Haplotype frequency estimation in the presence of genotyping errors

Several statistical methods have been proposed to estimate haplotype frequencies, either based on unrelated individuals or based on families. These estimates may yield insights on population genetics as well as associations between candidate regions and disease of interest. One limitation of the existing methods is that all these methods make the implicit assumption that there are no genotyping errors. However, genotyping errors are unavoidable in practice. Numerous methods have been developed to incorporate genotyping errors in genetic studies, but none to date have addressed the issues of haplotype inference in the presence of genotyping errors. In this article, we develop statistical methods for haplotype inference incorporating genotyping errors. We describe how our methods can be applied to analyze unrelated individuals as well as nuclear families. Our simulation results show that the proposed methods perform well in the presence of genotyping errors.     

Compound heterozygote for lipoprotein lipase deficiency: Ser----Thr244 and transition in 3'' splice site of intron 2 (AG----AA) in the lipoprotein lipase gene.

Cloning and sequencing of translated exons and intron-exon boundaries of the lipoprotein lipase gene in a patient of French descent who has the chylomicronemia syndrome revealed that he was a compound heterozygote for two nucleotide substitutions. One (TCC----ACC) leads to an amino acid substitution (Ser----Thr244), while the other alters the 3' splice site of intron 2 (AG----AA). The functional significance of the Thr244 amino acid substitution was established by in vitro expression in cultured mammalian cells.     

A missense (Asp250----Asn) mutation in the lipoprotein lipase gene in two unrelated families with familial lipoprotein lipase deficiency.

We have identified the molecular basis for familial lipoprotein lipase (LPL) deficiency in two unrelated families with the syndrome of familial hyperchylomicronemia. All 10 exons of the LPL gene were amplified from the two probands' genomic DNA by polymerase chain reaction. In family 1 of French descent, direct sequencing of the amplification products revealed that the patient was heterozygous for two missense mutations, Gly188----Glu (in exon 5) and Asp250----Asn (in exon 6). In family 2 of Italian descent, sequencing of multiple amplification products cloned in plasmids indicated that the patient was a compound heterozygote harboring two mutations, Arg243----His and Asp250----Asn, both in exon 6. Studies using polymerase chain reaction, restriction enzyme digestion (the Gly188----Glu mutation disrupts an Ava II site, the Arg243----His mutation, a Hha I site, and the Asp250----Asn mutation, a Taq I site), and allele-specific oligonucleotide hybridization confirmed that the patients were indeed compound heterozygous for the respective mutations. LPL constructs carrying the three mutations were expressed individually in Cos cells. All three mutant LPLs were synthesized and secreted efficiently; one (Asp250----Asn) had minimal (approximately 5%) catalytic activity and the other two were totally inactive. The three mutations occurred in highly conserved regions of the LPL gene. The fact that the newly identified Asp250----Asn mutation produced an almost totally inactive LPL and the location of this residue with respect to the three-dimensional structure of the highly homologous human pancreatic lipase suggest that Asp250 may be involved in a charge interaction with an alpha-helix in the amino terminal region of LPL. The occurrence of this mutation in two unrelated families of different ancestries (French and Italian) indicates either two independent mutational events affecting unrelated individuals or a common shared ancestral allele. Screening for the Asp250----Asn mutation should be included in future genetic epidemiology studies on LPL deficiency and familial combined hyperlipidemia.     

Differential effect of combined lipase deficiency (cld/cld) on human hepatic lipase and lipoprotein lipase secretion.

Combined lipase deficiency (cld) is a recessively inherited disorder in mice associated with a deficiency of LPL and hepatic lipase (HL) activity. LPL is synthesized in cld tissues but is retained in the endoplasmic reticulum (ER), whereas mouse HL (mHL) is secreted but inactive. In this study we investigated the effect of cld on the secretion of human HL (hHL) protein mass and activity. Differentiated liver cell lines were derived from cld mice and their normal heterozygous (het) littermates by transformation of hepatocytes with SV40 large T antigen. After transient transfection with lipase expression constructs, secretion of hLPL activity from cld cells was only 12% of that from het cells. In contrast, the rate of secretion of hHL activity and protein mass per unit of expressed hHL mRNA was identical for the two cell lines. An intermediate effect was observed for mHL, with a 46% reduction in secretion of activity from cld cells. The ER glucosidase inhibitor, castanospermine, decreased secretion of both hLPL and hHL from het cells by approximately 70%, but by only approximately 45% from cld cells. This is consistent with data suggesting that cld may result from a reduced concentration of the ER chaperone calnexin. In conclusion, our results demonstrate a differential effect of cld on hLPL, mHL, and hHL secretion, suggesting differential requirements for activation and exit of the enzymes from the ER.     

