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Moyamoya angiopathy is a cerebral vasculopathy causing progressive stenosis of the internal carotid arteries and the compensatory development of collateral blood vessels, leading to brain ischemia and an increased risk of cerebral haemorrhage. Although multiple non-genetic causes have been associated with moyamoya syndrome, it can also be associated with rare genetic syndromes. Moyamoya Disease 4, characterised by a short stature, hypergonadotropic hypogonadism and facial dysmorphism (MYMY4, OMIM #300845), also referred to as BRCC3-associated moyamoya syndrome, has so far been described in 11 individuals. Here, we describe a 23-year-old male presenting with moyamoya syndrome, global developmental delay and intellectual disability, epilepsy, short stature and dysmorphic features, who after > 17 years of uninformative diagnostics was diagnosed with BRCC3-associated moyamoya syndrome after clinical RNA-seq. Transcriptome analysis showed reduced expression of the likely disease-causing gene BRCC3 in patient-derived fibroblasts, which was subsequently found to be caused by a ~ 26 kb Xq28 deletion. We furthermore review all reported cases of BRCC3-associated moyamoya syndrome, further delineating this clinical entity., (© 2024 The Author(s). Clinical Genetics published by John Wiley & Sons Ltd.)

Background: The aim of this study was to evaluate the diagnostic yield of routine exome sequencing (ES) in fetuses with ultrasound anomalies., Methods: We performed a retrospective analysis of the ES results of 629 fetuses with isolated or multiple anomalies referred in 2019-2022. Variants in a gene panel consisting of approximately 3400 genes associated with multiple congenital anomalies and/or intellectual disability were analyzed. We used trio analysis and filtering for de novo variants, compound heterozygous variants, homozygous variants, X-linked variants, variants in imprinted genes, and known pathogenic variants., Results: Pathogenic and likely pathogenic variants (class five and four, respectively) were identified in 14.0% (88/629, 95% CI 11.5%-16.9%) of cases. In the current cohort, the probability of detecting a monogenetic disorder was ∼1:7 (88/629, 95% CI 1:8.7-1:5.9), ranging from 1:9 (49/424) in cases with one major anomaly to 1:5 (32/147) in cases with multiple system anomalies., Conclusions: Our results indicate that a notable number of fetuses (1:7) with ultrasound anomalies and a normal chromosomal microarray have a (likely) pathogenic variant that can be detected through prenatal ES. These results warrant implementation of exome sequencing in selected cases, including those with an isolated anomaly on prenatal ultrasound., (© 2024 The Author(s). Prenatal Diagnosis published by John Wiley & Sons Ltd.)

Background:  Potential underlying genetic variations of pectus excavatum (PE) are quite rare. Only one-fifth of PE cases are identified in the first decade of life and thus are of congenital origin. The objective of this study is to test if early-onset PE is more likely to be part of genetic variations than PE that becomes apparent during puberty or adolescence., Materials and Methods:  Children younger than 11 years who presented with PE to the outpatient clinic of the Department of Pediatric Surgery at our center between 2014 and 2020 were screened by two clinical geneticists separately. Molecular analysis was performed based on the differential diagnosis. Data of all young PE patients who already had been referred for genetic counseling were analyzed retrospectively., Results:  Pathogenic genetic variations were found in 8 of the 18 participants (44%): 3 syndromic disorders (Catel-Manzke syndrome and two Noonan syndromes), 3 chromosomal disorders (16p13.11 microduplication syndrome, 22q11.21 microduplication syndrome, and genetic gain at 1q44), 1 connective tissue disease (Loeys-Dietz syndrome), and 1 neuromuscular disorder (pathogenic variation in BICD2 gene)., Conclusion:  Early-onset PE is more likely to be part of genetic variations than PE that becomes apparent during puberty or adolescence. Referral for genetic counseling should therefore be considered., Trial Registration:  NCT05443113., Competing Interests: None declared., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution License, permitting unrestricted use, distribution, and reproduction so long as the original work is properly cited. (https://creativecommons.org/licenses/by/4.0/).)

