Nadine Hornig

The regulated retrotransposon transcriptome of mammalian cells

Although repetitive elements pervade mammalian genomes, their overall contribution to transcriptional activity is poorly defined. Here, as part of the FANTOM4 project, we report that 6–30% of cap-selected mouse and human RNA transcripts initiate within repetitive elements. Analysis of approximately 250,000 retrotransposon-derived transcription start sites shows that the associated transcripts are generally tissue specific, coincide with gene-dense regions and form pronounced clusters when aligned to full-length retrotransposon sequences. Retrotransposons located immediately 5′ of protein-coding loci frequently function as alternative promoters and/or express noncoding RNAs. More than a quarter of RefSeqs possess a retrotransposon in their 3′ UTR, with strong evidence for the reduced expression of these transcripts relative to retrotransposon-free transcripts. Finally, a genome-wide screen identifies 23,000 candidate regulatory regions derived from retrotransposons, in addition to more than 2,000 examples of bidirectional transcription. We conclude that retrotransposon transcription has a key influence upon the transcriptional output of the mammalian genome.

Recurrent mutation of the ID3 gene in Burkitt lymphoma identified by integrated genome, exome and transcriptome sequencing

Burkitt lymphoma is a mature aggressive B-cell lymphoma derived from germinal center B cells. Its cytogenetic hallmark is the Burkitt translocation t(8;14)(q24;q32) and its variants, which juxtapose the MYC oncogene with one of the three immunoglobulin loci. Consequently, MYC is deregulated, resulting in massive perturbation of gene expression. Nevertheless, MYC deregulation alone seems not to be sufficient to drive Burkitt lymphomagenesis. By whole-genome, whole-exome and transcriptome sequencing of four prototypical Burkitt lymphomas with immunoglobulin gene (IG)-MYC translocation, we identified seven recurrently mutated genes. One of these genes, ID3, mapped to a region of focal homozygous loss in Burkitt lymphoma. In an extended cohort, 36 of 53 molecularly defined Burkitt lymphomas (68%) carried potentially damaging mutations of ID3. These were strongly enriched at somatic hypermutation motifs. Only 6 of 47 other B-cell lymphomas with the IG-MYC translocation (13%) carried ID3 mutations. These findings suggest that cooperation between ID3 inactivation and IG-MYC translocation is a hallmark of Burkitt lymphomagenesis.

Linking promoters to functional transcripts in small samples with nanoCAGE and CAGEscan

Large-scale sequencing projects have revealed an unexpected complexity in the origins, structures and functions of mammalian transcripts. Many loci are known to produce overlapping coding and noncoding RNAs with capped 5′ ends that vary in size. Methods to identify the 5′ ends of transcripts will facilitate the discovery of new promoters and 5′ ends derived from secondary capping events. Such methods often require high input amounts of RNA not obtainable from highly refined samples such as tissue microdissections and subcellular fractions. Therefore, we developed nano–cap analysis of gene expression (nanoCAGE), a method that captures the 5′ ends of transcripts from as little as 10 ng of total RNA, and CAGEscan, a mate-pair adaptation of nanoCAGE that captures the transcript 5′ ends linked to a downstream region. Both of these methods allow further annotation-agnostic studies of the complex human transcriptome.

Enhancer of zeste homolog 2 overexpression has a role in the development of anaplastic thyroid carcinomas.

CONTEXT Enhancer of zeste homolog 2 (EZH2) is a histone lysine methyltransferase belonging to the polycomb group protein family. Overexpression of EZH2 has been found in several human malignancies including hematological and solid tumors. OBJECTIVES In this study we investigated the expression levels of EZH2 and its polycomb group protein partners in thyroid carcinoma tissues with different degrees of malignancy to identify potential new therapeutic targets for anaplastic thyroid carcinoma (ATC). RESULTS We show that high EZH2 expression levels are characteristic of undifferentiated ATC, whereas no significant changes were observed in well-differentiated papillary and follicular thyroid carcinomas as compared with normal thyroid. Knockdown of EZH2 in ATC cell lines results in cell growth inhibition, loss of anchorage-independent growth, migration, and invasion properties. Moreover, we demonstrate that EZH2 directly controls differentiation of ATC cells by silencing the thyroid specific transcription factor paired-box gene 8 (PAX8). CONCLUSIONS EZH2 is specifically overexpressed in ATC, and it directly contributes to transcriptional silencing of PAX8 gene and ATC differentiation.

A Recurrent Germline Mutation in the 5'UTR of the Androgen Receptor Causes Complete Androgen Insensitivity by Activating Aberrant uORF Translation.