Changes in the lipoprotein lipase (clearing-factor lipase) activity of white adipose tissue during development of the rat.

The lipoprotein lipase (clearing-factor lipase) activity of the white adipose tissue from rats aged between 1 and 145 days was determined. Five adipose-tissue sites (epididymal, uterine, subcutaneous, perirenal and intramuscular) together with serum concentrations of triacylglycerol, cholesterol and glucose were studied. The pattern of enzyme-activity change was remarkably similar in all the sites studied, although the growth of the tissues proceeded non-uniformly. After a peak of activity early in suckling, lipoprotein lipase activity fell to low values by 20 days of age. At weaning (21 days) the activity increased sharply and within 5 days high values were regained. The serum triacylglycerol and cholesterol concentrations were low at birth and reached peaks of concentration coincidentally with the minima of white-adipose-tissue lipoprotein lipase activities, seen late in suckling. The changes in enzyme activity were related to other metabolic changes in adipose tissue and with the known changes in plasma insulin concentrations occurring during development.     

The inhibition in vivo of lipoprotein lipase (clearing-factor lipase) activity by triton WR-1339.

1. Lipoprotein lipase activity was measured in heart homogenates and in heparin-releasable and non-releasable fractions of isolated perfused rat hearts, after the intravenous injection of Triton WR-1339. 2. In homogenates of hearts from starved, rats, lipoprotein lipase activity was significantly inhibited (P less than 0.001) 2h after the injection of Triton. This inhibition was restricted exclusively to the heparin-releasable fraction. Maximum inhibition occurred 30 min after the injection and corresponded to about 60% of the lipoprotein lipase activity that could be released from the heart during 30 s perfusion with heparin. 3. Hearts of Triton-treated starved rats were unable to take up and utilize 14C-labelled chylomicron triacylglycerol fatty acids, even though about 40% of heparin-releasable activity remained in the hearts. 4. It is concluded that Triton selectively inhibits the functional lipoprotein lipase, i.e. the enzyme directly involved in the hydrolysis of circulating plasma triacylglycerols. 5. Lipoprotein lipase activities measured in homogenates of soleus muscle of starved rats and adipose tissue of fed rats were decreased by 25 and 39% respectively after Triton injection. It is concluded that, by analogy with the heart, these Triton-inhibitable activities correspond to the functional lipoprotein lipase.     

Structure-activity and in vivo evaluation of a novel lipoprotein lipase (LPL) activator

Elevated triglycerides (TG) contribute towards increased risk for cardiovascular disease. Lipoprotein lipase (LPL) is an enzyme that is responsible for the metabolism of core triglycerides of very-low density lipoproteins (VLDL) and chylomicrons in the vasculature. In this study, we explored the structure-activity relationships of our lead compound (C10d) that we have previously identified as an LPL agonist. We found that the cyclopropyl moiety of C10d is not absolutely necessary for LPL activity. Several substitutions were found to result in loss of LPL activity. The compound C10d was also tested in vivo for its lipid lowering activity. Mice were fed a high-fat diet (HFD) for four months, and treated for one week at 10 mg/kg. At this dose, C10d exhibited in vivo biological activity as indicated by lower TG and cholesterol levels as well as reduced body fat content as determined by ECHO-MRI. Furthermore, C10d also reduced the HFD induced fat accumulation in the liver. Our study has provided insights into the structural and functional characteristics of this novel LPL activator.     

Comparative studies of zebrafish Danio rerio lipoprotein lipase (lpl) and hepatic lipase (lipc) genes belonging to the lipase gene family: evolution and expression pattern

In this study, bioinformatics analysis, tissue distribution and developmental expression pattern of lipoprotein lipase (lpl) and hepatic lipase (lipc) in zebrafish Danio rerio are reported. In adult D. rerio, lpl was highly expressed in liver. This is remarkably different from the tissue expression pattern of LPL in mammals, which is not detected in the adult liver. The expression of lipc was liver specific, which is consistent with that in mammals. During embryogenesis, lpl mRNA was increased gradually in concentration from 0·5 hpf (hour post fertilization) to 6 dpf (days post fertilization), but lipc was not expressed at the early stage of the embryo until 3 dpf. In situ hybridization further displayed the expression pattern of lpl mainly restricted to the head region including cells surrounding the mouth opening, branchial arches, pectoral fin and lateral line neuromast, whereas lipc was mainly restricted to the liver and part of head regions including lens. This lays a foundation for further investigation of lpl or lipc function and evolution in fishes.     

