Uracil DNA glycosylase (UNG) loss enhances DNA double strand break formation in human cancer cells exposed to pemetrexed.

Misincorporation of genomic uracil and formation of DNA double strand breaks (DSBs) are known consequences of exposure to TS inhibitors such as pemetrexed. Uracil DNA glycosylase (UNG) catalyzes the excision of uracil from DNA and initiates DNA base excision repair (BER). To better define the relationship between UNG activity and pemetrexed anticancer activity, we have investigated DNA damage, DSB formation, DSB repair capacity, and replication fork stability in UNG(+/+) and UNG(-/-) cells. We report that despite identical growth rates and DSB repair capacities, UNG(-/-) cells accumulated significantly greater uracil and DSBs compared with UNG(+/+) cells when exposed to pemetrexed. ChIP-seq analysis of γ-H2AX enrichment confirmed fewer DSBs in UNG(+/+) cells. Furthermore, DSBs in UNG(+/+) and UNG(-/-) cells occur at distinct genomic loci, supporting differential mechanisms of DSB formation in UNG-competent and UNG-deficient cells. UNG(-/-) cells also showed increased evidence of replication fork instability (PCNA dispersal) when exposed to pemetrexed. Thymidine co-treatment rescues S-phase arrest in both UNG(+/+) and UNG(-/-) cells treated with IC50-level pemetrexed. However, following pemetrexed exposure, UNG(-/-) but not UNG(+/+) cells are refractory to thymidine rescue, suggesting that deficient uracil excision rather than dTTP depletion is the barrier to cell cycle progression in UNG(-/-) cells. Based on these findings we propose that pemetrexed-induced uracil misincorporation is genotoxic, contributing to replication fork instability, DSB formation and ultimately cell death.

Menin molecular interactions: insights into normal functions and tumorigenesis.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disease predisposed by heterozygous germline mutations in the MEN1 tumor suppressor gene. Biallelic loss of MEN1 resulting from small mutation and/or loss of heterozygosity occurs in a large tissue spectrum of MEN1 tumors or non-hereditary tumors. Mouse models of MEN1 underexpression or overexpression have also supported the tumor-suppressor effect of the MEN1 gene. Menin, the 610-amino-acid protein encoded by MEN1, is expressed ubiquitously and found predominantly in the nucleus. Sequence analyses do not reveal motifs of known function other than two nuclear localization sequences. Menin has been found to partner in vitro with a variety of proteins that comprise transcription factors, DNA processing factors, DNA repair proteins, and cytoskeletal proteins. The diverse functions of menin interactors suggest roles for menin in multiple biological pathways. Inactivation of menin switches its JunD partner from a downstream action of growth suppression to growth promotion. This is a plausible mechanism for menin tumorigenesis.

Novel mutations in collagen VI genes: expansion of the Bethlem myopathy phenotype.

OBJECTIVE: To investigate the molecular basis of autosomal dominant limb-girdle muscular dystrophy (AD-LGMD) in three large new families. METHODS AND RESULTS: Genome-wide linkage was performed to show that the causative gene in all three families localized to chromosome 21q22.3 (Zmax = 10.3; theta = 0). This region contained the collagen VI alpha1 and alpha2 genes, which have been previously shown to harbor mutations causing a relatively mild congenital myopathy with contractures (Bethlem myopathy). Screening of the collagen VI alpha1 and alpha2 genes revealed novel, causative mutations in each family (COL6A1-K121R, G341D; COL6A2-D620N); two of these mutations were in novel regions of the proteins not previously associated with disease. Collagen VI is a ubiquitously expressed component of connective tissue; however, both limb-girdle muscular dystrophy and Bethlem myopathy patients show symptoms restricted to skeletal muscle. To address the muscle-specific symptoms resulting from collagen VI mutations, the authors studied three patient muscle biopsies at the molecular level (protein expression). A marked reduction of laminin beta1 protein in the myofiber basal lamina in all biopsies was found, although this protein was expressed normally in the neighboring capillary basal laminae. CONCLUSIONS: The authors' studies widen the clinical spectrum of Bethlem myopathy and suggest collagen VI etiology should be investigated in dominant limb-girdle muscular dystrophy. The authors hypothesize that collagen VI mutations lead to muscle-specific defects of the basal lamina, and may explain the muscle-specific symptoms of Bethlem and limb-girdle muscular dystrophy patients with collagen VI mutations.