Autosomal dominant polycystic kidney disease (ADPKD) resulting from pathogenic variants in PKD1 and PKD2 is the most common form of PKD, but other genetic causes tied to primary cilia function have been identified. Biallelic pathogenic variants in the serine/threonine kinase NEK8 cause a syndromic ciliopathy with extra-kidney manifestations. Here we identify NEK8 as a disease gene for ADPKD in 12 families. Clinical evaluation was combined with functional studies using fibroblasts and tubuloids from affected individuals. Nek8 knockout mouse kidney epithelial (IMCD3) cells transfected with wild type or variant NEK8 were further used to study ciliogenesis, ciliary trafficking, kinase function, and DNA damage responses. Twenty-one affected monoallelic individuals uniformly exhibited cystic kidney disease (mostly neonatal) without consistent extra-kidney manifestations. Recurrent de novo mutations of the NEK8 missense variant p.Arg45Trp, including mosaicism, were seen in ten families. Missense variants elsewhere within the kinase domain (p.Ile150Met and p.Lys157Gln) were also identified. Functional studies demonstrated normal localization of the NEK8 protein to the proximal cilium and no consistent cilia formation defects in patient-derived cells. NEK8-wild type protein and all variant forms of the protein expressed in Nek8 knockout IMCD3 cells were localized to cilia and supported ciliogenesis. However, Nek8 knockout IMCD3 cells expressing NEK8-p.Arg45Trp and NEK8-p.Lys157Gln showed significantly decreased polycystin-2 but normal ANKS6 localization in cilia. Moreover, p.Arg45Trp NEK8 exhibited reduced kinase activity in vitro. In patient derived tubuloids and IMCD3 cells expressing NEK8-p.Arg45Trp, DNA damage signaling was increased compared to healthy passage-matched controls. Thus, we propose a dominant-negative effect for specific heterozygous missense variants in the NEK8 kinase domain as a new cause of PKD., (Copyright © 2023 International Society of Nephrology. All rights reserved.)

Congenital diaphragmatic hernia (CDH) is a congenital structural anomaly in which the diaphragm has not developed properly. It may occur either as an isolated anomaly or with additional anomalies. It is thought to be a multifactorial disease in which genetic factors could either substantially contribute to or directly result in the developmental defect. Patients with aneuploidies, pathogenic variants or de novo Copy Number Variations (CNVs) impacting specific genes and loci develop CDH typically in the form of a monogenetic syndrome. These patients often have other associated anatomical malformations. In patients without a known monogenetic syndrome, an increased genetic burden of de novo coding variants contributes to disease development. In early years, genetic evaluation was based on karyotyping and SNP-array. Today, genomes are commonly analyzed with next generation sequencing (NGS) based approaches. While more potential pathogenic variants are being detected, analysis of the data presents a bottleneck-largely due to the lack of full appreciation of the functional consequence and/or relevance of the detected variant. The exact heritability of CDH is still unknown. Damaging de novo alterations are associated with the more severe and complex phenotypes and worse clinical outcome. Phenotypic, genetic-and likely mechanistic-variability hampers individual patient diagnosis, short and long-term morbidity prediction and subsequent care strategies. Detailed phenotyping, clinical follow-up at regular intervals and detailed registries are needed to find associations between long-term morbidity, genetic alterations, and clinical parameters. Since CDH is a relatively rare disorder with only a few recurrent changes large cohorts of patients are needed to identify genetic associations. Retrospective whole genome sequencing of historical patient cohorts using will yield valuable data from which today's patients and parents will profit Trio whole genome sequencing has an excellent potential for future re-analysis and data-sharing increasing the chance to provide a genetic diagnosis and predict clinical prognosis. In this review, we explore the pitfalls and challenges in the analysis and interpretation of genetic information, present what is currently known and what still needs further study, and propose strategies to reap the benefits of genetic screening., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Brosens, Peters, van Weelden, Bendixen, Brouwer, Sleutels, Bruggenwirth, van Ijcken, Veenma, Otter, Wijnen, Eggink, van Dooren, Reutter, Rottier, Schnater, Tibboel and de Klein.)

