Mutations in bile salt export pump (ABCB11) in two children with progressive familial intrahepatic cholestasis and cholangiocarcinoma.

Fatal peripheral cholangiocarcinoma developed in 2 girls with progressive familial intrahepatic cholestasis, ABCB11 mutations, and absent bile salt export pump (BSEP) expression. BSEP deficiency may cause cholangiocarcinoma through bile-composition shifts or bile-acid damage within cells capable of hepatocytic/cholangiocytic differentiation. This observation suggests the need for hepatobiliary-malignancy surveillance and early consideration for liver transplantation.

Hepatocanalicular bile salt export pump deficiency in patients with progressive familial intrahepatic cholestasis.

BACKGROUND & AIMS: Progressive familial intrahepatic cholestasis (PFIC), an inherited liver disease of childhood, is characterized by cholestasis and either normal or increased serum gamma-glutamyltransferase activity. Patients with normal gamma-glutamyltransferase activity have mutations of the FIC1 locus on chromosome 18q21 or mutations of the BSEP gene on chromosome 2q24. Also, patients with bile acid synthesis defects have low gamma-glutamyltransferase activity. We investigated expression of the bile salt export pump (BSEP) in liver samples from patients with a PFIC phenotype and correlated this with BSEP gene mutations. METHODS: BSEP and multidrug resistance protein 2 (MRP2) expressions were studied by immunohistochemistry in liver specimens of 28 patients and BSEP gene mutation analysis in 19 patients. Bile salt kinetics were studied in 1 patient. RESULTS: Sixteen of 28 liver samples showed no canalicular BSEP staining. Staining for MRP2 showed a normal canalicular pattern in all but 1 of these samples. Ten of 19 patients showed BSEP gene mutations; BSEP protein expression was lacking in all 10 patients. No mutations were found in 9 of 19 patients, and in all except 1, BSEP protein expression was normal. Bile salt concentration in bile of BSEP-negative/MRP2-positive PFIC patients was 0.2 +/- 0.2 mmol/L (n = 9; <1% of normal) and in BSEP-positive PFIC patients 18.1 +/- 9.9 mmol/L (n = 3; 40% of normal). The kinetic study confirmed the dramatic decrease of bile salt secretion in BSEP-negative patients. CONCLUSIONS: The findings show a close correlation between BSEP gene mutations and canalicular BSEP expression. Biliary secretion of bile salts is greatly reduced in BSEP-negative patients.

Gene structure of the human amiloride-sensitive epithelial sodium channel beta subunit.

ENaC functions in the transport of sodium ions across epithelial cells and consequently regulates blood volume and pressure. ENaC complex includes at least three different subunits, alpha, beta, and gamma, which are developmentally regulated and differentially controlled by aldosterone. In this study, we determined the exon-intron organization of the beta ENaC subunit by sequencing genomic DNA from three subjects from three different ethnic groups. The results showed that the coding region of the human betaENaC gene (SCNN1B) extends from exon 2 to exon 13. No polymorphism was observed in the examined subjects, indicating strict conservation of the coding region sequence. The introns of beta subunit gene are located at exactly the same positions as in the alpha and gamma subunits, although these proteins share only 26-32% sequence identity. These results thus elucidate the gene structure of the beta subunit and indicate that exon-intron architecture of the three genes encoding the three subunits of ENaC have remained highly conserved despite the divergence of their sequences.

A gene encoding a liver-specific ABC transporter is mutated in progressive familial intrahepatic cholestasis.

The progressive familial intrahepatic cholestases (PFIC) are a group of inherited disorders with severe cholestatic liver disease from early infancy. A subgroup characterized by normal serum cholesterol and gamma-glutamyltranspeptidase (gammaGT) levels is genetically heterogeneous with loci on chromosomes 2q (PFIC2) and 18q. The phenotype of the PFIC2-linked group is consistent with defective bile acid transport at the hepatocyte canalicular membrane. The PFIC2 gene has now been identified by mutations in a positional candidate, BSEP, which encodes a liver-specific ATP-binding cassette (ABC) transporter, sister of p-glycoprotein (SPGP). The product of the orthologous rat gene has been shown to be an effective bile acid transporter in vitro. These data provide evidence that SPGP is the human bile salt export pump (BSEP).

Absence of linkage of the epithelial sodium channel to hypertension in black Caribbeans.

Hypertensives of African origin have low-renin, sodium-sensitive blood pressure and respond poorly to treatment with angiotensin converting enzyme inhibitors. The epithelial sodium channel may be important in the pathogenesis of essential hypertension in this population. This is supported by the identification of mutations within this channel, which lead to excess sodium reabsorption and hypertension in Liddle's syndrome. In this study we tested whether there was linkage of the genes encoding the three subunits of the epithelial sodium channel to essential hypertension in 63 affected sibling pairs of West African origin from St. Vincent and the Grenadines. We found no support for linkage of the epithelial sodium channel to essential hypertension in this population. However, further studies will be needed in larger populations of African ancestry to exclude a contribution of the genes encoding the epithelial sodium channel to hypertension.

