The polymorphism at codon 54 of the FABP2 gene increases fat absorption in human intestinal explants.

Based on titration microcalorimetry and Caco-2 cell line transfection studies, it has been suggested that the A54T of the FABP2 gene plays a significant role in the assimilation of dietary fatty acids. However, reports were divergent with regard to the in vivo interaction between this polymorphism and postprandial lipemia. We therefore determined the influence of this intestinal fatty acid-binding protein polymorphism on intestinal fat transport using the human jejunal organ culture model, thus avoiding the interference of various circulating factors capable of metabolizing in vivo postprandial lipids. Analysis of DNA samples from 32 fetal intestines revealed 22 homozygotes for the wild-type Ala-54/Ala-54 genotype (0.83) and 10 heterozygotes for the polymorphic Thr-54/Ala-54 genotype (0.17). The Thr-encoding allele was associated with increased secretion of newly esterified triglycerides, augmented de novo apolipoprotein B synthesis, and elevated chylomicron output. On the other hand, no alterations were found in very low density lipoprotein and high density lipoprotein production, apolipoprotein A-I biogenesis, or microsomal triglyceride transfer protein mass and activity. Similarly, the alanine to threonine substitution at residue 54 did not result in changes in brush border hydrolytic activities (sucrase, glucoamylase, lactase, and alkaline phosphatase) or in glucose uptake or oxidation. Our data clearly document that the A54T polymorphism of FABP2 specifically influences small intestinal lipid absorption without modifying glucose uptake or metabolism. It is proposed that, in the absence of confounding factors such as environmental and genetic variables, the FABP2 polymorphism has an important effect on postprandial lipids in vivo, potentially influencing plasma levels of lipids and atherogenesis.

Determinants of lipid level variability in French-Canadian children with familial hypercholesterolemia.

The wide variability in the biochemical expression of familial hypercholesterolemia (FH) is only partly explained by mutational heterogeneity in the low density lipoprotein receptor (LDLR) gene. In the current study, we measured this biochemical variability in a group of children heterozygous for the >15-kb LDLR gene deletion (n=67) and examined the contribution of apolipoprotein (apo) E and B allelic variations to this phenotypic variability. Variances of total cholesterol (TC), LDL-C, and apoB concentrations and of the ratio of TC to high density lipoprotein cholesterol (HDL-C) were increased in FH subjects compared with controls. However, after taking the means into account, the coefficients of variation showed that the variability of LDL-C and apoB concentrations was smaller for FH than for controls and that the variability of TC and of the ratio TC to HDL-C was similar between both groups. The epsilon2/3 genotype was associated with lower mean TC, LDL-C, and apoB concentrations in FH. The magnitude of this effect was smaller in controls than in FH. Indeed, the percentages of total variance of TC, LDL-C, and apoB attributable to the apoE locus were 19.9%, 18.1%, and 11.8%, respectively, in FH cases and 5.9%, 7.4%, and 6.0%, respectively, in controls. We did not detect any effect of the apoB insertion/deletion polymorphism on lipid traits in FH children. However, in controls, we observed a strong interaction between apoE and apoB genotypes on apoB concentrations and on TC to HDL-C ratios. Our study reemphasizes the important role of apoE in lipid metabolism and illustrates that the effects of allelic variations on lipid traits are context dependent.

Human papillomavirus (HPV) study of 691 pathological specimens from Quebec by PCR-direct sequencing approach.

Human papillomaviruses (HPV) are etiological agents of cervical cancer. In order to address clinical demand for HPV detection and sequence typing, mostly in pre-cancerous cervical lesions, we applied our two-tier PCR-direct sequencing (PCR-DS) approach based on the use of both MY09/MY11 and GP5 + /GP6 + sets of primers. We tested 691 pathological specimens, all of which were biopsies, 75% of which were diagnosed histologically as cervical intraepithelial neoplasia (CIN) grades I-III. In total, 484 samples (70%) tested HPV-positive, yielding 531 HPV sequences from 47 HPV types, including two novel types. Four most frequently found HPV types accounted for 52.9% of all isolates: HPV6, 16, 11, and 31 (21.5%, 20.0%, 7.0%, and 4.5%, respectively). Some interesting results are the following: all currently known high-risk HPV (14 types) and low-risk HPV (6 types) were detected; HPV18 was not the 1st or 2nd but rather the 4th-5th most frequent high-risk HPV type; the highest detection rate for HPV (86%) among samples suspected to be HPV-infected was found in the youngest age group (0-10 years old), including 70% (44/63) "genital" HPV types; HPV types of undetermined cervical cancer risk represented 19% and of the total HPV isolates but were strongly increased in co-infections (36.5% of all isolates). To our knowledge, this is the largest sequencing-based study of HPV. The HPV types of unknown cancer risk, representing the majority of the known HPV types, 27 of the 47 types detected in this study, are not likely to play a major role in cervical cancer because their prevalence in CIN-I, II, and III declines from 16% to 8% to 2.5%. The two-tier PCR-DS method provides greater sensitivity than cycle sequencing using only one pair of primers. It could be used in a simple laboratory setting for quick and reliable typing of known and novel HPV from clinical specimens with fine sequence precision. It could also be applied to anti-cancer vaccine development.

