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1.  Ectopic MicroRNA-150-5p Transcription Sensitizes Glucocorticoid Therapy Response in MM1S Multiple Myeloma Cells but Fails to Overcome Hormone Therapy Resistance in MM1R Cells 
PLoS ONE  2014;9(12):e113842.
Glucocorticoids (GCs) selectively trigger cell death in the multiple myeloma cell line MM1S which express NR3C1/Glucocorticoid Receptor (GR) protein, but fail to kill MM1R cells which lack GR protein. Given recent demonstrations of altered microRNA profiles in a diverse range of haematological malignancies and drug resistance, we characterized GC inducible mRNA and microRNA transcription profiles in GC sensitive MM1S as compared to GC resistant MM1R cells. Transcriptome analysis revealed that GCs regulate expression of multiple genes involved in cell cycle control, cell organization, cell death and immunological disease in MM1S cells, which remain unaffected in MM1R cells. With respect to microRNAs, mir-150-5p was identified as the most time persistent GC regulated microRNA, out of 5 QPCR validated microRNAs (mir-26b, mir-125a-5p, mir-146-5p, mir-150-5p, and mir-184), which are GC inducible in MM1S but not in MM1R cells. Functional studies further revealed that ectopic transfection of a synthetic mir-150-5p mimics GR dependent gene expression changes involved in cell death and cell proliferation pathways. Remarkably, despite the gene expression changes observed, overexpression of mir-150-5p in absence of GCs did not trigger significant cytotoxicity in MM1S or MM1R cells. This suggests the requirement of additional steps in GC induced cell death, which can not be mimicked by mir-150-5p overexpression alone. Interestingly, a combination of mir-150-5p transfection with low doses GC in MM1S cells was found to sensitize therapy response, whereas opposite effects could be observed with a mir-150-5p specific antagomir. Although mir-150-5p overexpression did not substantially change GR expression levels, it was found that mir-150-5p evokes GR specific effects through indirect mRNA regulation of GR interacting transcription factors and hormone receptors, GR chaperones, as well as various effectors of unfolded protein stress and chemokine signalling. Altogether GC-inducible mir-150-5p adds another level of regulation to GC specific therapeutic responses in multiple myeloma.
doi:10.1371/journal.pone.0113842
PMCID: PMC4256227  PMID: 25474406
2.  Pharmacological Levels of Withaferin A (Withania somnifera) Trigger Clinically Relevant Anticancer Effects Specific to Triple Negative Breast Cancer Cells 
PLoS ONE  2014;9(2):e87850.
Withaferin A (WA) isolated from Withania somnifera (Ashwagandha) has recently become an attractive phytochemical under investigation in various preclinical studies for treatment of different cancer types. In the present study, a comparative pathway-based transcriptome analysis was applied in epithelial-like MCF-7 and triple negative mesenchymal MDA-MB-231 breast cancer cells exposed to different concentrations of WA which can be detected systemically in in vivo experiments. Whereas WA treatment demonstrated attenuation of multiple cancer hallmarks, the withanolide analogue Withanone (WN) did not exert any of the described effects at comparable concentrations. Pathway enrichment analysis revealed that WA targets specific cancer processes related to cell death, cell cycle and proliferation, which could be functionally validated by flow cytometry and real-time cell proliferation assays. WA also strongly decreased MDA-MB-231 invasion as determined by single-cell collagen invasion assay. This was further supported by decreased gene expression of extracellular matrix-degrading proteases (uPA, PLAT, ADAM8), cell adhesion molecules (integrins, laminins), pro-inflammatory mediators of the metastasis-promoting tumor microenvironment (TNFSF12, IL6, ANGPTL2, CSF1R) and concomitant increased expression of the validated breast cancer metastasis suppressor gene (BRMS1). In line with the transcriptional changes, nanomolar concentrations of WA significantly decreased protein levels and corresponding activity of uPA in MDA-MB-231 cell supernatant, further supporting its anti-metastatic properties. Finally, hierarchical clustering analysis of 84 chromatin writer-reader-eraser enzymes revealed that WA treatment of invasive mesenchymal MDA-MB-231 cells reprogrammed their transcription levels more similarly towards the pattern observed in non-invasive MCF-7 cells. In conclusion, taking into account that sub-cytotoxic concentrations of WA target multiple metastatic effectors in therapy-resistant triple negative breast cancer, WA-based therapeutic strategies targeting the uPA pathway hold promise for further (pre)clinical development to defeat aggressive metastatic breast cancer.
doi:10.1371/journal.pone.0087850
PMCID: PMC3912072  PMID: 24498382
3.  Echocardiographic integrated backscatter for detecting progression and regression of aortic valve calcifications in rats 
Background
Calcification is an independent predictor of mortality in calcific aortic valve disease (CAVD). The aim of this study was to evaluate the use of non-invasive, non-ionizing echocardiographic calibrated integrated backscatter (cIB) for monitoring progression and subsequent regression of aortic valvular calcifications in a rat model of reversible renal failure with CAVD, compared to histology.
