Lymphoblastoid cell lines (LCLs) derive from blood infected in vitro by Epstein–Barr virus and were used in several genetic, transcriptomic and epigenomic studies. Although few changes were shown between LCL and blood genotypes (SNPs) validating their use in genetics, more were highlighted for other genomic features and/or in their transcriptome and epigenome. This could render them less appropriate for these studies, notably when blood DNA could still be available. Here we developed a simple, high-throughput and cost-effective real-time PCR approach allowing to distinguish blood from LCL DNA samples based on the presence of EBV relative load and rearranged T-cell receptors γ and β. Our approach was able to achieve 98.5% sensitivity and 100% specificity on DNA of known origin (458 blood and 316 LCL DNA). It was further applied to 1957 DNA samples from the CEPH Aging cohort comprising DNA of uncertain origin, identifying 784 blood and 1016 LCL DNA. A subset of these DNA was further analyzed with an epigenetic clock indicating that DNA extracted from blood should be preferred to LCL for DNA methylation-based age prediction analysis. Our approach could thereby be a powerful tool to ascertain the origin of DNA in old collections prior to (epi)genomic studies.

The prognosis of patients with colorectal cancer is largely determined by the tumor stage. In this respect, colorectal cancer with lymph node metastases has the worst prognosis. Accordingly, there is considerable clinical interest in understanding the genetic mechanisms underlying metastasis formation. The short arm of chromosome 8 is often lost in colorectal cancer and has been associated with the advanced stages. A common region of deletion has been identified in 8p21, and we investigate here the localization of the putative tumor suppressor gene. A series of 683 sporadic microsatellite stability colorectal tumor samples has been genotyped on 12 microsatellite loci encompassing the common deleted region. Allelic losses were identified in 50% of the cases and 10 break points have been evidenced between D8S1734 and D8S1810, reducing the region of interest to D8S1771-D8S131. Among the 21 genes mapped in this interval, 14 candidate genes have been retained for the sequencing analysis of 48 tumors with 8p allelic loss. No mutation was found, suggesting more complex mechanisms of inactivation or side effects of chromosome arm 8q duplication, which might be up-regulating oncogenes not located within the deleted region.

Acute lymphoblastic leukemia is the most common malignancy in childhood. High-resolution allelotyping performed in our laboratory showed new chromosomal sites of nonrandom deletions. We have focused our work on 8q12 deletions, which we have found in about 4% of patients (eight of 205 informative cases). These deletions were of small size (less than 1 Mb) in all but one patient, and the deleted region common to all patients was delineated between two microsatellite markers (D8S1113 and D8S1763). This region was sequenced entirely from two overlapping bacterial artificial chromosomes. The common deleted region (120 kb) had a low GC content (37%), was composed more than 50% of LINE sequences, and contained only two candidate genes. The centromeric deletion borders were clustered within an interval of 33 kb between two microsatellite markers. This interval contains the first exon of an HMG-1-related gene (KIAA0808) and a putative gene, DL8q12, predicted to encode a protein with 231 amino acid residues with no homolog in protein databases. Analysis of the available mRNA from lymphoblastic cells of two patients with 8q12 deletions using common polymorphisms in the 3' UTR of KIAA0808 showed monoallelic expression of this gene. Identification of a biallelic polymorphism in the first exon of DL8q12 showed that this gene was deleted in two of four informative cases. Sequencing of the exons of both genes from all patients with 8q12 deletions did not show any mutation, which suggests that neither of these genes behaves as a classic tumor suppressor gene. (C) 2002 Wiley-Liss, Inc.

Loss of heterozygosity (LOH) represents the most frequent genetic alteration observed in hepatocellular carcinoma (HCC). Chromosome 16q is of particular interest as it exhibits LOH in 29% of HCC tumors and is frequently lost in breast, prostate, ovarian and gastric carcinomas. We genotyped 157 HCC tumors for 17 microsatellite markers distributed on chromosome 16q and determined a common region of LOH localized between the markers D16S518 and D16S504. By refining the boundaries of two interstitial LOH and two homozygous deletions, the critical region was delimited to 180 kb between D16S3096 and D16S3029. This region is located in intron 8 of the WWOX/FOR gene, but a search for mutations in all coding exons of this gene in 27 HCC tumors and cell lines did not reveal any tumor somatic alterations. Furthermore, by RT–PCR, no abnormal transcripts of this WWOX/FOR gene was detected in nine HCC cell lines. Finally, analysis of the p53 gene mutations with the clinical parameters of all tumors revealed that the two homozygous deletions have occurred in tumors presenting a R249S mutation. Our data revealed a relationship between chromosome 16q homozygous deletions and R249S p53 mutations in tumors where the patient had been exposed to Aflatoxin B1 (P=0.002). These results are consistent with a role of Aflatoxin B1 in the instability of chromosome 16q at the fragile site FRA16D. However, the nature of the specific gene that is altered during hepatocarcinogenesis remains to be elucidated.

Giant axonal neuropathy (GAN) is a rare autosomal recessive neurodegenerative disorder, characterised clinically by the development of chronic distal polyneuropathy during childhood, mental retardation, kinky or curly hair, skeletal abnormalities and, ultrastructurally, by axons in the central and peripheral nervous systems distended by masses of tightly woven neurofilaments. We recently localised the GAN locus in 16q24.1 to a 5-cM interval between the D16S507 and D16S511 markers by homozygosity mapping in three consanguineous Tunisian families. We have now established a contig-based physical map of the region comprising YACs and BACs where we have placed four genes, ten ESTs, three STSs and two additional microsatellite markers, and where we have identified six new SSCP polymorphisms and six new microsatellite markers. Using these markers, we have refined the position of our previous flanking recombinants. We also identified a shared haplotype between two Tunisian families and a small region of homozygosity in a Turkish family with distant consanguinity, both suggesting the occurrence of historic recombinations and supporting the conclusions based on the phase-known recombinations. Taken together, these results allow us to establish a transcription map of the region, and to narrow down the GAN position to a < 590 kb critical interval, an important step toward the identification of the defective gene.