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author:("rahe, Ralf")
1.  Most expression and splicing changes in myotonic dystrophy type 1 and type 2 skeletal muscle are shared with other muscular dystrophies 
Neuromuscular disorders : NMD  2013;24(3):227-240.
The prevailing pathomechanistic paradigm for myotonic dystrophy (DM) is that aberrant expression of embryonic/fetal mRNA/protein isoforms accounts for most aspects of the pleiotropic phenotype. To identify aberrant isoforms in skeletal muscle of DM1 and DM2 patients, we performed exon-array profiling and RT-PCR validation on the largest DM sample set to date, including Duchenne, Becker and tibial muscular dystrophy (NMD) patients as disease controls, and non-disease controls. Strikingly, most expression and splicing changes in DM patients were shared with NMD controls. Comparison between DM and NMD identified almost no significant differences. We conclude that DM1 and DM2 are essentially identical for dysregulation of gene expression, and DM expression changes represent a subset of broader spectrum dystrophic changes. We found no evidence for qualitative splicing differences between DM1 and DM2. While some DM-specific splicing differences exist, most of the DM splicing differences were also seen in NMD controls. SSBP3 exon 6 missplicing was observed in all diseased muscle and led to reduced protein. We conclude there is no widespread DM-specific spliceopathy in skeletal muscle and suggest that missplicing in DM (and NMD) may not be the driving mechanism for the muscle pathology, since the same pathways show expression changes unrelated to splicing.
PMCID: PMC3943873  PMID: 24332166
Myotonic dystrophy; DM1; DM2; aberrant isoform expression; missplicing
2.  Differences in aberrant expression and splicing of sarcomeric proteins in the myotonic dystrophies DM1 and DM2 
Acta neuropathologica  2010;119(4):465-479.
Aberrant transcription and mRNA processing of multiple genes due to RNA-mediated toxic gain-of-function has been suggested to cause the complex phenotype in myotonic dystrophies type 1 and 2 (DM1 and DM2). However, the molecular basis of muscle weakness and wasting and the different pattern of muscle involvement in DM1 and DM2 are not well understood. We have analyzed the mRNA expression of genes encoding muscle-specific proteins and transcription factors by microarray profiling and studied selected genes for abnormal splicing. A subset of the abnormally regulated genes was further analyzed at the protein level. TNNT3 and LDB3 showed abnormal splicing with significant differences in proportions between DM2 and DM1. The differential abnormal splicing patterns for TNNT3 and LDB3 appeared more pronounced in DM2 relative to DM1 and are among the first molecular differences reported between the two diseases. In addition to these specific differences, the majority of the analyzed genes showed an overall increased expression at the mRNA level. In particular, there was a more global abnormality of all different myosin isoforms in both DM1 and DM2 with increased transcript levels and a differential pattern of protein expression. Atrophic fibers in DM2 patients expressed only the fast myosin isoform, while in DM1 patients they co-expressed fast and slow isoforms. However, there was no increase of total myosin protein levels, suggesting that aberrant protein translation and/or turnover may also be involved.
PMCID: PMC4199327  PMID: 20066428
Myotonic dystrophy type 1 (DM1); Myotonic dystrophy type 2 (DM2); Skeletal muscle; Aberrant splicing; Microarray expression profiling
3.  Joint Effects of Germ-Line p53 Mutation, MDM2 SNP309, and Gender on Cancer Risk in Family Studies of Li-Fraumeni Syndrome 
Human genetics  2011;129(6):663-673.
Li-Fraumeni syndrome (LFS) is a rare familial cancer syndrome characterized by early cancer onset, diverse tumor types, and multiple primary tumors. Germ-line p53 mutations have been identified in most LFS families. A high-frequency genetic variant of single-nucleotide polymorphism (SNP) 309 in the MDM2 gene was recently confirmed to be a modifier of cancer risk in several case-control studies: substantially earlier cancer onset was observed in SNP309 G-allele carriers than in wild-type individuals by 7–16 years. However, risk analyses in family studies that jointly account for measured hereditary p53 mutations and SNP309 have not been evaluated. Here, we extended the statistical method that we recently developed to determine the combined effects of measured p53 mutations, SNP309, and gender and their interactions simultaneously. The method is structured for age-specific risk models based on Cox proportional hazards regression for censored age-of-onset traits. We analyzed the cancer incidence in 19 extended pedigrees with multiple germ-line p53 mutations ascertained through clinical LFS phenotype. The dataset consisted of 463 individuals with 129 p53 mutation carriers. Our analyses showed that the p53 germ-line mutation and its interaction with gender were strongly associated with familial cancer incidence and that the association between SNP309 and increased cancer risk was modest. In contrast with several outcomes in case-control studies, the interaction between SNP309 and p53 mutation was not statistically significant. The causal role of SNP309 in family studies was consistent with a previous finding that SNP309 G-alleles are associated with accelerated tumor formation in patients with sporadic and hereditary cancers.