Methods
28 male Wistar rats were prospectively followed during 21 weeks. Group 1 (N=14) was fed with a 0.5% adenine diet for 9 weeks to induce renal failure and CAVD. Group 2 (N=14) received a standard diet. At week 9, six animals of each group were killed. The remaining animals of group 1 (N=8) and group 2 (N=8) were kept on a standard diet for an additional 12 weeks. cIB of the aortic valve was calculated at baseline, 9 and 21 weeks, followed by measurement of the calcified area (Ca Area) on histology.
Results
At week 9, cIB values and Ca Area of the aortic valve were significantly increased in the adenine-fed rats compared to baseline and controls. After 12 weeks of adenine diet cessation, cIB values and Ca Area of group 1 decreased compared to week 9, while there was no longer a significant difference compared to age-matched controls of group 2.
Conclusions
cIB is a non-invasive tool allowing quantitative monitoring of CAVD progression and regression in a rat model of reversible renal failure, as validated by comparison with histology. This technique might become useful for assessing CAVD during targeted therapy.
doi:10.1186/1476-7120-11-4
PMCID: PMC3598438  PMID: 23351880
Aortic valve calcifications; Calcific aortic valve disease; Echocardiography; Integrated backscatter; Renal failure; Small animal
4.  Apoptosis in acquired and genetic hearing impairment 
Hearing Research  2011;281(1-2):18-27.
Apoptosis is an important physiological process. Normally, a healthy cell maintains a delicate balance between pro- and anti-apoptotic factors, allowing it to live and proliferate. It is thus not surprising that disturbance of this delicate balance may result in disease. It is a well known fact that apoptosis also contributes to several acquired forms of hearing impairment. Noise-induced hearing loss is the result of prolonged exposure to excessive noise, triggering apoptosis in terminally differentiated sensory hair cells. Moreover, hearing loss caused by the use of therapeutic drugs such as aminoglycoside antibiotics and cisplatin potentially may result in the activation of apoptosis in sensory hair cells leading to hearing loss due to the “ototoxicity” of the drugs. Finally, apoptosis is a key contributor to the development of presbycusis, age-related hearing loss. Recently, several mutations in apoptosis genes were identified as the cause of monogenic hearing impairment. These genes are TJP2, DFNA5 and MSRB3. This implies that apoptosis not only contributes to the pathology of acquired forms of hearing impairment, but also to genetic hearing impairment as well. We believe that these genes constitute a new functional class within the hearing loss field. Here, the contribution of apoptosis in the pathology of both acquired and genetic hearing impairment is reviewed.
doi:10.1016/j.heares.2011.07.002
PMCID: PMC3341727  PMID: 21782914
noise-induced hearing loss; presbycusis; monogenic hearing loss; ototoxicity; apoptosis
5.  The DFNA5 gene, responsible for hearing loss and involved in cancer, encodes a novel apoptosis-inducing protein 
DFNA5 was first identified as a gene causing autosomal dominant hearing loss (HL). Different mutations have been found, all exerting a highly specific gain-of-function effect, in which skipping of exon 8 causes the HL. Later reports revealed the involvement of the gene in different types of cancer. Epigenetic silencing of DFNA5 in a large percentage of gastric, colorectal and breast tumors and p53-dependent transcriptional activity have been reported, concluding that DFNA5 acts as a tumor suppressor gene in different frequent types of cancer. Despite these data, the molecular function of DFNA5 has not been investigated properly. Previous transfection studies with mutant DFNA5 in yeast and in mammalian cells showed a toxic effect of the mutant protein, which was not seen after transfection of the wild-type protein. Here, we demonstrate that DFNA5 is composed of two domains, separated by a hinge region. The first region induces apoptosis when transfected in HEK293T cells, the second region masks and probably regulates this apoptosis inducing capability. Moreover, the involvement of DFNA5 in apoptosis-related pathways in a physiological setting was demonstrated through gene expression microarray analysis using Dfna5 knockout mice. In view of its important role in carcinogenesis, this finding is expected to lead to new insights on the role of apoptosis in many types of cancer. In addition, it provides a new line of evidence supporting an important role for apoptosis in monogenic and complex forms of HL.
doi:10.1038/ejhg.2011.63
PMCID: PMC3179363  PMID: 21522185
tumor suppressor; hearing loss; apoptosis; dfna5; cancer; GSEA
6.  Amino acid 572 in TMC1: hot spot or critical functional residue for dominant mutations causing hearing impairment 
Journal of human genetics  2009;54(3):188-190.
