We identified two ATP8B1 mutations that resulted in an amino acid exchange (D70N and R867C) following DNA sequencing of 16 ICP cases. One variant was present in three of 182 ICP cases and the other was present in one case. Neither mutation was present in 120 parous controls. Thus it is possible that these variants play a role in the aetiology of ICP.
A recent report on ATP8B1
mutations in families with PFIC1 and BRIC revealed the presence of D70N as a compound heterozygote in combination with another missense mutation (1799G>A resulting in R600Q).19
It is possible that heterozygosity for D70N causes ICP without other features of BRIC. Alternatively, the women with D70N in the present study may have another abnormality in ATP8B1
that was not identified by sequencing the coding exons, or another ICP causing variant in a different gene. The most common ATP8B1
variant in BRIC, I661T, found in 79% of all BRIC cases with detectable ATP8B1
was not found in any of the ICP cases in this study, indicating that this BRIC associated variant did not cause ICP in the UK cases presented in this study. However, the data reported in our study cannot exclude I661T from having a role in the aetiology of ICP.
The function of FIC1, the ATP8B1
gene product, has not been established, nor has the mechanism by which variations in ATP8B1
result in cholestasis. It has been hypothesised that FIC1 is an aminophospholipid translocase which translocates phosphatidylserine from the outer to the inner leaflet of the canalicular plasma membrane.37
If this hypothesis is correct, this process could maintain the stable asymmetric distribution of phospholipids which is required for normal function of the transporters embedded therein. Thus a defect in FIC1 activity could influence the function of bile acid transporters without the molecule itself being involved in bile acid transport. Alternative functions for FIC1 have been proposed (for example, as a direct bile acid transporter or as a metal ion transporter).38
FIC1 is highly expressed in the small intestine and it has been proposed that mutations in PFIC1 patients may result in abnormal intestinal bile acid reabsorption.7
It has been shown that complete absence of ATP8B1
mRNA in the ileum of patients with PFIC leads to substantial downregulation of FXR, a nuclear receptor involved in the regulation of bile acid metabolism,39
and this is another possible mechanism that results in cholestasis. However, abnormal intestinal bile acid absorption alone is unlikely to explain why ATP8B1
mutations cause cholestasis in pregnancy as the main bile acids that are raised in ICP are primary bile acids.
The subfamily of P-type ATPases to which FIC1 belongs includes at least 10 more members based on similarities in the protein sequence and positioning of the functional domains.40
Evidence for a discrete function for FIC1 within this subfamily of P-type ATPases comes from the observation that its expression pattern is different, as it is one of the few family members not expressed in the brain.40
Also, FIC1 is the only member of the family which has to date been identified as causing disease.7,40
Residue R867 is localised within one of the “signature” motifs used to identify this subfamily of sequences.41
These diagnostic sequences were derived from alignments of 16 inferred protein sequences of subfamily members (from Schizosaccharomyces cerevisiae
, S pombe
, Caenorhabditis elegans
, Plasmodium falciparum
, and Drosophila melanogaster
) and the motif containing R867 is found in all 16 of them. This implies that it may play a role that is central to this type of molecule. Furthermore, R867 is found in the same position in all human subfamilies of amphipath transporters.40
The motif is not conserved in Ca2+
transporting P-type ATPases, indicating that its function may relate to substrate specificity. D70N results in the substitution of aspartic acid, which is a polar charged amino acid with a pKA of 3.65, with an asparagine, a polar uncharged amino acid. This leads to a change in the charge at neutral pH from negative to neutral at the respective position.
Although the D70N variant was not present in 120 parous controls in this study, it has been reported in two individuals of Caucasian origin.42
However, one of these was male and it is not stated whether the female carrier was parous. It is possible that D70N on its own is not of sufficiently high penetrance but leads to ICP only in combination with a second disadvantageous sequence variation or, alternatively, that the functional effect of the ATP8B1
variants reported in this study become clinically apparent as a consequence of the endocrine changes of pregnancy. Oestrogen treatment results in increased cholesterol content of membranes and reduced membrane fluidity, and this in turn reduces bile flow.43
Oestrogen treatment also causes reduced protein mass and RNA expression of Ntcp, the principal sinusoidal bile acid transporter, and other sinusoidal organic anion transporters, Oatp1, and Oatp2 in the rat,44–46
reduced protein mass of Oatp446
and reduced mRNA levels of murine mBsep.45
Either altered membrane fluidity, or reduced expression or function of the principal sinusoidal or canalicular bile acid transporters in pregnancy, in combination with loss of function of FIC1, could result in the development of cholestasis in a woman who is asymptomatic when not pregnant. It is also possible that the endocrine changes of pregnancy may influence the function of the mutant protein in a similar way to the K183R substitution in the thyrotrophin receptor that results in increased sensitivity to chorionic gonadotrophin in familial gestational hyperthyroidism.48
It remains to be seen whether the MRS findings are truly of significance as subject numbers were small. However, there have been many studies looking at the utilisation of 31
P MRS as a non-invasive technique for assessing liver disease, mainly in cirrhosis. These have reported good correlation with either an elevated PME resonance or decreased PDE resonance and functional capacity in cirrhotic livers.29–31
The ratio of PME to PDE has traditionally been viewed as an index of cell membrane turnover, the PME resonance including contributions from the precursors of phospholipid membrane synthesis, phosphocholine and phosphoethanolamine, as well as contributions from adenosine monophosphate and glycolytic intermediates.49
The PDE resonance contains information from membrane breakdown products, GPC and GPE, biliary phospholipid, and a contribution from endoplasmic reticulum.49
In women with a history of ICP, we observed an increase in %PDE, rather than the decrease seen in cirrhosis, a finding that has previously been reported in cholestatic liver transplant patients with chronic graft rejection.32
This has been interpreted as an increased or altered signal from biliary phospholipid, which probably underlies the findings in our patient group. It is possible that the abnormal %PDE signal is associated with an increase in the proportion of biliary phospholipid as a consequence of defective canalicular bile acid transport, and therefore a proportional reduction in biliary bile acid concentrations. The ICP cases also had a reduced PME/PDE ratio which may reflect increased cell membrane turnover.32
Although there were no differences in hepatic βNTP levels between cases and controls, it is intriguing that the two cases with D70N had lower levels than the other ICP cases.
It should be borne in mind that subjects in this study with 31P MRS abnormalities were not pregnant and were asymptomatic when the scans were performed. Therefore, the abnormal spectra may be indicative of underlying abnormalities of liver metabolism and biliary composition that only manifest as symptoms during pregnancy, but are not sufficiently severe to cause cholestasis in the non-pregnant state.
In summary, sequencing of the coding exons has revealed two ATP8B1 mutations in 16 ICP cases. The D70N mutation was present in another two affected women when 166 additional cases were screened for these variants. In vivo MRS studies of the liver in eight women with previous ICP, performed when they were not pregnant, demonstrated increased hepatic PDE and reduced PME/PDE values compared with normal controls. Two ICP cases with D70N had the lowest βNTP levels of the ICP cases measured.