PMCID: PMC4194062  PMID: 21305319
Muscle & nerve  2010;42(6):856-863.
Because of their central role in muscle development and maintenance, MEF2 family members represent excellent candidate effectors of the muscle pathology in myotonic dystrophy (DM). We investigated the expression and alternative splicing of all four MEF2 genes in muscle from neuromuscular disorder (NMD) patients, including DM1 and DM2. We observed MEF2A and MEF2C overexpression in all NMD muscle, including 12 MEF2-interacting genes. Exon 4 and 5 usage in MEF2A and MEF2C was different between DM and normal muscle, with DM showing the embryonic isoform. Similar splicing differences were observed in other NMD muscle. For MEF2C, missplicing was more pronounced in DM than in other dystrophies. Our data confirm dysregulation of MEF2A and MEF2C expression and splicing in several NMD, including DM. Our findings demonstrate that aberrant splicing in NMD is independent from expression of mutant repeats, and suggests that some aberrant splicing, even in DM, may be compensatory rather than primary.
PMCID: PMC4136472  PMID: 21104860
dysregulation; MADS-domain transcription enhancer factor 2; MEF2; myotonic dystrophy; splicing
Head & neck  2008;30(10):1273-1283.
Metastases are the primary cause of cancer treatment failure and death, yet metastatic mechanisms remain incompletely understood.
We studied the molecular basis of head and neck cancer metastasis by transcriptionally profiling 70 samples from 27 patients—matching normal adjacent tissue, primary tumor, and cervical lymph node metastases.
We identified tumor-associated expression signatures common to both primary tumors and metastases. Use of matching metastases revealed an additional 46 dysregulated genes associated solely with head and neck cancer metastasis. However, despite being metastasis-specific in our sample set, these 46 genes are concordant with genes previously discovered in primary tumors that metastasized.
Although our data and related studies show that most of the metastatic potential appears to be inherent to the primary tumor, they are also consistent with the notion that a limited number of additional clonal changes are necessary to yield the final metastatic cell(s), albeit in a variable temporal order.
PMCID: PMC4136479  PMID: 18642293
head and neck cancer; expression profiling; metastasis; gene signatures; oncogenomics
6.  High frequency of co-segregating CLCN1 mutations among myotonic dystrophy type 2 patients from Finland and Germany 
Journal of neurology  2008;255(11):1731-1736.
Based on previous reports the frequency of co-segregating recessive chloride channel (CLCN1) mutations in families with myotonic dystrophy type 2 (DM2) was suspected to be increased. We have studied the frequency of CLCN1 mutations in two separate patient and control cohorts from Germany and Finland, and for comparison in a German myotonic dystrophy type 1 (DM1) patient cohort. The frequency of heterozygous recessive chloride channel (CLCN1) mutations is disproportionally higher (5%) in currently diagnosed DM2 patients compared to 1.6% in the control population (p = 0.037), while the frequency in DM1 patients was the same as in the controls. Because the two genes segregate independently, the prevalence of CLCN1 mutations in the total DM2 patient population is, by definition, the same as in the control population. Our findings are, however, not based on the total DM2 population but on the currently diagnosed DM2 patients and indicate a selection bias in molecular diagnostic referrals. DM2 patients with co-segregating CLCN1 mutation have an increased likelihood to be referred for molecular diagnostic testing compared to DM2 patients without co-segregating CLCN1 mutation.
PMCID: PMC4079033  PMID: 18807109
myotonic dystrophy; co-segregation; CLCN1; genetic modifier; phenotype variation
7.  Altered expression and splicing of Ca2+ metabolism genes in myotonic dystrophies DM1 and DM2 
Neuropathology and applied neurobiology  2013;39(4):10.1111/j.1365-2990.2012.01289.x.
Myotonic dystrophy types 1 and 2 (DM1 and DM2) are multisystem disorders caused by similar repeat expansion mutations, with similar yet distinct clinical features. Aberrant splicing of multiple effector genes, as well as dysregulation of transcription and translation, have been suggested to underlie different aspects of the complex phenotypes in DM1 and DM2. Ca2+ plays a central role in both muscle contraction and control of gene expression, and recent expression profiling studies have indicated major perturbations of the Ca2+ signaling pathways in DM. Here we have further investigated the expression of genes and proteins involved in Ca2+ metabolism in DM patients, including Ca2+ channels and Ca2+ binding proteins.