Two different missense mutations, p.D572N and p.D572H, affecting the same nucleotide and codon of the TMC1 gene were earlier reported to cause autosomal dominant hearing impairment at locus DFNA36 in two North American families. No other dominant mutations of human TMC1 have been published. We ascertained a third North American family segregating autosomal dominant nonsyndromic hearing impairment at the DFNA36 locus. We identified the p.D572N mutation of TMC1 co-segregating with hearing loss in our study family. A comparative haplotype analysis of linked single nucleotide polymorphisms and short tandem repeats in the two families segregating p.D572N was not consistent with a founder effect. These findings can be explained in two ways. Either nucleotide 1714 is a hot spot for mutations or, alternatively, missense mutations at this site confer a specific pathogenic gain-of-function or dominant-negative effect.
doi:10.1038/jhg.2009.1
PMCID: PMC3431155  PMID: 19180119
D527N; DFNA36; hereditary hearing loss; hot spot; TMC1
7.  Short course radiotherapy with simultaneous integrated boost for stage I-II breast cancer, early toxicities of a randomized clinical trial 
Background
TomoBreast is a unicenter, non-blinded randomized trial comparing conventional radiotherapy (CR) vs. hypofractionated Tomotherapy (TT) for post-operative treatment of breast cancer. The purpose of the trial is to compare whether TT can reduce heart and pulmonary toxicity. We evaluate early toxicities.
Methods
The trial started inclusion in May 2007 and reached its recruitment in August 2011. Women with stage T1-3N0M0 or T1-2N1M0 breast cancer completely resected by tumorectomy (BCS) or by mastectomy (MA) who consented to participate were randomized, according to a prescribed computer-generated randomization schedule, between control arm of CR 25x2 Gy/5 weeks by tangential fields on breast/chest wall, plus supraclavicular-axillary field if node-positive, and sequential boost 8x2 Gy/2 weeks if BCS (cumulative dose 66 Gy/7 weeks), versus experimental TT arm of 15x2.8 Gy/3 weeks, including nodal areas if node-positive and simultaneous integrated boost of 0.6 Gy if BCS (cumulative dose 51 Gy/3 weeks). Outcomes evaluated were the pulmonary and heart function. Comparison of proportions used one-sided Fisher's exact test.
Results
By May 2010, 70 patients were randomized and had more than 1 year of follow-up. Out of 69 evaluable cases, 32 were assigned to CR (21 BCS, 11 MA), 37 to TT (20 BCS, 17 MA). Skin toxicity of grade ≥1 at 2 years was 60% in CR, vs. 30% in TT arm. Heart function showed no significant difference for left ventricular ejection fraction at 2 years, CR 4.8% vs. TT 4.6%. Pulmonary function tests at 2 years showed grade ≥1 decline of FEV1 in 21% of CR, vs. 15% of TT and decline of DLco in 29% of CR, vs. 7% of TT (P = 0.05).
Conclusions
There were no unexpected severe toxicities. Short course radiotherapy of the breast with simultaneous integrated boost over 3 weeks proved feasible without excess toxicities. Pulmonary tests showed a slight trend in favor of Tomotherapy, which will need confirmation with longer follow-up of patients.
Trail registration
ClinicalTrials.gov NCT00459628
doi:10.1186/1748-717X-7-80
PMCID: PMC3432009  PMID: 22656865
Early breast cancer; Hypofractionation; Simultaneous integrated boost (SIB); Image guided radiation treatment (IGRT); Intensity modulated radiotherapy (IMRT)
8.  Molecular diagnostics for congenital hearing loss including 15 deafness genes using a next generation sequencing platform 
BMC Medical Genomics  2012;5:17.
Background
Hereditary hearing loss (HL) can originate from mutations in one of many genes involved in the complex process of hearing. Identification of the genetic defects in patients is currently labor intensive and expensive. While screening with Sanger sequencing for GJB2 mutations is common, this is not the case for the other known deafness genes (> 60). Next generation sequencing technology (NGS) has the potential to be much more cost efficient. Published methods mainly use hybridization based target enrichment procedures that are time saving and efficient, but lead to loss in sensitivity. In this study we used a semi-automated PCR amplification and NGS in order to combine high sensitivity, speed and cost efficiency.
Results
In this proof of concept study, we screened 15 autosomal recessive deafness genes in 5 patients with congenital genetic deafness. 646 specific primer pairs for all exons and most of the UTR of the 15 selected genes were designed using primerXL. Using patient specific identifiers, all amplicons were pooled and analyzed using the Roche 454 NGS technology. Three of these patients are members of families in which a region of interest has previously been characterized by linkage studies. In these, we were able to identify two new mutations in CDH23 and OTOF. For another patient, the etiology of deafness was unclear, and no causal mutation was found. In a fifth patient, included as a positive control, we could confirm a known mutation in TMC1.
