosphate Dehydrogenase deficiency is one of the most common genetic diseases affecting an estimated 400 000 000 people worldwide. In Greece, where this disease is endemic an estimated 225 000 males and 92 000 females are affected . Hematological problems arising in the G-6-PD deficient populations from exposure to oxidizing xenobiotics have been well characterized ranging from hemolysis of red blood cells and hereditary non -spherocytic hemolytic anemia to sepsis and life threatening kernicterus in the newborn   Neonatal screening, for this disease, has long been established in many countries and the method most commonly used is the semi-quantitative method described by Beutler  or modifications to this method . This method was believed to discriminate between deficient (partial or total deficiency) and normal cases.
We provide evidence that this is not true because of the very low cut - off point of this method, which can only discover the totally deficient cases (less than 20% residual enzymatic activity) classifying the partially deficient neonates (residual enzymatic activity between 20 and 60%) as normal. This is extremely important because the missed partially deficient female neonates (female heterozygotes) are considerably more than the diagnosed ones while the risk of developing hematological problems from exposure to oxidizing agents is not neglectable . A quantitation of G6PD (using a fully quantitative G6PD screening kit) is proposed, employing the automated Haemoglobin (Hb) normalization  in order to resolve this problem.
Glucose-6-Phosphate Dehydrogenase (G-6-PD) deficiency is by far the most common genetic disorder affecting more than 310 000 people in Greece. Neonatal screening for G6PD deficiency has long been established  . From 1977 to 1998, 2 070 557 neonates have been screened for this deficiency at the Institute of Child Health in Athens (Institute of Child Health, Aghia Sophia Children Hospital, Athens, Greece) . The overall frequency for Greek neonates was found to be 4.5% for males and 1.84% for females. These percentages are annually confirmed by the screening center. The overall frequency for the Greek population is calculated to be 3.17% . It is evident that G6PD deficiency seems to affect more boys than girls a finding that, in older days, tended to characterize this disorder as a "male" disorder. Furthermore, Table 1 shows the genetic combinations that lead to either partial or total deficient individuals. It is well known that, although G6PD deficiency is an X-chromosome linked disorder it is not a recessive one . This is especially important for heterozygous females since both chromosomes may become active at one time or another, therefore the overall residual G6PD activity is between 10 - 60% of the normal. This is characterized as "partial deficiency" and may lead to hemolytic attacks even severe ones .
As shown, the combinations that lead to females homozygous or heterozygous for G6PD (total or partial G6PD deficiency) are more than the ones that result in deficient males. This is mainly because males can't inherit the mutated X (Xmut)chromosome from their father as females. In this table, each genetic combination is examined on its own and the result is expressed as a percentage for each sex. Thus, in the first combination, in which both the mother and the father have normal (not mutated) X-chromosomes, the percentage of males (or females) with normal activity is 100. In contrast, in the second combination, in which the mother has one mutated chromosome, the percentage of normal males is 50 while that of deficient males is also 50. Females derived from this combination can be either normal (50%) or partially deficient (50%). The percentages (scores) that lead to either total or partial deficiency are then added and shown as the total ratios for the two sexes.
Concerning total deficiency, the scores are 400 for males and 200 for females or 2:1. This ratio for total deficiency (males/ females = 2 : 1) is almost identical to the ratio obtained from over 2 000 000 neonates screened in Greece . Since males are always deficient they are always picked during screening. Hence the percentage of 4.5% deficient males can't be disputed and can be used as a basis for calculations. If we apply the ratios calculated in Table 1 (male / female = 2 : 1) to the percentage of deficient males (4.5%) we conclude that the totally deficient (homozygous) females should be a theoretical 2.25%. This is very close to the 1.84% figure which is the frequency observed after 20 years of screening. On the other hand, if we make the same calculations for the total number of affected females (heterozygous and homozygous) the theoretical figure should be 6.8%. This, if true, would mean that 3.7 deficient females correspond to each female detected. In Greece alone this would bring the overall population of females not diagnosed during the last 20 years to more than 49 500.
The procedure followed till now is the commonly used collection of Gurthie cards with dried blood spots from the neonates which are then tested using a modification of the classic semi-quantitative Beutler method  . This method, as used, has some major disadvantages, which could be described as time consuming, temperature limited and lack of adequate clinical information . The most important drawback of this method, which is the most commonly used worldwide, is its low cut-off limit. It was believed that this limit was high enough to discover even partially deficient females .
The aim of this study was to provide evidence that this is not so. In fact, the method currently employed only finds total G6PD deficiency cases and misses all partially deficient ones. Additionally we provide preliminary evidence that most (if not all) partially deficient female neonates are regularly classified as normals. Click on the Journal of Medical Screening icon below for the full article.
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Page last edited on 05/07/2005