Whole-Genome Sequencing Identified New Structural Variations in the DMD Gene That Cause Duchenne Muscular Dystrophy in Two Girls.

Dystrophinopathies are the most common muscle diseases, especially in men. In women, on the other hand, a manifestation of Duchenne muscular dystrophy is rare due to X-chromosomal inheritance. We present two young girls with severe muscle weakness, muscular dystrophies, and creatine kinase (CK) levels exceeding 10,000 U/L. In the skeletal muscle tissues, dystrophin staining reaction showed mosaicism. The almost entirely skewed X-inactivation in both cases supported the possibility of a dystrophinopathy. Despite standard molecular diagnostics (including multiplex ligation-dependent probe amplification (MLPA) and next generation sequencing (NGS) gene panel sequencing), the genetic cause of the girls' conditions remained unknown. However, whole-genome sequencing revealed two reciprocal translocations between their X chromosomes and chromosome 5 and chromosome 19, respectively. In both cases, the breakpoints on the X chromosomes were located directly within the DMD gene (in introns 54 and 7, respectively) and were responsible for the patients' phenotypes. Additional techniques such as Sanger sequencing, conventional karyotyping and fluorescence in situ hybridization (FISH) confirmed the disruption of DMD gene in both patients through translocations. These findings underscore the importance of accurate clinical data combined with histopathological analysis in pinpointing the suspected underlying genetic disorder. Moreover, our study illustrates the viability of whole-genome sequencing as a time-saving and highly effective method for identifying genetic factors responsible for complex genetic constellations in Duchenne muscular dystrophy (DMD).

Genetic Characterization of Rat Hepatic Stellate Cell Line PAV-1.

The rat hepatic stellate cell line PAV-1 was established two decades ago and proposed as a cellular model to study aspects of hepatic retinoic acid metabolism. This cell line exhibits a myofibroblast-like phenotype but also has the ability to store retinyl esters and synthesize retinoic acid from its precursor retinol. Importantly, when cultured with palmitic acid alone or in combination with retinol, the cells switch to a deactivated phenotype in which the proliferation and expression of profibrogenic marker genes are suppressed. Despite these interesting characteristics, the cell line has somehow fallen into oblivion. However, based on the fact that working with in vivo models is becoming increasingly complicated, genetically characterized established cell lines that mimic aspects of hepatic stellate cell biology are of fundamental value for biomedical research. To genetically characterize PAV-1 cells, we performed karyotype analysis using conventional chromosome analysis and multicolor spectral karyotyping (SKY), which allowed us to identify numerical and specific chromosomal alteration in PAV-1 cells. In addition, we used a panel of 31 species-specific allelic variant sites to define a unique short tandem repeat (STR) profile for this cell line and performed bulk mRNA-sequencing, showing that PAV-1 cells express an abundance of genes specific for the proposed myofibroblastic phenotype. Finally, we used Rhodamine-Phalloidin staining and electron microscopy analysis, which showed that PAV-1 cells contain a robust intracellular network of filamentous actin and process typical ultrastructural features of hepatic stellate cells.

Rat Hepatic Stellate Cell Line CFSC-2G: Genetic Markers and Short Tandem Repeat Profile Useful for Cell Line Authentication.