The 11 zinc finger (ZF) protein CTCF regulates topologically associating domain formation and transcription through selective binding to thousands of genomic sites. Here, we replaced endogenous CTCF in mouse embryonic stem cells with green-fluorescent-protein-tagged wild-type or mutant proteins lacking individual ZFs to identify additional determinants of CTCF positioning and function. While ZF1 and ZF8-ZF11 are not essential for cell survival, ZF8 deletion strikingly increases the DNA binding off-rate of mutant CTCF, resulting in reduced CTCF chromatin residence time. Loss of ZF8 results in widespread weakening of topologically associating domains, aberrant gene expression and increased genome-wide DNA methylation. Thus, important chromatin-templated processes rely on accurate CTCF chromatin residence time, which we propose depends on local sequence and chromatin context as well as global CTCF protein concentration., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

ANKRD17 is an ankyrin repeat-containing protein thought to play a role in cell cycle progression, whose ortholog in Drosophila functions in the Hippo pathway as a co-factor of Yorkie. Here, we delineate a neurodevelopmental disorder caused by de novo heterozygous ANKRD17 variants. The mutational spectrum of this cohort of 34 individuals from 32 families is highly suggestive of haploinsufficiency as the underlying mechanism of disease, with 21 truncating or essential splice site variants, 9 missense variants, 1 in-frame insertion-deletion, and 1 microdeletion (1.16 Mb). Consequently, our data indicate that loss of ANKRD17 is likely the main cause of phenotypes previously associated with large multi-gene chromosomal aberrations of the 4q13.3 region. Protein modeling suggests that most of the missense variants disrupt the stability of the ankyrin repeats through alteration of core structural residues. The major phenotypic characteristic of our cohort is a variable degree of developmental delay/intellectual disability, particularly affecting speech, while additional features include growth failure, feeding difficulties, non-specific MRI abnormalities, epilepsy and/or abnormal EEG, predisposition to recurrent infections (mostly bacterial), ophthalmological abnormalities, gait/balance disturbance, and joint hypermobility. Moreover, many individuals shared similar dysmorphic facial features. Analysis of single-cell RNA-seq data from the developing human telencephalon indicated ANKRD17 expression at multiple stages of neurogenesis, adding further evidence to the assertion that damaging ANKRD17 variants cause a neurodevelopmental disorder., (Copyright © 2021 American Society of Human Genetics. All rights reserved.)

Background: Neonatal jaundice is a common finding in newborns in Asia, including Indonesia. In some cases, the serum total bilirubin levels exceeds the 95th percentile for hours of life (neonatal hyperbilirubinemia). Severe neonatal hyperbilirubinemia (NH) could lead to kernicterus and neonatal death. Glucose-6-Phosphage Dehydrogenase (G6PD) genetic variations and deficiency have been reported in several studies to be associated with NH. This study aimed to analyze the G6PD genetic variations and its activity in neonates with and without hyperbilirubinemia in the Deutromalay Indonesian population., Methods: Deoxyribose Nucleic Acid (DNA) was isolated from peripheral blood of 116 and 115 healthy term neonates with and without hyperbilirubinemia. All infants underwent the following laboratory examinations: routine hematologic evaluation, Coombs test, G6PD activity measurement using the Randox kit method, and serum total bilirubin level. All exons of the G6PD gene were targeted for deep sequencing using MiSeq (Illumina). An association study of G6PD polymorphisms with NH was performed using PLINK., Results: The prevalence of G6PD deficiency in neonates with and without hyperbilirubinemia in Indonesian Deutromalay population were 1.72% (95% Confidence Interval (CI): 0.6-4.1%) and 1.74% (95% CI: 0.7-4.1%), respectively. The most common G6PD polymorphisms, i.e. rs1050757/c.* + 357A > G, rs2230037/c.1311C > T, and rs2071429/c.1365-13 T/IVS11, were identified. However, none of those polymorphisms and their haplotype were associated with NH (p > 0.05, Odds Ratio (OR) ~1.00). The prevalence of G6PD mutations in neonates with and without hyperbilirubinemia were 6.8% (95% CI: 2.3-11.5%) and 6.9% (95% CI: 2.3-11.6%), respectively. The most frequently identified G6PD mutation was the Viangchan variant (p.V291 M), which was followed by the Canton (p.R459L) and Vanua Lava (p.L128P) variants. Two novel mutations were identified both in case (p.V369A, p.I167F) and control (p.L474=, p.I36T) groups., Conclusion: The prevalence of G6PD deficiency is low in neonates with or without hyperbilirubinemia in Deutromalay Indonesian population. The majority of G6PD mutations identified among Indonesian Deutromalay population in this study are Viangchan, Canton and Vanua Lava variants.

