Genetics and Molecular Modeling of New Mutations of Familial Intrahepatic Cholestasis in a Single Italian Center

Familial intrahepatic cholestases (FICs) are a heterogeneous group of autosomal recessive disorders of childhood that disrupt bile formation and present with cholestasis of hepatocellular origin. Three distinct forms are described: FIC1 and FIC2, associated with low/normal GGT level in serum, which are caused by impaired bile salt secretion due to defects in ATP8B1 encoding the FIC1 protein and defects in ABCB11 encoding bile salt export pump protein, respectively; FIC3, linked to high GGT level, involves impaired biliary phospholipid secretion due to defects in ABCB4, encoding multidrug resistance 3 protein. Different mutations in these genes may cause either a progressive familial intrahepatic cholestasis (PFIC) or a benign recurrent intrahepatic cholestasis (BRIC). For the purposes of the present study we genotyped 27 children with intrahepatic cholestasis, diagnosed on either a clinical or histological basis. Two BRIC, 23 PFIC and 2 BRIC/PFIC were identified. Thirty-four different mutations were found of which 11 were novel. One was a 2Mb deletion (5’UTR- exon 18) in ATP8B1. In another case microsatellite analysis of chromosome 2, including ABCB11, showed uniparental disomy. Two cases were compound heterozygous for BRIC/PFIC2 mutations. Our results highlight the importance of the pathogenic role of novel mutations in the three genes and unusual modes of their transmission.     

FIC1, a P-type ATPase linked to cholestatic liver disease,has homologues (ATP8B2 and ATP8B3) expressed throughout the body

ATP8B1/FIC1 is a member of the Type IV P-type ATPase family, which function as ATP dependent aminophospholipid translocases (APLT). We identified two familial intrahepatic cholestasis type 1 (FIC1) homologues, ATP8B2 and ATP8B3, with 53% and 45% amino acid identity, respectively. The expression profile for each gene was determined using a 73-tissue human RNA expression array. The subfamily of FIC1-like proteins is expressed in a wide range of tissues. Given that mutations in FIC1 result in liver disease, these proteins may have important roles in other organs in which they are candidates for genetic and acquired diseases.     

FIC1, a P-type ATPase linked to cholestatic liver disease,has homologues (ATP8B2 and ATP8B3) expressed throughout the body

ATP8B1/FIC1 is a member of the Type IV P-type ATPase family, which function as ATP dependent aminophospholipid translocases (APLT). We identified two familial intrahepatic cholestasis type 1 (FIC1) homologues, ATP8B2 and ATP8B3, with 53% and 45% amino acid identity, respectively. The expression profile for each gene was determined using a 73-tissue human RNA expression array. The subfamily of FIC1-like proteins is expressed in a wide range of tissues. Given that mutations in FIC1 result in liver disease, these proteins may have important roles in other organs in which they are candidates for genetic and acquired diseases.     

Fine-resolution mapping by haplotype evaluation: the examples of PFIC1 and BRIC

Loci for two inherited liver diseases, benign recurrent intrahepatic cholestasis (BRIC) and progressive familial intrahepatic cholestasis type 1 (PFIC1), have previously been mapped to 18q21 by a search for shared haplotypes in patients in two isolated populations. This paper describes the use of further haplotype evaluation with a larger sample of patients for both disorders, drawn from several different populations. Our assessment places both loci in the same interval of less than 1 cM and has led to the discovery of the PFIC1/BRIC gene, FIC1; this discovery permits retrospective examination of the general utility of haplotype evaluation and highlights possible caveats regarding this method of genetic mapping. Received: 21 September 1998 / Accepted: 29 December 1998     

Localization of multiple pleiotropic genes for lipoprotein metabolism in baboons

We employed a novel approach to identify the key loci that harbor genes influencing lipoprotein metabolism in approximately 2,000 pedigreed baboons fed various diets differing in levels of fat and cholesterol. In this study, 126 overlapping traits related to both LDL and HDL metabolism were normalized and subjected to genome-wide linkage screening. As was expected, the traits were highly, but not completely, correlated. We exploited the information in these correlated traits by focusing on those genomic regions harboring quantitative trait loci (QTL) for multiple traits, reasoning that the more influential genes would impact a larger number of traits. This study identified five major QTL clusters (each with at least two significant logarithm of the odds scores >4.7), two of which had not been previously reported in baboons. One of these mapped to the baboon ortholog of human chromosome 1p32-p34 and influenced concentrations of LDL-cholesterol on Basal and high-fat, low-cholesterol diets. The other novel QTL cluster mapped to the baboon ortholog of human chromosome 12q13.13-q14.1 and influenced LDL size properties on high-fat, low-cholesterol and high-fat, high-cholesterol, but not Basal, diets. Confirming the value of this approach, three of the QTL clusters replicated published linkage findings for the same or similar traits.     