Hepatic stellate cells (HSCs) are also known as lipocytes, fat-storing cells, perisinusoidal cells, or Ito cells. These liver-specific mesenchymal cells represent about 5% to 8% of all liver cells, playing a key role in maintaining the microenvironment of the hepatic sinusoid. Upon chronic liver injury or in primary culture, these cells become activated and transdifferentiate into a contractile phenotype, i.e., the myofibroblast, capable of producing and secreting large quantities of extracellular matrix compounds. Based on their central role in the initiation and progression of chronic liver diseases, cultured HSCs are valuable in vitro tools to study molecular and cellular aspects of liver diseases. However, the isolation of these cells requires special equipment, trained personnel, and in some cases needs approval from respective authorities. To overcome these limitations, several immortalized HSC lines were established. One of these cell lines is CFSC, which was originally established from cirrhotic rat livers induced by carbon tetrachloride. First introduced in 1991, this cell line and derivatives thereof (i.e., CFSC-2G, CFSC-3H, CFSC-5H, and CFSC-8B) are now used in many laboratories as an established in vitro HSC model. We here describe molecular features that are suitable for cell authentication. Importantly, chromosome banding and multicolor spectral karyotyping (SKY) analysis demonstrate that the CFSC-2G genome has accumulated extensive chromosome rearrangements and most chromosomes exist in multiple copies producing a pseudo-triploid karyotype. Furthermore, our study documents a defined short tandem repeat (STR) profile including 31 species-specific markers, and a list of genes expressed in CFSC-2G established by bulk mRNA next-generation sequencing (NGS).

Detection of pericentric inversion with breakpoint in DMD by whole genome sequencing.

BACKGROUND: Dystrophinopathies caused by variants in the DMD gene are a well-studied muscle disease. The most common type of variant in DMD are large deletions. Very rarely reported forms of variants are chromosomal translocations, inversions and deep intronic variants (DIVs) because they are not detectable by standard diagnostic techniques (sequencing of coding sequence, copy number variant detection). This might be the reason that some clinically and histologically proven dystrophinopathy cases remain unsolved. METHODS: We used whole genome sequencing (WGS) to screen the entire DMD gene for variants in one of two brothers suffering from typical muscular dystrophy with strongly elevated creatine kinase levels. RESULTS: Although a pathogenic DIV could not be detected, we were able to identify a pericentric inversion with breakpoints in DMD intron 44 and Xq13.3, which could be confirmed by Sanger sequencing in the index as well as in his brother and mother. As this variation affects a major part of DMD it is most likely disease causing. CONCLUSION: Our findings elucidate that WGS is capable of detecting large structural rearrangements and might be suitable for the genetic diagnostics of dystrophinopathies in the future. In particular, inversions might be a more frequent cause for dystrophinopathies as anticipated and should be considered in genetically unsolved dystrophinopathy cases.

Genetic Characterization of Rat Hepatic Stellate Cell Line HSC-T6 for In Vitro Cell Line Authentication.

Immortalized hepatic stellate cells (HSCs) established from mouse, rat, and humans are valuable in vitro models for the biomedical investigation of liver biology. These cell lines are homogenous, thereby providing consistent and reproducible results. They grow more robustly than primary HSCs and provide an unlimited supply of proteins or nucleic acids for biochemical studies. Moreover, they can overcome ethical concerns associated with the use of animal and human tissue and allow for fostering of the 3R principle of replacement, reduction, and refinement proposed in 1959 by William M. S. Russell and Rex L. Burch. Nevertheless, working with continuous cell lines also has some disadvantages. In particular, there are ample examples in which genetic drift and cell misidentification has led to invalid data. Therefore, many journals and granting agencies now recommend proper cell line authentication. We herein describe the genetic characterization of the rat HSC line HSC-T6, which was introduced as a new in vitro model for the study of retinoid metabolism. The consensus chromosome markers, outlined primarily through multicolor spectral karyotyping (SKY), demonstrate that apart from the large derivative chromosome 1 (RNO1), at least two additional chromosomes (RNO4 and RNO7) are found to be in three copies in all metaphases. Additionally, we have defined a short tandem repeat (STR) profile for HSC-T6, including 31 species-specific markers. The typical features of these cells have been further determined by electron microscopy, Western blotting, and Rhodamine-Phalloidin staining. Finally, we have analyzed the transcriptome of HSC-T6 cells by mRNA sequencing (mRNA-Seq) using next generation sequencing (NGS).

Evolution of the Degenerated Y-Chromosome of the Swamp Guppy, Micropoecilia picta.

