Description of a novel missense mutation of glucose-6-phosphate dehydrogenase gene associated with asymptomatic high enzyme deficiency
Angelo Minucci, Paola Concolino, Mirca Antenucci, Concetta Santonocito, Franco Ameglio, Cecilia Zuppi, Bruno Giardina, Ettore Capoluongo
Laboratory of Clinical Molecular Biology, Department of Biochemistry and Clinical Biochemistry, Catholic University, Largo F. Vito 1, 00168, Rome, Italy
Received 5 March 2007; received in revised form 26 March 2007; accepted 29 March 2007
Available online 20 April 2007
We report a case of an asymptomatic young subject affected by severe deficiency of Glucose 6-phosphate dehydrogenase (G6PD) activity. A novel genetic mutation (G130A) in the third exon was found. We named this novel mutation the “G6PD RIGNANO variant”. These findings may contribute to a better knowledge of molecular epidemiology of the G6PD mutation and may represent an additional variant to be studied for a deep comprehension of in vivo compensation mechanisms of G6PD deficiency.
© 2007 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved
Keywords: Favism; G130A mutation; G6PD Rignano; Ala44Thr
Glucose-6-phosphate dehydrogenase (G6PD) is a highly polymorphic enzyme encoded by a human X-linked gene (Xq2.8) . This enzyme catalyses the first step of the pentose phosphate pathway converting glucose 6-phosphate to 6-phosphogluconate with production of NADPH. This deficiency is the most common human metabolic inborn error affecting more than 500 million people world wide [1–3]. The principal clinical signs of G6PD deficiency are an acute hemolytic anemia and jaundice induced by ingestion of oxidative drugs and/or broad beans. In Italy, the most common variant, associated with favism, is that called “Mediterranean”. Many variants of deficient G6PD have been described. Molecular studies have shown that the G6PD deficiencies are nearly always caused by single amino acid substitutions. In addition, it is very important to underline the fact that some of these variants (third type of variant)  may be asymptomatic in adult subjects (male and females), since they do not present hemolysis. These asymptomatic carriers are often discovered by means of routine screening or found in large population samples collected, as normal controls, for research purposes.
In addition, the generation of NADPH and the reduction of glutathione are variably impaired in these subjects, depending on the type of G6PD mutation. Mutations associated with chronic hemolysis tend to cluster near the NADP-binding domain of the G6PD gene, while those associated with acute intermittent hemolysis or no hemolysis are scattered in the remaining part of the gene [4,5].
Herein we report a case of severe in vitro G6PD deficiency, associated with a novel genetic mutation, discovered by chance in an asymptomatic subject born in the Italian Lazio Region.
Design and methods
A 21-year man, without history of favism or episodes of hemolysis was addressed to our Laboratory for a general screening finalized to the admission at Police School. In fact, before school admission, the Italian national protocols require the execution of a general screening for the following serum biochemical and blood hematological parameters: glucose, urea, creatinin, Na+, K+, Ca2+, lactate dehydrogenase, uric acid, ferritin, transferrin, iron, bilirubin, amylase, G6PD activity and finally whole blood count evaluation. This screening is necessary for a careful evaluation of the healthy status of these cadets. In addition, this subject reported that he had no problems eating fava beans or when treated with drugs contraindicated in favism.
All the above mentioned laboratory biochemical tests (performed by means of Olympus reagents on AU2700 instrumentation, Olympus SpA, Italy) resulted within the normal ranges, with the exception of total bilirubinemia (1.3 mg/dL; our normal values 1.1), direct bilirubinemia (0.5 mg/dL; our normal values b0.3) and G6PD activity (41.9 UI/Red blood cells; 1.46 UI/Hb). The modestly higher bilirubin value disappeared after one week and was not linked to other past hyperbilirubinemic serum levels.
G6PD activity was performed on a deleucocytated blood sample, following the Literature indications, and using a standard method for the enzymatic activity normalization .
In particular, the G6PD assay was performed by means of commercially available kit for in vitro diagnostic use (Trinity, Biotech). This commercial procedure is a spectrophotometric method which determines the G6PD activity measuring the formation of NADPH molecule (reading its increasing absorbance at 340 nm) . The red blood count (RBC), performed by XT-2100IR instrumentation (Dasit Srl, Milano, Italy), gave the following results: total RBC=5.26 million/ìL and Hemoglobin=15.9 g/dL.