Phospholipase D2 mediates signaling by ATPase class I type 8B membrane 1

Functional defects in ATPase class I type 8B membrane 1 (ATP8B1 or familial intrahepatic cholestasis 1, FIC1) lead to cholestasis by mechanism(s) that are not fully understood. One proposed pathophysiology involves aberrant signaling to the bile acid sensor, the farnesoid X receptor (FXR), via protein kinase C ζ (PKCζ). The following cell line-based studies investigated whether phospholipase D2 may transduce a signal from FIC1 to FXR. PLD2 gain of function led to activation of the bile salt export pump (BSEP) promoter, a well-characterized FXR response. BSEP activation by PLD2 could be blocked by abrogating either PKCζ or FXR signaling. PLD2 loss of function led to a reduction in BSEP promoter activity. In addition, a variety of proteins that are activated by FXR, including BSEP, were reduced in HepG2 cells treated with PLD2 siRNA. Similar effects were observed in freshly isolated human hepatocytes. Activation of BSEP by FIC1 gain of function was blocked when PLD2 but not PLD1 was silenced. Overexpression of wild-type but not Byler mutant FIC1 led to an increase in membrane associated PLD activity. An intermediate level of activation of PLD activity was induced when a benign recurrent intrahepatic cholestasis FIC1 mutant construct was expressed. These studies show that FIC1 signals to FXR via a signaling pathway including PLD2 and PKCζ.     

LRP5 sequence and polymorphisms in the baboon

Background  LRP5 is known to have an important relationship with bone density and a variety of other biological processes. Mapping to human chromosome 11q13.2, LRP5 shows considerable evolutionary conservation. Orthologs of this gene exist in many species, although comparison of human LRP5 with other non-human primates has not been performed until now.
Methods  We reported the complementary DNA (cDNA) sequence and deduced amino acid sequence for baboon LRP5 , and compared the baboon and human sequences. cDNA sequences for 21 baboons were examined to identify single-nucleotide polymorphisms (SNPs).
Results  Sequences of coding regions in human and baboon LRP5 showed 97– 99% homology. Twenty-five SNPs were identified in the coding region of baboon LRP5 .
Conclusion  The observed degree of coding sequence homology in LRP5 led us to expect that the baboon may serve as a useful model for future research into the role(s) of this gene in primate metabolic diseases.     

Pleiotropic QTL on chromosome 12q23-q24 influences triglyceride and high-density lipoprotein cholesterol levels: the HERITAGE family study

To determine whether a common quantitative trait locus (QTL) influences the variation of fasting triglyceride (TG) and high-density lipoprotein cholesterol (HDL-C) levels, we used a bivariate multipoint linkage analysis with 654 polymorphic markers in 99 white and 101 black families. The phenotypes were investigated under two conditions: at baseline and after a 20-week exercise training intervention. A maximum genome-wide bivariate LOD score of 3.0 (p = 0.00010) was found on chromosome 12q23-q24, located within the IGF1 gene (insulin-like growth factor 1, at 107 cM) for TG and HDL-C at baseline in whites. This bivariate linkage peak is considerably higher than the univariate linkage results at the same chromosome location for either trait (for TG, LOD = 2.07, p = 0.00108; for HDL-C, LOD = 2.04, p = 0.00101). The genetic correlations between baseline TG and HDL-C levels were -0.14 for the residual and -0.33 for the QTL components. Moreover, association analysis showed that TG, HDL-C, and IGF1 are significantly associated (p = 0.04). In conclusion, these results suggest that a QTL on chromosome 12q23-q24 influences the variation of plasma TG and HDL-C levels. Further investigation should confirm whether IGF1 or another nearby gene is responsible for the concomitant variation in TG and HDL-C levels.     

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.     

Strain Background Modifies Phenotypes in the ATP8B1-Deficient Mouse

Background

Mutations in ATP8B1 (FIC1) underlie cases of cholestatic disease, ranging from chronic and progressive (progressive familial intrahepatic cholestasis) to intermittent (benign recurrent intrahepatic cholestasis). The ATP8B1-deficient mouse serves as an animal model of human ATP8B1 deficiency.

