Autosomal dominant mutations affecting X inactivation choice in the mouse

X chromosome inactivation is the silencing mechanism eutherian mammals use to equalize the expression of X-linked genes between males and females early in embryonic development. In the mouse, genetic control of inactivation requires elements within the X inactivation center (Xic) on the X chromosome that influence the choice of which X chromosome is to be inactivated in individual cells. It has long been posited that unidentified autosomal factors are essential to the process. We have used chemical mutagenesis in the mouse to identify specific factors involved in X inactivation and report two genetically distinct autosomal mutations with dominant effects on X chromosome choice early in embryogenesis.     

Mutational inactivation of STAG2 causes aneuploidy in human cancer

Most cancer cells are characterized by aneuploidy, an abnormal number of chromosomes. We have identified a clue to the mechanistic origins of aneuploidy through integrative genomic analyses of human tumors. A diverse range of tumor types were found to harbor deletions or inactivating mutations of STAG2, a gene encoding a subunit of the cohesin complex, which regulates the separation of sister chromatids during cell division. Because STAG2 is on the X chromosome, its inactivation requires only a single mutational event. Studying a near-diploid human cell line with a stable karyotype, we found that targeted inactivation of STAG2 led to chromatid cohesion defects and aneuploidy, whereas in two aneuploid human glioblastoma cell lines, targeted correction of the endogenous mutant alleles of STAG2 led to enhanced chromosomal stability. Thus, genetic disruption of cohesin is a cause of aneuploidy in human cancer.     

A chicken transferrin gene in transgenic mice escapes X-chromosome inactivation

Mammalian X-chromosome inactivation involves a coordinate shutting down of physically linked genes. Several proposed models require the presence of specific sequences near genes to permit the spread of inactivation into these regions. If such models are correct, one might predict that heterologous genes transferred onto the X chromosome might lack the appropriate signal sequences and therefore escape inactivation. To determine whether a foreign gene inserted into the X chromosome is subject to inactivation, transgenic mice harboring 11 copies of the complete, 17-kilobase chicken transferrin gene on the X chromosome were used. Male mice hemizygous for this insert were bred with females bearing Searle's translocation, an X-chromosome rearrangement that is always active in heterozygous females (the unrearranged X chromosome is inactive). Female offspring bearing the Searle's translocation and the chicken transferrin gene had the same amount of chicken transferrin messenger RNA in liver as did transgenic male mice or transgenic female mice lacking the Searle's chromosome. This result shows that the inserted gene is not subject to X-chromosome inactivation and suggests that the inactivation process cannot spread over 187 kilobases of DNA in the absence of specific signal sequences required for inactivation.     

Mutations in CIC and FUBP1 contribute to human oligodendroglioma

Oligodendrogliomas are the second most common malignant brain tumor in adults and exhibit characteristic losses of chromosomes 1p and 19q. To identify the molecular genetic basis for this alteration, we performed exomic sequencing of seven tumors. Among other changes, we found that the CIC gene (homolog of the Drosophila gene capicua) on chromosome 19q was somatically mutated in six cases and that the FUBP1 gene [encoding far-upstream element (FUSE) binding protein] on chromosome 1p was somatically mutated in two tumors. Examination of 27 additional oligodendrogliomas revealed 12 and 3 more tumors with mutations of CIC and FUBP1, respectively, 58% of which were predicted to result in truncations of the encoded proteins. These results suggest a critical role for these genes in the biology and pathology of oligodendrocytes.     

Chromosome 17 deletions and p53 gene mutations in colorectal carcinomas

Previous studies have demonstrated that allelic deletions of the short arm of chromosome 17 occur in over 75% of colorectal carcinomas. Twenty chromosome 17p markers were used to localize the common region of deletion in these tumors to a region contained within bands 17p12 to 17p13.3. This region contains the gene for the transformation-associated protein p53. Southern and Northern blot hybridization experiments provided no evidence for gross alterations of the p53 gene or surrounding sequences. As a more rigorous test of the possibility that p53 was a target of the deletions, the p53 coding regions from two tumors were analyzed; these two tumors, like most colorectal carcinomas, had allelic deletions of chromosome 17p and expressed considerable amounts of p53 messenger RNA from the remaining allele. The remaining p53 allele was mutated in both tumors, with an alanine substituted for valine at codon 143 of one tumor and a histidine substituted for arginine at codon 175 of the second tumor. Both mutations occurred in a highly conserved region of the p53 gene that was previously found to be mutated in murine p53 oncogenes. The data suggest that p53 gene mutations may be involved in colorectal neoplasia, perhaps through inactivation of a tumor suppressor function of the wild-type p53 gene.     