The conspicuous colour sexual dimorphism of guppies has made them paradigmatic study objects for sex-linked traits and sex chromosome evolution. Both the X- and Y-chromosomes of the common guppy (Poecilia reticulata) are genetically active and homomorphic, with a large homologous part and a small sex specific region. This feature is considered to emulate the initial stage of sex chromosome evolution. A similar situation has been documented in the related Endler's and Oropuche guppies (P. wingei, P. obscura) indicating a common origin of the Y in this group. A recent molecular study in the swamp guppy (Micropoecilia. picta) reported a low SNP density on the Y, indicating Y-chromosome deterioration. We performed a series of cytological studies on M. picta to show that the Y-chromosome is quite small compared to the X and has accumulated a high content of heterochromatin. Furthermore, the Y-chromosome stands out in displaying CpG clusters around the centromeric region. These cytological findings evidently illustrate that the Y-chromosome in M. picta is indeed highly degenerated. Immunostaining for SYCP3 and MLH1 in pachytene meiocytes revealed that a substantial part of the Y remains associated with the X. A specific MLH1 hotspot site was persistently marked at the distal end of the associated XY structure. These results unveil a landmark of a recombining pseudoautosomal region on the otherwise strongly degenerated Y chromosome of M. picta. Hormone treatments of females revealed that, unexpectedly, no sexually antagonistic color gene is Y-linked in M. picta. All these differences to the Poecilia group of guppies indicate that the trajectories associated with the evolution of sex chromosomes are not in parallel.

Genetic Spectrum of Syndromic and Non-Syndromic Hearing Loss in Pakistani Families.

The current molecular genetic diagnostic rates for hereditary hearing loss (HL) vary considerably according to the population background. Pakistan and other countries with high rates of consanguineous marriages have served as a unique resource for studying rare and novel forms of recessive HL. A combined exome sequencing, bioinformatics analysis, and gene mapping approach for 21 consanguineous Pakistani families revealed 13 pathogenic or likely pathogenic variants in the genes GJB2, MYO7A, FGF3, CDC14A, SLITRK6, CDH23, and MYO15A, with an overall resolve rate of 61.9%. GJB2 and MYO7A were the most frequently involved genes in this cohort. All the identified variants were either homozygous or compound heterozygous, with two of them not previously described in the literature (15.4%). Overall, seven missense variants (53.8%), three nonsense variants (23.1%), two frameshift variants (15.4%), and one splice-site variant (7.7%) were observed. Syndromic HL was identified in five (23.8%) of the 21 families studied. This study reflects the extreme genetic heterogeneity observed in HL and expands the spectrum of variants in deafness-associated genes.

Biallelic TANGO1 mutations cause a novel syndromal disease due to hampered cellular collagen secretion.

The transport and Golgi organization 1 (TANGO1) proteins play pivotal roles in the secretory pathway. Full length TANGO1 is a transmembrane protein localised at endoplasmic reticulum (ER) exit sites, where it binds bulky cargo within the ER lumen and recruits membranes from the ER Golgi intermediate compartment to create an exit route for their export. Here we report the first TANGO1-associated syndrome in humans. A synonymous substitution that results in exon eight skipping in most mRNA molecules, ultimately leading to a truncated TANGO1 protein was identified as disease-causing mutation. The four homozygously affected sons of a consanguineous family display severe dentinogenesis imperfecta, short stature, various skeletal abnormalities, insulin-dependent diabetes mellitus, sensorineural hearing loss, and mild intellectual disability. Functional studies in HeLa and U2OS cells revealed that the corresponding truncated TANGO1 protein is dispersed in the ER and its expression in cells with intact endogenous TANGO1 impairs cellular collagen I secretion.

Independent Origin of XY and ZW Sex Determination Mechanisms in Mosquitofish Sister Species.

