Renal microRNA- and RNA-profiles in progressive chronic kidney disease.

BACKGROUND: MicroRNAs (miRNAs) contribute to chronic kidney disease (CKD) progression via regulating mRNAs involved in renal homeostasis. However, their association with clinical outcome remains poorly understood. MATERIALS AND METHODS: We performed miRNA and mRNA expression profiling on renal biopsy sections by qPCR (miRNA) and microarrays (mRNA) in a discovery (n = 43) and in a validation (n = 29) cohort. miRNAs differentiating stable and progressive cases were inversely correlated with putative target mRNAs, which were further characterized by pathway analysis using KEGG pathways. RESULTS: miR-30d, miR-140-3p, miR-532-3p, miR-194, miR-190, miR-204 and miR-206 were downregulated in progressive cases. These seven miRNAs correlated with upregulated 29 target mRNAs involved in inflammatory response, cell-cell interaction, apoptosis and intra-cellular signalling. In particular, miR-206 and miR-532-3p were associated with distinct biological processes via the expression of their target mRNAs: Reduced expression of miR-206 in progressive disease correlated with the upregulation of target mRNAs participating in inflammatory pathways (CCL19, CXCL1, IFNAR2, NCK2, PTK2B, PTPRC, RASGRP1 and TNFRSF25). Progressive cases also showed a lower expression of miR-532-3p and an increased expression of target transcripts involved in apoptosis pathways (MAP3K14, TNFRSF10B/TRAIL-R2, TRADD and TRAF2). In the validation cohort, we confirmed the decreased expression of miR-206 and miR-532-3p, and the inverse correlation of these miRNAs with the expression of nine of the 12 target genes. The levels of the identified miRNAs and the target mRNAs correlated with clinical parameters and histological damage indices. CONCLUSIONS: These results suggest the involvement of specific miRNAs and mRNAs in biological pathways associated with the progression of CKD.

Structural and numerical changes of chromosome X in patients with esophageal atresia.

Esophageal atresia with or without tracheoesophageal fistula (EA/TEF) is a relatively common birth defect often associated with additional congenital anomalies such as vertebral, anal, cardiovascular, renal and limb defects, the so-called VACTERL association. Yet, little is known about the causal genetic factors. Rare case reports of gastrointestinal anomalies in children with triple X syndrome prompted us to survey the incidence of structural and numerical changes of chromosome X in patients with EA/TEF. All available (n=269) karyotypes of our large (321) EA/TEF patient cohort were evaluated for X-chromosome anomalies. If sufficient DNA material was available, we determined genome-wide copy number profiles with SNP array and identified subtelomeric aberrations on the difficult to profile PAR1 region using telomere-multiplex ligation-dependent probe amplification. In addition, we investigated X-chromosome inactivation (XCI) patterns and mode of inheritance of detected aberrations in selected patients. Three EA/TEF patients had an additional maternally inherited X chromosome. These three female patients had normal random XCI patterns. Two male EA/TEF patients had small inherited duplications of the XY-linked SHOX (Short stature HOmeoboX-containing) locus. Patients were small for gestational age at birth (

Microhomology-mediated mechanisms underlie non-recurrent disease-causing microdeletions of the FOXL2 gene or its regulatory domain.

Genomic disorders are often caused by recurrent copy number variations (CNVs), with nonallelic homologous recombination (NAHR) as the underlying mechanism. Recently, several microhomology-mediated repair mechanisms--such as microhomology-mediated end-joining (MMEJ), fork stalling and template switching (FoSTeS), microhomology-mediated break-induced replication (MMBIR), serial replication slippage (SRS), and break-induced SRS (BISRS)--were described in the etiology of non-recurrent CNVs in human disease. In addition, their formation may be stimulated by genomic architectural features. It is, however, largely unexplored to what extent these mechanisms contribute to rare, locus-specific pathogenic CNVs. Here, fine-mapping of 42 microdeletions of the FOXL2 locus, encompassing FOXL2 (32) or its regulatory domain (10), serves as a model for rare, locus-specific CNVs implicated in genetic disease. These deletions lead to blepharophimosis syndrome (BPES), a developmental condition affecting the eyelids and the ovary. For breakpoint mapping we used targeted array-based comparative genomic hybridization (aCGH), quantitative PCR (qPCR), long-range PCR, and Sanger sequencing of the junction products. Microhomology, ranging from 1 bp to 66 bp, was found in 91.7% of 24 characterized breakpoint junctions, being significantly enriched in comparison with a random control sample. Our results show that microhomology-mediated repair mechanisms underlie at least 50% of these microdeletions. Moreover, genomic architectural features, like sequence motifs, non-B DNA conformations, and repetitive elements, were found in all breakpoint regions. In conclusion, the majority of these microdeletions result from microhomology-mediated mechanisms like MMEJ, FoSTeS, MMBIR, SRS, or BISRS. Moreover, we hypothesize that the genomic architecture might drive their formation by increasing the susceptibility for DNA breakage or promote replication fork stalling. Finally, our locus-centered study, elucidating the etiology of a large set of rare microdeletions involved in a monogenic disorder, can serve as a model for other clustered, non-recurrent microdeletions in genetic disease.

miRNA expression profiling: from reference genes to global mean normalization.