New method for automatic identification identification and typing of single and multiple superimposed human papillomavirus sequences.

This study of specimens of human papillomaviruses (HPV) through HPV-specific polymerase chain reaction (PCR), followed by direct sequencing, resulted in 11% (38/354) superimposed HPV sequences, signifying coinfection with more than one HPV type. To address the diagnostic problem that these superimposed ("degenerated," overlapping) sequences pose, the authors created a papillomavirus database in Microsoft Excel (Microsoft Corporation, Redmond, WA, U.S.A.) and Corel Quattro Pro 9 (Corel Corporation, Ottawa, Ontario, Canada) formats, retrievable from http://www2.crosswinds.net/ -crosswindswatson/index.html. This sequence database is simultaneously a search and comparison tool for quick (several seconds) typing of HPV from regular and "degenerated" sequencing results. Some of the advantages of the method are as follows: (1) superimposed HPV sequences that differ in length could be readily identified from a single input; (2) the search is restricted to the currently known 127 PV types, which speeds up the typing; (3) the most common HPV sequencing artifacts are included for quick detection; (4) there is no proprietary code and the database could be easily improved; (5) HPV sequence identification does not require internet connection; and (6) new HPV types could be easily detected. This method allowed resolution of all but 1 of 354 HPV-positive specimens. From 38 superimposed HPV sequences, this method identified one known HPV type (3 specimens), two HPV types (30 specimens) and three HPV types (4 specimens).

Direct human papillomavirus (HPV) sequencing method yields a novel HPV in a human immunodeficiency virus-positive Quebec woman and distinguishes a new HPV clade.

Papillomaviruses of supergroup A exhibit genital tropism and are best known as etiologic agents for benign and malignant cervical lesions in women. A polymerase chain reaction direct sequencing approach with P-33-labeled dideoxynucleotides was used to detect and type human papillomaviruses (HPVs) in cervical biopsies. A novel sequence was found in condylomatous specimens from a human immunodeficiency virus-positive French Canadian woman. The viral gene L1 was sequenced completely, yielding a novel HPV type of supergroup A, named JC9710. This is related to a previously described HPV type from New Mexico, CP8061, and to Colobus monkey papillomavirus 1. Sequence similarity searches and phylogenetic analyses with different software packages clustered the three viral types as a new clade, for which the next available number, A15, was proposed.

Familial lipoprotein lipase deficiency in infancy: clinical, biochemical, and molecular study.

OBJECTIVES: To describe the characteristics of lipoprotein lipase (LPL)-deficient patients seen in infancy and to evaluate the safety and efficacy of severe fat restriction. METHODS: Children <1 year old presenting with chylomicronemia between 1972 and 1995 were identified, and their clinical courses were reviewed retrospectively. RESULTS: LPL deficiency was demonstrated in 16 infants who presented with irritability (n = 7), lower intestinal bleeding (n = 2), pallor, anemia, or splenomegaly (n = 5), and a family history or fortuitous discovery (n = 2). All plasma samples were lactescent at presentation. Chylomicronemia responded rapidly to dietary fat restriction, and it was possible to maintain satisfactory metabolic control for a prolonged period of time. Only 1 adolescent girl had an episode of pancreatitis associated with the use of oral contraceptives. No persistent adverse effects on growth were seen. We obtained abnormal values for serum iron, alkaline phosphatase, and total calcium. CONCLUSIONS: The presentation of LPL deficiency is heterogeneous during infancy. Close dietary monitoring is required to avoid nutritional deficiencies. Estrogen therapy should be avoided in LPL-deficient patients.

JC9813-A putative novel human papillomavirus identified by PCR-DS.

Papillomaviruses consist of more than 130 viral types described so far. Most of them are human papillomaviruses (HPV) of supergroup A, demonstrating ano-genital tropism and characterized as etiological agents for benign and malignant cervical lesions in women. A PCR-direct sequencing (PCR-DS) approach with P-33 labeled dideoxynucleotides was used to detect and type human papillomaviruses in cervical biopsies. One novel sequence was identified in a LSIL (low-grade squamous intraepithelial lesions) specimen from an HIV-positive English Canadian patient. The structure of the viral gene L1 was determined, yielding a putative novel HPV type of supergroup A (clade A8) named JC9813.

A two-tier polymerase chain reaction direct sequencing method for detecting and typing human papillomaviruses in pathological specimens.

An in-house polymerase chain reaction direct sequencing (PCR-DS) approach for HPV detection and typing was developed, taking advantage of two widely used pairs of human papillomavirus (HPV)-specific PCR primers, MY09/MY11 and GP5/GP6, and 33P-labeled dideoxynucleotides. In this study, 105 pathological specimens were examined: 89% were diagnosed as cervical intraepithelial neoplasia (CIN) grade I-III, 76.2% were HPV-positive by PCR-DS. The PCR using GP5/GP6 (first tier) and MY09/MY11 primers (second tier for the GP5/GP6-negative samples) detected additional 15%-25% HPV-positive samples compared with each pair used separately. Direct sequencing was then used to type the HPV. A readout of a sequence as short as 34 nucleotides within a specific region in the L1 gene is sufficient to type known or novel sequences. Because of its high sensitivity and cost-effectiveness, the two-tier PCR-DS was adopted by the authors as the current method of choice for HPV diagnosis with ultimate sequence precision.

