Jesús M. Martín-Campos

A genomewide exploration suggests a new candidate gene at chromosome 11q23 as the major determinant of plasma homocysteine levels: results from the GAIT project.

Homocysteine (Hcy) plasma level is an independent risk marker for venous thrombosis, myocardial infarction, stroke, congestive heart failure, osteoporotic fractures, and Alzheimer disease. Hcy levels are determined by the interaction of genetic and environmental factors. The genetic basis is still poorly understood, since only the MTHFR 677 C-->T polymorphism has been consistently associated with plasma Hcy levels. We conducted a genomewide linkage scan for genes affecting variation in plasma Hcy levels in 398 subjects from 21 extended Spanish families. A variance-components linkage method was used to analyze the data. The strongest linkage signal (LOD score of 3.01; genomewide P = .035) was found on chromosome 11q23, near marker D11S908, where a candidate gene involved in the metabolism of Hcy (the nicotinamide N-methyltransferase gene [NNMT]) is mapped. Haplotype analyses of 10 single-nucleotide polymorphisms within this gene found one haplotype associated with plasma Hcy levels (P = .0003). Our results, to our knowledge, represent the first genomic scan for quantitative variation in Hcy plasma levels. They strongly suggest that the NNMT gene could be a major genetic determinant of plasma Hcy levels in Spanish families. Since this gene encodes an enzyme involved in Hcy synthesis, this finding would be consistent with known biochemical pathways. These data could be relevant in determining the relationships between Hcy level, cardiovascular disease, osteoporosis, and Alzheimer disease.

Molecular profiling related to poor prognosis in thyroid carcinoma. Combining gene expression data and biological information.

Undifferentiated and poorly differentiated thyroid tumors are responsible for more than half of thyroid cancer patient deaths in spite of their low incidence. Conventional treatments do not obtain substantial benefits, and the lack of alternative approaches limits patient survival. Additionally, the absence of prognostic markers for well-differentiated tumors complicates patient-specific treatments and favors the progression of recurrent forms. In order to recognize the molecular basis involved in tumor dedifferentiation and identify potential markers for thyroid cancer prognosis prediction, we analysed the expression profile of 44 thyroid primary tumors with different degrees of dedifferentiation and aggressiveness using cDNA microarrays. Transcriptome comparison of dedifferentiated and well-differentiated thyroid tumors identified 1031 genes with >2-fold difference in absolute values and false discovery rate of <0.15. According to known molecular interaction and reaction networks, the products of these genes were mainly clustered in the MAPkinase signaling pathway, the TGF-β signaling pathway, focal adhesion and cell motility, activation of actin polymerization and cell cycle. An exhaustive search in several databases allowed us to identify various members of the matrix metalloproteinase, melanoma antigen A and collagen gene families within the upregulated gene set. We also identified a prognosis classifier comprising just 30 transcripts with an overall accuracy of 95%. These findings may clarify the molecular mechanisms involved in thyroid tumor dedifferentiation and provide a potential prognosis predictor as well as targets for new therapies.

Overexpression of Human Apolipoprotein A-II in Transgenic Mice Does Not Impair Macrophage-Specific Reverse Cholesterol Transport In Vivo

BACKGROUND Overexpression of human apolipoprotein (apo) A-II in transgenic mice induces high-density lipoprotein (HDL) deficiency, and increased atherosclerosis susceptibility only when fed an atherogenic diet. This may, in part, be caused by impairment in reverse cholesterol transport (RCT). METHODS AND RESULTS [3H]cholesterol-labeled macrophages were injected intraperitoneally into mice maintained on a chow diet or an atherogenic diet. Plasma [3H]cholesterol did not differ from human apoA-II transgenic and control mice at 24 or 48 hours after the label injection. On the chow diet, human apoA-II transgenic mice presented increased [3H]cholesterol in liver (1.3-fold) and feces (6-fold) compared with control mice (P<0.05). The magnitude of macrophage-specific RCT did not differ between transgenic and control mice fed the atherogenic diet. CONCLUSIONS Human apoA-II maintains effective RCT from macrophages to feces in vivo despite an HDL deficiency. These findings suggest that the increased atherosclerotic lesions observed in apoA-II transgenic mice fed an atherogenic diet are not caused by impairment in macrophage-specific RCT.