Fish are known for the outstanding variety of their sex determination mechanisms and sex chromosome systems. The western (Gambusia affinis) and eastern mosquitofish (G. holbrooki) are sister species for which different sex determination mechanisms have been described: ZZ/ZW for G. affinis and XX/XY for G. holbrooki Here, we carried out restriction-site associated DNA (RAD-) and pool sequencing (Pool-seq) to characterize the sex chromosomes of both species. We found that the ZW chromosomes of G. affinis females and the XY chromosomes of G. holbrooki males correspond to different linkage groups, and thus evolved independently from separate autosomes. In interspecific hybrids, the Y chromosome is dominant over the W chromosome, and X is dominant over Z. In G. holbrooki, we identified a candidate region for the Y-linked melanic pigmentation locus, a rare male phenotype that constitutes a potentially sexually antagonistic trait and is associated with other such characteristics, e.g., large body size and aggressive behavior. We developed a SNP-based marker in the Y-linked allele of GIPC PDZ domain containing family member 1 (gipc1), which was linked to melanism in all tested G. holbrooki populations. This locus represents an example for a color locus that is located in close proximity to a putative sex determiner, and most likely substantially contributed to the evolution of the Y.

Genome-wide DNA methylation analysis of colorectal adenomas with and without recurrence reveals an association between cytosine-phosphate-guanine methylation and histological subtypes.

Aberrant methylation of DNA is supposed to be a major and early driver of colonic adenoma development, which may result in colorectal cancer (CRC). Although gene methylation assays are used already for CRC screening, differential epigenetic alterations of recurring and nonrecurring colorectal adenomas have yet not been systematically investigated. Here, we collected a sample set of formalin-fixed paraffin-embedded colorectal low-grade adenomas (n = 72) consisting of primary adenomas without and with recurrence (n = 59), recurrent adenomas (n = 10), and normal mucosa specimens (n = 3). We aimed to unveil differentially methylated CpG positions (DMPs) across the methylome comparing not only primary adenomas without recurrence vs primary adenomas with recurrence but also primary adenomas vs recurrent adenomas using the Illumina Human Methylation 450K BeadChip array. Unsupervised hierarchical clustering exhibited a significant association of methylation patterns with histological adenoma subtypes. No significant DMPs were identified comparing primary adenomas with and without recurrence. Despite that, a total of 5094 DMPs (false discovery rate <0.05; fold change >10%) were identified in the comparisons of recurrent adenomas vs primary adenomas with recurrence (674; 98% hypermethylated), recurrent adenomas vs primary adenomas with and without recurrence (241; 99% hypermethylated) and colorectal adenomas vs normal mucosa (4179; 46% hypermethylated). DMPs in cytosine-phosphate-guanine (CpG) islands were frequently hypermethylated, whereas open sea- and shelf-regions exhibited hypomethylation. Gene ontology analysis revealed enrichment of genes associated with the immune system, inflammatory processes, and cancer pathways. In conclusion, our methylation data could assist in establishing a more robust and reproducible histological adenoma classification, which is a prerequisite for improving surveillance guidelines.

Epigenetic signatures of Werner syndrome occur early in life and are distinct from normal epigenetic aging processes.

Werner Syndrome (WS) is an adult-onset segmental progeroid syndrome. Bisulfite pyrosequencing of repetitive DNA families revealed comparable blood DNA methylation levels between classical (18 WRN-mutant) or atypical WS (3 LMNA-mutant and 3 POLD1-mutant) patients and age- and sex-matched controls. WS was not associated with either age-related accelerated global losses of ALU, LINE1, and α-satellite DNA methylations or gains of rDNA methylation. Single CpG methylation was analyzed with Infinium MethylationEPIC arrays. In a correspondence analysis, atypical WS samples clustered together with the controls and were clearly separated from classical WS, consistent with distinct epigenetic pathologies. In classical WS, we identified 659 differentially methylated regions (DMRs) comprising 3,656 CpG sites and 613 RefSeq genes. The top DMR was located in the HOXA4 promoter. Additional DMR genes included LMNA, POLD1, and 132 genes which have been reported to be differentially expressed in WRN-mutant/depleted cells. DMRs were enriched in genes with molecular functions linked to transcription factor activity and sequence-specific DNA binding to promoters transcribed by RNA polymerase II. We propose that transcriptional misregulation of downstream genes by the absence of WRN protein contributes to the variable premature aging phenotypes of WS. There were no CpG sites showing significant differences in DNA methylation changes with age between WS patients and controls. Genes with both WS- and age-related methylation changes exhibited a constant offset of methylation between WRN-mutant patients and controls across the entire analyzed age range. WS-specific epigenetic signatures occur early in life and do not simply reflect an acceleration of normal epigenetic aging processes.