We used patient muscle biopsies to analyze mRNA expression and splicing of genes by microarray expression profiling and RT-PCR. We studied protein expression by immunohistochemistry and immunoblotting.
Most of the genes studied showed mRNA up-regulation in expression profiling. When analyzed by immunohistochemistry the Ca2+ release channel ryanodine receptor was reduced in DM1 and DM2, as was calsequestrin 2, a sarcoplasmic reticulum lumen Ca2+ storage protein. Abnormal splicing of ATP2A1 was more pronounced in DM2 than DM1.
We observed abnormal mRNA and protein expression in DM affecting several proteins involved in Ca2+ metabolism, with some differences between DM1 and DM2. Our protein expression studies are suggestive of a post-transcriptional defect(s) in the myotonic dystrophies.
PMCID: PMC3882430  PMID: 22758909
Myotonic dystrophy type 1 (DM1); myotonic dystrophy type 2 (DM2); skeletal muscle; calcium metabolism
8.  PTBP1-dependent Regulation of USP5 Alternative RNA Splicing Plays a Role in Glioblastoma Tumorigenesis 
Molecular carcinogenesis  2011;51(11):895-906.
Aberrant RNA splicing is thought to play a key role in tumorigenesis. The assessment of its specific contributions is limited by the complexity of information derived from genome-wide array-based approaches. We describe how performing splicing factor-specific comparisons using both tumor and cell line datasets may more readily identify physiologically relevant tumor-specific splicing events. Affymetrix exon array data derived from glioblastoma (GBM) tumor samples with defined PTBP1 levels were compared with data from U251 GBM cells with and without PTBP1 knockdown. This comparison yielded overlapping gene sets that comprised only a minor fraction of each dataset. The identification of a novel GBM-specific splicing event involving the USP5 gene led us to further examine its role in tumorigenesis. In GBM, USP5 generates a shorter isoform 2 through recognition of a 5′ splice site within exon 15. Production of the USP5 isoform 2 was strongly correlated with PTBP1 expression in GBM tumor samples and cell lines. Splicing regulation was consistent with the presence of an intronic PTBP1 binding site and could be modulated through antisense targeting of the isoform 2 splice site to force expression of isoform 1 in GBM cells. The forced expression of USP5 isoform 1 in two GBM cell lines inhibited cell growth and migration, implying an important role for USP5 splicing in gliomagenesis. These results support a role for aberrant RNA splicing in tumorigenesis and suggest that changes in relatively few genes may be sufficient to drive the process.
PMCID: PMC3319169  PMID: 21976412
Exon Array; USP5; isopeptidase-T; GBM
9.  Population frequency of myotonic dystrophy: higher than expected frequency of myotonic dystrophy type 2 (DM2) mutation in Finland 
Myotonic dystrophy (DM) is the most common adult-onset muscular dystrophy with an estimated prevalence of 1/8000. There are two genetically distinct types, DM1 and DM2. DM2 is generally milder with more phenotypic variability than the classic DM1. Our previous data on co-segregation of heterozygous recessive CLCN1 mutations in DM2 patients indicated a higher than expected DM2 prevalence. The aim of this study was to determine the DM2 and DM1 frequency in the general population, and to explore whether the DM2 mutation functions as a modifier in other neuromuscular diseases (NMD) to account for unexplained phenotypic variability. We genotyped 5535 Finnish individuals: 4532 normal blood donors, 606 patients with various non-myotonic NMD, 221 tibial muscular dystrophy patients and their 176 healthy relatives for the DM2 and DM1 mutations. We also genotyped an Italian idiopathic non-myotonic proximal myopathy cohort (n=93) for the DM2 mutation. In 5496 samples analyzed for DM2, we found three DM2 mutations and two premutations. In 5511 samples analyzed for DM1, we found two DM1 mutations and two premutations. In the Italian cohort, we identified one patient with a DM2 mutation. We conclude that the DM2 mutation frequency is significantly higher in the general population (1/1830; P-value=0.0326) than previously estimated. The identification of DM2 mutations in NMD patients with clinical phenotypes not previously associated with DM2 is of particular interest and is in accord with the high overall prevalence. On the basis of our results, DM2 appears more frequent than DM1, with most DM2 patients currently undiagnosed with symptoms frequently occurring in the elderly population.
PMCID: PMC3137497  PMID: 21364698
myotonic dystrophy; mutation frequency; prevalence; population
10.  Methylation of the Candidate Biomarker TCF21 Is Very Frequent Across A Spectrum of Early Stage Non-Small Cell Lung Cancers 
Cancer  2010;117(3):606-617.
