Glória Isidro

Loss of Heterozygosity at Chromosome 9p21 in Primary Neuroblastomas: Evidence for Two Deleted Regions

The genes responsible for the development of neuroblastoma following in vivo deletion or mutation are largely unknown. We have performed loss of heterozygosity studies on a series of 24 Portuguese primary neuroblastomas using 6 polymorphic markers located at chromosome 9p21 spanning the p16/MTS1/CDKN2, p15/MTS2/CDKN2B, and the interferon alpha and beta genes. Loss of heterozygosity was observed in 4 of the 24 tumors (17%), a somewhat lower percentage than a previous study that identified patients by a mass screening program. A correlation was also observed between 9p21 LOH and 1p36 LOH in our group of tumors. Two distinct regions of 9p21 deletion were observed: one located in the region adjacent to the markers D9S162 and D9S1747 and a second located centromerically of the p16 gene near the D9S171 marker. The latter region is exclusive of the p16 gene. This result suggests the presence of at least one other tumor suppressor gene at 9p21, apart from the p16 and p15 genes, which may be of importance to the development of neuroblastoma.

Is prophylactic colectomy indicated in patients with MYH-associated polyposis?

Objectives  The MYH gene has recently been associated with multiple colorectal tumours. It participates in the DNA base‐excision‐repair, avoiding mutations in other genes, namely the APC and Ki‐ras. Recently, biallelic MYH mutations have been described in patients with attenuated polyposis and in 7.5% with classic polyposis and no detectable APC mutation. The aim of this study was to analyse the incidence of germ‐line MYH mutations in selected Portuguese families recorded in a hereditary tumour registry and to evaluate the risk of colorectal cancer in this syndrome.

Insertion of a short Alu sequence into the hMSH2 gene following a double cross over next to sequences with chi homology.

Alu repeat sequences and other multiple copy repetitive elements are present throughout the human genome and are active in promoting recombination. It is believed that reverse transcription of transcribed Alu repeats followed by chromosomal integration has been responsible for the wide dispersion and high copy number of these sequences. During studies on the hMSH2 gene we have used RT-PCR to amplify from peripheral blood lymphocytes a cDNA species in which 553 base pairs of hMSH2 cDNA have been deleted to be replaced by a short 36 base pair Alu sequence as a result of a genomic insertion/deletion event. The 36 base pair Alu insert is homologous to a 26 base pair Alu sequence previously implicated in the promotion of recombination and contains the GCTGG motif which is part of the prokaryotic chi sequence. A second chi-like sequence is also located within the deleted hMSH2 region. Both chi-like sequences are located within 4 bp of the two 4-bp regions of cross over containing the insertion/deletion breakpoints. This suggest that a double recombination event has occurred, providing direct evidence for the recombinogenic activity of this Alu element. Furthermore, it suggests that chi-like sequences may define recombination hotspots as in prokaryotes.

A missense mutation in the APC tumor suppressor gene disrupts an ASF/SF2 splicing enhancer motif and causes pathogenic skipping of exon 14

A missense mutation at codon 640 in the APC gene was identified in a familial adenomatous polyposis (FAP) patient, however, its pathological consequence remained unclear. Here we found that this missense mutation interferes at the nucleotide level with an exonic splicing regulatory element and leads to aberrant splicing of the mutant APC transcript rather than exerting its effect through the observed amino acid change. Analysis of the patient RNA revealed complete skipping of exon 14 in transcripts from the mutant APC allele, leading to a frameshift and a premature stop codon. When cloned into a splicing reporter minigene and transfected into colorectal cell lines, the exon 14 point mutation c.1918C>G (pR640G) was found sufficient to cause the observed exon skipping. Bioinformatic analysis predicted the mutation to change SRp55, hnRNP A1 or ASF/SF2 splicing factor binding sites. Using RNA interference methodology these predictions were experimentally validated and revealed that only ASF/SF2 was required for exon 14 inclusion. These research data identify APC mutation c.1918C>G (pR640G) as pathogenic and indicate a mechanism involving disruption of an ASF/SF2 exonic splicing enhancer element. The results allow genetic diagnosis of a hereditary tumour predisposition but also illustrate the need to complement in silico prediction by splicing reporter assays.