Conclusions
We have developed an assay that holds great promise as a tool for screening patients with familial autosomal recessive nonsyndromal hearing loss (ARNSHL). For the first time, an efficient, reliable and cost effective genetic test, based on PCR enrichment, for newborns with undiagnosed deafness is available.
doi:10.1186/1755-8794-5-17
PMCID: PMC3443074  PMID: 22607986
Deafness; Next generation sequencing; PCR based enrichment; Genetic diagnostics
9.  Two Iranian Families with a Novel Mutation in GJB2 Causing Autosomal Dominant Nonsyndromic Hearing Loss 
Mutations in GJB2, encoding connexin 26 (Cx26), cause both autosomal dominant and autosomal recessive nonsyndromic hearing loss at the DFNA3 and DFNB1 loci, respectively. Most of the over 100 described GJB2 mutations cause autosomal recessive nonsyndromic hearing loss. Only a minority has been associated with autosomal dominant hearing loss. In this study, we present two families with autosomal dominant nonsyndromic hearing loss caused by a novel mutation in GJB2 (p.Asp46Asn). Both families were ascertained from the same village in northern Iran consistent with a founder effect. This finding implicates the D46N missense mutation in Cx26 as a common cause of deafness in this part of Iran mandating mutation screening of GJB2 for D46N in all persons with hearing loss who originate from this geographic region.
doi:10.1002/ajmg.a.33209
PMCID: PMC3080436  PMID: 21484990
connexin 26; D46N; autosomal dominant nonsyndromic hearing loss; DFNA3; Iran
10.  DFNA8/12 Caused by TECTA Mutations is the Most Identified Subtype of Non-syndromic Autosomal Dominant Hearing Loss 
Human Mutation  2011;32(7):825-834.
The prevalence of DFNA8/DFNA12 (DFNA8/12), a type of autosomal dominant non-syndromic hearing loss (ADNSHL), is unknown as comprehensive population-based genetic screening has not been conducted. We therefore completed unbiased screening for TECTA mutations in a Spanish cohort of 372 probands from ADNSHL families. Three additional families (Spanish, Belgian and English) known to be linked to DFNA8/12 were also included in the screening. In an additional cohort of 835 American ADNSHL families, we preselected 73 probands for TECTA screening based on audiometric data. In aggregate, we identified 23 TECTA mutations in this process. Remarkably 20 of these mutations are novel, more than doubling the number of reported TECTA ADNSHL mutations from 13 to 33. Mutations lie in all domains of the α-tectorin protein, including those for the first time identified in the entactin domain, the vWFD1, vWFD2 and vWFD3 repeats, and the D1-D2 and TIL2 connectors. While the majority are private mutations, four of them – p.Cys1036Tyr, p.Cys1837Gly, p.Thr1866Met and p.Arg1890Cys – were observed in more than one unrelated family. For two of these mutations founder effects were also confirmed. Our data validate previously observed genotype-phenotype correlations in DFNA8/12 and introduce new correlations. Specifically, mutations in the N-terminal region of α-tectorin (entactin domain, vWFD1 and vWFD2) lead to mid frequency NSHL, a phenotype previously associated only with mutations in the ZP domain. Collectively, our results indicate that DFNA8/12 hearing loss is a frequent type of ADNSHL.
doi:10.1002/humu.21512
PMCID: PMC3326665  PMID: 21520338
DFNA8; DFNA12; TECTA; mid-frequency hearing loss; high-frequency hearing loss
11.  The splicing mutant of the human tumor suppressor protein DFNA5 induces programmed cell death when expressed in the yeast Saccharomyces cerevisiae 
DFNA5 was first identified as a gene responsible for autosomal dominant deafness. Different mutations were found, but they all resulted in exon 8 skipping during splicing and premature termination of the protein. Later, it became clear that the protein also has a tumor suppression function and that it can induce apoptosis. Epigenetic silencing of the DFNA5 gene is associated with different types of cancers, including gastric and colorectal cancers as well as breast tumors. We introduced the wild-type and mutant DFNA5 allele in the yeast Saccharomyces cerevisiae. The expression of the wild-type protein was well tolerated by the yeast cells, although the protein was subject of degradation and often deposited in distinct foci when cells entered the diauxic shift. In contrast, cells had problems to cope with mutant DFNA5 and despite an apparent compensatory reduction in expression levels, the mutant protein still triggered a marked growth defect, which in part can be ascribed to its interaction with mitochondria. Consistently, cells with mutant DFNA5 displayed significantly increased levels of ROS and signs of programmed cell death. The latter occurred independently of the yeast caspase, Mca1, but involved the mitochondrial fission protein, Fis1, the voltage-dependent anion channel protein, Por1 and the mitochondrial adenine nucleotide translocators, Aac1 and Aac3. Recent data proposed DFNA5 toxicity to be associated to a globular domain encoded by exon 2–6. We confirmed these data by showing that expression of solely this domain confers a strong growth phenotype. In addition, we identified a point mutant in this domain that completely abrogated its cytotoxicity in yeast as well as human Human Embryonic Kidney 293T cells (HEK293T). Combined, our data underscore that the yeast system offers a valuable tool to further dissect the apoptotic properties of DFNA5.
