Christina M. Fleischmann

Mutations in PTF1A cause pancreatic and cerebellar agenesis.

Individuals with permanent neonatal diabetes mellitus usually present within the first three months of life and require insulin treatment. We recently identified a locus on chromosome 10p13–p12.1 involved in permanent neonatal diabetes mellitus associated with pancreatic and cerebellar agenesis in a genome-wide linkage search of a consanguineous Pakistani family. Here we report the further linkage analysis of this family and a second family of Northern European descent segregating an identical phenotype. Positional cloning identified the mutations 705insG and C886T in the gene PTF1A, encoding pancreas transcription factor 1α, as disease-causing sequence changes. Both mutations cause truncation of the expressed PTF1A protein C-terminal to the basic-helix-loop-helix domain. Reporter-gene studies using a minimal PTF1A deletion mutant indicate that the deleted region defines a new domain that is crucial for the function of this protein. PTF1A is known to have a role in mammalian pancreatic development, and the clinical phenotype of the affected individuals implicated the protein as a key regulator of cerebellar neurogenesis. The essential role of PTF1A in normal cerebellar development was confirmed by detailed neuropathological analysis of Ptf1a−/− mice.

Comprehensive analysis of the contribution of germline MYH variation to early-onset colorectal cancer.

Mutations in the base excision repair gene MYH have recently been shown to confer recessive susceptibility to colorectal adenomas and carcinomas. To evaluate the contribution of germline MYH mutations to early‐onset colorectal cancer, we screened a series of 358 unselected early‐onset cases for germline changes in the coding sequence of the gene. Two cases harbored biallelic germline mutations (0.6%; 95% CI = 0.06–2.0) and 8 single MYH mutations (2.2%; 95% CI = 0.9–4.4). Both cases harboring biallelic MYH mutations had multiple polyps but not profuse polyposis. All cases had distally sited tumors. No biallelic mutations were detected among 354 controls. These results confirm that biallelic MYH mutations confer susceptibility to colorectal cancer but are unlikely to account for more than 3% of early‐onset colorectal cancer.

Inherited variants in MYH are unlikely to contribute to the risk of lung carcinoma

The base excision repair gene MYH protects against damage to DNA from reactive oxygen species, which are commonly found in cigarette smoke. Inherited mutations in MYH predispose to colorectal adenomas and carcinomas that show a characteristic pattern of somatic G:C→T:A mutations in the APC gene. A similar pattern of somatic mutations in the TP53 gene is reported in smoking-related lung cancers. We therefore tested whether germline changes in MYH may also contribute to the development of lung cancer by screening for variants in 276 patients with lung carcinoma and 106 normal controls. No patients harboured truncating mutations in MYH and only a single patient was a carrier for the G382D missense mutation. We identified three common coding region (V22M, Q324H and S501F) and intronic (157+30A>G, 462+35G>A and 1435−40G>C) variants, but none were over-represented in the patient samples, indicating that MYH variants are unlikely to predispose significantly to the risk of lung cancer.

Contribution of the CHEK2 1100delC variant to risk of multiple colorectal adenoma and carcinoma

Aneuploidy is a characteristic of a subset of colorectal tumours. CHEK2 (also known as CHK2) is one of the cell cycle checkpoint genes coding for a family of proteins that sense damage in eukaryotic cells. Germline variation in CHEK2 has recently been shown to confer cancer susceptibility. Heterozygous mutations have been identified in patients with TP53-negative Li-Fraumeni syndrome. Furthermore, the CHEK2 1100delC variant carried by 1% of the population has been shown to act as a low penetrance allele for both breast and prostate cancers. To further our knowledge about the contribution of CHEK2 1100delC to cancer incidence we have analysed a series of 149 patients with multiple colorectal adenomas some of whom developed colorectal cancer. The CHEK2 1100delC allele was not over-represented in cases suggesting that this variant is not associated with an increased risk of colorectal disease.

Evaluation of xeroderma pigmentosum XPA, XPC, XPD, XPF, XPB, XPG and DDB2 genes in familial early-onset lung cancer predisposition.

Epidemiological data has implicated heterozygosity for xeroderma pigmentosum (XP) as a risk factor for lung cancer. XP has 8 known complementation groups, 7 of which are caused by mutations in genes encoding components of the nucleotide excision repair (NER) pathway. To formally investigate the role of XP‐related NER genes in lung cancer susceptibility, we screened germline DNA from 92 familial early‐onset lung cancer patients for mutations in all coding regions and intron–exon boundaries of XPA, XPC, XPD, XPF, XPB, XPG and DDB2. Forty‐one exonic variants were identified. Twenty‐four were nonsynonymous, of which 14 were previously documented polymorphisms. Ten missense variants had not been previously described; none of which were detected in germline DNA from 278 cancer‐free controls. Two of the novel missense changes are predicted to be functionally deleterious. Our findings are compatible with XP heterozygosity being a risk factor for lung cancer susceptibility.