Bidirectional transcriptional activity of PGK-neomycin and unexpected embryonic lethality in heterozygote chimeric knockout mice.

In an effort to create a conventional knockout mouse model for multiple endocrine neoplasia type 1 (MEN1), we targeted disruption of the mouse Men1 gene through homologous recombination in ES cells. Men1 exons 2-4 were replaced by a PGK-neomycin cassette inserted in the opposite direction of Men1 transcription (Men1(MSK/+)). Unexpectedly, the Men1 conventional knockout was lethal in heterozygous, chimeric animals. Analysis of embryos revealed late gestational lethality with some embryos showing omphalocele. This was a very surprising phenotype, given that humans and mice that are heterozygotes for loss of function mutations in MEN1 are phenotypically normal except for a risk of endocrine tumors. Northern analysis of Men1(MSK/+) embryonic stem cell RNA revealed the presence of an abundant, novel transcript of 2.1 kb, in addition to the expected wild-type transcripts of 2.7 kb and 3.1 kb. RT-PCR analysis identified this aberrant transcript as arising from the antisense strand of the PGK promoter. We hypothesize that this transcript is producing either a toxic effect at the RNA level, or a dominant negative effect through the production of an amino-terminal truncated protein product. This example serves as a cautionary reminder that mouse knockouts using PGK-neo may sometimes display phenotypes that reflect more than just the loss of function of the targeted gene.

A mouse model of multiple endocrine neoplasia, type 1, develops multiple endocrine tumors.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant cancer syndrome, characterized primarily by multiple tumors in the parathyroid glands, endocrine pancreas, and anterior pituitary. Other tumors, including gastrinoma, carcinoid, adrenal cortical tumors, angiofibroma, collagenoma, and lipoma, also occur in some patients. Individuals with MEN1 almost always have loss-of-function mutations in the MEN1 gene on chromosome 11, and endocrine tumors arising in these patients usually show somatic loss of the remaining wild-type allele. To examine the role of MEN1 in tumor formation, a mouse model was generated through homologous recombination of the mouse homolog Men1. Homozygous mice die in utero at embryonic days 11.5-12.5, whereas heterozygous mice develop features remarkably similar to those of the human disorder. As early as 9 months, pancreatic islets show a range of lesions from hyperplasia to insulin-producing islet cell tumors, and parathyroid adenomas are also frequently observed. Larger, more numerous tumors involving pancreatic islets, parathyroids, thyroid, adrenal cortex, and pituitary are seen by 16 months. All of the tumors tested to date show loss of the wild-type Men1 allele, further supporting its role as a tumor suppressor gene.

A novel ryanodine receptor gene mutation causing both cores and rods in congenital myopathy.

BACKGROUND: Central core disease (CCD) and nemaline rod myopathy are generally considered two genetically and histologically distinct disorders. CCD is defined by the presence of well-demarcated round cores within most myofibers. Nemaline rod myopathy is distinguished by the presence of characteristic nemaline bodies within myofibers. The simultaneous occurrence of both cores and rods in the same muscle biopsy has been described, but no gene mutations have been reported yet for this condition. OBJECTIVE: To describe a family containing 16 affected individuals in six generations with an autosomal dominant congenital myopathy that shows clinical and histologic features of both CCD and nemaline myopathy, and to determine the genetic etiology and protein composition of the cores/rods in this family. METHODS AND RESULTS: The results of linkage analyses excluded involvement of the two autosomal dominant nemaline myopathy loci on chromosome 1, but were consistent with a localization of the disease gene at the CCD locus on chromosome 19q13.1 (ryanodine receptor). SSCP analysis and DNA sequencing identified a novel Thr4637Ala mutation in the transmembrane region of the ryanodine receptor protein. Immunofluorescence studies of patient muscle biopsies showed the central cores to stain for ryanodine receptor. CONCLUSIONS: These data suggest that the occurrence of nemaline bodies can be a secondary feature of CCD, and that genetic studies on previously reported core/rod families should be targeted to the ryanodine receptor locus. The results of the immunofluorescence studies suggest that the cores contain excess abnormal ryanodine receptor protein.

