Mutations in SEC63 cause autosomal dominant polycystic liver disease.

Mutations in PRKCSH, encoding the beta-subunit of glucosidase II, an N-linked glycan-processing enzyme in the endoplasmic reticulum (ER), cause autosomal dominant polycystic liver disease. We found that mutations in SEC63, encoding a component of the protein translocation machinery in the ER, also cause this disease. These findings are suggestive of a role for cotranslational protein-processing pathways in maintaining epithelial luminal structure and implicate noncilial ER proteins in human polycystic disease.

Quantitation of serum angiopoietin-like proteins 3 and 4 in a Finnish population sample.

We have developed and validated quantitative ELISAs for human angiopoietin-like (ANGPTL)3 and 4 and correlated their serum levels with parameters of lipid and carbohydrate metabolism. For this study, we used a random subsample of the Health 2000 Health Examination Survey consisting of 125 men and 125 women, aged 30-94 years. The anthropometric and biochemical parameters of subjects were characterized in detail. ANGPTL 3 and 4 levels were determined using the developed ELISAs. The intra- and inter-assay coefficients of variation for the assays were less than 15%. The average serum concentration of ANGPTL3 was 368 +/- 168 ng/ml (mean +/- SD) and for ANGPTL4 it was 18 +/- 23 ng/ml (mean +/- SD). ANGPTL4 serum levels displayed high variability between individuals ranging from 2 to 158 ng/ml. In post-heparin plasma, both ANGPTL 3 and 4 were increased. Low levels of ANGPTL3 were associated with decreased HDL-cholesterol and increased triglyceride levels. ANGPTL4 levels were positively correlated with FFAs (P = 0.044) and waist-hip ratio (P = 0.016). The developed ELISAs will be important tools to clarify the role of ANGPTL 3 and 4 in human energy metabolism and partitioning of triglycerides between sites of storage (adipose tissue) and oxidation (skeletal and cardiac muscle).

Isolated polycystic liver disease genes define effectors of polycystin-1 function.

Dominantly inherited isolated polycystic liver disease (PCLD) consists of liver cysts that are radiologically and pathologically identical to those seen in autosomal dominant polycystic kidney disease, but without clinically relevant kidney cysts. The causative genes are known for fewer than 40% of PCLD index cases. Here, we have used whole exome sequencing in a discovery cohort of 102 unrelated patients who were excluded for mutations in the 2 most common PCLD genes, PRKCSH and SEC63, to identify heterozygous loss-of-function mutations in 3 additional genes, ALG8, GANAB, and SEC61B. Similarly to PRKCSH and SEC63, these genes encode proteins that are integral to the protein biogenesis pathway in the endoplasmic reticulum. We inactivated these candidate genes in cell line models to show that loss of function of each results in defective maturation and trafficking of polycystin-1, the central determinant of cyst pathogenesis. Despite acting in a common pathway, each PCLD gene product demonstrated distinct effects on polycystin-1 biogenesis. We also found enrichment on a genome-wide basis of heterozygous mutations in the autosomal recessive polycystic kidney disease gene PKHD1, indicating that adult PKHD1 carriers can present with clinical PCLD. These findings define genetic and biochemical modulators of polycystin-1 function and provide a more complete definition of the spectrum of dominant human polycystic diseases.

Isolated polycystic liver disease genes define effectors of polycystin-1 function.