Pathological exon skipping in an HNPCC proband with MLH1 splice acceptor site mutation

One of the most commonly mutated mismatch repair genes in human nonpolyposis colorectal cancer (HNPCC) is MLH1. We identified a splice site mutation in MLH1 in a colorectal cancer proband (T‐to‐A at position −11 of intron 1 splice acceptor) and investigated its functional consequences by RT‐PCR, using lymphocyte mRNA from the proband, two noncarrying siblings, and one unrelated individual. Subcloning of PCR products followed by sequencing of individual clones revealed increased transcript heterogeneity in the mutation carrier, attributable to the presence of a variety of mRNA forms lacking exon 2, or combinations of exons 2, 4, 6, 9, and 10. The full‐length transcript subcloned from the mutation carrier was detected with a much reduced frequency, suggesting that only the wild‐type allele produced functional MLH1 mRNA. The three noncarriers expressed some previously described transcripts and several novel variants, but none that lacked exon 2. The results are consistent with the hypothesis that this splice site mutation causes skipping of MLH1 exon 2 in a large proportion of mRNA transcripts derived from the mutated allele. Such an observation strengthens the case for identifying the mutation as pathogenic in this HNPCC family, which is of interest given the rarity of exon skipping defects resulting from splice acceptor site mutations outside the invariant AG dinucleotide.

Naturally occurring splicing variants of the hMSH2 gene containing nonsense codons identify possible mRNA instability motifs within the gene coding region.

We have identified certain unusually spliced cDNA species following PCR amplification of peripheral blood lymphocyte (PBL) mRNA from the hMSH2 gene. A naturally occurring transcript containing a nonsense codon due to the skipping of 5 exons was amplified from PBLs of several healthy individuals. A feature of this and another unusual splicing product was the presence of sequence motifs which bore significant similarity to mRNA instability determinants in the region immediately downstream of the stop codon. In particular, the rare tetranucleotide GAUG, previously identified in yeast as being of critical importance to the rapid degradation of nonsense-containing mRNAs was situated 23 base pairs downstream of the stop codon. Furthermore the region downstream of the stop codon was A:U rich and contained 2 copies of the AUUUA motif. As other forms of alternative splicing would not result in the same juxtaposition of stop codons and instability motifs, we suggest that the stop codons may have been deliberately introduced by the splicing process for their proximity to these destabilising motifs, and that splicing may play a role in channeling mRNAs into degradative pathways. These results are consistent with the hypothesis that nuclear factors may scan pre-mRNAs prior to splicing.

Four Novel MSH2 / MLH1 Gene Mutations in Portuguese HNPCC Families

Hereditary non‐polyposis colorectal cancer (HNPCC) is considered to be determined by germline mutations in the mismatch repair (MMR) genes, especially MSH2 and MLH1. While screening for mutations in these two genes in HNPCC portuguese families, 3 previously unreported MSH2 and 1 MLH1 mutations have been identified in families meeting strict Amsterdam criteria. Hum Mutat 15:116, 2000.

P73 EXPRESSION IN NEUROBLASTOMA: A ROLE IN THE BIOLOGY OF ADVANCED TUMORS?

p73, a recently identified gene showing high homology to p53 and mapping to 1p36.33, was presented as a candidate gene for neuroblastoma. In this study the authors evaluate the levels and allelic nature of p73 expression in primary neuroblastomas using reverse transcription-polymerase chain reaction-restriction fragment length polymorphism strategies based on intragenic polymorphisms. From 32 neuroblastoma patients, 11 were heterozygous for the p73 polymorphisms analyzed. p73 expression was found to be low in the correspondent tumors and while all 6 stages 1 and 2 tumors presented biallelic expression, 4 out of the 5 stage 4 tumors showed only one active p73 allele. Analysis of blood samples from 8 healthy donors and 4 neuroblastoma patients revealed much higher levels of p73 expression, and exclusively of biallelic nature. These results are supportive of a role for p73 in the biology of neuroblastoma, particularly in some advanced tumors. Nevertheless, the G81A/C91T polymorphism, previously implicated in regulating the expression of p73, did not show any significant association with neuroblastoma development.