The transcription factor TCF21 is involved in mesenchymal-to-epithelial differentiation and was shown to be aberrantly hypermethylated in lung and head and neck cancers. Because of its reported high frequency of hypermethylation in lung cancer, we sought to characterize the stages and types of non-small cell lung cancer (NSCLC) that are hypermethylated and to define the frequency of hypermethylation and associated “second hits”.
We determined TCF21 promoter hypermethylation in 105 NSCLC including various stages and histologies in smokers and nonsmokers. Additionally, we examined TCF21 loss-of-heterozygosity and mutational status. We also assayed 22 cancer cell lines from varied tissue origins. We validated and expanded our NSCLC results by examining TCF21 immunohistochemical expression on a tissue microarray containing 300 NSCLC cases.
Overall, 81% of NSCLC samples showed TCF21 promoter hypermethylation and 84% showed decreased TCF21 protein expression. Multivariate analysis showed that TCF21 expression, although below normal in both histologies, was lower in adenocarcinoma than squamous cell carcinoma, and was not independently correlated with gender, smoking and EGFR mutation status, or clinical outcome. Cell lines from other cancer types also showed frequent TCF21 promoter hypermethylation.
Hypermethylation and decreased expression of TCF21 were tumor-specific and very frequent in all NSCLC, even early-stage disease, thus making TCF21 a potential candidate methylation biomarker for early-stage NSCLC screening. TCF21 hypermethylation in a variety of tumor cell lines suggests it may also be a valuable methylation biomarker in other tumor types.
PMCID: PMC3023841  PMID: 20945327
TCF21; methylation; biomarker; lung cancer; screening
11.  Singular Value Decomposition-based Alternative Splicing Detection 
Altered alternative splicing has been identified as an important factor in tumorigenesis. The Affymetrix exon tiling array is designed for detecting alternative splicing events in a transcriptome-wide fashion; however, there are currently few analysis tools that are well studied for effective detection of alternative splicing events. We propose a new screening procedure based on singular value decomposition (SVD) of the residual matrix from a robust additive model fit to probe selection region (PSR) data. With this approach, we analyze the exon tiling array data from a brain cancer study conducted at the M. D. Anderson Cancer Center, and show that the proposed SVD-based approach is able to better accommodate outlying measures and capitalize on the multidimensional group-by-PSR gene expression profiles for more effective detection of group-specific alternative splicing events as well as the PSRs that are most likely associated with the alternative splicing. Lab validation confirmed some of our findings, but the list of candidates detected with our proposed method may provide a better signpost to guide further investigations.
PMCID: PMC2841408  PMID: 20305737
Alternative Splicing; Exon tiling array; Median regression; Probe selection region; Robust SVD
12.  Splicing factors PTBP1 and PTBP2 promote proliferation and migration of glioma cell lines 
Brain  2009;132(8):2277-2288.
Polypyrimidine tract-binding protein 1 (PTBP1) is a multi-functional RNA-binding protein that is aberrantly overexpressed in glioma. PTBP1 and its brain-specific homologue polypyrimidine tract-binding protein 2 (PTBP2) regulate neural precursor cell differentiation. However, the overlapping and non-overlapping target transcripts involved in this process are still unclear. To determine why PTBP1 and not PTBP2 would promote glial cell-derived tumours, both PTBP1 and PTBP2 were knocked down in the human glioma cell lines U251 and LN229 to determine the role of these proteins in cell proliferation, migration, and adhesion. Surprisingly, removal of both PTBP1 and PTBP2 slowed cell proliferation, with the double knockdown having no additive effects. Decreased expression of both proteins individually and in combination inhibited cell migration and increased adhesion of cells to fibronectin and vitronectin. A global survey of differential exon expression was performed following PTBP1 knockdown in U251 cells using the Affymetrix Exon Array to identify PTBP1-specific splicing targets that enhance gliomagenesis. In the PTBP1 knockdown, previously determined targets were unaltered in their splicing patterns. A single gene, RTN4 (Nogo) had significantly enhanced inclusion of exon 3 when PTBP1 was removed. Overexpression of the splice isoform containing exon 3 decreased cell proliferation to a similar degree as the removal of PTBP1. These results provide the first evidence that RNA-binding proteins affect the invasive and rapid growth characteristics of glioma cell lines. Its actions on proliferation appear to be mediated, in part, through alternative splicing of RTN4.
