Molecular characterization of familial hypercholesterolemia in Spain.

Familial hypercholesterolemia (FH), characterized by isolated elevation of plasmatic low-density lipoprotein (LDL) cholesterol and premature coronary heart disease (CHD), is associated with mutations in three major genes: LDL receptor (LDLR), apolipoprotein B (APOB) and proprotein convertase subtilisin/kexin 9 (PCSK9). We have analyzed 5430 Spanish index cases and 2223 relatives since 2004 with LIPOchip(®) genetic diagnostic platform, a microarray for the detection of Spanish common mutations in these three genes, including copy number variation (CNV) in LDLR, followed by sequencing analysis of the coding regions of LDLR and exon 26 of APOB, when the result is negative. Samples were received from hospitals of all around Spain. The preferred clinical criterion to diagnose FH was Dutch Lipid Clinic Network (DLCN) score. Our results show that there is a broad spectrum of mutations in the LDLR gene in Spain since about 400 different mutations were detected, distributed along almost the whole LDLR gene. Mutations in APOB (mainly p.Arg3527Gln) covered 6.5% of positive cases and only one PCSK9 mutation was detected. We found correlation between more severe mutations and the clinical diagnosis but also that 28% of FH patients harboring mutations do not have a definite clinical diagnosis. This study analyzes the mutation spectrum in Spain, remarks the importance of genetic diagnosis of FH patients, as well as the cascade screening, and shows how it is being carried out in Spain.

Results of the 2003-2004 GEP-ISFG collaborative study on mitochondrial DNA: focus on the mtDNA profile of a mixed semen-saliva stain.

We report here a review of the seventh mitochondrial DNA (mtDNA) exercise undertaken by the Spanish and Portuguese working group (GEP) of the International Society for Forensic Genetics (ISFG) corresponding to the period 2003-2004. Five reference bloodstains from five donors (M1-M5), a mixed stain of saliva and semen (M6), and a hair sample (M7) were submitted to each participating laboratory for nuclear DNA (nDNA; autosomal STR and Y-STR) and mtDNA analysis. Laboratories were asked to investigate the contributors of samples M6 and M7 among the reference donors (M1-M5). A total of 34 laboratories reported total or partial mtDNA sequence data from both, the reference bloodstains (M1-M5) and the hair sample (M7) concluding a match between mtDNA profiles of M5 and M7. Autosomal STR and Y-STR profiling was the preferred strategy to investigate the contributors of the semen/saliva mixture (M6). Nuclear DNA profiles were consistent with a mixture of saliva from the donor (female) of M4 and semen from donor M5, being the semen (XY) profile the dominant component of the mixture. Strikingly, and in contradiction to the nuclear DNA analysis, mtDNA sequencing results yield a more simple result: only the saliva contribution (M4) was detected, either after preferential lysis or after complete DNA digestion. Some labs provided with several explanations for this finding and carried out additional experiments to explain this apparent contradictory result. The results pointed to the existence of different relative amounts of nuclear and mtDNAs in saliva and semen. We conclude that this circumstance could strongly influence the interpretation of the mtDNA evidence in unbalanced mixtures and in consequence lead to false exclusions. During the GEP-ISFG annual conference a validation study was planned to progress in the interpretation of mtDNA from different mixtures.

Ancestral origins of the prion protein gene D178N mutation in the Basque Country.

Fatal familial insomnia (FFI) and familial Creutzfeldt-Jakob disease (fCJD) are familial prion diseases with autosomal dominant inheritance of the D178N mutation. FFI has been reported in at least 27 pedigrees around the world. Twelve apparently unrelated FFI and fCJD pedigrees with the characteristic D178N mutation have been reported in the Prion Diseases Registry of the Basque Country since 1993. The high incidence of familial prion diseases in this region may reflect a unique ancestral origin of the chromosome carrying this mutation. In order to investigate this putative founder effect, we developed "happy typing", a new approach to the happy mapping method, which consists of the physical isolation of large haploid genomic DNA fragments and their analysis by the Polymerase Chain Reaction in order to perform haplotypic analysis instead of pedigree analysis. Six novel microsatellite markers, located in a 150-kb genomic segment flanking the PRNP gene were characterized for typing haploid DNA fragments of 285 kb in size. A common haplotype was found in patients from the Basque region, strongly suggesting a founder effect. We propose that "happy typing" constitutes an efficient method for determining disease-associated haplotypes, since the analysis of a single affected individual per pedigree should provide sufficient evidence.

P14ARF gene alterations in human hepatocellular carcinoma.