Identification of two novel LDL receptor gene defects in French-Canadian pediatric population: mutational analysis and biochemical studies.

Familial hypercholesterolemia (FH) is at least twofold more prevalent in French Canadians from Québec than in most Western populations. Although our recent data confirmed this high frequency of heterozygous FH in our pediatric population with hypercholesterolemia, none of the five established molecular defects for the French-Canadian population was detected in 29% of the unrelated French-Canadian children characterized by a persistent increase in LDL (low density lipoprotein receptor) cholesterol and a positive parental history of hyperlipidemia (Assouline et al., 1995). To probe for new mutations, six of these molecularly undiagnosed children were investigated as index patients. By using single-strand conformation polymorphism analysis and DNA sequencing, two novel mutations were identified in two of these subjects: (1) 7-base pair (bp) duplication following nucleotide 681 (according to the cDNA sequence) in exon 4 (681ins7), which causes a frameshift, the introduction of a stop at codon 208, and premature chain termination, and (2) A to G change in exon 8 substituting a tyrosine for a cysteine at amino acid 354 (Y354C). A third subject carried the recently reported exon 10 mutation (Y468X), whereas the remaining three patients demonstrated various known polymorphisms with no effect on gene product. Rapid molecular assays were developed to detect the two new mutations as well as the Y468X mutation. Screening of our cohort showed heterozygosity in 1/88, in 2/88, and in 2/88 of patients for the 681ins7, the Y354C, and the Y468X mutations, respectively.

Chronic sodium benzoate therapy in children with inborn errors of urea synthesis: effect on carnitine metabolism and ammonia nitrogen removal.

Sodium benzoate (SB) therapy is known to increase ammonia (NH3) nitrogen elimination via conjugation with glycine and excretion as urinary hippurate. In 16 children with inborn errors of urea synthesis we studied two issues: (1) the effect of chronic SB administration upon carnitine metabolism and (2) the efficacy of chronic SB therapy as measured by the molar ratio of hippurate excretion to SB intake. Measurements were performed during elective hospitalizations when the patients were in stable metabolic condition. We found that chronic SB therapy is not associated with a constant level of hippurate elimination and that interindividual and intraindividual variability may result in irregular removal of NH3 nitrogen. This variability may be due to various factors including the formation of small quantities of benzoylcarnitine, which was detected in the plasma of three of four patients receiving SB and carnitine therapy and in one of two patients on SB therapy without carnitine supplementation. The ratios of acyl to free carnitine were elevated in both plasma and urine in patients not receiving carnitine supplementation, but were normal in patients receiving supplementation.

Familial hypercholesterolemia: molecular, biochemical, and clinical characterization of a French-Canadian pediatric population.

BACKGROUND: Familial hypercholesterolemia (FH) is a dominantly-inherited disorder attributable to a defect in the low-density lipoprotein (LDL) receptor gene. Five mutations at this locus have been identified in French-Canadians. In children, it may be difficult to clinically distinguish FH from other forms of polygenic or monogenic hyperlipidemia. Therefore, our objectives were to define the molecular basis of our subjects' hypercholesterolemia, to characterize their biochemical phenotype in relation to the underlying molecular defect, and to assess their response to chronic dietary therapy. METHODS: We studied 88 unrelated French-Canadian children with a persistent increase in LDL cholesterol and a parental history of hyperlipidemia. Baseline and end-of-diet lipid and apolipoprotein levels were measured. Mutational analysis at the LDL receptor gene locus was performed. RESULTS: Heterozygosity for the common French-Canadian LDL receptor gene > 10-kb deletion was found in 57% of subjects (group 1), 14% carried one of the other four previously characterized LDL receptor gene mutations (group 2), and none of the five molecular defects tested was detected in 29% (group 3). Total cholesterol, LDL cholesterol, and apolipoprotein B baseline levels were similar among these three groups but significantly higher than in control subjects. However, there was wide interindividual variability even among those carrying the same mutation. Significantly lower baseline levels of high-density lipoprotein cholesterol and apolipoprotein A1 were found in group 1 compared with group 3 and the controls. The response to diet was similar among the three groups with an average reduction in the mean level of total cholesterol of 4.4%. CONCLUSIONS: The frequency of proven FH heterozygotes (71%) was remarkable in the pediatric population studied. Our data suggest that, in children, a persistent primary increase in LDL cholesterol associated with a parental history of hyperlipidemia is a good predictor of an underlying monogenic disorder as opposed to a polygenic disorder, at least in French-Canadians. Only molecular analysis allowed us to unequivocally define the cause of our patients' hypercholesterolemia. Most children with familial hyperlipidemia did not reach desirable plasma lipid levels solely under diet therapy.