PMCID: PMC2724917  PMID: 19506066
PTBP1; PTBP2; RTN4; glioma; RNA splicing
13.  Effects of MDM2, MDM4 and TP53 Codon 72 Polymorphisms on Cancer Risk in a Cohort Study of Carriers of TP53 Germline Mutations 
PLoS ONE  2010;5(5):e10813.
Previous studies have shown that MDM2 SNP309 and p53 codon 72 have modifier effects on germline P53 mutations, but those studies relied on case-only studies with small sample sizes. The impact of MDM4 polymorphism on tumor onset in germline mutation carriers has not previously been studied.
Methodology/Principal Findings
We analyzed 213 p53 germline mutation carriers including 168(78.9%) affected with cancer and 174 who had genotypic data. We analyzed time to first cancer using Kaplan-Meier and Cox proportional hazards methods, comparing risks according to polymorphism genotypes. For MDM2 SNP309, a significant difference of 9.0 years in the average age of cancer diagnosis was observed between GG/GT and TT carriers (18.6 versus 27.6 years, P = 0.0087). The hazards ratio was 1.58 (P = 0.03) comparing risks among individuals with GG/GT to risk among TT, but this effect was only significant in females (HR = 1.60, P = 0.02). Compared to other genotypes, P53 codon 72 PP homozygotes had a 2.24 times (P = 0.03) higher rate for time to develop cancer. We observed a multiplicative joint effect of MDM2 and p53 codon72 polymorphism on risk. The MDM4 polymorphism had no significant effects.
Our results suggest that the MDM2 SNP309 G allele is associated with cancer risk in p53 germline mutation carriers and accelerates time to cancer onset with a pronounced effect in females. A multiplicative joint effect exists between the MDM2 SNP309 G allele and the p53 codon 72 G allele in the risk of cancer development. Our results further define cancer risk in carriers of germline p53 mutations.
PMCID: PMC2877078  PMID: 20520810
14.  ZNF9 Activation of IRES-Mediated Translation of the Human ODC mRNA Is Decreased in Myotonic Dystrophy Type 2 
PLoS ONE  2010;5(2):e9301.
Myotonic dystrophy types 1 and 2 (DM1 and DM2) are forms of muscular dystrophy that share similar clinical and molecular manifestations, such as myotonia, muscle weakness, cardiac anomalies, cataracts, and the presence of defined RNA-containing foci in muscle nuclei. DM2 is caused by an expansion of the tetranucleotide CCTG repeat within the first intron of ZNF9, although the mechanism by which the expanded nucleotide repeat causes the debilitating symptoms of DM2 is unclear. Conflicting studies have led to two models for the mechanisms leading to the problems associated with DM2. First, a gain-of-function disease model hypothesizes that the repeat expansions in the transcribed RNA do not directly affect ZNF9 function. Instead repeat-containing RNAs are thought to sequester proteins in the nucleus, causing misregulation of normal cellular processes. In the alternative model, the repeat expansions impair ZNF9 function and lead to a decrease in the level of translation. Here we examine the normal in vivo function of ZNF9. We report that ZNF9 associates with actively translating ribosomes and functions as an activator of cap-independent translation of the human ODC mRNA. This activity is mediated by direct binding of ZNF9 to the internal ribosome entry site sequence (IRES) within the 5′UTR of ODC mRNA. ZNF9 can activate IRES-mediated translation of ODC within primary human myoblasts, and this activity is reduced in myoblasts derived from a DM2 patient. These data identify ZNF9 as a regulator of cap-independent translation and indicate that ZNF9 activity may contribute mechanistically to the myotonic dystrophy type 2 phenotype.
PMCID: PMC2823779  PMID: 20174632
15.  DEAR1 Is a Dominant Regulator of Acinar Morphogenesis and an Independent Predictor of Local Recurrence-Free Survival in Early-Onset Breast Cancer 
PLoS Medicine  2009;6(5):e1000068.
Ann Killary and colleagues describe a new gene that is genetically altered in breast tumors, and that may provide a new breast cancer prognostic marker.
Breast cancer in young women tends to have a natural history of aggressive disease for which rates of recurrence are higher than in breast cancers detected later in life. Little is known about the genetic pathways that underlie early-onset breast cancer. Here we report the discovery of DEAR1 (ductal epithelium–associated RING Chromosome 1), a novel gene encoding a member of the TRIM (tripartite motif) subfamily of RING finger proteins, and provide evidence for its role as a dominant regulator of acinar morphogenesis in the mammary gland and as an independent predictor of local recurrence-free survival in early-onset breast cancer.