MicroRNAs (miRNAs) are an important class of gene regulators, acting on several aspects of cellular function such as differentiation, cell cycle control, and stemness. These master regulators constitute an invaluable source of biomarkers, and several miRNA signatures correlating with patient diagnosis, prognosis, and response to treatment have been identified. Within this exciting field of research, whole-genome RT-qPCR-based miRNA profiling in combination with a global mean normalization strategy has proven to be the most sensitive and accurate approach for high-throughput miRNA profiling (Mestdagh et al., Genome Biol 10:R64, 2009). In this chapter, we summarize the power of the previously described global mean normalization method in comparison to the multiple reference gene normalization method using the most stably expressed small RNA controls. In addition, we compare the original global mean method to a modified global mean normalization strategy based on the attribution of equal weight to each individual miRNA during normalization. This modified algorithm is implemented in Biogazelle's qbasePLUS software and is presented here for the first time.

Expanding the spectrum of FOXC1 and PITX2 mutations and copy number changes in patients with anterior segment malformations.

PURPOSE: Anterior segment dysgenesis (ASD) comprises a heterogeneous group of developmental abnormalities that affect several structures of the anterior segment of the eye. The main purpose of this study was to assess the proportion of FOXC1 and PITX2 mutations and copy number changes in 80 probands with ASD. METHODS: The patients were examined for FOXC1 and PITX2 copy number changes and mutations using MLPA (multiplex ligation-dependent probe amplification) and direct sequencing. Subsequently, the identified copy number changes were fine-mapped using high-resolution microarrays. In the remaining mutation-negative patients, sequencing of the FOXC1 andPITX2 3' untranslated regions (UTRs) and three other candidate genes (P32, PDP2, and FOXC2) was performed. RESULTS: Thirteen FOXC1 and eight PITX2 mutations were identified, accounting for 26% (21/80) of the cases. In addition, six FOXC1 and five PITX2 deletions were found, explaining 14% (11/80) of the cases. The smallest FOXC1 and PITX2 deletions were 5.4 and 1.6 kb in size, respectively. Six patients carrying FOXC1 deletions presented with variable extraocular phenotypic features such as hearing defects (in 4/6) and mental retardation (in 2/6). No further genetic defects were found in the remaining mutation-negative patients. CONCLUSIONS: FOXC1 and PITX2 genetic defects explain 40% of our large ASD cohort. The current spectrum of intragenic FOXC1 and PITX2 mutations was extended considerably, the identified copy number changes were fine mapped, the smallest FOXC1 and PITX2 deletions reported so far were identified, and the need for dedicated copy number screening of the FOXC1 and PITX2 genomic landscape was emphasized. This study is unique in that sequence and copy number changes were screened simultaneously in both genes.

Improved molecular diagnostics of idiopathic short stature and allied disorders: quantitative polymerase chain reaction-based copy number profiling of SHOX and pseudoautosomal region 1.

CONTEXT: Short stature has an incidence of three in 100 in children. Reliable molecular genetic testing may be crucial in the context of beneficial disease management. Deletions spanning or surrounding the SHOX gene account for a significant proportion of patients with idiopathic short stature (ISS) and allied disorders, such as Leri-Weill dyschondrosteosis. OBJECTIVE: Several shortcomings of current strategies for copy number profiling of the SHOX region prompted us to develop an improved test for molecular diagnostics of the SHOX region. DESIGN AND RESULTS: We introduced a quantitative PCR (qPCR)-based copy number profiling test, consisting of 11 amplicons targeting clinically relevant regions, i.e. the SHOX gene and regulatory regions. To ensure an optimal sensitivity and specificity, this test was validated in 32 controls and 18 probands with previously identified copy number changes. In addition, 152 probands with SHOX-associated phenotypes were screened, revealing 10 novel copy number changes. CONCLUSION: This highly validated qPCR test supersedes other approaches for copy number screening of the SHOX region in terms of reliability, accuracy, and cost efficiency. In addition, another strong point is the fact that it can be easily implemented in any standard equipped molecular laboratory. Our qPCR-based test is highly recommended for molecular diagnostics of idiopathic short stature and allied disorders.

Functional exploration of the adult ovarian granulosa cell tumor-associated somatic FOXL2 mutation p.Cys134Trp (c.402C>G).