Purpose: Using genome-wide noninvasive prenatal screening (NIPS), we detected a 20-megabase specific deletion starting at 10q25 in eight pregnancies. The deletion could not be confirmed by invasive testing. Since all 10(q25→qter) deletions started close to the FRA10B fragile site in 10q25, we investigated whether the pregnant women were indeed carriers of FRA10B., Methods: We performed NIPS analysis for all autosomes using single-read sequencing. Analysis was done with the WISECONDOR algorithm. Culture of blood lymphocytes with bromodeoxyuridine was used to detect FRA10B expansions. Fluorescence in situ hybridization and array analysis were used to find maternal and/or fetal deletions., Results: We confirmed the presence of a FRA10B expansion in all four tested mothers. Fluorescence in situ hybridization and array analysis confirmed the presence of a maternal mosaic deletion of 10(q25→qter)., Conclusion: The recurring 10(q25→qter) deletion detected with NIPS is a false-positive result caused by a maternal low-level mosaic deletion associated with FRA10B expansions. This has important consequences for clinical follow-up, as invasive procedures are unnecessary. Expanded maternal FRA10B repeats should be added to the growing group of variants in the maternal genome that may cause false-positive NIPS results.

Neonatal hyperbilirubinemia (NH) is a common finding in newborn babies in Indonesia. Common and rare variants of UGT1A1 have been known to contribute to NH etiology. This study aims to identify UGT1A1 genetic variation and haplotype associated with NH in Indonesian population. DNA was isolated from 116 cases and 115 controls and a targeted-deep sequencing approach was performed on the promoter, UTRs, and exonic regions of UGT1A1 . Determining association of common variants and haplotype analysis were performed using PLINK and Haploview. Ten and 4 rare variants were identified in cases and controls, respectively. The UGT1A1 rare variants frequency in cases (5.17%) was higher than that in controls (1.7%). Four of those rare variants in cases (p.Ala61Thr, p.His300Arg, p.Lys407Asn, and p.Tyr514Asn) and three in controls (p.Tyr79X, p.Ala346Val, and p.Thr412Ser) are novel variants. The frequencies of p.Gly71Arg, p.Pro229Gln, and TA 7 common variants were not significantly different between cases and controls. A haplotype, consisting of 3 major alleles of 3' UTRs common variants (rs8330C>G, rs10929303C>T, and rs1042640C>G), was associated with NH incidence ( p = 0.025) in this population. Using targeted-deep sequencing and haplotype analysis, we identified novel UGT1A1 rare variants and disease-associated haplotype in NH in Indonesian population.

The ability of bacteria and fungi to communicate with each other is a remarkable aspect of the microbial world. It is recognized that volatile organic compounds (VOCs) act as communication signals, however the molecular responses by bacteria to fungal VOCs remain unknown. Here we perform transcriptomics and proteomics analyses of Serratia plymuthica PRI-2C exposed to VOCs emitted by the fungal pathogen Fusarium culmorum. We find that the bacterium responds to fungal VOCs with changes in gene and protein expression related to motility, signal transduction, energy metabolism, cell envelope biogenesis, and secondary metabolite production. Metabolomic analysis of the bacterium exposed to the fungal VOCs, gene cluster comparison, and heterologous co-expression of a terpene synthase and a methyltransferase revealed the production of the unusual terpene sodorifen in response to fungal VOCs. These results strongly suggest that VOCs are not only a metabolic waste but important compounds in the long-distance communication between fungi and bacteria.