Unbalanced segregation of a paternal t(9;11)(p24.3;p15.4) translocation causing familial Beckwith-Wiedemann syndrome: a case report.

BACKGROUND: The vast majority of cases with Beckwith-Wiedemann syndrome (BWS) are caused by a molecular defect in the imprinted chromosome region 11p15.5. The underlying mechanisms include epimutations, uniparental disomy, copy number variations, and structural rearrangements. In addition, maternal loss-of-function mutations in CDKN1C are found. Despite growing knowledge on BWS pathogenesis, up to 20% of patients with BWS phenotype remain without molecular diagnosis. CASE PRESENTATION: Herein, we report an Iranian family with two females affected with BWS in different generations. Bisulfite pyrosequencing revealed hypermethylation of the H19/IGF2: intergenic differentially methylated region (IG DMR), also known as imprinting center 1 (IC1) and hypomethylation of the KCNQ1OT1: transcriptional start site (TSS) DMR (IC2). Array CGH demonstrated an 8 Mb duplication on chromosome 11p15.5p15.4 (205,827-8,150,933) and a 1 Mb deletion on chromosome 9p24.3 (209,020-1,288,114). Chromosome painting revealed that this duplication-deficiency in both patients is due to unbalanced segregation of a paternal reciprocal t(9;11)(p24.3;p15.4) translocation. CONCLUSIONS: This is the first report of a paternally inherited unbalanced translocation between the chromosome 9 and 11 short arms underlying familial BWS. Copy number variations involving the 11p15.5 region are detected by the consensus diagnostic algorithm. However, in complex cases which do not only affect the BWS region itself, characterization of submicroscopic chromosome rearrangements can assist to estimate the recurrence risk and possible phenotypic outcomes.

Targeted Molecular Analysis in Adrenocortical Carcinomas: A Strategy Toward Improved Personalized Prognostication.

Context: Adrenocortical carcinoma (ACC) has a heterogeneous prognosis, and current medical therapies have limited efficacy in its advanced stages. Genome-wide multiomics studies identified molecular patterns associated with clinical outcome. Objective: Here, we aimed at identifying a molecular signature useful for both personalized prognostic stratification and druggable targets, using methods applicable in clinical routine. Design: In total, 117 tumor samples from 107 patients with ACC were analyzed. Targeted next-generation sequencing of 160 genes and pyrosequencing of 4 genes were applied to formalin-fixed, paraffin-embedded (FFPE) specimens to detect point mutations, copy number alterations, and promoter region methylation. Molecular results were combined with clinical/histopathological parameters (tumor stage, age, symptoms, resection status, and Ki-67) to predict progression-free survival (PFS). Results: In addition to known driver mutations, we detected recurrent alterations in genes not previously associated with ACC (e.g., NOTCH1, CIC, KDM6A, BRCA1, BRCA2). Best prediction of PFS was obtained integrating molecular results (more than one somatic mutation, alterations in Wnt/ß-catenin and p53 pathways, high methylation pattern) and clinical/histopathological parameters into a combined score (P < 0.0001, χ2 = 68.6). Accuracy of prediction for early disease progress was 83.3% (area under the receiver operating characteristic curve: 0.872, 95% confidence interval 0.80 to 0.94). Furthermore, 17 potentially targetable alterations were found in 64 patients (e.g., in CDK4, NOTCH1, NF1, MDM2, and EGFR and in DNA repair system). Conclusions: This study demonstrates that molecular profiling of FFPE tumor samples improves prognostication of ACC beyond clinical/histopathological parameters and identifies new potential drug targets. These findings pave the way to precision medicine in this rare disease.

