Debby M.E.I. Hellebrekers

N-Myc Downstream-Regulated Gene 4 (NDRG4): A Candidate Tumor Suppressor Gene and Potential Biomarker for Colorectal Cancer

BACKGROUND Identification of hypermethylated tumor suppressor genes in body fluids is an appealing strategy for the noninvasive detection of colorectal cancer. Here we examined the role of N-Myc downstream-regulated gene 4 (NDRG4) as a novel tumor suppressor and biomarker in colorectal cancer. METHODS NDRG4 promoter methylation was analyzed in human colorectal cancer cell lines, colorectal tissue, and noncancerous colon mucosa by using methylation-specific polymerase chain reaction (PCR) and bisulfite sequencing. NDRG4 mRNA and protein expression were studied using real-time-PCR and immunohistochemistry, respectively. Tumor suppressor functions of NDRG4 were examined by colony formation, cell proliferation, and migration and invasion assays in colorectal cancer cell lines that were stably transfected with an NDRG4 expression construct. Quantitative methylation-specific PCR was used to examine the utility of NDRG4 promoter methylation as a biomarker in fecal DNA from 75 colorectal cancer patients and 75 control subjects. All P values are two-sided. RESULTS The prevalence of NDRG4 promoter methylation in two independent series of colorectal cancers was 86% (71/83) and 70% (128/184) compared with 4% (2/48) in noncancerous colon mucosa (P < .001). NDRG4 mRNA and protein expression were decreased in colorectal cancer tissue compared with noncancerous colon mucosa. NDRG4 overexpression in colorectal cancer cell lines suppressed colony formation (P = .014), cell proliferation (P < .001), and invasion (P < .001). NDRG4 promoter methylation analysis in fecal DNA from a training set of colorectal cancer patients and control subjects yielded a sensitivity of 61% (95% confidence interval [CI] = 43% to 79%) and a specificity of 93% (95% CI = 90% to 97%). An independent test set of colorectal cancer patients and control subjects yielded a sensitivity of 53% (95% CI = 39% to 67%) and a specificity of 100% (95% CI = 86% to 100%). CONCLUSIONS NDRG4 is a candidate tumor suppressor gene in colorectal cancer whose expression is frequently inactivated by promoter methylation. NDRG4 promoter methylation is a potential biomarker for the noninvasive detection of colorectal cancer in stool samples.

GATA4 and GATA5 are Potential Tumor Suppressors and Biomarkers in Colorectal Cancer

Purpose: The transcription factors GATA4 and GATA5 are involved in gastrointestinal development and are inactivated by promoter hypermethylation in colorectal cancer. Here, we evaluated GATA4/5 promoter methylation as potential biomarkers for noninvasive colorectal cancer detection, and investigated the role of GATA4/5 in colorectal cancer. Experimental Design: Promoter methylation of GATA4/5 was analyzed in colorectal tissue and fecal DNA from colorectal cancer patients and healthy controls using methylation-specific PCR. The potential function of GATA4/5 as tumor suppressors was studied by inducing GATA4/5 overexpression in human colorectal cancer cell lines. Results:GATA4/5 methylation was observed in 70% (63/90) and 79% (61/77) of colorectal carcinomas, respectively, and was independent of clinicopathologic features. Methylation frequencies in normal colon tissues from noncancerous controls were 6% (5 of 88, GATA4; P < 0.001) and 13% (13 of 100, GATA5; P < 0.001). GATA4/5 overexpression suppressed colony formation (P < 0.005), proliferation (P < 0.001), migration (P < 0.05), invasion (P < 0.05), and anchorage-independent growth (P < 0.0001) of colorectal cancer cells. Examination of GATA4 methylation in fecal DNA from two independent series of colorectal cancer patients and controls yielded a sensitivity of 71% [95% confidence interval (95% CI), 55-88%] and specificity of 84% (95% CI, 74–95%) for colorectal cancer detection in the training set, and a sensitivity of 51% (95% CI, 37–65%) and specificity of 93% (95% CI, 84-100%) in the validation set. Conclusions: Methylation of GATA4/5 is a common and specific event in colorectal carcinomas, and GATA4/5 exhibit tumor suppressive effects in colorectal cancer cells in vitro. GATA4 methylation in fecal DNA may be of interest for colorectal cancer detection.

Methylation-Specific PCR Unraveled

Methylation‐specific PCR (MSP) is a simple, quick and cost‐effective method to analyze the DNA methylation status of virtually any group of CpG sites within a CpG island. The technique comprises two parts: (1) sodium bisulfite conversion of unmethylated cytosines to uracil under conditions whereby methylated cytosines remains unchanged and (2) detection of the bisulfite induced sequence differences by PCR using specific primer sets for both unmethylated and methylated DNA. This review discusses the critical parameters of MSP and presents an overview of the available MSP variants and the (clinical) applications.