Germ line p53 mutations in a familial syndrome of breast cancer, sarcomas, and other neoplasms

Familial cancer syndromes have helped to define the role of tumor suppressor genes in the development of cancer. The dominantly inherited Li-Fraumeni syndrome (LFS) is of particular interest because of the diversity of childhood and adult tumors that occur in affected individuals. The rarity and high mortality of LFS precluded formal linkage analysis. The alternative approach was to select the most plausible candidate gene. The tumor suppressor gene, p53, was studied because of previous indications that this gene is inactivated in the sporadic (nonfamilial) forms of most cancers that are associated with LFS. Germ line p53 mutations have been detected in all five LFS families analyzed. These mutations do not produce amounts of mutant p53 protein expected to exert a trans-dominant loss of function effect on wild-type p53 protein. The frequency of germ line p53 mutations can now be examined in additional families with LFS, and in other cancer patients and families with clinical features that might be attributed to the mutation.     

Exome sequencing of head and neck squamous cell carcinoma reveals inactivating mutations in NOTCH1

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. To explore the genetic origins of this cancer, we used whole-exome sequencing and gene copy number analyses to study 32 primary tumors. Tumors from patients with a history of tobacco use had more mutations than did tumors from patients who did not use tobacco, and tumors that were negative for human papillomavirus (HPV) had more mutations than did HPV-positive tumors. Six of the genes that were mutated in multiple tumors were assessed in up to 88 additional HNSCCs. In addition to previously described mutations in TP53, CDKN2A, PIK3CA, and HRAS, we identified mutations in FBXW7 and NOTCH1. Nearly 40% of the 28 mutations identified in NOTCH1 were predicted to truncate the gene product, suggesting that NOTCH1 may function as a tumor suppressor gene rather than an oncogene in this tumor type.     

Clonal analysis of human colorectal tumors

The clonal composition of human colorectal tumors was studied by means of restriction fragment length polymorphisms (RFLPs). First, X-linked RFLPs were used to examine the pattern of X chromosome inactivation in colorectal tumors of females. All 50 tumors examined showed monoclonal patterns of X chromosome inactivation; these tumors included 20 carcinomas as well as 30 adenomas of either familial or spontaneous type. Second, RFLPs of autosomes were used as clonal markers to detect the somatic loss or gain of specific chromosomal sequences in colorectal tumors. Among other changes, it was found that somatic loss of chromosome 17p sequences occurred in over 75 percent of the carcinomas examined, but such loss was rare in adenomas. These data support a monoclonal origin for colorectal neoplasms, and suggest that a gene on the short arm of chromosome 17 may be associated with progression from the benign to the malignant state.     

X-Chromosome inactivation in cloned mouse embryos

To study whether cloning resets the epigenetic differences between the two X chromosomes of a somatic female nucleus, we monitored X inactivation in cloned mouse embryos. Both X chromosomes were active during cleavage of cloned embryos, followed by random X inactivation in the embryo proper. In the trophectoderm (TE), X inactivation was nonrandom with the inactivated X of the somatic donor being chosen for inactivation. When female embryonic stem cells with two active X chromosomes were used as donors, random X inactivation was seen in the TE and embryo. These results demonstrate that epigenetic marks can be removed and reestablished on either X chromosome during cloning. Our results also suggest that the epigenetic marks imposed on the X chromosomes during gametogenesis, responsible for normal imprinted X inactivation in the TE, are functionally equivalent to the marks imposed on the chromosomes during somatic X inactivation.     

Concerted nonsyntenic allelic loss in human colorectal carcinoma

Familial polyposis coli (FPC) is caused by an autosomal dominant gene on chromosome 5, and it has been proposed that colorectal cancer in the general population arises from loss or inactivation of the FPC gene, analogous to recessive tumor genes in retinoblastoma and Wilms' tumor. Since allelic loss can be erroneously scored in nonhomogeneous samples, tumor cell populations were first microdissected from 24 colorectal carcinomas, an additional nine cancers were engrafted in nude mice, and nuclei were flow-sorted from an additional two. Of 31 cancers informative for chromosome 5 markers, only 6 (19%) showed loss of heterozygosity of chromosome 5 alleles, compared to 19 of 34 (56%) on chromosome 17, and 17 of 33 (52%) on chromosome 18. Therefore, it appears that (i) FPC is a true dominant for adenomatosis but not a common recessive gene for colon cancer; and (ii) simple Mendelian models involving loss of alleles at a single locus may be inappropriate for understanding common human solid tumors.     