Background: CTCF is a highly conserved and essential zinc finger protein expressed in virtually all cell types. In conjunction with cohesin, it organizes chromatin into loops, thereby regulating gene expression and epigenetic events. The function of CTCFL or BORIS, the testis-specific paralog of CTCF, is less clear., Results: Using immunohistochemistry on testis sections and fluorescence-based microscopy on intact live seminiferous tubules, we show that CTCFL is only transiently present during spermatogenesis, prior to the onset of meiosis, when the protein co-localizes in nuclei with ubiquitously expressed CTCF. CTCFL distribution overlaps completely with that of Stra8, a retinoic acid-inducible protein essential for the propagation of meiosis. We find that absence of CTCFL in mice causes sub-fertility because of a partially penetrant testicular atrophy. CTCFL deficiency affects the expression of a number of testis-specific genes, including Gal3st1 and Prss50. Combined, these data indicate that CTCFL has a unique role in spermatogenesis. Genome-wide RNA expression studies in ES cells expressing a V5- and GFP-tagged form of CTCFL show that genes that are downregulated in CTCFL-deficient testis are upregulated in ES cells. These data indicate that CTCFL is a male germ cell gene regulator. Furthermore, genome-wide DNA-binding analysis shows that CTCFL binds a consensus sequence that is very similar to that of CTCF. However, only ~3,700 out of the ~5,700 CTCFL- and ~31,000 CTCF-binding sites overlap. CTCFL binds promoters with loosely assembled nucleosomes, whereas CTCF favors consensus sites surrounded by phased nucleosomes. Finally, an ES cell-based rescue assay shows that CTCFL is functionally different from CTCF., Conclusions: Our data suggest that nucleosome composition specifies the genome-wide binding of CTCFL and CTCF. We propose that the transient expression of CTCFL in spermatogonia and preleptotene spermatocytes serves to occupy a subset of promoters and maintain the expression of male germ cell genes.

The use of bacterial artificial chromosomes (BACs) provides a consistent and high targeting efficiency of homologous recombination in embryonic stem (ES) cells, facilitated by long stretches of sequence homology. Here, we introduce a BAC targeting method which employs restriction fragment length polymorphisms (RFLPs) in targeted polymorphic C57BL/6/Cast/Ei F1 mouse ES cell lines to identify properly targeted ES cell clones. We demonstrate that knockout alleles can be generated either by targeting of an RFLP located in the open reading frame thereby disrupting the RFLP and ablating gene function, or by introduction of a transcription stop cassette that prematurely stops transcription of an RFLP located downstream of the stop cassette. With both methods we have generated Rnf12 heterozygous knockout ES cells, which were identified by allele specific PCR using genomic DNA or cDNA as a template. Our results indicate that this novel strategy is efficient and precise, by combining a high targeting efficiency with a convenient PCR based readout and reliable detection of correct targeting events.

Background: CCCTC binding factor (CTCF) is a highly conserved zinc finger protein, which is involved in chromatin organization, local histone modifications, and RNA polymerase II-mediated gene transcription. CTCF may act by binding tightly to DNA and recruiting other proteins to mediate its various functions in the nucleus. To further explore the role of this essential factor, we used a mass spectrometry-based approach to screen for novel CTCF-interacting partners., Results: Using biotinylated CTCF as bait, we identified upstream binding factor (UBF) and multiple other components of the RNA polymerase I complex as potential CTCF-interacting partners. Interestingly, CTCFL, the testis-specific paralog of CTCF, also binds UBF. The interaction between CTCF(L) and UBF is direct, and requires the zinc finger domain of CTCF(L) and the high mobility group (HMG)-box 1 and dimerization domain of UBF. Because UBF is involved in RNA polymerase I-mediated ribosomal (r)RNA transcription, we analyzed CTCF binding to the rDNA repeat. We found that CTCF bound to a site upstream of the rDNA spacer promoter and preferred non-methylated over methylated rDNA. DNA binding by CTCF in turn stimulated binding of UBF. Absence of CTCF in cultured cells resulted in decreased association of UBF with rDNA and in nucleolar fusion. Furthermore, lack of CTCF led to reduced binding of RNA polymerase I and variant histone H2A.Z near the rDNA spacer promoter, a loss of specific histone modifications, and diminished transcription of non-coding RNA from the spacer promoter., Conclusions: UBF is the first common interaction partner of CTCF and CTCFL, suggesting a role for these proteins in chromatin organization of the rDNA repeats. We propose that CTCF affects RNA polymerase I-mediated events globally by controlling nucleolar number, and locally by regulating chromatin at the rDNA spacer promoter, similar to RNA polymerase II promoters. CTCF may load UBF onto rDNA, thereby forming part of a network that maintains rDNA genes poised for transcription.