Dominantly inherited isolated polycystic liver disease (PCLD) consists of liver cysts that are radiologically and pathologically identical to those seen in autosomal dominant polycystic kidney disease, but without clinically relevant kidney cysts. The causative genes are known for fewer than 40% of PCLD index cases. Here, we have used whole exome sequencing in a discovery cohort of 102 unrelated patients who were excluded for mutations in the 2 most common PCLD genes, PRKCSH and SEC63, to identify heterozygous loss-of-function mutations in 3 additional genes, ALG8, GANAB, and SEC61B. Similarly to PRKCSH and SEC63, these genes encode proteins that are integral to the protein biogenesis pathway in the endoplasmic reticulum. We inactivated these candidate genes in cell line models to show that loss of function of each results in defective maturation and trafficking of polycystin-1, the central determinant of cyst pathogenesis. Despite acting in a common pathway, each PCLD gene product demonstrated distinct effects on polycystin-1 biogenesis. We also found enrichment on a genome-wide basis of heterozygous mutations in the autosomal recessive polycystic kidney disease gene PKHD1, indicating that adult PKHD1 carriers can present with clinical PCLD. These findings define genetic and biochemical modulators of polycystin-1 function and provide a more complete definition of the spectrum of dominant human polycystic diseases.

Polycystic liver and kidney diseases.

There have been remarkable advances in research on polycystic liver and kidney diseases recently, covering cloning of new genes, refining disease classifications, and advances in understanding more about the molecular pathology of these diseases. Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary disease affecting kidneys. It affects 1/400 to 1/1000 live births and accounts for 5% of the end stage renal disease in the United States and Europe, and is caused by gene defects in the PKD1 or PKD2 genes. Compared to ADPKD, polycystic liver disease (PCLD) is a milder disease and does not lower life expectancy. Both diseases are usually adult-onset diseases. Defects in genes, which code the hepatocystin and SEC63 proteins, have just recently been found to cause PCLD. It now seems that ADPKD is caused by malfunction of the primary cilia, a cell organ sensing fluid movement, and that PCLD is a sequel from defects in protein processing. Autosomal recessive polycystic kidney disease (ARPKD) belongs to a group of congenital hepatorenal fibrocystic syndromes. All ARPKD patients have a gene defect in a gene called PKHD1, the protein product of which localizes to primary cilia. We summarize the present clinical and molecular knowledge of these diseases in this review.

Polycystic liver disease is genetically heterogeneous: clinical and linkage studies in eight Finnish families.

BACKGROUND/AIMS: Polycystic liver disease (PCLD), a dominantly inherited condition separate from polycystic kidney disease (PKD), has recently been found to be linked to a locus on chromosome 19p13.2-13.1 in two North American families. Our aim was to study whether there is clinical or genetic heterogeneity within PCLD families. METHODS: We collected clinical data of eight Finnish PCLD families and performed both linkage analysis and an extended admixture test. We used genetic markers located on chromosome 19p13.2-13.1 and, in addition, on the three known PKD loci on chromosomes 4q21-q23 (PKD2), 6p21 (ARPKD) and 16p13.3-p13.12 (PKD1). RESULTS: There were a total of 33 PCLD patients among which the severity of the disease varied greatly even within families. Seven patients had disabling symptoms requiring cyst decompression while ten patients were found only when the symptomless family members were studied by abdominal ultrasound. When genetic homogeneity was assumed, the PCLD locus on chromosome 19p13.2-13.1 was excluded but when genetic heterogeneity was allowed, five families out of seven showed linkage to that locus. All three PKD loci were excluded. CONCLUSIONS: Most Finnish PCLD families are linked to chromosome 19p13.2-13.1 but there exists also a second PCLD locus yet to be found.

Quantitation of the active and low-active forms of human plasma phospholipid transfer protein by ELISA.