INTRODUCTION: The molecular status of the p14(ARF) gene has not been fully elucidated in hepatocellular carcinoma (HCC). This study was performed to determine genetic and epigenetic alterations in the p14(ARF) tumor suppressor gene and their effect on HCC progression. METHODS: The status of p14 was evaluated in 117 HCC tumoral nodules and 110 corresponding non-tumor tissues by loss of heterozygosity at the 9p21-22 region, homozygous deletions, single strand conformation polymorphism-polymerase chain reaction mutational analysis and methylation-specific polymerase chain reaction. RESULTS: The most frequent inactivation mechanism was hypermethylation of the promoter region, which was found in 41.9% of tumor samples and in 19.1% of non-tumor samples. Loss of heterozygosity at the 9p21 region was detected in 27.3% and 10% of tumor and non-tumor tissues, respectively. Homozygous deletions and mutations were less common events in hepatocarcinogenesis. We found 5.9% of the tumor cases with exon 2 homozygous deletions and 3.4% of the cases with mutations. We described a silent mutation in codon 42 of exon 1beta for the first time. No association was found between inactivation of p14(ARF) and clinicopathological characteristics or prognosis. CONCLUSION: We can conclude that p14(ARF) is frequently and early altered in HCC, being the main cause of inactivation promoter hypermethylation. Our results suggest that the p14(ARF) gene plays an important role in the pathogenesis of hepatocellular carcinoma.

p16INK4A gene alterations are not a prognostic indicator for survival in patients with hepatocellular carcinoma undergoing curative hepatectomy.

BACKGROUND AND AIM: Hepatocellular carcinoma (HCC) is a common malignancy worldwide that is highly associated with chronic hepatitis B or C infection and cirrhosis. The tumor suppressor gene p16INK4A is an important component of the cell cycle and inactivation of the gene has been found in a variety of human cancers. The present study was performed to determine genetic and epigenetic alterations in the p16INK4A tumor suppressor gene and the effect of these on HCC progression. METHODS: The status of p16INK4A was evaluated in 117 HCC tumoral nodules and 110 corresponding peritumoral tissues by loss of heterozigosity (LOH) at the 9p21-22 region, homozygous deletions, single-strand conformation polymorphism-polymerase chain reaction (PCR) mutational analysis and methylation specific PCR. RESULTS: The most frequent inactivation mechanism was hypermethylation of the promoter region, which was found in 63.2% of the tumor samples and in 28.2% of the peritumoral samples. Loss of heterozygosity at the 9p21 region was detected in 27.3% and 10% of tumor and peritumoral tissues, respectively. Homozygous deletions and mutations were less common events in hepatocarcinogenesis. The authors found 5.9% of the tumor cases with exon 2 homozygous deletions and 8.6% with mutations. Two polymorphisms were detected, one at codon 148 (GCG --> ACG, Ala --> Thr) in three cases and the other in exon 3 at 540 bp (34.2% of the samples). No association was found between inactivation of p16INK4A and clinicopathological characteristics or prognosis. CONCLUSION: p16INK4A is altered frequently and early in HCC, being the predominant mechanism of inactivation promoter hypermethylation. The present results suggest that the p16INK4A gene plays an important role in the pathogenesis of HCC.

No association between GSTP1 gene aberrant promoter methylation and prognosis in surgically resected hepatocellular carcinoma patients from the Basque Country (Northern Spain).

AIMS: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, being linked etiologically to several factors. Glutathione-S-transferases (GSTs) are a family of enzymes that play an important role in detoxification. Hypermethylation of regulatory sequences at glutathione-S-transferase pi class gene (GSTP1) has been found in different human tumor types. In this study, we have studied the methylation status of the GSTP1 promoter region in patients from the Basque Country (Northern Spain) by methylation-specific PCR (MSP). METHODS AND RESULTS: GSTP1 aberrant promoter methylation was present in 24 of 117 (20.5%) tumor samples being associated with late stages of tumor progression. Patients with multiple HCCs showed different patterns of methylation, which could suggest a different clonal origin of multicentric HCC or different degrees of differentiation. No effect on disease-free survival or overall survival was observed in patients with GSTP1 methylated who underwent curative resection. CONCLUSIONS: We can conclude that GSTP1 promoter CpG island methylation appears to be a less common event during hepatocarcinogenesis in European populations than in Asian populations, being associated with late stages of tumor progression. These findings could also be useful to provide new therapeutic strategies through the use of demethylating agents.

Frequent loss of p53 codon 72 Pro variant in hepatitis C virus-positive carriers with hepatocellular carcinoma.

Codon 72 exon 4 polymorphism of the p53 gene has been implicated in cancer risk and it has been suggested that it may have an impact on the clinical outcome of the disease. Our objective was to evaluate the association between p53 polymorphism at codon 72 and hepatocellular carcinoma. The p53 codon 72 genotype was examined in 97 biopsy samples from 67 Basque patients histologically diagnosed with hepatocellular carcinoma. Blood samples collected from 111 Basque residents were examined as a control group. The polymorphism was examined by both single strand conformation polymorphism analysis and allele specific polymerase chain reaction. Fisher's exact test was used to evaluate the data. The results showed that there were no statistically significant differences in the frequency of codon 72 polymorphism genotype between patients with liver cancer and healthy controls. We found a frequent loss of proline allele in hepatitis C virus (HCV)-positive carriers. In conclusion, the lack of a significant relationship between this polymorphism and risk of hepatocellular carcinoma suggests that it does not predispose towards hepatocarcinogenesis in this population. We suggest that the frequent loss of the proline allele in HCV-associated carcinogenesis of the liver plays some role in hepatocarcinogenesis.