Inhibition of lipoprotein lipolysis by polyarginine and evaluation of the mechanism of its interaction with lipoprotein lipase

It was found that polyarginine (Mr 40 000-60 000) is a strong inhibitor of the lipoprotein lipase activity in vivo and in vitro. The inhibitory effect in vivo was observed after a single intravenous injection of 0.85-3.5 mg/kg to rabbits, that in vitro at the polypeptide concentration of greater than or equal to 2.5 micrograms/ml. Within the first few hours after intravenous injection of polyarginine hyperlipidemia occurred with an obvious increase in the plasma triglyceride and VLDL fractions and a slight decrease of the LDL and HDL fractions. These changes typical for reduced lipoprotein lipolysis were due to the formation of a polyarginine-heparin complex, on the one hand, and to the formation of a polyarginine-enzyme complex devoid of the lipolytic properties, on the other. The inhibitory effect of polyarginine on lipoprotein lipase is related to the whole polypeptide molecule or its large fragment, since arginine and metformine (bi-guanidine compound) have no effect on the enzyme activity.     

Development of the Handy Bio-Strand and its application to genotyping of OPRM1 (A118G)

We previously developed a three-dimensional microarray system, the Bio-Strand, which exhibits advantages in automated DNA analysis in combination with our Magtration Technology. In the current study, we have developed a compact system for the Bio-Strand, the Handy Bio-Strand, which consists of several tools for the preparation of Bio-Strand Tip, hybridization, and detection. Using the Handy Bio-Strand, we performed single nucleotide polymorphism (SNP) genotyping of OPRM1 (A118G) by allele-specific oligonucleotide competitive hybridization (ASOCH). DNA fragments containing SNP sites were amplified from genomic DNA by PCR and then were fixed on a microporous nylon thread. Thus, prepared Bio-Strand Tip was hybridized with allele-specific Cy5 probes (<15mer), on which the SNP site was designed to be located in the center. By optimizing the amount of competitors, the selectivity of Cy5 probes increased without a drastic signal decrease. OPRM1 (A118G) genotypes of 23 human genomes prepared from whole blood samples were determined by ASOCH using the Handy Bio-Strand. The results were perfectly consistent with those determined by PCR direct sequencing. ASOCH using the Handy Bio-Strand would be a very simple and reliable method for SNP genotyping for small laboratories and hospitals.     

Accuracy of haplotype frequency estimation for biallelic loci, via the expectation-maximization algorithm for unphased diploid genotype data

Haplotype analyses have become increasingly common in genetic studies of human disease because of their ability to identify unique chromosomal segments likely to harbor disease-predisposing genes. The study of haplotypes is also used to investigate many population processes, such as migration and immigration rates, linkage-disequilibrium strength, and the relatedness of populations. Unfortunately, many haplotype-analysis methods require phase information that can be difficult to obtain from samples of nonhaploid species. There are, however, strategies for estimating haplotype frequencies from unphased diploid genotype data collected on a sample of individuals that make use of the expectation-maximization (EM) algorithm to overcome the missing phase information. The accuracy of such strategies, compared with other phase-determination methods, must be assessed before their use can be advocated. In this study, we consider and explore sources of error between EM-derived haplotype frequency estimates and their population parameters, noting that much of this error is due to sampling error, which is inherent in all studies, even when phase can be determined. In light of this, we focus on the additional error between haplotype frequencies within a sample data set and EM-derived haplotype frequency estimates incurred by the estimation procedure. We assess the accuracy of haplotype frequency estimation as a function of a number of factors, including sample size, number of loci studied, allele frequencies, and locus-specific allelic departures from Hardy-Weinberg and linkage equilibrium. We point out the relative impacts of sampling error and estimation error, calling attention to the pronounced accuracy of EM estimates once sampling error has been accounted for. We also suggest that many factors that may influence accuracy can be assessed empirically within a data set-a fact that can be used to create "diagnostics" that a user can turn to for assessing potential inaccuracies in estimation.     