doi:10.3389/fonc.2012.00077
PMCID: PMC3404532  PMID: 22848872
DFNA5; Saccharomyces cerevisiae; yeast; cell death; apoptosis; hearing impairment; tumor suppressor
14.  High Frequency of the p.R34X Mutation in the TMC1 Gene Associated with Nonsyndromic Hearing Loss Is Due to Founder Effects 
Founder mutations, particularly 35delG in the GJB2 gene, have to a large extent contributed to the high frequency of autosomal recessive nonsyndromic hearing loss (ARNSHL). Mutations in transmembrane channel-like gene 1 (TMC1) cause ARNSHL. The p.R34X mutation is the most frequent known mutation in the TMC1 gene. To study the origin of this mutation and determine whether it arose in a common ancestor, we analyzed 21 polymorphic markers spanning the TMC1 gene in 11 unrelated individuals from Algeria, Iran, Iraq, Lebanon, Pakistan, Tunisia, and Turkey who carry this mutation. In nine individuals, we observed significant linkage disequilibrium between p.R34X and five polymorphic markers within a 220 kb interval, suggesting that p.R34X arose from a common founder. We estimated the age of this mutation to be between 1075 and 1900 years, perhaps spreading along the third Hadramaout population movements during the seventh century. A second founder effect was observed in Turkish and Lebanese individuals with markers in a 920 kb interval. Screening for the TMC1 p.R34X mutation is indicated in the genetic evaluation of persons with ARNSHL from North African and Southwest Asia.
doi:10.1089/gtmb.2009.0174
PMCID: PMC2936956  PMID: 20373850
15.  A genome-wide association study for age-related hearing impairment in the Saami 
This study aimed at contributing to the elucidation of the genetic basis of age-related hearing impairment (ARHI), a common multifactorial disease with an important genetic contribution as demonstrated by heritability studies. We conducted a genome-wide association study (GWAS) in the Finnish Saami, a small, ancient, genetically isolated population without evidence of demographic expansion. The choice of this study population was motivated by its anticipated higher extent of LD, potentially offering a substantial power advantage for association mapping. DNA samples and audiometric measurements were collected from 352 Finnish Saami individuals, aged between 50 and 75 years. To reduce the burden of multiple testing, we applied principal component (PC) analysis to the multivariate audiometric phenotype. The first three PCs captured 80% of the variation in hearing thresholds, while maintaining biologically important audiometric features. All subjects were genotyped with the Affymetrix 100 K chip. To account for multiple levels of relatedness among subjects, as well as for population stratification, association testing was performed using a mixed model. We summarised the top-ranking association signals for the three traits under study. The top-ranked SNP, rs457717 (P-value 3.55 × 10−7), was associated with PC3 and was localised in an intron of the IQ motif-containing GTPase-activating-like protein (IQGAP2). Intriguingly, the SNP rs161927 (P-value 0.000149), seventh-ranked for PC1, was positioned immediately downstream from the metabotropic glutamate receptor-7 gene (GRM7). As a previous GWAS of a European and Finnish sample set already suggested a role for GRM7 in ARHI, this study provides further evidence for the involvement of this gene.
doi:10.1038/ejhg.2009.234
PMCID: PMC2987344  PMID: 20068591
Saami; isolated population; mixed model; genome-wide association study; age-related hearing impairment; presbycusis
16.  Genome-wide SNP analysis reveals no gain in power for association studies of common variants in the Finnish Saami 
The Saami from Fennoscandia are believed to represent an ancient, genetically isolated population with no evidence of population expansion. Theoretical work has indicated that under this demographic scenario, extensive linkage disequilibrium (LD) is generated by genetic drift. Therefore, it has been suggested that the Saami would be particularly suited for genetic association studies, offering a substantial power advantage and allowing more economic study designs. However, no study has yet assessed this claim. As part of a GWAS for a complex trait, we evaluated the relative power for association studies of common variants in the Finnish Saami. LD patterns in the Saami were very similar to those in the non-African HapMap reference panels. Haplotype diversity was reduced and, on average, levels of LD were higher in the Saami as compared with those in the HapMap panels. However, using a ‘hidden' SNP approach we show that this does not translate into a power gain in association studies. Contrary to earlier claims, we show that for a given set of common SNPs, genomic coverage attained in the Saami is similar to that in the non-African HapMap panels. Nevertheless, the reduced haplotype diversity could potentially facilitate gene identification, especially if multiple rare variants play a role in disease etiology. Our results further indicate that the HapMap is a useful resource for genetic studies in the Saami.