A mouse model of the aniridia-Wilms tumor deletion syndrome

Deletion of chromosome 11p13 in humans produces the WAGR syndrome, consisting of aniridia (an absence or malformation of the iris), Wilms tumor (nephroblastoma), genitourinary malformations, and mental retardation. An interspecies backcross between Mus musculus/domesticus and Mus spretus was made in order to map the homologous chromosomal region in the mouse genome and to define an animal model of this syndrome. Nine evolutionarily conserved DNA clones from proximal human 11p were localized on mouse chromosome 2 near Small-eyes (Sey), a semidominant mutation that is phenotypically similar to aniridia. Analysis of Dickie's Small-eye (SeyDey), a poorly viable allele that has pleiotropic effects, revealed the deletion of three clones, f3, f8, and k13, which encompass the aniridia (AN2) and Wilms tumor susceptibility genes in man. Unlike their human counterparts, SeyDey/+ mice do not develop nephroblastomas. These findings suggest that the Small-eye defect is genetically equivalent to human aniridia, but that loss of the murine homolog of the Wilms tumor gene is not sufficient for tumor initiation. A comparison among Sey alleles suggests that the AN2 gene product is required for induction of the lens and nasal placodes.     

Mutations in SDHD, a mitochondrial complex II gene, in hereditary paraganglioma

Hereditary paraganglioma (PGL) is characterized by the development of benign, vascularized tumors in the head and neck. The most common tumor site is the carotid body (CB), a chemoreceptive organ that senses oxygen levels in the blood. Analysis of families carrying the PGL1 gene, described here, revealed germ line mutations in the SDHD gene on chromosome 11q23. SDHD encodes a mitochondrial respiratory chain protein-the small subunit of cytochrome b in succinate-ubiquinone oxidoreductase (cybS). In contrast to expectations based on the inheritance pattern of PGL, the SDHD gene showed no evidence of imprinting. These findings indicate that mitochondria play an important role in the pathogenesis of certain tumors and that cybS plays a role in normal CB physiology.     

Ectopic expression of germline genes drives malignant brain tumor growth in Drosophila

Model organisms such as the fruit fly Drosophila melanogaster can help to elucidate the molecular basis of complex diseases such as cancer. Mutations in the Drosophila gene lethal (3) malignant brain tumor cause malignant growth in the larval brain. Here we show that l(3)mbt tumors exhibited a soma-to-germline transformation through the ectopic expression of genes normally required for germline stemness, fitness, or longevity. Orthologs of some of these genes were also expressed in human somatic tumors. In addition, inactivation of any of the germline genes nanos, vasa, piwi, or aubergine suppressed l(3)mbt malignant growth. Our results demonstrate that germline traits are necessary for tumor growth in this Drosophila model and suggest that inactivation of germline genes might have tumor-suppressing effects in other species.     

Tumor suppressor genes: the puzzle and the promise

Tumor suppressor genes are wild-type alleles of genes that play regulatory roles in cell proliferation, differentiation, and other cellular and systemic processes. It is their loss or inactivation that is oncogenic. The first evidence of tumor suppressor genes appeared in the early 1970s, but only within the past few years has a wealth of new information illuminated the central importance of these genes. Two or more different suppressor genes may be inactivated in the same tumors, and the same suppressors may be inactive in different tumor types (for example, lung, breast, and colon). The suppressor genes already identified are involved in cell cycle control, signal transduction, angiogenesis, and development, indicating that they contribute to a broad array of normal and tumor-related functions. It is proposed that tumor suppressor genes provide a vast untapped resource for anticancer therapy.     

Heterozygous germ line hCHK2 mutations in Li-Fraumeni syndrome

The hCHK2 gene encodes the human homolog of the yeast Cds1 and Rad53 G2 checkpoint kinases, whose activation in response to DNA damage prevents cellular entry into mitosis. Here, it is shown that heterozygous germ line mutations in hCHK2 occur in Li-Fraumeni syndrome, a highly penetrant familial cancer phenotype usually associated with inherited mutations in the TP53 gene. These observations suggest that hCHK2 is a tumor suppressor gene conferring predisposition to sarcoma, breast cancer, and brain tumors, and they also provide a link between the central role of p53 inactivation in human cancer and the well-defined G2 checkpoint in yeast.     

Genetic origin of mutations predisposing to retinoblastoma

Retinoblastoma is one of several human tumors to which predisposition can be inherited. Molecular genetic analysis of several nonheritable cases has led to the hypothesis that this tumor develops after the occurrence of specific mitotic events involving human chromosome 13. These events reveal initial predisposing recessive mutations. Evidence is presented that similar chromosomal events occur in tumors from heritable cases. The chromosome 13 found in the tumors was the one carrying the predisposing germline mutation and not the homolog containing the wild-type allele at the Rb-1 locus. These results suggest a new approach for identifying recessive mutant genes that lead to cancer and a conceptual basis for accurate prenatal predictions of cancer predisposition.