Genetic and epigenetic changes in clonal descendants of irradiated human fibroblasts.

To study delayed genetic and epigenetic radiation effects, which may trigger radiation-induced carcinogenesis, we have established single-cell clones from irradiated and non-irradiated primary human fibroblasts. Stable clones were endowed with the same karyotype in all analyzed metaphases after 20 population doublings (PDs), whereas unstable clones displayed mosaics of normal and abnormal karyotypes. To account for variation in radiation sensitivity, all experiments were performed with two different fibroblast strains. After a single X-ray dose of 2 Gy more than half of the irradiated clones exhibited radiation-induced genome instability (RIGI). Irradiated clones displayed an increased rate of loss of chromosome Y (LOY) and copy number variations (CNVs), compared to controls. CNV breakpoints clustered in specific chromosome regions, in particular 3p14.2 and 7q11.21, coinciding with common fragile sites. CNVs affecting the FHIT gene in FRA3B were observed in independent unstable clones and may drive RIGI. Bisulfite pyrosequencing of control clones and the respective primary culture revealed global hypomethylation of ALU, LINE-1, and alpha-satellite repeats as well as rDNA hypermethylation during in vitro ageing. Irradiated clones showed further reduced ALU and alpha-satellite methylation and increased rDNA methylation, compared to controls. Methylation arrays identified several hundred differentially methylated genes and several enriched pathways associated with in vitro ageing. Methylation changes in 259 genes and the MAP kinase signaling pathway were associated with delayed radiation effects (after 20 PDs). Collectively, our results suggest that both genetic (LOY and CNVs) and epigenetic changes occur in the progeny of exposed cells that were not damaged directly by irradiation, likely contributing to radiation-induced carcinogenesis. We did not observe epigenetic differences between stable and unstable irradiated clones. The fact that the DNA methylation (DNAm) age of clones derived from the same primary culture varied greatly suggests that DNAm age of a single cell (represented by a clone) can be quite different from the DNAm age of a tissue. We propose that DNAm age reflects the emergent property of a large number of individual cells whose respective DNAm ages can be highly variable.

Hereditary hearing loss SNP-microarray pilot study.

OBJECTIVES: Despite recent advancements in diagnostic tools, the genomic landscape of hereditary hearing loss remains largely uncharacterized. One strategy to understand genome-wide aberrations includes the analysis of copy number variation that can be mapped using SNP-microarray technology. A growing collection of literature has begun to uncover the importance of copy number variation in hereditary hearing loss. This pilot study underpins a larger effort that involves the stage-wise analysis of hearing loss patients, many of whom have advanced to high-throughput sequencing analysis. DATA DESCRIPTION: Our data originate from the Infinium HumanOmni1-Quad v1.0 SNP-microarrays (Illumina) that provide useful markers for genome-wide association studies and copy number variation analysis. This dataset comprises a cohort of 108 individuals (99 with hearing loss, 9 normal hearing family members) for the purpose of understanding the genetic contribution of copy number variations to hereditary hearing loss. These anonymized SNP-microarray data have been uploaded to the NCBI Gene Expression Omnibus and are intended to benefit other investigators interested in aggregating platform-matched array patient datasets or as part of a supporting reference tool for other laboratories to better understand recurring copy number variations in other genetic disorders.

Chromosome Banding in Amphibia. XXXV. Highly Mobile Nucleolus Organizing Regions in Craugastor fitzingeri (Anura, Craugastoridae).

