Ototoxicity is a frequent adverse event of cisplatin treatment. No therapy is currently available for cisplatin-induced ototoxicity. A systematic review of experimental animal studies and in vitro experiments was conducted to evaluate gene therapy as a potential future therapeutic option.

Eligible studies were identified through searches of electronic databases Ovid MEDLINE, Ovid MEDLINE In-Process, Embase, PubMed, Biosis Previews, Scopus, ISI Web of Science, and The Cochrane Library.

Articles obtained from the search were independently reviewed by 2 authors using specific criteria to identify experimental animal studies and in vitro experiments conducted to evaluate gene therapy for cisplatin-induced ototoxicity. No restriction was applied to publication dates or languages.

Data extracted included experiment type, cell type, species, targeted gene, gene expression, method, administration, inner ear site evaluated, outcome measures for cytotoxicity, and significant results.

Fourteen articles were included in this review. In vitro and in vivo experiments have been performed to evaluate the potential of gene expression manipulation for cisplatin-induced ototoxicity. Twelve different genes were targeted including NTF3, GDNF, HO-1, XIAP, Trpv1, BCL2, Otos, Nfe2l2, Nox1, Nox3, Nox4, and Ctr1. All of the included articles demonstrated a benefit of gene therapy on cytotoxicity caused by cisplatin.

Experimental animal studies and in vitro experiments have demonstrated the efficacy of gene therapy for cisplatin-induced ototoxicity. However, further investigation regarding safety, immunogenicity, and consequences of genetic manipulation in the inner ear tissues must be completed to develop future therapeutic options.

Many tumors exhibit elevated chromosome mis-segregation termed chromosome instability (CIN), which is likely to be a potent driver of tumor progression and drug resistance. Causes of CIN are poorly understood but probably include prior genome tetraploidization, centrosome amplification and mitotic checkpoint defects. This study identifies epigenetic alteration of the centromere as a potential contributor to the CIN phenotype. The centromere controls chromosome segregation and consists of higher-order repeat (HOR) alpha-satellite DNA packaged into two chromatin domains: the kinetochore, harboring the centromere-specific H3 variant centromere protein A (CENP-A), and the pericentromeric heterochromatin, considered important for cohesion. Perturbation of centromeric chromatin in model systems causes CIN. As cancer cells exhibit widespread chromatin changes, we hypothesized that pericentromeric chromatin structure could also be affected, contributing to CIN. Cytological and chromatin immunoprecipitation and PCR (ChIP-PCR)-based analyses of HT1080 cancer cells showed that only one of the two HORs on chromosomes 5 and 7 incorporate CENP-A, an organization conserved in all normal and cancer-derived cells examined. Contrastingly, the heterochromatin marker H3K9me3 (trimethylation of H3 lysine 9) mapped to all four HORs and ChIP-PCR showed an altered pattern of H3K9me3 in cancer cell lines and breast tumors, consistent with a reduction on the kinetochore-forming HORs. The JMJD2B demethylase is overexpressed in breast tumors with a CIN phenotype, and overexpression of exogenous JMJD2B in cultured breast epithelial cells caused loss of centromere-associated H3K9me3 and increased CIN. These findings suggest that impaired maintenance of pericentromeric heterochromatin may contribute to CIN in cancer and be a novel therapeutic target.

Association studies on gene polymorphisms of neurotransmitter systems have hypothesized an involvement of dopamine receptors in susceptibility to schizophrenia. However, structural and morphological abnormalities in different brain regions of schizophrenic patients support neurodevelopmental etiology for schizophrenia and neurotrophic factor genes could be candidates for genetic studies. The glial cell line-derived neurotrophic factor (GDNF) is a neurotrophic and potential differentiation factor for dopaminergic systems. We have performed, in an Italian sample, an association study on 3' UTR (AGG)n repeat in GDNF gene. Our results have evidenced a difference in the allele frequencies between patients and controls (CLUMP (T1) chi2 = 17.365, df = 9, P = 0.043) and the (AGG)n > or = 15 alleles (Fisher Exact Test (two side) chi2 = 11.818, df = 1, P = 0.0003) were more present in the controls group. Similarity, the carriers of (AGG)n > or = 15 (OR = 0.176 95% CI: 0.060-0.520) were more present in the same group. These results support that the (AGG)n > or = 15 alleles could be protective factors against schizophrenia and thus they suggest a possible involvement of GDNF gene in the genetic liability to illness.