Identification of a locus for progressive familial intrahepatic cholestasis PFIC2 on chromosome 2q24.

Progressive familial intrahepatic cholestasis (PFIC; OMIM 211600) is the second most common familial cholestatic syndrome presenting in infancy. A locus has previously been mapped to chromosome 18q21-22 in the original Byler pedigree. This chromosomal region also harbors the locus for benign recurrent intrahepatic cholestasis (BRIC) a related phenotype. Linkage analysis in six consanguineous PFIC pedigrees from the Middle East has previously excluded linkage to chromosome 18q21-22, indicating the existence of locus heterogeneity within the PFIC phenotype. By use of homozygosity mapping and a genome scan in these pedigrees, a locus designated "PFIC2" has been mapped to chromosome 2q24. A maximum LOD score of 8.5 was obtained in the interval between marker loci D2S306 and D2S124, with all families linked.

Locus heterogeneity in progressive familial intrahepatic cholestasis.

Progressive familial intrahepatic cholestasis (PFIC or Byler disease) is a rare autosomal recessive form of severe and fatal cholestatic liver disease. A locus for PFIC has recently been mapped to chromosome 18q21-q22 in the original Byler pedigree. This region harbours the locus for a related phenotype, benign recurrent intrahepatic cholestasis (BRIC), suggesting that these traits are allelic. Linkage analysis was undertaken in five consanguineous PFIC pedigrees from Saudi Arabia using marker loci (D18S69, D18S41, D18S64, D18S38, D18S42, D18S55, D18S68, and D18S61) which span the Byler disease/BRIC region on 18q21-q22. In this family set the disease locus was excluded from this region, showing that locus heterogeneity exists for the PFIC phenotype.

A novel splice-site mutation in the gamma subunit of the epithelial sodium channel gene in three pseudohypoaldosteronism type 1 families.

Pseudohypoaldosteronism type 1 (PHA1, OMIM 264350) is an uncommon inherited disorder characterized by salt-wasting and end-organ unresponsiveness to mineralocorticoids. A complete genome search using homozygosity mapping in eleven consanguineous families with PHA1 provided conclusive evidence of linkage with heterogeneity. The disease locus mapped to chromosome 16p12.2-13.11 in six families and to 12p13.1-pter in the other five families. These two chromosomal regions harbour the genes encoding the three subunits of the human amiloride sensitive epithelial sodium channel (hENaC): SCNN1B and SCNN1G on 16p and SCNN1A on 12p. Our linkage results have been further supported by the recent report of mutations in the alpha and beta subunit genes in PHA1 patients. We now report the identification of a 3' splice site mutation in SCNN1G (318-1 G-->A) in three families showing linkage to 16p. Abnormal splicing results with the production of two messenger RNAs, one arising from activation of an adjacent cryptic splice site and the other from skipping of the downstream exon. The two corresponding mutant gamma hENaC subunits are predicted to have three highly conserved amino acids in the extracellular domain replaced by a novel amino acid (KYS106-108-->N) and truncation from 649 to 134 amino acids respectively. These three families all originate from the Indian sub-continent and the probands have severe generalized PHA. They share a common haplotype which suggests the presence of a founder mutation in this sub-population.

Localisation of pseudohypoaldosteronism genes to chromosome 16p12.2-13.11 and 12p13.1-pter by homozygosity mapping.

Pseudohypoaldosteronism type 1 (PHA1, OMIM 264350) is a rare Mendelian disorder characterised by end-organ unresponsiveness to mineralocorticoids. Most steroid hormone insensitivity syndromes arise from mutations in the corresponding receptor, but available genetic evidence is against involvement of the mineralocorticoid receptor gene, MLR, in PHA1. A complete genome scan for PHA1 genes was undertaken using homozygosity mapping in 11 consanguineous families. Conclusive evidence of linkage with heterogeneity was obtained with a maximum two-locus admixture lod score of 9.9. The disease locus mapped to chromosome 16p12.2-13.11 in six families and to 12p13.1-pter in the other five families. The two chromosomal regions harbour genes for subunits of the amiloride-sensitive epithelial sodium channel: SCNN1B and SCNN1G on 16p and SCNN1A on 12p. Liddle's syndrome of hypertension and pseudoaldosteronism has been shown to arise from mutations in SCNN1B and SCNN1G. These results strongly suggest that PHA1 and Liddle's syndrome are allelic variants caused by mutations in genes encoding subunits of this sodium channel. These genes are of broad biological interest both in relation to sodium and water homeostasis in mammals and by virtue of their homology to the mec genes of Caenorhabditis elegans involved in mechanosensitivity and neuronal degeneration.