Methods and Findings
Suppression subtractive hybridization identified DEAR1 as a novel gene mapping to a region of high-frequency loss of heterozygosity (LOH) in a number of histologically diverse human cancers within Chromosome 1p35.1. In the breast epithelium, DEAR1 expression is limited to the ductal and glandular epithelium and is down-regulated in transition to ductal carcinoma in situ (DCIS), an early histologic stage in breast tumorigenesis. DEAR1 missense mutations and homozygous deletion (HD) were discovered in breast cancer cell lines and tumor samples. Introduction of the DEAR1 wild type and not the missense mutant alleles to complement a mutation in a breast cancer cell line, derived from a 36-year-old female with invasive breast cancer, initiated acinar morphogenesis in three-dimensional (3D) basement membrane culture and restored tissue architecture reminiscent of normal acinar structures in the mammary gland in vivo. Stable knockdown of DEAR1 in immortalized human mammary epithelial cells (HMECs) recapitulated the growth in 3D culture of breast cancer cell lines containing mutated DEAR1, in that shDEAR1 clones demonstrated disruption of tissue architecture, loss of apical basal polarity, diffuse apoptosis, and failure of lumen formation. Furthermore, immunohistochemical staining of a tissue microarray from a cohort of 123 young female breast cancer patients with a 20-year follow-up indicated that in early-onset breast cancer, DEAR1 expression serves as an independent predictor of local recurrence-free survival and correlates significantly with strong family history of breast cancer and the triple-negative phenotype (ER−, PR−, HER-2−) of breast cancers with poor prognosis.
Our data provide compelling evidence for the genetic alteration and loss of expression of DEAR1 in breast cancer, for the functional role of DEAR1 in the dominant regulation of acinar morphogenesis in 3D culture, and for the potential utility of an immunohistochemical assay for DEAR1 expression as an independent prognostic marker for stratification of early-onset disease.
Editors' Summary
Each year, more than one million women discover that they have breast cancer. This type of cancer begins when cells in the breast that line the milk-producing glands or the tubes that take the milk to the nipples (glandular and ductal epithelial cells, respectively) acquire genetic changes that allow them to grow uncontrollably and to move around the body (metastasize). The uncontrolled division leads to the formation of a lump that can be detected by mammography (a breast X-ray) or by manual breast examination. Breast cancer is treated by surgical removal of the lump or, if the cancer has started to spread, by removal of the whole breast (mastectomy). Surgery is usually followed by radiotherapy or chemotherapy. These “adjuvant” therapies are designed to kill any remaining cancer cells but can make patients very ill. Generally speaking, the outlook for women with breast cancer is good. In the US, for example, nearly 90% of affected women are still alive five years after their diagnosis.
Why Was This Study Done?
Although breast cancer is usually diagnosed in women in their 50s or 60s, some women develop breast cancer much earlier. In these women, the disease is often very aggressive. Compared to older women, young women with breast cancer have a lower overall survival rate and their cancer is more likely to recur locally or to metastasize. It would be useful to be able to recognize those younger women at the greatest risk of cancer recurrence so that they could be offered intensive surveillance and adjuvant therapy; those women at a lower risk could have gentler treatments. To achieve this type of “stratification,” the genetic changes that underlie breast cancer in young women need to be identified. In this study, the researchers discover a gene that is genetically altered (by mutations or deletion) in early-onset breast cancer and then investigate whether its expression can predict outcomes in women with this disease.
What Did the Researchers Do and Find?
The researchers used “suppression subtractive hybridization” to identify a new gene in a region of human Chromosome 1 where loss of heterozygosity (LOH; a genetic alteration associated with cancer development) frequently occurs. They called the gene DEAR1 (ductal epithelium-associated RING Chromosome 1) to indicate that it is expressed in ductal and glandular epithelial cells and encodes a “RING finger” protein (specifically, a subtype called a TRIM protein; RING finger proteins such as BRCA1 and BRCA2 have been implicated in early cancer development and in a large fraction of inherited breast cancers). DEAR1 expression was reduced or lost in several ductal carcinomas in situ (a local abnormality that can develop into breast cancer) and advanced breast cancers, the researchers report. Furthermore, many breast tumors carried DEAR1 missense mutations (genetic changes that interfere with the normal function of the DEAR1 protein) or had lost both copies of DEAR1 (the human genome contains two copies of most genes). To determine the function of DEAR1, the researchers replaced a normal copy of DEAR1 into a breast cancer cell that had a mutation in DEAR1. They then examined the growth of these genetically manipulated cells in special three-dimensional cultures. The breast cancer cells without DEAR1 grew rapidly without an organized structure while the breast cancer cells containing the introduced copy of DEAR1 formed structures that resembled normal breast acini (sac-like structures that secrete milk). In normal human mammary epithelial cells, the researchers silenced DEAR1 expression and also showed that without DEAR1, the normal mammary cells lost their ability to form proper acini. Finally, the researchers report that DEAR1 expression (detected “immunohistochemically”) was frequently lost in women who had had early-onset breast cancer and that the loss of DEAR1 expression correlated with reduced local recurrence-free survival, a strong family history of breast cancer and with a breast cancer subtype that has a poor outcome.