doi:10.1038/ejhg.2009.210
PMCID: PMC2987317  PMID: 19935831
Saami; genome-wide association studies; linkage disequilibrium; population isolates
17.  A NOVEL DFNB1 DELETION ALLELE SUPPORTS THE EXISTENCE OF A DISTANT CIS-REGULATORY REGION THAT CONTROLS GJB2 AND GJB6 EXPRESSION 
Clinical genetics  2010;78(3):267-274.
Eleven affected members of a large German-American family segregating recessively inherited, congenital, non-syndromic sensorineural hearing loss (SNHL) were found to be homozygous for the common 35delG mutation of GJB2, the gene encoding the gap junction protein Connexin 26. Surprisingly, four additional family members with bilateral profound SNHL carried only a single 35delG mutation. Previously, we demonstrated reduced expression of both GJB2 and GJB6 mRNA from the allele carried in trans with that bearing the 35delG mutation in these four persons. Using array comparative genome hybridization (arrayCGH), we have now identified on this allele a deletion of 131.4 kb whose proximal breakpoint lies more than 100 kb upstream of the transcriptional start sites of GJB2 and GJB6. This deletion, del(chr13:19,837,344-19,968,698), segregates as a completely penetrant DFNB1 allele in this family. It is not present in 528 persons with SNHL and monoallelic mutation of GJB2 or GJB6, nor have we identified any other candidate pathogenic copy number variation by arrayCGH in a subset of 10 such persons. Characterization of distant GJB2/GJB6 cis-regulatory regions evidenced by this allele may be required to find the ‘missing’ DFNB1 mutations that are believed to exist.
doi:10.1111/j.1399-0004.2010.01387.x
PMCID: PMC2919588  PMID: 20236118
Connexin 26; connexin 30; DFNB1; gene expression regulation; GJB2; GJB6; sensorineural hearing loss; sequence deletion
18.  Multiple enhancers located in a 1-Mb region upstream of POU3F4 promote expression during inner ear development and may be required for hearing 
Human Genetics  2010;128(4):411-419.
POU3F4 encodes a POU-domain transcription factor required for inner ear development. Defects in POU3F4 function are associated with X-linked deafness type 3 (DFN3). Multiple deletions affecting up to ~900-kb upstream of POU3F4 are found in DFN3 patients, suggesting the presence of essential POU3F4 enhancers in this region. Recently, an inner ear enhancer was reported that is absent in most DFN3 patients with upstream deletions. However, two indications suggest that additional enhancers in the POU3F4 upstream region are required for POU3F4 function during inner ear development. First, there is at least one DFN3 deletion that does not eliminate the reported enhancer. Second, the expression pattern driven by this enhancer does not fully recapitulate Pou3f4 expression in the inner ear. Here, we screened a 1-Mb region upstream of the POU3F4 gene for additional cis-regulatory elements and searched for novel DFN3 mutations in the identified POU3F4 enhancers. We found several novel enhancers for otic vesicle expression. Some of these also drive expression in kidney, pancreas and brain, tissues that are known to express Pou3f4. In addition, we report a new and smallest deletion identified so far in a DFN3 family which eliminates 3.9 kb, comprising almost exclusively the previous reported inner ear enhancer. We suggest that multiple enhancers control the expression of Pou3f4 in the inner ear and these may contribute to the phenotype observed in DFN3 patients. In addition, the novel deletion demonstrates that the previous reported enhancer, although not sufficient, is essential for POU3F4 function during inner ear development.