A subset of patients with monogenic disorders lacks disease causing mutations in the protein coding region of the corresponding gene. Here we describe a recurrent germline mutation found in two unrelated patients with complete androgen insensitivity syndrome (CAIS) generating an upstream open reading frame (uORF) in the 5’ untranslated region (5’-UTR) of the androgen receptor (AR) gene. We show in patient derived primary genital skin fibroblasts as well as in cell-based reporter assays that this mutation severely impacts AR function by reducing AR protein levels without affecting AR mRNA levels. Importantly, the newly generated uORF translates into a polypeptide and the expression level of this polypeptide inversely correlates with protein translation from the primary ORF of the AR thereby providing a model for AR-5′UTR mediated translational repression. Our findings not only add a hitherto unrecognized genetic cause to complete androgen insensitivity but also underline the importance of 5′UTR mutations affecting uORFs for the pathogenesis of monogenic disorders in general.

Determination of 17OHPreg and DHEAS by LCMSMS: Impact of Age, Sex, Pubertal Stage and BMI on the Δ5-steroid-pathway

Background Dehydroepiandrosterone sulfate (DHEAS) and 17-hydroxypregnenolone (17OHPreg) are important for understanding the Δ5 pathway (e.g., in adrenarche and obesity). Although mass spectrometry has become the state-of-the-art method for quantifying steroids, there are few comprehensive age-, sex-, and pubertal stage-specific reference ranges for children. Aims To develop a sensitive and reliable ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for simultaneous quantification of DHEAS and 17OHPreg and to establish entire age-, sex- and pubertal stage-specific reference ranges in children. Methods A total of 684 children, 453 (243 female, 210 male) with normal body mass index (BMI; <90th) and 231 (132 female, 99 male) obese subjects (>97th), were categorized into 11 age groups, and age- and Tanner stage (PH)-specific reference ranges were determined. Results The limit of detection was 0.05 nmol/L for 17OHPreg and 0.5 nmol/L for DHEAS. Levels of both steroids declined after the neonatal period. Comparisons with RIA assays (Siemens, Munich, Germany) (DHEAS) and an in-house kit (17OHPreg) revealed 0.95 and 0.93, respectively, as coefficients of determination. Although DHEAS-generally higher in boys-increased continuously starting at 3 to 6 years, 17OHPreg remained largely constant. In obese patients, both were significantly elevated, also in part after alignment to Tanner stages (PH). Conclusions UPLC-MS/MS is sensitive and reliable for quantifying DHEAS and 17OHPreg. Our data support differential maturation of CYP17 during adrenarche with successively increasing 17,20-lyase activity but largely constant 17α-hydroxylation activity. Endocrine interpretation of 17OHPreg and DHEAS must consider differential patterns for age, sex, pubertal stage, and BMI.

Epigenetic Repression of Androgen Receptor Transcription in Mutation-Negative Androgen Insensitivity Syndrome (AIS Type II)

Context Inactivating mutations within the AR gene are present in only ~40% of individuals with clinically and hormonally diagnosed androgen insensitivity syndrome (AIS). Previous studies revealed the existence of an AR gene mutation-negative group of patients with AIS who have compromised androgen receptor (AR) function (AIS type II). Objective To investigate whether AIS type II can be due to epigenetic repression of AR transcription. Design Quantification of AR mRNA and AR proximal promoter CpG methylation levels in genital skin-derived fibroblasts (GFs) derived from patients with AIS type II and control individuals. Setting University hospital endocrine research laboratory. Patients GFs from control individuals (n = 11) and patients with AIS type II (n = 14). Main Outcome Measure(s) Measurement of AR mRNA and AR promoter CpG methylation as well as activity of AR proximal promoter in vitro. Results Fifty-seven percent of individuals with AIS type II (n = 8) showed a reduced AR mRNA expression in their GFs. A significant inverse correlation was shown between AR mRNA abundance and methylation at two consecutive CpGs within the proximal AR promoter. Methylation of a 158-bp-long region containing these CpGs was sufficient to severely reduce reporter gene expression. This region was bound by the runt related transcription factor 1 (RUNX1). Ectopic expression of RUNX1 in HEK293T cells was able to inhibit reporter gene expression through this region. Conclusions Aberrant CpGs methylation within the proximal AR promoter plays an important role in the control of AR gene expression and may result in AIS type II. We suggest that transcriptional modifiers, such as RUNX1, could play roles therein offering new perspectives for understanding androgen-mediated endocrine diseases.