Enhanced in vivo sensitivity to interferon with in vitro resistant B16 tumor cells in mice

Mouse B16 melanoma cells rapidly develop resistance to the antiproliferative effects of interferon α (IFNα) and interferon β (IFNβ) when they are exposed to the interferons in vitro. This resistance was characterized to be non-genetic and dose-dependent, and does not alter other IFN-induced effects such as antiviral effects and elevation of 2′,5′-oligoadenylate synthetase activity in IFN-treated cells. The study of these IFN-resistant cells has been extended to an in vivo tumor model. Resistance, if it occurred in vivo, did not adversely affect the survival of IFN-treated mice. Further, IFN-treated mice inoculated with B16 cells that were resistant in vitro (B16αres cells) survived significantly longer than IFN-treated mice inoculated with B16 cells that were sensitive in vitro. The IFN-treated B16αres-inoculated mice had a significantly higher cure rate as well. The prolonged survival of the mice bearing B16αres cell tumors did not seem to be caused by the slower growth rate of the B16αres cells, since experiments performed with a tenfold higher B16αres cell inoculum and a tenfold lower B16 cell inoculum did not show any change in the survival pattern. It is clear that in vitro resistant B16αres cells are more sensitive to antitumor effects induced by IFN in vivo than in vitro sensitive B16 cells.

Potentiation of interferon action by mixtures of recombinant DNA-derived human interferons

Natural and essentially pure recombinant DNA-derived HuIFN-alpha and HuIFN-gamma were examined for their relative abilities to potentiate interferon action. Potentiation of human interferons antiviral and antiproliferative activities were studied. The essentially pure recombinant DNA-derived human interferons were found to be as effective in their potentiating interactions as their natural counterparts. The results demonstrate that it is the human interferons themselves which interact to potentiate human interferons varied activities.

Potentiating effect of murine interferon-γ-containing lymphokine preparations on the antiviral and antiproliferative effects of murine interferon-α/β: identification of the potentiation factor as murine interferon-γ itself

Abstract Mixed preparations of murine interferon-γ (MuIFN-γ) and murine interferon-α/β (MuIFN-α/β) have been shown to induce more than additive levels of antiviral protection, when compared to those induced by these interferons given separately. MuIFN-γ preparations contain many lymphokines and several of these as well as MuIFN-γ itself may participate in this potentiation. In the present study, natural as well as three recombinant DNA-derived MuIFN-γs, in combination with antibody to MuIFN-γ have been employed to examine the precise role of MuIFN-γ. The antiviral effect was examined with a single cycle virus yield reduction assay and the antiproliferative effect with a colony formation inhibition assay. Recombinant DNA-derived MuIFN-γ was as effective as natural MuIFN-γ at participating in the potentiation of both the antiviral and antiproliferative activities. Antibody to MuIFN-γ effectively blocked the potentiation of both the antiviral and the antiproliferative activities of natural and recombinant DNA-derived MuIFN-γs. Since the recombinant DNA-derived preparations from E. colie can be assumed not to contain mammalian proteins other than MuIFN-γ, the data conclusively demonstrate that the potentiation factor in MuIFN-γ preparations is MuIFN-γ itself.

Lack of mda-6/WAF1/CIP1-mediated inhibition of cyclin-dependent kinases in interferon-α resistant murine B16 melanoma cells

Previously we demonstrated that IFN-alpha augments mda-6/WAF1 and inhibits cyclin-dependent kinases in a p53-independent fashion in B 16 murine melanoma cells. On the other hand, IFN-gamma activates p53 expression without affecting the mda-6/WAF1 system. Combination of the two IFNs is additive. B16 cells acquire IFN-alpha resistant but IFN-gamma sensitive phenotype after long term IFN-alpha treatment (B16alpha cells). Here we demonstrate the absence of mda-6/WAF1-associated repression of cyclin-dependent kinases, but the existence of p53-dependent c-myc inhibition in IFN-gamma-treated B16alpha cells. Clearly, selective desensitization of IFN-alpha related growth regulation does not influence the IFN-gamma associated pathway. Our results further support the coexistence of distinct growth regulatory mechanisms in B16 cells that can be activated by different IFN-types independently of each other.

Mutations in the candidate tumour suppressor gene FLJ12973 on chromosome 15q15 are rare in colorectal cancer

Allele imbalance at chromosome 15q14-q22 is seen in a high proportion of sporadic colorectal cancers encompassing the colorectal adenoma and carcinoma susceptibility locus. The FLJ12973 gene, which has recently been identified as a candidate tumour suppressor, maps to 15q15 and encodes a WD-repeat protein with structural similarity to the small subunit of the xeroderma pigmentosum E (XP-E) complex. To examine the proposition that FLJ12973 is involved in colorectal cancer we analysed 31 tumours for sequence variation. No missense changes or pathogenic mutations--truncating or splice site--were detected in any of the tumours. While epigenetic effects on FLJ12973 cannot be excluded, these results show that it is not a common target for mutations in colorectal cancers.