Parkinson's disease is the second most common neurodegenerative disorder after Alzheimer's disease. Most cases are sporadic, however familial cases do exist. We examined 12 families with familial Parkinson's disease ascertained at the Movement Disorder clinic at the Oregon Health Sciences University for genetic linkage to a number of candidate loci. These loci have been implicated in familial Parkinson's disease or in syndromes with a clinical presentation that overlaps with parkinsonism, as well as potentially in the pathogenesis of the disease.

The examined loci were PARK3, Parkin, DRD (dopa-responsive dystonia), FET1 (familial essential tremor), BDNF (brain-derived neurotrophic factor), GDNF (glial cell line-derived neurotrophic factor), Ret, DAT1 (the dopamine transporter), Nurr1 and Synphilin-1. Linkage to the alpha-synuclein gene and the Frontotemporal dementia with parkinsonism locus on chromosome 17 had previously been excluded in the families included in this study. Using Fastlink, Genehunter and Simwalk both parametric and model-free non-parametric linkage analyses were performed.

In the multipoint parametric linkage analysis lod scores were below -2 for all loci except FET1 and Synphilin-1 under an autosomal dominant model with incomplete penetrance. Using non-parametric linkage analysis there was no evidence for linkage, although linkage could not be excluded. A few families showed positive parametric and non-parametric lod scores indicating possible genetic heterogeneity between families, although these scores did not reach any degree of statistical significance.

We conclude that in these families there was no evidence for linkage to any of the loci tested, although we were unable to exclude linkage with both parametric and non-parametric methods.

The glial cell line-derived neurotrophic factor (GDNF) is an important neurotrophic and potential differentiation factor for dopaminergic systems. Both the dopamine theory and the neurodevelopmental hypothesis of schizophrenia suggest that alterations of GDNF functions could be involved in the pathogenesis of schizophrenia. Using polymerase chain reaction and single strand conformational polymorphism analysis, we searched for polymorphisms in the GDNF gene in 50 patients with schizophrenia. No evidence was obtained, however, for the presence of polymorphisms in the DNA sequence encoding GDNF mature peptide in our patients. We then examined a trinucleotide repeat (AGG)(n) polymorphism in the 3'-UTR of the GDNF gene for allelic association in a Japanese sample of 99 schizophrenic patients and 98 control subjects. There was no significant difference in the overall distribution of the allele between the two groups. When each allele was examined separately, the allele (AGG)(10) was more common in schizophrenic patients than in control subjects, but this finding was not significant when multiple testing was taken into account in the analysis. Overall, we obtained no solid evidence for the involvement of the GDNF gene in the pathogenesis of schizophrenia, although further studies in larger numbers of subjects will be required to conclude whether the trinucleotide repeat polymorphism is associated with the development of schizophrenia.

To address the regulation of glial cell line-derived neurotrophic factor (GDNF) gene expression, we have isolated 5' extended cDNAs, cloned the human GDNF gene, and characterized the promoter. GDNF-encoding 5' extended cDNAs containing a novel exon were isolated via reverse transcription-polymerase chain reaction (RT-PCR) of mRNA from human fetal kidney and adult skeletal muscle. The GDNF gene was isolated from a human genomic library in a P1 bacteriophage vector. Analysis of the 5' flanking sequence revealed a promoter that lacks a CCAAT-box motif and is GC rich. Consensus binding sites for a variety of transcription factors have been identified in the promoter. Promoter/reporter plasmids have been constructed by fusion of the promoter and a portion of exon I to a luciferase gene. The promoter/reporter construct and a number of promoter deletions were transiently transfected into two human cell lines known to express GDNF. Multiple enhancer and silencer regions were revealed as well as a minimal promoter sufficient for basal transcription. Finally, a RT-PCR assay, specific for transcripts originating from this GDNF promoter, was developed and used to show that this promoter is active in vivo. The results suggest GDNF is regulated in a complex manner.

Human SK-N-AS neuroblastoma and U-87MG glioblastoma cell lines were found to secrete relatively high levels of glial cell line-derived neurotrophic factor (GDNF). In response to growth factors, cytokines, and pharmacophores, the two cell lines differentially regulated GDNF release. A 24-hr exposure to tumor necrosis factor-alpha (TNFalpha; 10 ng/ml) or interleukin-1beta (IL-1,; 10 ng/ml) induced GDNF release in U-87MG cells, but repressed GDNF release from SK-N-AS cells. Fibroblast growth factors (FGF)-1, -2, and -9 (50 ng/ml), the prostaglandins PGA2, PGE2, and PGI2 (10 microM), phorbol 12,13-didecanoate (PDD; 10 nM), okadaic acid (10 nM), dexamethasone (1 microM), and vitamin D3 (1 microm) also differentially effected GDNF release from U-87MG and SK-N-AS cells. A result shared by both cell lines, was a two- to threefold increase in GDNF release by db-cAMP (1 mM), or forskolin (10 microM). In general, analysis of steady-state GDNF mRNA levels correlated with changes in extracellular GDNF levels in U-87MG cells but remained static in SK-N-AS cells. The data suggest that human GDNF synthesis/release can be regulated by numerous factors, signaling through multiple and diverse secondary messenger systems. Furthermore, we provide evidence of differential regulation of human GDNF synthesis/release in cells of glial (U-87MG) and neuronal (SK-N-AS) origin.