Differentiation of naive CD4+ cells into Th2 cells is accompanied by chromatin remodeling at the Th2 cytokine locus allowing the expression of the IL-4, IL-5, and IL-13 genes. In this report, we investigated the role in Th2 differentiation of the transcription regulator CCCTC-binding factor (CTCF). Chromatin immunoprecipitation analysis revealed multiple CTCF binding sites in the Th2 cytokine locus. Conditional deletion of the Ctcf gene in double-positive thymocytes allowed development of peripheral T cells, but their activation and proliferation upon anti-CD3/anti-CD28 stimulation in vitro was severely impaired. Nevertheless, when TCR signaling was circumvented with phorbol ester and ionomycin, we observed proliferation of CTCF-deficient T cells, enabling the analysis of Th2 differentiation in vitro. We found that in CTCF-deficient Th2 polarization cultures, transcription of IL-4, IL-5, and IL-13 was strongly reduced. By contrast, CTCF deficiency had a moderate effect on IFN-gamma production in Th1 cultures and IL-17 production in Th17 cultures was unaffected. Consistent with a Th2 cytokine defect, CTCF-deficient mice had very low levels of IgG1 and IgE in their serum, but IgG2c was close to normal. In CTCF-deficient Th2 cultures, cells were polarized toward the Th2 lineage, as substantiated by induction of the key transcriptional regulators GATA3 and special AT-rich binding protein 1 (SATB1) and down-regulation of T-bet. Also, STAT4 expression was low, indicating that in the absence of CTCF, GATA3 still operated as a negative regulator of STAT4. Taken together, these findings show that CTCF is essential for GATA3- and SATB1-dependent regulation of Th2 cytokine gene expression.

The 11-zinc finger protein CCCTC-binding factor (CTCF) is a highly conserved protein, involved in imprinting, long-range chromatin interactions and transcription. To investigate its function in vivo, we generated mice with a conditional Ctcf knockout allele. Consistent with a previous report, we find that ubiquitous ablation of the Ctcf gene results in early embryonic lethality. Tissue-specific inactivation of CTCF in thymocytes specifically hampers the differentiation of alphabeta T cells and causes accumulation of late double-negative and immature single-positive cells in the thymus of mice. These cells are normally large and actively cycling, and contain elevated amounts of CTCF. In Ctcf knockout animals, however, these cells are small and blocked in the cell cycle due to increased expression of the cyclin-CDK inhibitors p21 and p27. Taken together, our results show that CTCF is required in a dose-dependent manner and is involved in cell cycle progression of alphabeta T cells in the thymus. We propose that CTCF positively regulates cell growth in rapidly dividing thymocytes so that appropriate number of cells are generated before positive and negative selection in the thymus.

In genomic imprinting, one of the two parental alleles of an autosomal gene is silenced epigenetically by a cis-acting mechanism. A bidirectional silencer for a 400-kilobase region that contains three imprinted, maternally expressed protein-coding genes (Igf2r/Slc22a2/Slc22a3) has been shown by targeted deletion to be located in a sequence of 3.7 kilobases, which also contains the promoter for the imprinted, paternally expressed non-coding Air RNA. Expression of Air is correlated with repression of all three genes on the paternal allele; however, Air RNA overlaps just one of these genes in an antisense orientation. Here we show, by inserting a polyadenylation signal that truncates 96% of the RNA transcript, that Air RNA is required for silencing. The truncated Air allele maintains imprinted expression and methylation of the Air promoter, but shows complete loss of silencing of the Igf2r/Slc22a2/Slc22a3 gene cluster on the paternal chromosome. Our results indicate that non-coding RNAs have an active role in genomic imprinting.