What Do These Findings Mean?
These findings indicate that genetic alteration and loss of expression of DEAR1 are common in breast cancer. Although laboratory experiments may not necessarily reflect what happens in people, the results from the three-dimensional culture of breast epithelial cells suggest that DEAR1 may regulate the normal acinar structure of the breast. Consequently, loss of DEAR1 expression could be an early event in breast cancer development. Most importantly, the correlation between DEAR1 expression and both local recurrence in early-onset breast cancer and a breast cancer subtype with a poor outcome suggests that it might be possible to use DEAR1 expression to identify women with early-onset breast cancer who have an increased risk of local recurrence so that they get the most appropriate treatment for their cancer.
Additional Information
Please access these Web sites via the online version of this summary at
This study is further discussed in a PLoS Medicine Perspective by Senthil Muthuswamy
The US National Cancer Institute provides detailed information for patients and health professionals on all aspects of breast cancer, including information on genetic alterations in breast cancer (in English and Spanish)
The MedlinePlus Encyclopedia provides information for patients about breast cancer; MedlinePlus also provides links to many other breast cancer resources (in English and Spanish)
The UK charities Cancerbackup (now merged with MacMillan Cancer Support) and Cancer Research UK also provide detailed information about breast cancer
PMCID: PMC2673042  PMID: 19536326
16.  Genome-Wide Hypomethylation in Head and Neck Cancer Is More Pronounced in HPV-Negative Tumors and Is Associated with Genomic Instability 
PLoS ONE  2009;4(3):e4941.
Loss of genome-wide methylation is a common feature of cancer, and the degree of hypomethylation has been correlated with genomic instability. Global methylation of repetitive elements possibly arose as a defense mechanism against parasitic DNA elements, including retrotransposons and viral pathogens. Given the alterations of global methylation in both viral infection and cancer, we examined genome-wide methylation levels in head and neck squamous cell carcinoma (HNSCC), a cancer causally associated with human papilloma virus (HPV). We assayed global hypomethylation levels in 26 HNSCC samples, compared with their matched normal adjacent tissue, using Pyrosequencing-based methylation assays for LINE repeats. In addition, we examined cell lines derived from a variety of solid tumors for LINE and SINE (Alu) repeats. The degree of LINE and Alu hypomethylation varied among different cancer cell lines. There was only moderate correlation between LINE and Alu methylation levels, with the range of variation in methylation levels being greater for the LINE elements. LINE hypomethylation was more pronounced in HPV-negative than in HPV-positive tumors. Moreover, genomic instability, as measured by genome-wide loss-of-heterozygosity (LOH) single nucleotide polymorphism (SNP) analysis, was greater in HNSCC samples with more pronounced LINE hypomethylation. Global hypomethylation was variable in HNSCC. Its correlation with both HPV status and degree of LOH as a surrogate for genomic instability may reflect alternative oncogenic pathways in HPV-positive versus HPV-negative tumors.
PMCID: PMC2654169  PMID: 19293934
17.  High quality copy number and genotype data from FFPE samples using Molecular Inversion Probe (MIP) microarrays 
A major challenge facing DNA copy number (CN) studies of tumors is that most banked samples with extensive clinical follow-up information are Formalin-Fixed Paraffin Embedded (FFPE). DNA from FFPE samples generally underperforms or suffers high failure rates compared to fresh frozen samples because of DNA degradation and cross-linking during FFPE fixation and processing. As FFPE protocols may vary widely between labs and samples may be stored for decades at room temperature, an ideal FFPE CN technology should work on diverse sample sets. Molecular Inversion Probe (MIP) technology has been applied successfully to obtain high quality CN and genotype data from cell line and frozen tumor DNA. Since the MIP probes require only a small (~40 bp) target binding site, we reasoned they may be well suited to assess degraded FFPE DNA. We assessed CN with a MIP panel of 50,000 markers in 93 FFPE tumor samples from 7 diverse collections. For 38 FFPE samples from three collections we were also able to asses CN in matched fresh frozen tumor tissue.
Using an input of 37 ng genomic DNA, we generated high quality CN data with MIP technology in 88% of FFPE samples from seven diverse collections. When matched fresh frozen tissue was available, the performance of FFPE DNA was comparable to that of DNA obtained from matched frozen tumor (genotype concordance averaged 99.9%), with only a modest loss in performance in FFPE.