BACKGROUND: The somatic mutation in the FOXL2 gene c.402C>G (p.Cys134Trp) has recently been identified in the vast majority of adult ovarian granulosa cell tumors (OGCTs) studied. In addition, this mutation seems to be specific to adult OGCTs and is likely to be a driver of malignant transformation. However, its pathogenic mechanisms remain elusive. METHODOLOGY/PRINCIPAL FINDINGS: We have sequenced the FOXL2 open reading frame in a panel of tumor cell lines (NCI-60, colorectal carcinoma cell lines, JEG-3, and KGN cells). We found the FOXL2 c.402C>G mutation in the adult OGCT-derived KGN cell line. All other cell lines analyzed were negative for the mutation. In order to gain insights into the pathogenic mechanism of the p.Cys134Trp mutation, the subcellular localization and mobility of the mutant protein were studied and found to be no different from those of the wild type (WT). Furthermore, its transactivation ability was in most cases similar to that of the WT protein, including in conditions of oxidative stress. A notable exception was an artificial promoter known to be coregulated by FOXL2 and Smad3, suggesting a potential modification of their interaction. We generated a 3D structural model of the p.Cys134Trp variant and our analysis suggests that homodimer formation might also be disturbed by the mutation. CONCLUSIONS/SIGNIFICANCE: Here, we confirm the specificity of the FOXL2 c.402C>G mutation in adult OGCTs and begin the exploration of its molecular significance. This is the first study demonstrating that the p.Cys134Trp mutant does not have a strong impact on FOXL2 localization, solubility, and transactivation abilities on a panel of proven target promoters, behaving neither as a dominant-negative nor as a loss-of-function mutation. Further studies are required to understand the specific molecular effects of this outstanding FOXL2 mutation.

Accurate and objective copy number profiling using real-time quantitative PCR.

Copy number changes are known to be involved in numerous human genetic disorders. In this context, qPCR-based copy number screening may serve as the method of choice for targeted screening of the relevant disease genes and their surrounding regulatory landscapes. qPCR has many advantages over alternative methods, such as its low consumable and instrumentation costs, fast turnaround and assay development time, high sensitivity and open format (independent of a single supplier). In this chapter we provide all relevant information for a successfully implement of qPCR-based copy number analysis. We emphasize the significance of thorough in silico and empirical validation of the primers, the need for a well thought-out experiment design, and the importance of quality controls along the entire workflow. Furthermore, we suggest an appropriate and practical way to calculate copy numbers and to objectively interpret the results. The provided guidelines will most certainly improve the quality and reliability of your qPCR-based copy number screening.

Disease-causing 7.4 kb cis-regulatory deletion disrupting conserved non-coding sequences and their interaction with the FOXL2 promotor: implications for mutation screening.

To date, the contribution of disrupted potentially cis-regulatory conserved non-coding sequences (CNCs) to human disease is most likely underestimated, as no systematic screens for putative deleterious variations in CNCs have been conducted. As a model for monogenic disease we studied the involvement of genetic changes of CNCs in the cis-regulatory domain of FOXL2 in blepharophimosis syndrome (BPES). Fifty-seven molecularly unsolved BPES patients underwent high-resolution copy number screening and targeted sequencing of CNCs. Apart from three larger distant deletions, a de novo deletion as small as 7.4 kb was found at 283 kb 5' to FOXL2. The deletion appeared to be triggered by an H-DNA-induced double-stranded break (DSB). In addition, it disrupts a novel long non-coding RNA (ncRNA) PISRT1 and 8 CNCs. The regulatory potential of the deleted CNCs was substantiated by in vitro luciferase assays. Interestingly, Chromosome Conformation Capture (3C) of a 625 kb region surrounding FOXL2 in expressing cellular systems revealed physical interactions of three upstream fragments and the FOXL2 core promoter. Importantly, one of these contains the 7.4 kb deleted fragment. Overall, this study revealed the smallest distant deletion causing monogenic disease and impacts upon the concept of mutation screening in human disease and developmental disorders in particular.

Identification of copy number variants associated with BPES-like phenotypes.

Blepharophimosis-Ptosis-Epicanthus inversus syndrome (BPES) is a well-characterized rare syndrome that includes an eyelid malformation associated with (type I) or without premature ovarian failure (type II). Patients with typical BPES have four major characteristics: blepharophimosis, ptosis, epicanthus inversus and telecanthus. Mutations in the FOXL2 gene, encoding a forkhead transcription factor, are responsible for the majority of both types of BPES. However, many patients with BPES-like features, i.e., having at least two major characteristics of BPES, have an unidentified cause. Here, we report on a group of 27 patients with BPES-like features, but without an identified genetic defect in the FOXL2 gene or flanking region. These patients were analyzed with whole-genome high-density arrays in order to identify copy number variants (CNVs) that might explain the BPES-like phenotype. In nine out of 27 patients (33%) CNVs not previously described as polymorphisms were detected. Four of these patients displayed psychomotor retardation as an additional clinical characteristic. In conclusion, we demonstrate that BPES-like phenotypes are frequently caused by CNVs, and we emphasize the importance of whole-genome copy number screening to identify the underlying genetic causes of these phenotypes.