Imprinted maternal-allele-specific expression of the mouse insulin-like growth-factor type 2 receptor (Igf2r) gene depends on a 3.7-kb element named region 2, located in the second intron of the gene. Region 2 carries a maternal-allele-specific methylation imprint and contains an imprinted CpG island promoter (Air) that expresses a noncoding antisense RNA from the paternal inherited allele only. Here, we use transgenes to test the minimal requirements for imprinting of Air and to test if the action of region 2 is restricted to Igf2r. Transgenes up to 9 kb with Air as a single promoter are expressed but not imprinted. When coupled to the Igf2r CpG island promoter on a 44-kb transgene, Air was imprinted in one of three lines. However, Air on a 4.6-kb fragment is also imprinted in 2 of 14 lines when inserted in an intron of an adenine phosphoribosyltransferase (Aprt) transgene, and in one line, the imprinted methylation and expression of Air have been transferred onto the Aprt CpG island promoter. These data suggest that a dual CpG island promoter setting may facilitate Air imprinting as a short transgene and also show that Air can transfer imprinting onto other genes. However, for reliable Air imprinting, elements are necessary that are located outside a 44-kb region spanning the Air-Igf2r promoters.

In the mouse, expression of an antisense Igf2r RNA (Air) is correlated with Igf2r repression on the paternal allele. One of the possible models for Igf2r repression could be through promoter competition or through the action of the Air RNA, in, e.g., transcriptional interference or repressor binding. These models predict the conservation of AIR RNA in human samples with monoallelic IGF2R expression and the production of AIR RNA in first-trimester human tissues. However, by strand-specific RT-PCR and by ribonuclease protection assay we have not detected any AIR RNA in first-trimester placental tissue samples, not even in samples that downregulate IGF2R expression in an allele-specific manner. This indicates that in contrast to the mouse, allelic IGF2R repression in the developing human placenta does not correlate with AIR expression., (Copyright 2001 Academic Press.)

Receptor-mediated activation of phosphatidylcholine phosphatidohydrolase or phospholipase D (PLD) was studied in Chinese hamster ovary (CHO) cells expressing the cholecystokinin-A (CCK-A) receptor. Cells were labelled with [3H]myristic acid for 24 h and PLD-catalysed [3H]phosphatidylethanol formation was measured in the presence of 1% (v/v) ethanol. Cholecystokinin-(26-33)-peptide amide (CCK8) increased PLD activity both time- and dose-dependently. Maximal activation of protein kinase C (PKC) with 1 microM PMA or sustained elevation of the cytosolic free Ca2+ concentration ([Ca2+]i) with 1 microM thapsigargin increased PLD activity to 50% and 70% of the maximal value obtained with CCK8 respectively. The stimulatory effects of CCK8, PMA and thapsigargin were abolished in cells in which PKC was downregulated or inhibited by chelerythrine. PMA/Ca2+-stimulated PLD activity was absent in a homogenate of PKC-downregulated cells but could be restored upon addition of purified rat brain PKC. CCK8-induced PLD activation was inhibited by 90% in the absence of external Ca2+, demonstrating that receptor-mediated activation of PKC in itself does not significantly add to PLD activation but requires a sustained increase in [Ca2+]i. Taken together, the results presented demonstrate that, in CHO-CCK-A cells, receptor-mediated PLD activation is completely dependent on PKC, but that the extent to which PLD becomes activated depends largely, if not entirely, on the magnitude and duration of the agonist-induced increase in [Ca2+]i.

The gonad-inhibiting hormone (GIH) is produced in the eyestalk X-organ sinus gland complex of male and female lobsters, and plays a prominent role in the regulation of reproduction, e.g. inhibition of vitellogenesis in female animals. To study this neurohormone at the mRNA level, we cloned and sequenced a cDNA which encodes GIH in the lobster Homarus americanus. The structure of preproGIH consists of a signal peptide and the GIH peptide itself. A comparative analysis revealed that lobster GIH, together with crab molt-inhibiting hormone, belongs to a separate group of the crustacean hyperglycemic hormone (CHH) peptide family which seems to be unique for crustaceans. Expression studies showed that GIH mRNA is expressed in the eyestalk, indicating that the neuroendocrine center in this optic structure is the only source of GIH. As this center modulates the other (neuro)endocrine organs in crustaceans, it is postulated that GIH regulates production and release of hormones involved in reproduction/molting processes.