Electronic supplementary material
The online version of this article (doi:10.1007/s00439-010-0864-x) contains supplementary material, which is available to authorized users.
doi:10.1007/s00439-010-0864-x
PMCID: PMC2939330  PMID: 20668882
19.  Phenotypic variability of patients homozygous for the GJB2 mutation 35delG cannot be explained by the influence of one major modifier gene 
Hereditary hearing loss is a very heterogeneous trait, with 46 gene identifications for nonsyndromic hearing loss. Mutations in GJB2 cause up to half of all cases of severe-to-profound congenital autosomal recessive nonsyndromic hearing loss, with 35delG being the most frequent mutation in Caucasians. Although a genotype-phenotype correlation has been established for most GJB2 genotypes, the hearing loss of 35delG homozygous patients is mild-to-profound. We hypothesize that this phenotypic variability is at least partly caused by the influence of modifier genes. By performing a whole-genome association study on 35delG homozygotes, we sought to identify modifier genes. The association study was performed by comparing the genotypes of mild/moderate cases and profound cases. The first analysis included a pooling-based whole-genome association study of a first set of 255 samples by using both the Illumina 550K and Affymetrix 500K chips. This analysis resulted in a ranking of all analysed SNPs according to their p-values. The top 250 most significantly associated SNPs were genotyped individually in the same sample set. All 192 SNPs that still had significant p-values were genotyped in a second independent set of 297 samples for replication. The significant p-values were replicated in nine SNPs, with combined p-values between 3×10−3 and 1×10−4. This study suggests that the phenotypic variability in 35delG homozygous patients cannot be explained by the effect of one major modifier gene. Significantly associated SNPs may reflect a small modifying effect on the phenotype. Increasing the power of the study will be of the greatest importance to confirm these results.
doi:10.1038/ejhg.2008.201
PMCID: PMC2883287  PMID: 18985073
Hereditary hearing loss; connexin 26; 35delG; association study; modifier gene
20.  Forty-six genes causing nonsyndromic hearing impairment: which ones should be analyzed in DNA diagnostics? 
Mutation research  2008;681(2-3):189-196.
Hearing impairment is the most common sensory disorder, present in 1 of every 500 newborns. With 46 genes implicated in nonsyndromic hearing loss, it is also an extremely heterogeneous trait. Here, we categorize for the first time all mutations reported in nonsyndromic deafness genes, both worldwide and more specifically in Caucasians. The most frequent genes implicated in autosomal recessive nonsyndromic hearing loss are GJB2, which is responsible for more than half of cases, followed by SLC26A4, MYO15A, OTOF, CDH23 and TMC1. None of the genes associated with autosomal dominant nonsyndromic hearing loss accounts for a preponderance of cases. Mutations are somewhat more frequently reported in WFS1, KCNQ4, COCH and GJB2. Only a minority of these genes is currently included in genetic diagnostics, the selection criteria typically reflecting: 1) high frequency as a cause of deafness (i.e. GJB2); 2) association with another recognizable feature (i.e. SLC26A4 and enlarged vestibular aqueduct); or 3) a recognizable audioprofile (i.e. WFS1). New and powerful DNA sequencing technologies have been developed over the past few years, but have not yet found their way into DNA diagnostics. Implementing these technologies is likely to happen within the next 5 years, and will cause a breakthrough in terms of power and cost efficiency. It will become possible to analyze most - if not all - deafness genes, as opposed to one or a few genes currently. This ability will greatly improve DNA diagnostics, provide epidemiological data on gene-based mutation frequencies, and reveal novel genotype-phenotype correlations.
doi:10.1016/j.mrrev.2008.08.002
PMCID: PMC2847850  PMID: 18804553
Hereditary hearing loss; ARNSHL; ADNSHL; GJB2; gene frequencies; genetic counselling
21.  Function and expression pattern of nonsyndromic deafness genes 
Current molecular medicine  2009;9(5):546-564.
Hearing loss is the most common sensory disorder, present in 1 of every 500 newborns. To date, 46 genes have been identified that cause nonsyndromic hearing loss, making it an extremely heterogeneous trait. This review provides a comprehensive overview of the inner ear function and expression pattern of these genes. In general, they are involved in hair bundle morphogenesis, form constituents of the extracellular matrix, play a role in cochlear ion homeostasis or serve as transcription factors. During the past few years, our knowledge of genes involved in hair bundle morphogenesis has increased substantially. We give an up-to-date overview of both the nonsyndromic and Usher syndrome genes involved in this process, highlighting proteins that interact to form macromolecular complexes. For every gene, we also summarize its expression pattern and impact on hearing at the functional level. Gene-specific cochlear expression is summarized in a unique table by structure/cell type and is illustrated on a cochlear cross-section, which is available online via the Hereditary Hearing Loss Homepage. This review should provide auditory scientists the most relevant information for all identified nonsyndromic deafness genes.
PMCID: PMC2840995  PMID: 19601806
Hereditary hearing loss; ARNSHL; ADNSHL; hair bundle; ion homeostasis; cochlear expression pattern
22.  GRM7 variants confer susceptibility to age-related hearing impairment 
Human Molecular Genetics  2008;18(4):785-796.