On the basis of these results, we suggested this cadet to be subjected to the G6PD genetic test. After informed consent was given, genomic DNA was extracted from the proband's blood sample (High pure PCR Template Preparation kit, Roche).
Therefore, we performed a preliminary genetic screening including only the most frequent G6PD mutations affecting the Italian population (eight mutations). Since this test resulted negative, we decided to sequence the entire G6PD gene. The direct sequencing of the whole gene was consequently performed following the literature indications . The NC-000023 genetic reference was employed for the primer design and for the sequence alignment. All sequence amplicons were run on ABI Prims 3100 Avant (Applied Biosystems) using the Big Dye terminator technique.
The direct sequencing analysis of exons and intron–exon junctions, performed on the proband, did not show the presence of any mutation, with the exception of the remaining exon3 which presented the G130A (Ala44Thr) mutation (see Fig. 1).
This missense mutation was in close proximity to the G6PD coenzyme binding domain. In particular, this domain corresponds to the last amino acid of the GxxGDLx fingerprint coenzyme binding domain (residues 38–44) . To confirm this mutation, we also tested the mother and the brother of this individual. The same results were obtained on the 45-year old mother (who gave G130A heterozygous) and from the second son. As expected, both individuals presented an impaired G6PD activity (see Table 1). The RBC and the hemoglobin values, for the proband's mother and brother, were 4.7 million/ìL, 5.10 million/ìL, 15.0 g/dL and 14.7 g/dL, respectively. In each assay, a normal control sample (included in the kit) was employed, with a mean value of 1360±89 obtained in our Laboratory. A deeper familial genetic analysis was not possible to perform, due to the fact that the maternal grandfathers died and no other relatives were available.
Values of G6PD activity assayed in patients and normal controls
| Individuals || Activity (UI/L) || UI/RBC || UI/Hb |
| Proband || 221 || 41.9 || 1.46 |
| Mother || 560 || 117 || 3.7 |
| Brother || 234 || 45.8 || 1.59 |
| Normal control * || 1410 || – || – |
This normal control was always used before starting the analytical procedure.
Normal G6PD ranges for our Laboratory: UI/RBC=220–550; UI/Hb=7–18.
* The normal control sample (namely Normal level) is included in the commercially kit and consisted in a lyophilized sample containing G6PD enzyme in a stabilized human red cell hemolysate base.
More than 300 G6PD variants have been defined [1,2]. Most of them are considered sporadic but various occur at a high frequency. G6PD variants are generally subdivided into three different categories in relationship with the type of hemolysis determined by single specific mutations. The most common endemic variants are generally associated with acute intermittent hemolytic anemia, while those producing chronic hemolytic anemia are very rare. In this case, the degree of hemolysis is highly variable, ranging from mild to transfusion dependent cases . The third type of variant is associated with no evident risk of hemolysis .
Although the protective role of G6PD deficiency against malaria is still under debate, the geographic distribution of gene frequency of these deficient variants overlaps closely with the prevalence of malaria .
In the last years, various studies reported the discovery of novel mutations associated with different degrees of G6PD deficiency. In particular, some of these have indeed been reported as asymptomatic cases .
In the present paper, we report a case of asymptomatic patient with high in vitro G6PD deficiency, carrying a novel inherited mutation at position G130A (Ala44Thr), which we called as “G6PD RIGNANO variant”, from the name of a small town named Rignano Flaminio (Lazio, Italy). In fact, the mother presented a family name closely restricted to this geographic area of Italy. Thus, we can hypothesize the presence of a founder effect in this area. These data may be interesting considering that they may represent another condition to be studied for a better comprehension of G6PD biology and molecular epidemiology.
A possible limit of this paper may be represented by the fact that we have not assayed the pyruvate kinase activity (PK), since this method is not available in our Laboratory. In particular, the G6PD/PK ratio is reported as a reliable method able to differentiate G6PD-heterozygous individuals from the normal population .
Further studies are necessary for a better knowledge of the following data: (1) the prevalence of this novel mutation in the Central Italian areas that in the past suffered from malaria; (2) the possible mechanisms of compensation of G6PD deficiency, resulting in an apparent absence of the typical clinical features of favism disease.
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