Structural and functional analysis of APOA5 mutations identified in patients with severe hypertriglyceridemia

During the diagnosis of three unrelated patients with severe hypertriglyceridemia, three APOA5 mutations [p.(Ser232_Leu235)del, p.Leu253Pro, and p.Asp332ValfsX4] were found without evidence of concomitant LPL, APOC2, or GPIHBP1 mutations. The molecular mechanisms by which APOA5 mutations result in severe hypertriglyceridemia remain poorly understood, and the functional impairment/s induced by these specific mutations was not obvious. Therefore, we performed a thorough structural and functional analysis that included follow-up of patients and their closest relatives, measurement of apoA-V serum concentrations, and sequencing of the APOA5 gene in 200 nonhyperlipidemic controls. Further, we cloned, overexpressed, and purified both wild-type and mutant apoA-V variants and characterized their capacity to activate LPL. The interactions of recombinant wild-type and mutated apoA-V variants with liposomes of different composition, heparin, LRP1, sortilin, and SorLA/LR11 were also analyzed. Finally, to explore the possible structural consequences of these mutations, we developed a three-dimensional model of full-length, lipid-free human apoA-V. A complex, wide array of impairments was found in each of the three mutants, suggesting that the specific residues affected are critical structural determinants for apoA-V function in lipoprotein metabolism and, therefore, that these APOA5 mutations are a direct cause of hypertriglyceridemia.

A novel germline mutation in exon 5 of the multiple endocrine neoplasia type 1 gene

Abstract The autosomal dominant multiple endocrine neoplasia type 1 (MEN1) syndrome is characterized by neoplasia of parathyroids, anterior pituitary, and gastrointestinal and pancreatic neuroendocrine tissues. Recently the gene responsible for the MEN1 syndrome has been identified on chromosome region 11q13. Most of the described mutations are nucleotide substitutions and small deletions affecting exons 2 and 3, causing protein truncation. Only one mutation in exon 5 has been found, and this corresponds to a MEN1 sporadic case. Small insertions are also rare. We studied a MEN1 family composed of five members, two of whom were clinically affected. We found a new germline 1 basepair insertional mutation affecting the exon 5 of the MEN1 gene in the two members affected in this MEN1 family.

Molecular Pathology of Multiple Endocrine Neoplasia Type I: Two Novel Germline Mutation; and Updated Classification of Mutations Affecting MEN1 Gene

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by the combined development of tumors in several endocrine glands and other tissues. The MEN1 gene was recently identified and isolated by positional cloning. This gene was screened in two unrelated MEN1 Spanish kindreds (with four affected members and seven asymptomatic members) using single-strand conformation polymorphism, DNA sequencing, and restriction enzyme analysis. Two novel germline mutations were identified: a missense in exon 2 (H139R) and a splice-site in intron 9 (1461-2A>C). These findings allowed us to identify the MEN1 carriers among the seven asymptomatic members analyzed. An updated review of the mutations and polymorphisms found in the analysis of the MEN1 gene is provided. The report of all germline mutations causing MEN1 and easy access to this updated information are both of special diagnostic interest, because this greatly facilitates the task of attributing the disorder to a specific mutation found in a given MEN1 family. This is especially helpful in the critical differentiation of missense mutations from nonsynonymous polymorphisms that fit the pattern of segregation of the disease, but do not cause it.

Genetic, clinical, and biochemical analysis of unrelated Spanish families with multiple endocrine neoplasia type I.

OBJECTIVE (1) To study seven unrelated Spanish families with multiple endocrine neoplasia type I (MEN I), describing clinical features and investigating the presence of germline mutations in the MEN1 gene, and (2) to establish reference values for pancreatic polypeptide and gastrin after a standardized test meal in a healthy control group, analyzing the usefulness of this test for detecting neuroendocrine gastroenteropancreatic tumors in subjects with MEN I. METHODS Two or three generations of 7 kindreds with MEN I, consisting of a total of 39 individual family members, were investigated. Three of the families were subjected only to genetic analysis, and the other four families were also assessed clinically. A group of 23 healthy control subjects were also studied. RESULTS Mutations in the MEN1 gene were found in six of the seven families studied. Of the 4 families studied clinically, 12 family members were genetically affected. In these study subjects, hyperparathyroidism, adrenal adenomas, neuroendocrine gastroenteropancreatic tumors, and pituitary adenomas developed in 100%, 50%, 16%, and 12%, respectively. All demonstrated pancreatic tumors were associated with abnormal results after a test meal, but 75% of them also showed high basal hormonal measurements. CONCLUSION Analysis of the MEN1 gene decreases the total number of subjects who need to undergo repeated clinical and biochemical studies, but genetic mutations are not detected in all families with MEN I. Hyperparathyroidism is the most common manifestation of the syndrome, but the presence of adrenal adenomas has probably been underestimated. Ingestion of a standardized test meal for stimulation of gastrin and pancreatic polypeptide could be a complementary procedure for diagnosing gastroenteropancreatic tumors in selected patients with MEN I in whom basal gastrin and pancreatic polypeptide levels are normal.