Methodology/Principal Findings

We investigated the effect of genetic background on phenotypes of ATP8B1-deficient and wild-type mice, using C57Bl/6 (B6), 129, and (B6-129) F1 strain backgrounds. B6 background resulted in greater abnormalities in ATP8B1-deficient mice than did 129 and/or F1 background. ATP8B1-deficient pups of B6 background gained less weight. In adult ATP8B1-deficient mice at baseline, those of B6 background had lower serum cholesterol levels, higher serum alkaline phosphatase levels, and larger livers. After challenge with cholate-supplemented diet, these mice exhibited higher serum alkaline phosphatase and bilirubin levels, greater weight loss and larger livers. ATP8B1-deficient phenotypes in mice of F1 and 129 backgrounds are usually similar, suggesting that susceptibility to manifestations of ATP8B1 deficiency may be recessive. We also detected differences in hepatobiliary phenotypes between wild-type mice of differing strains.

Conclusions/Significance

Our results indicate that the ATP8B1-deficient mouse in a B6 background may be a better model of human ATP8B1 deficiency and highlight the importance of informed background strain selection for mouse models of liver disease.     

A genomewide linkage scan for quantitative trait loci influencing the craniofacial complex in baboons (Papio hamadryas spp.)

Numerous studies have detected significant contributions of genes to variation in development, size, and shape of craniofacial traits in a number of vertebrate taxa. This study examines 43 quantitative traits derived from lateral cephalographs of 830 baboons (Papio hamadryas) from the pedigreed population housed at the Southwest National Primate Research Center. Quantitative genetic analyses were conducted using the SOLAR analytic platform, a maximum-likelihood variance components method that incorporates all familial information for parameter estimation. Heritability estimates were significant and of moderate to high magnitude for all craniofacial traits. Additionally, 14 significant quantitative trait loci (QTL) were identified for 12 traits from the three developmental components (basicranium, splanchnocranium, and neurocranium) of the craniofacial complex. These QTL were found on baboon chromosomes (and human orthologs) PHA1 (HSA1), PHA 2 (HSA3), PHA4 (HSA6), PHA11 (HSA12), PHA13 (HSA2), PHA16 (HSA17), and PHA17 (HSA13) (PHA, P. hamadryas; HSA, Homo sapiens). This study of the genetic architecture of the craniofacial complex in baboons provides the groundwork needed to establish the baboon as an animal model for the study of genetic and nongenetic influences on craniofacial variation.     

Identification of candidate genes encoding an LDL-C QTL in baboons

Cardiovascular disease (CVD) is the leading cause of death in developed countries, and dyslipidemia is a major risk factor for CVD. We previously identified a cluster of quantitative trait loci (QTL) on baboon chromosome 11 for multiple, related quantitative traits for serum LDL-cholesterol (LDL-C). Here we report differentially regulated hepatic genes encoding an LDL-C QTL that influences LDL-C levels in baboons. We performed hepatic whole-genome expression profiling for LDL-C-discordant baboons fed a high-cholesterol, high-fat (HCHF) diet for seven weeks. We detected expression of 117 genes within the QTL 2-LOD support interval. Three genes were differentially expressed in low LDL-C responders and 8 in high LDL-C responders in response to a HCHF diet. Seven genes (ACVR1B, CALCOCO1, DGKA, ERBB3, KRT73, MYL6B, TENC1) showed discordant expression between low and high LDL-C responders. To prioritize candidate genes, we integrated miRNA and mRNA expression profiles using network tools and found that four candidates (ACVR1B, DGKA, ERBB3, TENC1) were miRNA targets and that the miRNAs were inversely expressed to the target genes. Candidate gene expression was validated using QRT-PCR and Western blotting. This study reveals candidate genes that influence variation in LDL-C in baboons and potential genetic mechanisms for further investigation.     