Glial cell line-derived neurotrophic factor (GDNF), a distant member of the TGF-beta superfamily, is a survival factor for various neurons, making it a potential therapeutic agent for neurodegenerative disorders. Here we present the genomic structure and characterization of the promoter of the human GDNF (hGDNF) gene. It contains three exons coding for a cDNA of 4.6 kb including large 5'- and 3'-untranslated regions (UTRs). The 3'-UTR contains a polymorphic AGG repeat that appears not to be expanded in patients suffering from different neurodegenerative disorders. RT-PCR results in at least three different hGDNF transcripts including one that lacks exon 2. Transient expression experiments reveal that exon 2 is essential for proper cellular processing to yield a secreted form of hGDNF, whereas expression of exon 3 alone is sufficient to code for a mature form of hGDNF retained within the cell. Our data show that the hGDNF gene is driven by a TATA-containing promoter preceding exon 1. A second promoter element has been mapped to a region 5' of exon 2. Both promoters are in close proximity to CpG islands covering exons 1 and 2. Using luciferase as a reporter gene, the TATA-containing hGDNF promoter facilitates a 20- to 40-fold increase in transcription when compared with a corresponding promoterless construct, whereas the second promoter confers only weak activity. Furthermore, fibroblast growth factor 2, tetradecanoyl 12-phorbol acetate, an inflammatory agent, and cAMP increase promoter activity, suggesting that GDNF transcriptional regulation is a target of exogenous signals.

Glial cell line-derived neurotrophic factor (GDNF) is a potent survival factor for nigrostriatal dopaminergic, central cholinergic, and motoneurons. GDNF also prevents the neuronal loss in experimental animal models for Parkinson's disease (PD). We have now investigated the GDNF gene for possible mutations in a group of nonfamilial PD and other patients. By cleavase fragment length polymorphism (CFLP) analysis and direct sequencing of the full coding region of GDNF gene we found a novel GDNF sequence variant in 1 of 30 PD patients. The alteration does not change the predicted amino acid sequence and it was also found in 1 of 20 patients without PD, suggesting that it represents a polymorphism in the gene. No other sequence variations were found. We conclude therefore that mutations in the GDNF coding region are not commonly contributing to the pathogenesis of PD.

Hirschsprung's disease (HD) is a relatively common cause of intestinal obstruction in the newborn, characterized by the absence of autonomic ganglion cells in the terminal bowel. Existence of familial cases indicates that genetic factors may be involved in the etiology of some cases of HD. Different inheritance patterns observed in subsets of HD families or kindreds, and the detection of different chromosome aberrations in some HD patients, suggest genetic heterogeneity of HD. Recent expansion of molecular genetics has identified multiple susceptibility genes of HD. These include the RET gene, the glial cell-derived neurotrophic factor gene, the endothelin-B receptor gene, and endothelin-3 gene. Furthermore, some other genes or genetic factors are speculated to be implicated in the development of HD, and it is believed that multiple factors play a role in disease development in some cases. Taken together, these data suggest and may explain the complexity of the etiology of HD. This review focuses on recent advances in our understanding of the genetic aspects of HD.

Causative germline missense mutations in the RET proto-oncogene have been associated with over 92% of families with the inherited cancer syndrome multiple endocrine neoplasia type 2 (MEN 2). MEN 2A is characterized primarily by medullary thyroid carcinoma (MTC) and pheochromocytoma, both tumors of neural crest origin. Parathyroid hyperplasia or adenoma is also seen in MEN 2A, but rarely in MEN 2B, which has additional stigmata, including a marfanoid habitus, mucosal neuromas, and ganglioneuromatosis of the gastrointestinal tract. In familial MTC, MTC is the only lesion present. Somatic RET mutations have also been identified in a subset of sporadic MTCs, pheochromocytomas, and rarely, small cell lung cancer, but not in sporadic parathyroid hyperplasias/adenomas or other neuroendocrine tumors. Glial cell line-derived neurotrophic factor (GDNF) and its receptor molecule GDNFR-alpha, have recently been identified as members of the RET ligand binding complex. Therefore, the genes encoding both GDNF and GDNFR-alpha are excellent candidates for a role in the pathogenesis of those MEN 2 families and sporadic neuroendocrine tumors without RET mutations. No mutations were found in the coding region of GDNF in DNA samples from 9 RET mutation negative MEN 2 individuals (comprising 6 distinct families), 12 sporadic MTCs, 17 sporadic cases of parathyroid adenoma, and 10 small cell lung cancer cell lines. Therefore, we find no evidence that mutation within the coding regions of GDNF plays a role in the genesis of MEN 2 and sporadic neuroendocrine tumors.