Unique PABP2 mutations in "Cajuns" suggest multiple founders of oculopharyngeal muscular dystrophy in populations with French ancestry.

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset autosomal dominant myopathy found world-wide, but with the highest incidence in French-Canadians. Short GCG expansions in the poly(A) binding protein 2 (PABP2) gene were identified recently as the molecular basis for OPMD in French-Canadians. All French-Canadian cases of OPMD have been traced to a single founder couple [Bouchard, 1997: Neuromuscul Disord 7(Suppl):S5-S11]. Cultural links between French-Canadians and Cajuns suggest that this same founder couple may have transmitted the OPMD mutation to Cajuns as well. To determine if OPMD patients from Louisiana share a founder effect with French-Canadian families, we collected blood samples and muscle biopsies from several Cajuns with OPMD for mutation and linkage studies. We found a unique 'GCA GCG GCG' insertion mutation in Cajuns. Consistent with these sequence data, we identified a disease haplotype in our Cajun families that is different from the ancestral haplotype defined in French-Canadians. These data prove that different founders introduced the PABP2 mutation to Cajuns and French-Canadians and lend support to emerging genealogical data suggesting that French-Canadians and Cajuns represent distinct immigrant groups from France.

nimO, an Aspergillus gene related to budding yeast Dbf4, is required for DNA synthesis and mitotic checkpoint control.

The nimO predicted protein of Aspergillus nidulans is related structurally and functionally to Dbf4p, the regulatory subunit of Cdc7p kinase in budding yeast. nimOp and Dbf4p are most similar in their C-termini, which contain a PEST motif and a novel, short-looped Cys2-His2 zinc finger-like motif. DNA labelling and reciprocal shift assays using ts-lethal nimO18 mutants showed that nimO is required for initiation of DNA synthesis and for efficient progression through S phase. nimO18 mutants abrogated a cell cycle checkpoint linking S and M phases by segregating their unreplicated chromatin. This checkpoint defect did not interfere with other checkpoints monitoring spindle assembly and DNA damage (dimer lesions), but did prevent activation of a DNA replication checkpoint. The division of unreplicated chromatin was accelerated in cells lacking a component of the anaphase-promoting complex (bimEAPC1), consistent with the involvement of nimO and APC/C in separate checkpoint pathways. A nimO deletion conferred DNA synthesis and checkpoint defects similar to nimO18. Inducible nimO alleles lacking as many as 244 C-terminal amino acids supported hyphal growth, but not asexual development, when overexpressed in a ts-lethal nimO18 strain. However, the truncated alleles could not rescue a nimO deletion, indicating that the C terminus is essential and suggesting some type of interaction among nimO polypeptides.

"De novo" duplication Xq23-->Xq26 of paternal origin in a girl with a mildly affected phenotype.

We report a de novo dup(X)(q23-->q26) in a 3-year-old girl with growth retardation, developmental delay, and minor anomalies. X-inactivation in lymphocytes by BRDU labeling showed the abnormal X was late replicating. The androgen receptor assay (HAR) demonstrated a skewed methylation (88.8%) of the paternal allele and a 11.2% methylation of the maternal allele. These data, which suggest the duplication was paternally inherited, are the first parental-origin identification of a duplication Xq. The mild phenotype of the patient may be related to the size and region of the duplication, the low percentage of a dup(X) active detected by the HAR assay, or a combination of these mechanisms.

Asymptomatic dystrophinopathy.

A 4-year-old girl was referred for evaluation for a mild but persistent serum aspartate aminotransferase (AST) elevation detected incidentally during routine blood screening for a skin infection. Serum creatine kinase activity was found to be increased. Immunohistochemical study for dystrophin in her muscle biopsy showed results consistent with a carrier state for muscular dystrophy. Molecular work-up showed the proposita to be a carrier of a deletion mutation of exon 48 of the dystrophin gene. Four male relatives also had the deletion mutation, yet showed no clinical symptoms of muscular dystrophy (age range 8-58 yrs). Linkage analysis of the dystrophin gene in the family showed a spontaneous change of an STR45 allele, which could be due to either an intragenic double recombination event, or CA repeat length mutation leading to identical size alleles. To our knowledge, this is the first documentation of an asymptomatic dystrophinopathy in multiple males of advanced age. Based on molecular findings, this family would be given a diagnosis of Becker muscular dystrophy. This diagnosis implies the development of clinical symptoms, even though this family is clearly asymptomatic. This report underscores the caution which must be exercized when giving presymptomatic diagnoses based on molecular studies.