Progressive familial intrahepatic cholestasis (PFIC): evidence for genetic heterogeneity by exclusion of linkage to chromosome 18q21–q22

Progressive familial intrahepatic cholestasis (PFIC) is the second most common form of familial intrahepatic cholestasis. The genes for PFIC and for a milder form of the disease, benign recurrent intrahepatic cholestasis (BRIC), were recently mapped to a 19-cM region on chromosome 18q21–q22. The results suggest that PFIC and BRIC are allelic diseases. We have studied 11 Swedish patients from eight families with clinical and biochemical features consistent with PFIC. The families were genotyped for markers D18S69, D18S64, D18S55 and D18S68, spanning the PFIC candidate region. Unexpectedly, the segregation of haplotypes excluded the entire region in three families, and no indications for shared haplotypes were found in the patients of the six remaining families. A four-point linkage analysis of all families excluded linkage from D18S69 to D18S55 (Z_max < –5). Thus, our data strongly suggest the presence of a second, yet unknown, locus for PFIC. The results indicate that great care should be taken when using 18q markers for prenatal diagnosis and genetic counseling for the disease. Received: 12 February 1997 / Accepted: 11 April 1997     

Quantitative trait loci on chromosomes 1, 2, 3, 4, 8, 9, 11, 12, and 18 control variation in levels of T and B lymphocyte subpopulations

Lymphocyte subpopulation levels are used for prognosis and monitoring of a variety of human diseases, especially those with an infectious etiology. As a primary step to defining the major gene variation underlying these phenotypes, we conducted the first whole-genome screen for quantitative variation in lymphocyte count, CD4 T cell, CD8 T cell, B cell, and natural killer cell numbers, as well as CD4:CD8 ratio. The screen was performed in 15 of the CEPH families that form the main human genome genetic project mapping resource. Quantitative-trait loci (QTLs) that account for significant proportions of the phenotypic variance of lymphocyte subpopulations were detected on chromosomes 1, 2, 3, 4, 8, 9, 11, 12, and 18. The most significant QTL found was for CD4 levels on chromosome 8 (empirical P=.00005). Two regions of chromosome 4 showed significant linkage to CD4:CD8 ratio (empirical P=.00007 and P=.003). A QTL for the highly correlated measures of CD4 and CD19 levels colocalized at 18q21 (both P=.003). Similarly, a shared region of chromosome 1 was linked to CD8 and CD19 levels (P=.0001 and P=.002, respectively). Several of the identified chromosome regions are likely to harbor polymorphic candidate genes responsible for these important human phenotypes. Their discovery has important implications for understanding the generation of the immune repertoire and understanding immune-system homeostasis. More generally, these data show the power of an integrated human gene-mapping approach for heritable molecular phenotypes, using large pedigrees that have been extensively genotyped.     

Comparative mapping of a region on chromosome 10 containing QTL for reproduction in swine

Several quantitative trait loci (QTL) for important reproductive traits (age of puberty, ovulation rate, nipple number and plasma FSH) have been identified on the long arm of porcine chromosome 10. Bi-directional chromosome painting has shown that this region is homologous to human chromosome 10p. Because few microsatellite or type I markers have been placed on SSC10, we wanted to increase the density of known ESTs mapped in this region of the porcine genome. Genes were chosen for their position on human chromosome 10, sequence availability from the TIGR pig gene indices, and their potential as a candidate gene. The PCR primers were designed to amplify across introns or 3'-UTR to maximize single nucleotide polymorphism (SNP) discovery. Parents of the mapping population (one sire and seven dams) were amplified and sequenced to find informative markers. The SNPs were genotyped using primer extension and mass spectrometry. These amplification products were also used to probe a BAC library (RPCI-44, Roswell Park Cancer Institute) for positive clones and screened for microsatellites. Six genes from human chromosome 10p (AKR1C2, PRKCQ, ITIH2, ATP5C1, PIP5K2A and GAD2) were mapped in the MARC swine mapping population. Gene order was conserved within these markers from centromere to telomere of porcine chromosome 10q, as compared with human chromosome 10p. Four of these genes (PIP5K2A, ITIH2, GAD2 and AKR1C2), which map under QTL, are potential candidate genes. Identification of porcine homologues near important QTL and development of a comparative map for this chromosome will allow further fine- mapping and positional cloning of candidate genes affecting reproductive traits.     

A new genetic locus, Bevi, on human chromosome 6 which controls the replication of baboon type C virus in human cells.