Molecular diagnosis of lecithin: cholesterol acyltransferase deficiency in a presymptomatic proband.

Abstract We report the molecular diagnosis of a lecithin : cholesterol acyltransferase deficiency in a 12-year old proband with a high-density lipoprotein deficiency. The increased percentage of free cholesterol in plasma and high-density lipoprotein indicated an inherited lecithin : cholesterol acyltransferase deficiency as the underlying cause. This diagnosis was confirmed by a low plasma lecithin : cholesterol acyltransferase activity and a combination of genetic analyses which demonstrated compound heterozygosity for two mutations in the lecithin : cholesterol acyltransferase gene of the proband. One was a previously unreported 2 bp deletion leading to a stop signal in codon 77 and the other a point mutation causing Arg 135 → Gln transition. To our knowledge, this is the first diagnosis of lecithin : cholesterol acyltransferase deficiency in a pre-symptomatic patient. Whether the proband will develop signs of complete lecithin : cholesterol acyltransferase deficiency or the milder form (Fish Eye Disease) is uncertain, although the former possibility is more likely. The risk of premature atherosclerosis conferred by lecithin : cholesterol acyltransferase deficiency is not well established. The proband will need to be carefully monitored in the future.

Autosomal dominant hypercholesterolemia in Catalonia: Correspondence between clinical-biochemical and genetic diagnostics in 967 patients studied in a multicenter clinical setting

BACKGROUND Autosomal dominant hypercholesterolemia (ADH) is associated with mutations in the low-density lipoprotein (LDL) receptor (LDLR), apolipoprotein B (APOB), and proprotein convertase subtilisin/kexin 9 (PCSK9) genes, and it is estimated to be greatly underdiagnosed. The most cost-effective strategy for increasing ADH diagnosis is a cascade screening from mutation-positive probands. OBJECTIVE The objective of this study was to evaluate the results from 2008 to 2016 of ADH genetic analysis performed in our clinical laboratory, serving most lipid units of Catalonia, a Spanish region with approximately 7.5 million inhabitants. METHODS After the application of the Dutch Lipid Clinic Network (DLCN) clinical diagnostic score for ADH, this information and blood or saliva from 23 different lipid clinic units were investigated in our laboratory. DNA was screened for mutations in LDLR, APOB, and PCSK9, using the DNA-array LIPOchip, the next-generation sequencing SEQPRO LIPO RS platform, and multiplex ligation-dependent probe amplification (MLPA). The Simon Broome Register Group (SBRG) criteria was calculated and analyzed for comparative purposes. RESULTS A total of 967 unrelated samples were analyzed. From this, 158 pathogenic variants were detected in 356 patients. The main components of the DLCN criteria associated with the presence of mutation were plasma LDL cholesterol (LDLc), age, and the presence of tendinous xanthomata. The contribution of family history to the diagnosis was lower than in other studies. DLCN and SBRG were similarly useful for predicting the presence of mutation. CONCLUSION In a real clinical practice, multicenter setting in Catalonia, the percentage of positive genetic diagnosis in patients potentially affected by ADH was 38.6%. The DLCN showed a relatively low capacity to predict mutation detection but a higher one for ruling out mutation.

A rare STAP1 mutation incompletely associated with familial hypercholesterolemia

Autosomal dominant hypercholesterolemia, being referred to as familial hypercholesterolemia (FH), is mainly due to defective LDL receptor (LDLR) function, but is also associated with variants in genes encoding APOB (LDLR ligand) and PCSK9, the catabolic regulator of LDLR. The signal-transducing adaptor family member 1 (STAP1) gene has been recently linked to FH. We describe the case of a 56-year-old male patient found to have hypercholesterolemia at age 34, but who did not continue follow-up nor received treatment with lipid-lowering drugs. At age 55 he suffered a myocardial infarction. A systematic NGS analysis did not show point mutations in the LDLR, APOB, LDLRAP1, or PCSK9 genes, nor large rearrangements of the LDLR gene, but revealed the heterozygous missense variant rs199787258 of STAP1 (c.526C > T; p.Pro176Ser). This variant was also found in heterozygosis in the two siblings of the index case, who also had hypercholesterolemia, but did not cosegregate in his progeny. A bioinformatics analysis and available structural information predicts p.Pro176Ser as the most damaging of all STAP1 missense variants associated with familial hypercholesterolemia. Our findings confirm and extend the linkage between STAP1 variants and FH, and point to an important role of this adaptor protein within a signaling pathway that affects cholesterol homeostasis.