MIP technology can be used to generate high quality CN and genotype data in FFPE as well as fresh frozen samples.
PMCID: PMC2649948  PMID: 19228381
18.  Myotonic Dystrophy Type 2 Found in Two of Sixty-Three Persons Diagnosed as Having Fibromyalgia 
Arthritis and rheumatism  2008;58(11):3627-3631.
Because of its high prevalence, fibromyalgia (FM) is a major general health issue. Myotonic dystrophy type 2 (DM2) is a recently described autosomal-dominant multisystem disorder. Besides variable proximal muscle weakness, myotonia, and precocious cataracts, muscle pain and stiffness are prominent presenting features of DM2. After noting that several of our mutation-positive DM2 patients had a previous diagnosis of FM, suggesting that DM2 may be misdiagnosed as FM, we invited 90 randomly selected patients diagnosed as having FM to undergo genetic testing for DM2. Of the 63 patients who agreed to participate, 2 (3.2%) tested positive for the DM2 mutation. Their cases are described herein. DM2 was not found in any of 200 asymptomatic controls. We therefore suggest that the presence of DM2 should be investigated in a large sample of subjects diagnosed as having FM, and clinicians should be aware of overlap in the clinical presentation of these 2 distinct disorders.
PMCID: PMC2585600  PMID: 18975316
19.  Global analysis of aberrant pre-mRNA splicing in glioblastoma using exon expression arrays 
BMC Genomics  2008;9:216.
Tumor-predominant splice isoforms were identified during comparative in silico sequence analysis of EST clones, suggesting that global aberrant alternative pre-mRNA splicing may be an epigenetic phenomenon in cancer. We used an exon expression array to perform an objective, genome-wide survey of glioma-specific splicing in 24 GBM and 12 nontumor brain samples. Validation studies were performed using RT-PCR on glioma cell lines, patient tumor and nontumor brain samples.
In total, we confirmed 14 genes with glioma-specific splicing; seven were novel events identified by the exon expression array (A2BP1, BCAS1, CACNA1G, CLTA, KCNC2, SNCB, and TPD52L2). Our data indicate that large changes (> 5-fold) in alternative splicing are infrequent in gliomagenesis (< 3% of interrogated RefSeq entries). The lack of splicing changes may derive from the small number of splicing factors observed to be aberrantly expressed.
While we observed some tumor-specific alternative splicing, the number of genes showing exclusive tumor-specific isoforms was on the order of tens, rather than the hundreds suggested previously by in silico mining. Given the important role of alternative splicing in neural differentiation, there may be selective pressure to maintain a majority of splicing events in order to retain glial-like characteristics of the tumor cells.
PMCID: PMC2410136  PMID: 18474104
20.  A draft annotation and overview of the human genome 
Genome Biology  2001;2(7):research0025.1-research0025.18.
The recent draft assembly of the human genome provides a unified basis for describing genomic structure and function. The draft is sufficiently accurate to provide useful annotation, enabling direct observations of previously inferred biological phenomena.
We report here a functionally annotated human gene index placed directly on the genome. The index is based on the integration of public transcript, protein, and mapping information, supplemented with computational prediction. We describe numerous global features of the genome and examine the relationship of various genetic maps with the assembly. In addition, initial sequence analysis reveals highly ordered chromosomal landscapes associated with paralogous gene clusters and distinct functional compartments. Finally, these annotation data were synthesized to produce observations of gene density and number that accord well with historical estimates. Such a global approach had previously been described only for chromosomes 21 and 22, which together account for 2.2% of the genome.
We estimate that the genome contains 65,000-75,000 transcriptional units, with exon sequences comprising 4%. The creation of a comprehensive gene index requires the synthesis of all available computational and experimental evidence.
PMCID: PMC55322  PMID: 11516338
21.  A draft annotation and overview of the human genome 
Genome Biology  2001;2(3):preprint0001.1-preprint0001.39.
The recent draft assembly of the human genome provides a unified basis for describing genomic structure and function. The draft is sufficiently accurate to provide useful annotation, enabling direct observations of previously-inferred biological phenomena. We report a functionally annotated human gene index placed directly on the genome. The index is based on the integration of public transcript, protein, and mapping information, supplemented with computational prediction. Such a global approach has been described only for chromosomes 21 and 22, which together account for 2.2% of the genome. We estimate that the genome contains 65,000-75,000 transcriptional units, with exonic sequences comprising 4%.
PMCID: PMC4073052

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