Relative efficiency of haplotype frequency estimation in sibships and nuclear families compared to unrelated individuals

The problem of estimating haplotype frequencies from unphased single nucleotide polymorphism (SNP) genotype data in sibships with and without parents is considered. We focus on the Fisher information of the haplotype frequencies of the parents in order to correctly deal with the dependence of haplotypes within sibships. We compare these Fisher information matrices with those obtained for unrelated individuals and study the relative efficiency of sibships with and without parents compared to unrelated individuals in estimating haplotype frequencies. Crudely summarizing, the second sib contributes half the information of the first, except for rare haplotypes, when the second sib counts almost as one. We argue that the relative efficiencies can also be used to correct for dependence in the calculation of standard errors after initially ignoring the dependence in the estimation phase.     

Effect of carbonyl cyanide m-chlorophenylhydrazone (CCCP) on the dimerization of lipoprotein lipase

Lipoprotein lipase (LPL), an enzyme playing the central role in triglyceride metabolism, is a glycoprotein and a homodimer of identical subunits. Dimerization and proper processing of oligosaccharide chains are important maturation steps in post-translational regulation of enzyme activity. Indirect evidences suggest that dimerization of LPL occurs in endoplasmic reticulum (ER) or Golgi. In this study, we investigated the dimerization status of LPL in 3T3-L1 adipocytes, using sucrose density gradient ultracentrifugation and carbonyl cyanide m-chlorophenylhydrazone (CCCP), an inhibitor of ER-Golgi protein transport. In the presence of CCCP, no increase of cellular LPL activity was detected during 2 h of recovery period after the depletion of LPL with heparin and cycloheximide. Only endoglycosidase H (endo H)-sensitive subunits were found in CCCP-treated cells after endo H digestion, suggesting that inactive LPL was retained in ER. In the presence of castanospermine, an inhibitor of ER glucosidase I, LPL subunits of both control and CCCP-treated cells had same molecular weight, indicating that complete oligosaccharides were transferred to LPL subunits in the presence of CCCP. In sucrose density gradient ultracentrifugation, all the LPL protein synthesized in the presence of CCCP was found at the dimeric fractions as in control cells. Most of LPL protein in control cells showed high affinity for heparin, and there was no difference between the control and CCCP-treated cells. These results suggest that dimerization and acquisition of high affinity for heparin of LPL can occur in ER of CCCP-treated cells without acquisition of catalytic activity.     

Identification of factors regulating lipoprotein lipase catalyzed hydrolysis in rats with the aid of monoacid-rich lipoprotein preparations(1)

To identify the substrate specificity and regulatory factors in lipoprotein lipase (LPL) catalyzed hydrolysis of triacylglycerol-rich lipoprotein, monoacid-rich lipoproteins were used to study the kinetic parameters of LPL. Feeding growing rats with diets rich in palmitic acid (16:0), oleic acid (18:1) or linoleic acid (18:2) for 10 days increased the corresponding acid content in the triacylglycerols of the lipoproteins. Force-feeding the monoacid-rich triacylglycerols, particularly 16:0 or 18:1, increased the respective fatty acid content in both chylomicrons and VLDLs. Major apolipoproteins and lipid compositions were essentially similar among all lipoproteins differing in monoacid species, except for apo A-IV. The Vmax of LPL for 16:0-rich chylomicrons and VLDLs were higher than for 18:1- or 18:2-rich lipoproteins. Order parameter (S), an indicator of the surface fluidity of lipoproteins, decreased with the chain length and unsaturation of monoacid in similar manner as the Vmax. The Vmax of LPL increased linearly (P < 0.05) with an increase in either the palmitic acid content of the lipoprotein triacylglycerols or order parameter (S) of the lipoproteins. The order parameter (S) and Vmax of LPL were higher in 16:0 triacylglycerol emulsions with apo B than with 18:1 or 18:2 triacylglycerols. The apo A-IV in triacylglycerol emulsions stimulated Vmax of LPLs in the presence of apo B and apo C-II. The binding of apo A-IV to 16:0 triacylglycerol emulsions was higher than to other triacylglycerol emulsions. These findings suggest that lipoprotein catalysis by LPL is modulated by the 16:0 level in the lipoprotein triacylglycerol, which affects the surface fluidity and apo A-IV content of lipoproteins.