De novo der(X)t(X;10)(q26;q21) with features of distal trisomy 10q: case report of paternal origin identified by late replication with BrdU and the human androgen receptor assay (HAR).

We describe an 11 year old girl with a de novo unbalanced t(X;10) that resulted in a deletion of Xq26-->Xqter and a trisomy of 10q21-->10qter. Her clinical features were of distal trisomy 10q, but she lacked the cardiovascular and renal malformations observed in duplications of 10q24-->10qter and had only moderate mental retardation. X inactivation was assessed on peripheral blood lymphocytes by late replication with BrdU (LR) and the human androgen receptor assay (HAR). By LR the der(X) was inactive without spreading to 10q21-->10qter in all cells. The HAR assay showed skewed methylation of the paternal allele (90%). The correlation of HAR and LR suggests that the der(X) was paternally inherited and is consistent with data from other de novo balanced and unbalanced X;autosome translocations detected in females. This is the first report of parental origin of a de novo trisomy 10q.

Genetic counseling of isolated carriers of Duchenne muscular dystrophy.

It has recently become possible to detect female carriers of Duchenne muscular dystrophy with no affected male relative in the family. These "isolated carriers" represent about 10% of women with high serum creatine phosphokinase (CPK) levels and clinical evidence of a muscle disease. Most isolated carriers ascertained by clinical and/or CPK levels and diagnosed by dystrophin immunostaining of muscle biopsy show symptoms of a muscular dystrophy, and often carry the diagnosis of recessive "limb-girdle muscular dystrophy" prior to dystrophin analysis. It has been difficult to offer genetic counseling and prenatal diagnosis for Duchenne muscular dystrophy in the families of these isolated carriers, largely due to the difficulty in determining which of the dystrophin alleles segregating in the family harbors the mutation in the heterozygote. Here we report genetic counseling of three isolated carriers and their families. In two cases, prenatal diagnosis of at-risk pregnancies was conducted. We determined X inactivation patterns and inheritance of X chromosomes in each family, and used this information to define the at-risk dystrophin gene. In all three families, the mutation was a de novo event, two in the paternal germ-line, and one in the maternal germ-line. In each case we show that sibs of the heterozygous woman are at population risk, while pregnancies of each propositus are at high risk. Our results show that accurate genetic counseling and prenatal diagnosis can be offered to these families.

The Aspergillus nidulans bimE (blocked-in-mitosis) gene encodes multiple cell cycle functions involved in mitotic checkpoint control and mitosis.

The bimE (blocked-in-mitosis) gene appears to function as a negative mitotic regulator because the recessive bimE7 mutation can override certain interphase-arresting treatments and mutations, causing abnormal induction of mitosis. We have further investigated the role of bimE in cell cycle checkpoint control by: (1) coordinately measuring mitotic induction and DNA content of bimE7 mutant cells; and (2) analyzing epistasis relationships between bimE7 and 16 different nim mutations. A combination of cytological and flow cytometric techniques was used to show that bimE7 cells at restrictive temperature (44 degrees C) undergo a normal, although somewhat slower cell cycle prior to mitotic arrest. Most bimE7 cells were fully reversible from restrictive temperature arrest, indicating that they are able to enter mitosis normally, and therefore require bimE function in order to finish mitosis. Furthermore, epistasis studies between bimE7 and mutations in cdc2 pathway components revealed that the induction of mitosis caused by inactivation of bimE requires functional p34cdc2 kinase, and that mitotic induction by bimE7 depends upon several other nim genes whose functions are not yet known. The involvement of bimE in S phase function and mitotic checkpoint control was suggested by three lines of evidence. First, at restrictive temperature the bimE7 mutation slowed the cell cycle by delaying the onset or execution of S phase. Second, at permissive temperature (30 degrees C) the bimE7 mutation conferred enhanced sensitivity to the DNA synthesis inhibitor hydroxyurea. Finally, the checkpoint linking M phase to the completion of S phase was abolished when bimE7 was combined with two nim mutations that cause arrest in G1 or S phase. A model for bimE function based on these findings is presented.