A 7-year cytogenetic study on the leaf litter frog Craugastor fitzingeri from Costa Rica and Panama revealed the existence of highly mobile nucleolus organizing regions (NORs) in their genomes. Silver (Ag)-staining of the active NORs demonstrated an exceptional interindividual pattern of NOR distribution at the telomeres of the chromosomes. All individuals examined showed a different and specific NOR location in their karyotypes. Furthermore, intraindividual variation in the NOR sites was found. This observation suggested the existence of mobile NORs in C. fitzingeri. Confirmation of this phenomenon was possible by systematic FISH analysis using an 18S + 28S rDNA probe. The extremely variable number and position of the NORs in C. fitzingeri is best explained by highly mobile NORs that move freely between the telomeres of the chromosomes. These transpositions must occur preferentially in premeiotic, meiotic, or postmeiotic stages, but also at a lower incidence in the somatic tissues of the animals. It is hypothesized that transposable (mobile) elements are closely linked to the NORs or are inserted into the major 18S + 28S rDNA spacers of C. fitzingeri. When such transposable elements spread by transpositions, they can carry with them complete or partial NORs. The present study provides detailed information on various differential chromosome banding techniques, in situ hybridization experiments, chromosomal hypermethylation patterns, determination of the genome size, and analyses of restriction fragment length polymorphisms of the DNA.

Analysis of global DNA methylation changes in primary human fibroblasts in the early phase following X-ray irradiation.

Epigenetic alterations may contribute to the generation of cancer cells in a multi-step process of tumorigenesis following irradiation of normal body cells. Primary human fibroblasts with intact cell cycle checkpoints were used as a model to test whether X-ray irradiation with 2 and 4 Gray induces direct epigenetic effects (within the first cell cycle) in the exposed cells. ELISA-based fluorometric assays were consistent with slightly reduced global DNA methylation and hydroxymethylation, however the observed between-group differences were usually not significant. Similarly, bisulfite pyrosequencing of interspersed LINE-1 repeats and centromeric α-satellite DNA did not detect significant methylation differences between irradiated and non-irradiated cultures. Methylation of interspersed ALU repeats appeared to be slightly increased (one percentage point; p = 0.01) at 6 h after irradiation with 4 Gy. Single-cell analysis showed comparable variations in repeat methylation among individual cells in both irradiated and control cultures. Radiation-induced changes in global repeat methylation, if any, were much smaller than methylation variation between different fibroblast strains. Interestingly, α-satellite DNA methylation positively correlated with gestational age. Finally, 450K methylation arrays mainly targeting genes and CpG islands were used for global DNA methylation analysis. There were no detectable methylation differences in genic (promoter, 5' UTR, first exon, gene body, 3' UTR) and intergenic regions between irradiated and control fibroblast cultures. Although we cannot exclude minor effects, i.e. on individual CpG sites, collectively our data suggest that global DNA methylation remains rather stable in irradiated normal body cells in the early phase of DNA damage response.

Genetics of Tinnitus: Still in its Infancy.

Tinnitus is the perception of a phantom sound that affects between 10 and 15% of the general population. Despite this considerable prevalence, treatments for tinnitus are presently lacking. Tinnitus exhibits a diverse array of recognized risk factors and extreme clinical heterogeneity. Furthermore, it can involve an unknown number of auditory and non-auditory networks and molecular pathways. This complex combination has hampered advancements in the field. The identification of specific genetic factors has been at the forefront of several research investigations in the past decade. Nine studies have examined genes in a case-control association approach. Recently, a genome-wide association study has highlighted several potentially significant pathways that are implicated in tinnitus. Two twin studies have calculated a moderate heritability for tinnitus and disclosed a greater concordance rate in monozygotic twins compared to dizygotic twins. Despite the more recent data alluding to genetic factors in tinnitus, a strong association with any specific genetic locus is lacking and a genetic study with sufficient statistical power has yet to be designed. Future research endeavors must overcome the many inherent limitations in previous study designs. This review summarizes the previously embarked upon tinnitus genetic investigations and summarizes the hurdles that have been encountered. The identification of candidate genes responsible for tinnitus may afford gene based diagnostic approaches, effective therapy development, and personalized therapeutic intervention.