Hirschsprung disease (HSCR, aganglionic megacolon) is a common congenital malformation leading to bowel obstruction, with an incidence of 1/5,000 live births. It is characterized by the absence of intrinsic ganglion cells in the myenteric and submucosal plexuses along variable lengths of the gastrointestinal tract. As enteric neurons are derived from the vagal neural crest, HSCR is regarded as a neurocristopathy. On the basis of a skewed sex-ratio (M/F = 4/1) and a risk to relatives much higher than the incidence in the general population, HSCR has long been regarded as a sex-modified multifactorial disorder. Accordingly, segregation analysis suggested an incompletely penetrant dominant inheritance in HSCR families with aganglionosis extending beyond the sigmoid colon. We and others have mapped a dominant gene for HSCR to chromosome 10q11.2 and have ascribed the disease to mutations in the RET proto-oncogene. However, the lack of genotype-phenotype correlation, the low penetrance and the sex-dependent effect of RET mutations supported the existence of one or more modifier gene(s) in familial HSCR. In addition, thus far, RET mutations only accounted for 50% and 15-20% of familial and sporadic HSCR patients, respectively. RET encodes a tyrosine kinase receptor whose ligand was unknown. Recently, the Glial cell line-derived neurotrophic factor (GDNF) has been identified to be a ligand for RET. Moreover, Gdnf-/- knockout mutant mice display congenital intestinal aganglionosis and renal agenesis, a phenotype very similar to the Ret-/- mouse. These data prompted us to hypothesize that mutations of the gene encoding GDNF could either cause or modulate the HSCR phenotype in some cases.

Hirschsprung disease (HSCR), or congenital aganglionic megacolon, is the most common cause of congenital bowel obstruction with an incidence of 1 in 5000 live births. HSCR may be inherited as a single gene disorder with reduced penetrance or as a multigenic trait. HSCR mutations have been identified in the RET receptor tyrosine kinase, endothelin-B receptor (EDNRB) and its physiological ligand, endothelin 3 (EDN3). Although RET's ligand has remained elusive, it is expected to be an extracellular neurotrophic molecule expressed in the developing gut and kidney mesenchyme, based on the phenotypes of intestinal aganglionosis and renal agenesis observed in homozygous RET knockout (Ret -/-) mice. The glial cell line-derived neurotrophic factor (GDNF) is such a molecule. Recently, mice carrying two null alleles for Gdnf were shown to exhibit phenotypes remarkably similar to Ret-/- animals. We screened 106 unrelated HSCR patients for mutations in GDNF by direct sequencing. We identified one familial mutation in a HSCR patient with a known de novo RET mutation and malrotation of the gut. No haplotype sharing was evident in any of 36 HSCR kindreds typed for microsatellite markers surrounding GDNF on human chromosome 5p. Our data suggest that GDNF is a minor contributor to human HSCR susceptibility and that loss of its function in enteric neurogenesis may be compensated for by other neurotrophic factors or via other pathways. However, it may be that in rare instances, RET and GDNF mutations act in concert to produce an enteric phenotype.

Karyotypes with an interstitial deletion and a marker chromosome formed from the deleted segment are rare. We identified such a rearrangement in a newborn infant, who presented with macrocephaly, asymmetric square skull, minor facial anomalies, omphalocele, inguinal hernias, hypospadias, and club feet. The karyotype 46,XY,del(5) (pter --> p13::cen --> qter)/47,XY,+dicr(5)(:p13 --> cen::p13 --> cen), del(5)(pter --> p13::cen --> qter) was identified by banding studies and FISH analysis in the peripheral lymphocytes. One breakpoint on the del(5) maps distal to GDNF, and FISH analysis using an alpha-satellite probe suggests that the proximal breakpoint maps within the centromere. The dicentric r(5) consists of two copies of the segment deleted in the del(5), resulting in trisomy of proximal 5p (5p13-cen). The phenotype of the propositus is compared with other trisomy 5p cases and possible mechanisms for the generation of this unique chromosomal rearrangement are discussed.