Angiostatic activity of DNA methyltransferase inhibitors

Inhibitors of DNA methyltransferases (DNMT) and histone deacetylases can reactivate epigenetically silenced tumor suppressor genes and thereby decrease tumor cell growth. Little, however, is known on the effects of these compounds in endothelial cell biology and tumor angiogenesis. Here, we show that the DNMT inhibitors 5-aza-2′-deoxycytidine and zebularine markedly decrease vessel formation in different tumor models. We show that DNMT inhibitors are antiproliferative for tumor-conditioned endothelial cells, without affecting endothelial cell apoptosis and migration. Furthermore, these compounds inhibit angiogenesis in vitro and in vivo as shown by inhibition of endothelial cells sprouting in a three-dimensional gel and inhibition of microvessel formation in the chorioallantoic membrane, respectively. 5-Aza-2′-deoxycytidine, as well as the histone deacetylase inhibitor trichostatin A, reactivates the growth-inhibiting genes TSP1, JUNB, and IGFBP3, which are suppressed in tumor-conditioned endothelial cells. Despite enhanced DNMT activity and increased overall genomic methylation levels in tumor-conditioned endothelial cells, silencing of these genes seemed not to be regulated by direct promoter hypermethylation. For IGFBP3, gene expression in endothelial cells correlated with histone H3 acetylation patterns. In conclusion, our data show that DNMT inhibitors have angiostatic activity in addition to their inhibitory effects on tumor cells. This dual action of these compounds makes them promising anticancer therapeutics. [Mol Cancer Ther 2006;5(2)467–75]

PGD and heteroplasmic mitochondrial DNA point mutations: a systematic review estimating the chance of healthy offspring

BACKGROUND Mitochondrial disorders are often fatal multisystem disorders, partially caused by heteroplasmic mitochondrial DNA (mtDNA) point mutations. Prenatal diagnosis is generally not possible for these maternally inherited mutations because of extensive variation in mutation load among embryos and the inability to accurately predict the clinical expression. The aim of this study is to investigate if PGD could be a better alternative, by investigating the existence of a minimal mutation level below which the chance of an embryo being affected is acceptably low, irrespective of the mtDNA mutation. METHODS We performed a systematic review of muscle mutation levels, evaluating 159 different heteroplasmic mtDNA point mutations derived from 327 unrelated patients or pedigrees, and reviewed three overrepresented mtDNA mutations (m.3243A>G, m.8344A>G and m.8993T>C/G) separately. RESULTS Mutation levels were included for familial mtDNA point mutations only, covering all affected (n = 195) and unaffected maternal relatives (n = 19) from 137 pedigrees. Mean muscle mutation levels were comparable between probands and affected maternal relatives, and between affected individuals with tRNA- versus protein-coding mutations. Using an estimated a priori prevalence of being affected in pedigrees of 0.477, we calculated that a 95% or higher chance of being unaffected was associated with a muscle mutation level of 18% or less. At a mutation level of 18%, the predicted probability of being affected is 0.00744. The chance of being unaffected was lower only for the m.3243A>G mutation (P < 0.001). Most carriers of mtDNA mutations will have oocytes with mutation levels below this threshold. CONCLUSIONS Our data show, for the first time, that carriers of heteroplasmic mtDNA mutations will have a fair chance of having healthy offspring, by applying PGD. Nevertheless, our conclusions are partly based on estimations and, as indicated, do not provide absolute certainty. Carriers of mtDNA should be informed about these constraints.

De novo mtDNA point mutations are common and have a low recurrence risk

Background Severe, disease-causing germline mitochondrial (mt)DNA mutations are maternally inherited or arise de novo. Strategies to prevent transmission are generally available, but depend on recurrence risks, ranging from high/unpredictable for many familial mtDNA point mutations to very low for sporadic, large-scale single mtDNA deletions. Comprehensive data are lacking for de novo mtDNA point mutations, often leading to misconceptions and incorrect counselling regarding recurrence risk and reproductive options. We aim to study the relevance and recurrence risk of apparently de novo mtDNA point mutations. Methods Systematic study of prenatal diagnosis (PND) and recurrence of mtDNA point mutations in families with de novo cases, including new and published data. ‘De novo’ based on the absence of the mutation in multiple (postmitotic) maternal tissues is preferred, but mutations absent in maternal blood only were also included. Results In our series of 105 index patients (33 children and 72 adults) with (likely) pathogenic mtDNA point mutations, the de novo frequency was 24.6%, the majority being paediatric. PND was performed in subsequent pregnancies of mothers of four de novo cases. A fifth mother opted for preimplantation genetic diagnosis because of a coexisting Mendelian genetic disorder. The mtDNA mutation was absent in all four prenatal samples and all 11 oocytes/embryos tested. A literature survey revealed 137 de novo cases, but PND was only performed for 9 (including 1 unpublished) mothers. In one, recurrence occurred in two subsequent pregnancies, presumably due to germline mosaicism. Conclusions De novo mtDNA point mutations are a common cause of mtDNA disease. Recurrence risk is low. This is relevant for genetic counselling, particularly for reproductive options. PND can be offered for reassurance.

