Cardiac defects are infrequent findings in individuals with 8p23.1 genomic duplications containing GATA4.

BACKGROUND: The GATA4 gene is critical to regulating myocardial differentiation and function. Haploinsufficiency of GATA4 is strongly associated with congenital heart defects (CHD). However, it is inconclusive whether duplicated GATA4 causes CHD. METHODS AND RESULTS: We evaluated 1645 consecutive pediatric patients with various developmental disorders by high-resolution microarray-based comparative genomic hybridization and found 8 probands and 2 relatives with pathogenic genomic imbalances containing GATA4. Four probands contain an ≈4.0-Mb interstitial duplication of 8p23.1 flanked by the 2 olfactory receptor gene clusters REPD and REPP, representing 0.24% (4/1645) of the patients analyzed. None of the 4 patients has CHD or any other heart diseases and 1 mother who transmitted the duplication to her child has a history of aortic stenosis. Two patients who carry multiple genomic abnormalities, including a duplication containing GATA4, have complex CHD. Only 1 of the 3 individuals carrying genomic deletion containing GATA4 has atrial septal and ventricular septal defects. CONCLUSIONS: Cardiac defects are infrequent findings in individuals with 8p23.1 genomic duplications containing GATA4. A 0.24% detection rate of this duplication in this study is significantly higher than previously estimated. Observation in 2 patients with multiple genomic abnormalities and complex CHD is consistent with a 2-hit model that emphasizes accumulative effects of >1 insult to the genome, leading to a visible or more severe clinical manifestation. Haploinsufficient GATA4 may show variable expressivity with a wide spectrum of clinical findings, including CHD.

16p13.11 duplication is a risk factor for a wide spectrum of neuropsychiatric disorders.

The chromosome 16p13.11 heterozygous deletion is associated with a diverse array of neuropsychiatric disorders including intellectual disabilities, autism, schizophrenia, epilepsy and attention-deficit hyperactivity disorder. However the clinical significance of its reciprocal duplication is not clearly defined yet. We evaluated 1645 consecutive pediatric patients with various developmental disorders by high-resolution microarray-based comparative genomic hybridization and identified four deletions and eight duplications within the 16p13.11 region, representing ∼0.73% (12/1645) of the patients analyzed. Recurrent clinical features in these patients include mental retardation/intellectual disability, autism, seizure, dysmorphic feature or multiple congenital anomalies. Our data expand the spectrum of the clinical findings in patients with these genomic abnormalities and provide further support for the pathogenic involvement of this duplication in patients who carry them.

MicroRNA 21 blocks apoptosis in mouse periovulatory granulosa cells.

MicroRNAs (miRNAs) play important roles in many developmental processes, including cell differentiation and apoptosis. Transition of proliferative ovarian granulosa cells to terminally differentiated luteal cells in response to the ovulatory surge of luteinizing hormone (LH) involves rapid and pronounced changes in cellular morphology and function. MicroRNA 21 (miR-21, official symbol Mir21) is one of three highly LH-induced miRNAs in murine granulosa cells, and here we examine the function and temporal expression of Mir21 within granulosa cells as they transition to luteal cells. Granulosa cells were transfected with blocking (2'-O-methyl) and locked nucleic acid (LNA-21) oligonucleotides, and mature Mir21 expression decreased to one ninth and one twenty-seventh of its basal expression, respectively. LNA-21 depletion of Mir21 activity in cultured granulosa cells induced apoptosis. In vivo, follicular granulosa cells exhibit a decrease in cleaved caspase 3, a hallmark of apoptosis, 6 h after the LH/human chorionic gonadotropin surge, coincident with the highest expression of mature Mir21. To examine whether Mir21 is involved in regulation of apoptosis in vivo, mice were treated with a phospho thioate-modified LNA-21 oligonucleotide, and granulosa cell apoptosis was examined. Apoptosis increased in LNA-21-treated ovaries, and ovulation rate decreased in LNA-21-treated ovaries, compared with their contralateral controls. We have examined a number of Mir21 apoptotic target transcripts identified in other systems; currently, none of these appear to play a role in the induction of ovarian granulosa cell apoptosis. This study is the first to implicate the antiapoptotic Mir21 (an oncogenic miRNA) as playing a clear physiologic role in normal tissue function.

Quantitative RT-PCR methods for mature microRNA expression analysis.

This chapter describes two methods to measure expression of mature miRNA levels using qRT-PCR. The first method uses stem-loop RT primers to produce cDNA for specific miRNAs, a technique that our laboratory has modified to increase the number of miRNAs being reverse transcribed within a single RT reaction from one (as suggested by the manufacturer) to five. The second method uses a modified oligo(dT) technique to reverse transcribe all transcripts within an RNA sample; therefore, target miRNA and normalizing mRNA can be analyzed from the same RT reaction. We examined the level of miRNA-132, a miRNA known to be upregulated in granulosa cells following hCG treatment, using both of these methods. Data were normalized to GAPDH or snU6 and evaluated by DeltaDeltaCt and standard curve analysis. There was no significant difference (P > 0.05) in miRNA-132 expression between the stem-loop and modified oligo(dT) RT methods indicating that both are statistically equivalent. However, from a technical point of view, the modified oligo(dT) method was less time consuming and required only a single RT reaction to reverse transcribe both miRNA and mRNA.

Hormonal regulation of MicroRNA expression in periovulatory mouse mural granulosa cells.

MicroRNAs (miRNAs) mediate posttranscriptional gene regulation by binding to the 3' untranslated region of messenger RNAs to either inhibit or enhance translation. The extent and hormonal regulation of miRNA expression by ovarian granulosa cells and their role in ovulation and luteinization is unknown. In the present study, miRNA array analysis was used to identify 212 mature miRNAs as expressed and 13 as differentially expressed in periovulatory granulosa cells collected before and after an ovulatory dose of hCG. Two miRNAs, Mirn132 and Mirn212 (also known as miR-132 and miR-212), were found to be highly upregulated following LH/hCG induction and were further analyzed. In vivo and in vitro temporal expression analysis by quantitative RT-PCR confirmed that LH/hCG and cAMP, respectively, increased transcription of the precursor transcript as well as the mature miRNAs. Locked nucleic acid oligonucleotides complementary to Mirn132 and Mirn212 were shown to block cAMP-mediated mature miRNA expression and function. Computational analyses indicated that 77 putative mRNA targets of Mirn132 and Mirn212 were expressed in ovarian granulosa cells. Furthermore, upon knockdown of Mirn132 and Mirn212, a known target of Mirn132, C-terminal binding protein 1, showed decreased protein levels but no change in mRNA levels. The following studies are the first to describe the extent of miRNA expression within ovarian granulosa cells and the first to demonstrate that LH/hCG regulates the expression of select miRNAs, which affect posttranscriptional gene regulation within these cells.