MDR3 mutations associated with intrahepatic and gallbladder cholesterol cholelithiasis: an update

Background: The recurrent microlithiasis represents one of the most frequent clinical forms of lithiasis of the bile ducts. This affection is characterized by the presence of cholesterolic microgallstones on hepatic canaliculars, and belongs to a heterogeneous group of autosomal recessive liver disorders. Radiological diagnosis can be confirmed by analysis of MDR3 gene, coding a protein involved in physiologic translocation of phospholipids in bile. Discovery of MDR3 mutations is of particular interest, since normally associated with good effectiveness of medication by ursodesoxycholic acid. AIM: To review MDR3 mutations in humans associated with recurrent cholesterol microlithiasis and to suggest a practical approach for MDR3 gene analysis. Results: 48 mutations of MDR3 gene have been reported in humans to date, from which 43 (89.5%) in the coding region, and 5 splice site mutations have been associated to cholesterol cholelithiasis. 21 (43.8%) of the 43 precited mutations are located in only 8 exons on 28, near transmembrane or nucleotide binding domains of the protein. From the 22 remaining described mutations, 9 (18.8%) are restricted to exon 14. We suggest therefore to start analysis of MDR3 gene by screening exons 6, 7, 9, 10, 12, 14, 17, 23 and 24 with an appropriate protocol in this diagnosis associated with effective treatment. In conclusion such therapeutic orientation is valuable, since recurrent cholesterolic microlithiasis occurs relatively early in life, and by the fact that recurrence of symptoms may occur despite cholecystectomy, or shock-wave therapy

Characterization of a novel BCHE "silent" allele: point mutation (p.Val204Asp) causes loss of activity and prolonged apnea with suxamethonium.

Butyrylcholinesterase deficiency is characterized by prolonged apnea after the use of muscle relaxants (suxamethonium or mivacurium) in patients who have mutations in the BCHE gene. Here, we report a case of prolonged neuromuscular block after administration of suxamethonium leading to the discovery of a novel BCHE variant (c.695T>A, p.Val204Asp). Inhibition studies, kinetic analysis and molecular dynamics were undertaken to understand how this mutation disrupts the catalytic triad and determines a "silent" phenotype. Low activity of patient plasma butyrylcholinesterase with butyrylthiocholine (BTC) and benzoylcholine, and values of dibucaine and fluoride numbers fit with heterozygous atypical silent genotype. Electrophoretic analysis of plasma BChE of the proband and his mother showed that patient has a reduced amount of tetrameric enzyme in plasma and that minor fast-moving BChE components: monomer, dimer, and monomer-albumin conjugate are missing. Kinetic analysis showed that the p.Val204Asp/p.Asp70Gly-p.Ala539Thr BChE displays a pure Michaelian behavior with BTC as the substrate. Both catalytic parameters Km = 265 µM for BTC, two times higher than that of the atypical enzyme, and a low Vmax are consistent with the absence of activity against suxamethonium. Molecular dynamic (MD) simulations showed that the overall effect of the mutation p.Val204Asp is disruption of hydrogen bonding between Gln223 and Glu441, leading Ser198 and His438 to move away from each other with subsequent disruption of the catalytic triad functionality regardless of the type of substrate. MD also showed that the enzyme volume is increased, suggesting a pre-denaturation state. This fits with the reduced concentration of p.Ala204Asp/p.Asp70Gly-p.Ala539Thr tetrameric enzyme in the plasma and non-detectable fast moving-bands on electrophoresis gels

Radius of gyration of BChE in p.Val204Asp mutant and the usual enzyme.

<p>Along the MD trajectory size of usual BChE is maintained at the same level while for the mutant it gradually increases what indicates transition to a molten globule state.</p

Catalytic properties of patient and parents’s butyrylcholinesterase with BTC as the substrate at pH 7.0 and 25°C (average values from duplicate measurements ± standard deviation).

<p>litterature values <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101552#pone.0101552-Masson2" target="_blank">[12]</a>: a and b) k_cat = V_max/[E] = 24 000±10 000 min⁻¹; b*: substrate activation factor (see Eq 1).</p

Hydrogen bonding network in p.Val204Asp BChE formed during MD simulation.

<p>Histidine 438 faces away from Serine 198, thus disrupting the function of the catalytic triad. This conformational change explains why mutation V204D causes the BChE enzyme to lose activity.</p

Pedigree of the family with BChE activities and genotypes.

<p>BChE activities were measured on Cobas® 6000 system. Results of genotyping are given as electropherograms. Sequence analyses reveal (A) an heterozygous mutation c.695T>A (p.Val204Asp). This mutation is observed in the proband and in patient I-1; (B) an heterozygous mutation c.293A>G (p.Asp70Gly, atypical variant). This mutation is observed in the proband and in patient I-2 and (C) an heterozygous mutation c.1699G>A (p.Ala539Thr, Kalow variant, electrophorogram not shown). This mutation is observed in the proband and in patient I-2.</p

Model of BChE catalyzed-hydrolysis of substrates.

<p>Model of BChE catalyzed-hydrolysis of substrates.</p

Activity stained polyacrylamide gels of plasma BChE samples.

<p>Nondenaturing gel stained for BChE activity. Plasma samples (5 µl per lane) were genotype UU, wild-type usual control; genotype US, the father (I-1) heterozygous for the silent variant; genotype UAK, the mother (I-2) heterozygous for the atypical/K variant; and genotype AKS, the son heterozygous (II-1) for the atypical/K variant and silent BChE. The majority of the plasma BChE is a tetramer (C4). Minor activity bands are for monomer (C1), a BChE monomer- albumin dimer (C2), and a BChE dimer (C3). The AKS plasma has weaker staining intensity for tetrameric BChE (C4) and no apparent minor bands of BChE activity.</p

Oligonucleotide primers used in PCR for the amplification of the <i>BCHE</i> gene.

<p>Oligonucleotide primers used in PCR for the amplification of the <i>BCHE</i> gene.</p

Evolutionary conservation of the region of BChE including in the variant site p.Val204Asp.

<p>Evolutionary conservation of the region of BChE including in the variant site p.Val204Asp.</p