Human plasma contains two forms of phospholipid transfer protein (PLTP), one catalytically active [high-activity PLTP (HA-PLTP)] and the other a low-activity (LA-PLTP) form. We present here a PLTP ELISA that allows not only for accurate measurement of PLTP concentration in plasma but also of the distribution of both LA- and HA-PLTP. To achieve similar immunoreactivity of the two PLTP forms, a denaturing sample pretreatment with 0.5% SDS was required. Distribution of LA- and HA-PLTP in plasma was assessed using size-exclusion chromatography, Heparin-Sepharose chromatography, anti-PLTP immunoaffinity chromatography, and dextran sulfate-CaCl2 precipitation. All four methods demonstrated that approximately 60% of plasma PLTP represents LA-PLTP and 40% represents HA-PLTP. According to the modified ELISA, the total serum PLTP concentration in a random Finnish population sample (n = 80) was 5.81 +/- 1.33 mg/l (mean +/- SD) (range, 2.78-10.06 mg/l) and the mean activity was 5.84 +/- 1.39 micromol/ml/h (range, 3.21-11.15 micromol/ml/h). To quantitate both forms of PLTP in sera from this sample, we combined dextran sulfate-CaCl2 precipitation with the modified PLTP ELISA. The HA-PLTP mass (mean, 1.87 +/- 0.85 mg/l) correlated significantly with serum PLTP activity, whereas that of LA-PLTP (mean, 3.94 +/- 1.4 mg/l) showed no correlation with phospholipid transfer activity.

Active and low-active forms of serum phospholipid transfer protein in a normal Finnish population sample.

Human serum phospholipid transfer protein (PLTP) exists as a catalytically active (HA-PLTP) and a low-active (LA-PLTP) form. In this study, the association of PLTP activity and the concentrations of both forms with lipid and carbohydrate parameters were investigated. In a random Finnish population sample, serum PLTP concentration (n=250) was 6.56 +/- 1.45 mg/l, the mean lipoprotein-independent (PLTPexo) phospholipid transfer activity was 6.59 +/- 1.66 micromol/ml/h, and the mean lipoprotein-dependent (PLTPendo) activity was 1.37 +/- 0.29 micromol/ml/h. Of the serum PLTP concentration, approximately 46% was in a catalytically active form. HA-PLTP concentration correlated positively with serum PLTPexo activity (r=0.380, P <0.001), HDL cholesterol (r=0.291, P <0.001), and apolipoprotein A-I (r=0.187, P <0.01). Of the potential regulatory factors for PLTP, apolipoprotein E showed a weak positive correlation with serum PLTPexo (r=0.154, P <0.05) and PLTPendo (r=0.192, P <0.01) activity but not with PLTP concentration. Weak associations were also observed between PLTP parameters and determinants of glucose homeostasis (glucose, insulin, and homeostasis model assessment for insulin resistance). The present data on PLTP activity and concentration reveal novel connections of the two PLTP forms to lipid and carbohydrate metabolism.

Abnormal hepatocystin caused by truncating PRKCSH mutations leads to autosomal dominant polycystic liver disease.

Mutations in protein kinase C substrate 80K-H (PRKCSH), encoding for the protein hepatocystin, cause autosomal dominant polycystic liver disease (PCLD), which is clinically characterized by the presence of multiple liver cysts. PCLD has been documented in families from Europe (Netherlands, Belgium, Finland) as well as from the United States. In this article, we report results from extensive mutational analysis of the PRKCSH gene in a group of 14 PCLD families and 65 singleton cases of Dutch and Finnish descent with multiple simple liver cysts. We identified PRKCSH mutations in 12 families and in 3 sporadic cases. In 8 of 10 Finnish families we detected the 1437+2delTG splice-site mutation. In Dutch families, we found 2 other mutations that affect correct splicing of PRKCSH: 292+1 G>C (2 families) and 1338-2 A>G (1 family). In another Dutch family, we detected a novel deletion (374-375delAG) in exon 6, predicting an abnormal shortened protein. Investigation of the carrier haplotypes identified a common founder chromosome in unrelated individuals in each of the 3 identified splice-site mutations. In 2 Finnish families with dominantly inherited PCLD, and in 62 of 65 sporadic cases with multiple simple liver cysts, we failed to demonstrate any PRKCSH mutation. This corroborates the notion that autosomal dominant PCLD is genetically heterogeneous. In conclusion, we propose that, on the basis of our results, genetic screening for PRKCSH gene mutations should be limited to patients either with a positive family history for PCLD or who have severe polycystic liver disease.