G6PD gene sequencing revealed the mutation c.637G>T (p.V213L) which is known as the Gastonia, Marion, or Minnesota variant and is clinically associated with CNSHA [8
]. This mutation is located at the dimer interface and disrupts dimerization causing decreased enzyme activity and a marked decline in intravascular erythrocyte survival [8
]. Another mutation, not previously reported in the literature or the Human Genome Mutation Database, c.1037A>T (p.N346I) was also identified in exon 9 of the G6PD gene, located in a hydrophilic loop at the tetramer interface (). In our patient, the hydrophobic side chain of isoleucine substitutes the hydrophilic side chain of asparagine, potentially disrupting the tetramer formation. This mutation, which we propose to name G6PD Cincinnati, affects a highly conserved amino acid residue of the enzyme and therefore it is unlikely to be an asymptomatic polymorphism (). Search in NCI human SNP database (http://www.ncbi.nlm.nih.gov/projects/SNP/snp_gf.cgi
) querying genotypes from a population of 870 individuals revealed no polymorphisms at this location. Of interest, the glutamic acid at the position 347 (E347), just next to N346 (), participates in one of the salt bridges between dimers to stabilize the tetramer, as deduced by crystallography studies [10
]. While several of the class I mutations are located at the dimer interface, there is one more class I mutation at the tetramer interface (E274K named G6PD Cleveland) that has been described so far [10
Figure 2 A. G6PD protein sequences for the species shown were obtained from the NCBI nucleotide database (accession numbers NP_001035810, NP_032088, NP_001080019, XP_699168, P12646, AAT93017, and NP_416366 respectively). Sequence alignment was performed using (more ...)
Immunoblotting for G6PD after native gel electrophoresis was performed in the patient's blood sample and control specimens (). Bands at the expected molecular weights for the G6PD monomer, dimer, and tetramer were seen in the control specimen. The quantity of monomer was similar between patient and control, while the patient's G6PD dimer and tetramer were significantly decreased. Although it is considered unlikely that a substantial amount of monomer exists normally, because NADP in the RBCs induces dimerization [5
], about one-fifth of the total enzyme in the control sample was in monomeric form. This may have been caused by processing of the RBC lysate to achieve hemoglobin depletion, which was necessary to allow immunoblotting at an area otherwise overwhelmed by an excessive amount of hemoglobin tetramer (M.W. 68 kDa). G6PD deficiency can be caused either by a reduced number of G6PD molecules with normal catalytic activity or by a normal number of molecules with decreased catalytic activity or by a combination of these two mechanisms [5
]. The number of G6PD molecules declines due to accelerated breakdown rather than decreased rate of synthesis [12
]. From the immunoblot in , we conclude that decreased amount of the enzyme along with decreased activity due to impaired oligomer formation contribute to the phenotype of class I G6PD deficiency in our patient.
We cannot exclude the possibility that the severe presentation of our patient could have been caused by the G6PD Gastonia mutation alone causing overwhelming hemolysis due to oxidative stress caused by perinatal complications. However, the concurrent G6PD Cincinnati mutation may have aggravated the phenotype, as it has been previously described with multiple G6PD mutations [13
]. Neonatal hemolysis causing progressive cholestasis, hepatosplenomegaly, and liver dysfunction has been previously reported in association with hemolytic conditions, such as pyruvate kinase deficiency [14
], Rh incompatibility [15
], hereditary pyropoikilocytosis [16
], and recently G6PD deficiency [17
]. The common pathophysiology proposed in these clinical disorders is bilirubin-load out of proportion to the rate of choleresis in the neonatal liver, leading to cholestasis and hepatitis. Complete work-up needs to be performed in a case of neonatal obstructive jaundice to rule-out sepsis, congenital infections, inborn errors of metabolism, and primary liver disease. However, when serum AST is disproportionately higher than serum ALT, the possibility of extensive hemolysis needs to be considered. And as it is frequently true in pediatrics, a complete family history can be very helpful.