Somatic cell hybrids derived from seven independent fusions between mouse X human and hamster X human parental cells were examined for their ability to support the replication of the baboon endogenous type C virus. These hybrids preferentially segregated human chromosomes while retaining rodent chromosomes, as demonstrated by karyotypic and isozyme analysis. A total of 41 primary colonies and 33 secondary subclones were analyzed for viral replication, as well as for the presence of enzyme structural gene markers for 19 of 23 human chromosomes. A syntenic association was seen between the ability of the baboon type C virus to infect and replicate in hybrid cultures and the expression of human malic enzyme-1 (assigned to human chromosome 6). Analysis of 86 highly segregated subclones derived from cells preinfected with baboon type C virus showed that the continued production of baboon type C virus segregated concordantly with the expression of three enzyme genes assigned to human chromosome 6 (malic enzyme-1, phosphoglucomutase-3 and superoxide dismutase-2). Subclones of infected hybrids which lost chromosome 6 and failed to release virus also failed to synthesize the virus-coded major structural protein p30. No syntenic association between baboon virus expression and any of 18 other human chromosomes was observed. These studies define a new gene (designated Bevi) on human chromosome 6 which dominantly controls the replication of baboon type C virus. The data suggest that Bevi may be a preferred integration site for the baboon type C DNA provirus in the human genome.     

Characterization of the porcine FABP5 gene and its association with the FAT1 QTL in an Iberian by Landrace cross

We have characterized and mapped the porcine fatty acid binding protein 5, epidermal (FABP5) gene. According to linkage and RH mapping, this gene is located close to the FABP4 (fatty acid binding protein 4, adipocyte) gene on swine chromosome 4. We resequenced 4.7 kb of the FABP5 gene in the parental population of an Iberian x Landrace cross (IBMAP), identifying seven SNPs arranged in two distinct FABP5 haplotypes. QTL and association analyses in the IBMAP population showed that this gene is strongly associated with fat deposition. QTL and haplotype analysis revealed that both FABP4 and FABP5 (clustered in mammals) are major candidate genes for the FAT1 QTL; the most likely position for the FAT1 QTL is between these two genes. Finally, our results suggest the presence of more than one QTL affecting fatness traits on porcine chromosome 4.     

Genome‐wide association study for jumping performances in French sport horses

A genome‐wide association study was performed to identify single nucleotide polymorphisms (SNPs) associated with jumping performances of warmbloods in France. The 999 horses included in the study for jumping performances were sport horses [mostly Selle Français (68%), Anglo‐Arabians (13%) and horses from the other European studbooks]. Horses were genotyped using the Illumina EquineSNP50 BeadChip. Of the 54 602 SNPs available on this chip, 44 424 were retained after quality testing. Phenotypes were obtained by deregressing official breeding values for jumping competitions to use all available information, that is, the performances of each horse as well as those of its relatives. Two models were used to test the effects of the genotypes on deregressed phenotypes: a single‐marker mixed model and a haplotype‐based mixed model (significant: P < 1E‐05; suggestive: P < 1E‐04). Both models included a polygenic effect to take into account familial structures. For jumping performances, one suggestive quantitative trait locus (QTL) located on chromosome 1 (BIEC2_31196 and BIEC2_31198) was detected with both models. This QTL explains 0.7% of the phenotypic variance. RYR2, a gene encoding a major calcium channel in cardiac muscle in humans and mice, is located 0.55 Mb from this potential QTL.     

Determinants of variation in serum paraoxonase enzyme activity in baboons

Paraoxonase (PON), an HDL-associated enzyme, is one of many circulating antioxidants thought to play a vital protective role. To better understand the determinants of quantitative variation in serum PON activity, we assayed PON in samples from 611 pedigreed baboons fed three diets. PON was measured enzymatically; the main determinant of variation was genetic and consisted of at least three components: two loci detected by linkage analyses and a residual polygenic component. Multipoint linkage analyses gave peak log of the odds (LOD) scores on the baboon homolog of human chromosome 7q21-22 (near PON1, the structural gene) of 9.1 on the low-cholesterol, high-fat diet and 4.1 on the high-cholesterol, high-fat diet (genome-wide P values were 1 x 10(-8) and 0.0018, respectively). Surprisingly, a second locus on the baboon homolog of human chromosome 12q13 gave a LOD score of 2.9 on the high-cholesterol, high-fat diet (genome-wide P value was 0.032). We identified several significant covariates, including age, sex, diet, and apolipoprotein A-I concentrations. We estimate that 53% of total trait variation in baboons is explained by genes and 17% by covariates, thus accounting for approximately 70% of total variation in baboon PON. Although the generation of free radicals is influenced primarily by environmental factors, our findings suggest strong genetic regulation of one component in the antioxidant defense system that plays a major role in susceptibility to atherosclerosis.