Novel pathogenic SLC25A46 splice-site mutation causes an optic atrophy spectrum disorder

The inherited optic neuropathies comprise a group of genetically heterogeneous disorders causing optic nerve dysfunction. In some cases, optic neuropathies are associated with cerebellar atrophy which mainly affects the vermis. Here, we describe a Moroccan girl of consanguineous parents with optic atrophy and cerebellar atrophy. Exome sequencing revealed a novel homozygous mutation (c.283+3G>T) in the donor splice site for exon 1 of SLC25A46. RNA analysis revealed that an alternative splice site within exon 1 was used leading to a premature termination codon within exon 2. SLC25A46 mRNA expression showed there is no wild‐type transcript present in the patient and the mutant transcript does not undergo nonsense‐mediated mRNA decay. Futhermore, we observed c.283+3G>T SLC25A46 mutation induces mitochondrial fragmentation. An additional 10 patients with optic atrophy and cerebellar atrophy, which were negative for mtDNA and OPA1 variants, were tested for pathogenic mutations in the SLC25A46 gene. However, no additional variants were identified. Our findings confirm the recent report of pathogenic SLC25A46 mutations as a novel cause for optic atrophy spectrum disorder.

Large scale mtDNA sequencing reveals sequence and functional conservation as major determinants of homoplasmic mtDNA variant distribution

The mitochondrial DNA (mtDNA) is highly variable, containing large numbers of pathogenic mutations and neutral polymorphisms. The spectrum of homoplasmic mtDNA variation was characterized in 730 subjects and compared with known pathogenic sites. The frequency and distribution of variants in protein coding genes were inversely correlated with conservation at the amino acid level. Analysis of tRNA secondary structures indicated a preference of variants for the loops and some acceptor stem positions. This comprehensive overview of mtDNA variants distinguishes between regions and positions which are likely not critical, mainly conserved regions with pathogenic mutations and essential regions containing no mutations at all.

Pathogenic CWF19L1 variants as a novel cause of autosomal recessive cerebellar ataxia and atrophy.

Autosomal recessive cerebellar ataxia (ARCA) is a group of neurological disorders characterized by degeneration or abnormal development of the cerebellum and spinal cord. ARCA is clinically and genetically highly heterogeneous, with over 20 genes involved. Exome sequencing of a girl with ARCA from non-consanguineous Dutch parents revealed two pathogenic variants c.37G>C; p.D13H and c.946A>T; p.K316* in CWF19L1, a gene with an unknown function, recently reported to cause ARCA in a Turkish family. Sanger sequencing showed that the c.37G>C variant was inherited from the father and the c.946A>T variant from the mother. Pathogenicity was based on the damaging effect on protein function as the c.37G>C variant changed the highly conserved, negatively charged aspartic acid to the positively charged histidine and the c.946A>T variant introduced a premature stop codon. In addition, 27 patients with ARCA were tested for pathogenic variants in CWF19L1, however, no pathogenic variants were identified. Our data confirm CWF19L1 as a novel but rare gene causing ARCA.

NDUFA9 point mutations cause a variable mitochondrial complex I assembly defect

Mitochondrial respiratory chain complex I consists of 44 different subunits and contains 3 functional modules: the Q‐, the N‐ and the P‐module. NDUFA9 is a Q‐module subunit required for complex I assembly or stability. However, its role in complex I biogenesis has not been studied in patient fibroblasts. So far, a single patient carrying an NDUFA9 variant with a severe neonatally fatal phenotype has been reported. Via exome sequencing, we identified a novel homozygous NDUFA9 missense variant in another patient with a milder phenotype including childhood‐onset progressive generalized dystonia and axonal peripheral neuropathy. We performed complex I assembly analysis using primary skin fibroblasts of both patients. Reduced complex I abundance and an accumulation of Q‐module subassemblies were present in both patients but more pronounced in the severe clinical phenotype patient. The latter displayed additional accumulation of P‐module subassemblies, which was not present in the milder‐phenotype patient. Lentiviral complementation of both patient fibroblast cell lines with wild‐type NDUFA9 rescued complex I deficiency and the assembly defects. Our report further characterizes the phenotypic spectrum of NDUFA9 deficiency and demonstrates that the severity of the clinical phenotype correlates with the severity of the effects of the different NDUFA9 variants on complex I assembly.