Multiples of Median-Transformed, Normalized Reference Ranges of Steroid Profiling Data Independent of Age, Sex, and Units

Background/Aims: The high complexity of pediatric reference ranges across age, sex, and units impairs clinical application and comparability of steroid hormone data, e.g., in congenital adrenal hyperplasia (CAH). We developed a multiples-of-median (MoM) normalization tool to overcome this major drawback in pediatric endocrinology. Methods: Liquid chromatography tandem mass spectrometry data comprising 10 steroid hormones representing 905 controls (555 males, 350 females, 0 to > 16 years) from 2 previous datasets were MoM transformed across age and sex. Twenty-three genetically proven CAH patients were included (21-hydroxylase deficiency [21OHD], n = 19; 11β-hydroxylase deficiency [11OHD], n = 4). MoM cutoffs for single steroids predicting 21OHD and 11OHD were computed and validated through new, independent patients (21OHD, n = 8; adrenal cortical carcinoma, n = 6; obesity, n = 40). Results: 21OHD and 11OHD patients showed disease-typical, easily recognizable MoM patterns independent of age, sex, and concentration units. Two single-steroid cutoffs indicated 21OHD: 3.87 MoM for 17-hydroxyprogesterone (100% sensitivity and 98.83% specificity) and 12.28 MoM for 21-deoxycortisol (94.74% sensitivity and 100% specificity). A cutoff of 13.18 MoM for 11-deoxycortisol indicated 11OHD (100% sensitivity and 100% specificity). Conclusions: Age- and sex-independent MoMs are straightforward for a clinically relevant display of multi-steroid patterns. In addition, defined single-steroid MoMs can serve alone as predictors of 21OHD and 11OHD. Finally, MoM transformation offers substantial enhancement of routine and scientific steroid hormone data exchange due to improved comparability.

Next generation sequencing and functional characterization of the androgen receptor (AR) gene in patients with androgen insensitivity syndrome (AIS) and controls

Androgen insensitivity syndrome (AIS) is characterized by a partial to complete lack of genital virilization in genetically male individuals. It is classically caused by inactivating mutations in the coding region of the X-chromosomal androgen receptor (AR) gene. However, up to two-third of the patients with a clinically established diagnosis of AIS lack a detectable molecular cause to date. As conventional sequencing is restricted to the AR coding exons, we set up a next generation sequencing (NGS) approach of the entire AR-gene locus for a comprehensive AR mutation analysis in patients with AIS. To this purpose, DNA was extracted from cultured genital skin fibroblasts (GSF = scrotum, foreskin, labia) of 80 patients with known and presumed AIS, two patients with 17sHSDIII deficiency, six patients with 5alpha-reductase deficiency and 15 control males. Patients were suspected to have AIS based on clinical findings, pathological androgen binding, reduced AR expression in GSF or a combination of these. The AR-sequencing library was produced using a capture-based method (Haloplex; Agilent). The target sequence included the coding region, the UTRs, 90% of the intron sequences as well as a 9kb upstream and 5kb downstream sequence. Sequencing was performed on a Miseq benchtop sequencer (Illumina). Alignment to the hg19 reference genome and single nucleotide polymorphism (SNP) calling was performed by the MiSeq-Reporter software (Illumina). Targeted NGS confirmed AR mutations in all patients with mutations previously identified by Sanger sequencing. Additional SNPs were detected in and outside the AR-coding region. In order to understand the functional impact of the SNPs, they were further tested for the ability of the endogenous AR to induce transcription of the AR target gene Apolipoprotein D (APOD-Assay). In conclusion we show that NGS is a valid method for AR-sequencing in presumed AIS and allows in combination with the APOD-Assay a refined classification of AIS.

Hormonal Control of Fetal Sex Development

Sexual differentiation of genital anatomy in the male occurs under strict hormonal control of the fetal testes and is confined to a developmental time window between the 8th and 12th week of gestation. Wolffian ducts will develop into epididymis, vasa deferentia and prostatic urethra in response to paracrine actions of testosterone produced by the Leydig cells, while Mullerian ducts will perish due to Anti-Mullerian-Hormone (AMH) produced by the Sertoli cells. External genital virilisation is characterized by fusion of the genital midline and growth of the genital tubercle involving local intracellular biosynthesis of dihydrotestosterone from testosterone, forming scrotum and penis respectively. Androgens transduce their developmental signals via the intracellular androgen receptor which is expressed in the fetal genitalia. However, the early fetal molecular androgen response programs, that is, the genes and transcripts involved, are still unknown. There is evidence of long-term programming of functional reproductive properties during this time window which may be reflected by persistent changes in the transcriptome and the methylome as documented in postnatal human genital tissues. The absence of testosterone and AMH is sufficient for anatomically female genital development in the fetus.