Age-related hearing impairment (ARHI), or presbycusis, is the most prevalent sensory impairment in the elderly. ARHI is a complex disease caused by an interaction between environmental and genetic factors. Here we describe the results of the first whole genome association study for ARHI. The study was performed using 846 cases and 846 controls selected from 3434 individuals collected by eight centers in six European countries. DNA pools for cases and controls were allelotyped on the Affymetrix 500K GeneChip® for each center separately. The 252 top-ranked single nucleotide polymorphisms (SNPs) identified in a non-Finnish European sample group (1332 samples) and the 177 top-ranked SNPs from a Finnish sample group (360 samples) were confirmed using individual genotyping. Subsequently, the 23 most interesting SNPs were individually genotyped in an independent European replication group (138 samples). This resulted in the identification of a highly significant and replicated SNP located in GRM7, the gene encoding metabotropic glutamate receptor type 7. Also in the Finnish sample group, two GRM7 SNPs were significant, albeit in a different region of the gene. As the Finnish are genetically distinct from the rest of the European population, this may be due to allelic heterogeneity. We performed histochemical studies in human and mouse and showed that mGluR7 is expressed in hair cells and in spiral ganglion cells of the inner ear. Together these data indicate that common alleles of GRM7 contribute to an individual's risk of developing ARHI, possibly through a mechanism of altered susceptibility to glutamate excitotoxicity.
doi:10.1093/hmg/ddn402
PMCID: PMC2638831  PMID: 19047183
24.  Phenotypic variability of patients homozygous for the GJB2 mutation 35delG cannot be explained by the influence of one major modifier gene 
Hereditary hearing loss (HL) is a very heterogeneous trait, with 46 gene identifications for non-syndromic HL. Mutations in GJB2 cause up to half of all cases of severe-to-profound congenital autosomal recessive non-syndromic HL, with 35delG being the most frequent mutation in Caucasians. Although a genotype–phenotype correlation has been established for most GJB2 genotypes, the HL of 35delG homozygous patients is mild to profound. We hypothesise that this phenotypic variability is at least partly caused by the influence of modifier genes. By performing a whole-genome association (WGA) study on 35delG homozygotes, we sought to identify modifier genes. The association study was performed by comparing the genotypes of mild/moderate cases and profound cases. The first analysis included a pooling-based WGA study of a first set of 255 samples by using both the Illumina 550K and Affymetrix 500K chips. This analysis resulted in a ranking of all analysed single-nucleotide polymorphisms (SNPs) according to their P-values. The top 250 most significantly associated SNPs were genotyped individually in the same sample set. All 192 SNPs that still had significant P-values were genotyped in a second independent set of 297 samples for replication. The significant P-values were replicated in nine SNPs, with combined P-values between 3 × 10−3 and 1 × 10−4. This study suggests that the phenotypic variability in 35delG homozygous patients cannot be explained by the effect of one major modifier gene. Significantly associated SNPs may reflect a small modifying effect on the phenotype. Increasing the power of the study will be of greatest importance to confirm these results.
doi:10.1038/ejhg.2008.201
PMCID: PMC2883287  PMID: 18985073
hereditary hearing loss; connexin 26; 35delG; association study; modifier gene
25.  Variations in HSP70 genes associated with noise-induced hearing loss in two independent populations 
Noise-induced hearing loss (NIHL) is one of the most important occupational health hazards. Millions of people worldwide are exposed daily to harmful levels of noise. NIHL is a complex disease resulting from an interaction between genetic and environmental factors. Although the environmental risk factors have been studied extensively, little is known about the genetic factors. Heat-shock proteins (HSPs) are induced after exposure to severe noise. When first induced by exposure to moderate sound levels, they can protect the ear from damage from excessive noise exposure. This protection is highly variable between individuals. An association of HSP70 genes with NIHL has been described by Yang et al (2006) in a Chinese sample set of noise-exposed workers. In this study, three polymorphisms (rs1043618, rs1061581 and rs2227956) in HSP70-1, HSP70-2 and HSP70-hom, respectively, were genotyped in 206 Swedish and 238 Polish DNA samples of noise-exposed subjects and analyzed. One SNP, rs2227956 in HSP70-hom, resulted in a significant association with NIHL in both sample sets. In addition, rs1043618 and rs1061581 were significant in the Swedish sample set. Analysis of the haplotypes composed of the three SNPs revealed significant associations between NIHL and haplotype GAC in both sample sets and with haplotype CGT in the Swedish sample set. In conclusion, this study replicated the association of HSP70 genes with NIHL in a second and third independent noise-exposed sample set, hereby adding to the evidence that HSP70 genes may be NIHL susceptibility genes.
doi:10.1038/ejhg.2008.172
PMCID: PMC2986160  PMID: 18813331
noise-induced hearing loss; HSP70; association study; replication

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