A regulatory path associated with X-linked intellectual disability and epilepsy links KDM5C to the polyalanine expansions in ARX.

Intellectual disability (ID) and epilepsy often occur together and have a dramatic impact on the development and quality of life of the affected children. Polyalanine (polyA)-expansion-encoding mutations of aristaless-related homeobox (ARX) cause a spectrum of X-linked ID (XLID) diseases and chronic epilepsy, including infantile spasms. We show that lysine-specific demethylase 5C (KDM5C), a gene known to be mutated in XLID-affected children and involved in chromatin remodeling, is directly regulated by ARX through the binding in a conserved noncoding element. We have studied altered ARX carrying various polyA elongations in individuals with XLID and/or epilepsy. The changes in polyA repeats cause hypomorphic ARX alterations, which exhibit a decreased trans-activity and reduced, but not abolished, binding to the KDM5C regulatory region. The altered functioning of the mutants tested is likely to correlate with the severity of XLID and/or epilepsy. By quantitative RT-PCR, we observed a dramatic Kdm5c mRNA downregulation in murine Arx-knockout embryonic and neural stem cells. Such Kdm5c mRNA diminution led to a severe decrease in the KDM5C content during in vitro neuronal differentiation, which inversely correlated with an increase in H3K4me3 signal. We established that ARX polyA alterations damage the regulation of KDM5C expression, and we propose a potential ARX-dependent path acting via chromatin remodeling.

Insight into IKBKG/NEMO locus: report of new mutations and complex genomic rearrangements leading to incontinentia pigmenti disease.

Incontinentia pigmenti (IP) is an X-linked-dominant Mendelian disorder caused by mutation in the IKBKG/NEMO gene, encoding for NEMO/IKKgamma, a regulatory protein of nuclear factor kappaB (NF-kB) signaling. In more than 80% of cases, IP is due to recurrent or nonrecurrent deletions causing loss-of-function (LoF) of NEMO/IKKgamma. We review how the local architecture of the IKBKG/NEMO locus with segmental duplication and a high frequency of repetitive elements favor de novo aberrant recombination through different mechanisms producing genomic microdeletion. We report here a new microindel (c.436_471delinsT, p.Val146X) arising through a DNA-replication-repair fork-stalling-and-template-switching and microhomology-mediated-end-joining mechanism in a sporadic IP case. The LoF mutations of IKBKG/NEMO leading to IP include small insertions/deletions (indel) causing frameshift and premature stop codons, which account for 10% of cases. We here present 21 point mutations previously unreported, which further extend the spectrum of pathologic variants: 14/21 predict LoF because of premature stop codon (6/14) or frameshift (8/14), whereas 7/21 predict a partial loss of NEMO/IKKgamma activity (two splicing and five missense). We review how the analysis of IP-associated IKBKG/NEMO hypomorphic mutants has contributed to the understanding of the pathophysiological mechanism of IP disease and has provided important information on affected NF-kB signaling. We built a locus-specific database listing all IKBKG/NEMO variants, accessible at http://IKBKG.lovd.nl.

Genomic architecture at the Incontinentia Pigmenti locus favours de novo pathological alleles through different mechanisms.

IKBKG/NEMO gene mutations cause an X-linked, dominant neuroectodermal disorder named Incontinentia Pigmenti (IP). Located at Xq28, IKBKG/NEMO has a unique genomic organization, as it is part of a segmental duplication or low copy repeat (LCR1-LCR2, >99% identical) containing the gene and its pseudogene copy (IKBKGP). In the opposite direction and outside LCR1, IKBKG/NEMO partially overlaps G6PD, whose mutations cause a common X-linked human enzymopathy. The two LCRs in the IKBKG/NEMO locus are able to recombine through non-allelic homologous recombination producing either a pathological recurrent exon 4-10 IKBKG/NEMO deletion (IKBKGdel) or benign small copy number variations. We here report that the local high frequency of micro/macro-homologies, tandem repeats and repeat/repetitive sequences make the IKBKG/NEMO locus susceptible to novel pathological IP alterations. Indeed, we describe the first two independent instances of inter-locus gene conversion, occurring between the two LCRs, that copies the IKBKGP pseudogene variants into the functional IKBKG/NEMO, causing the de novo occurrence of p.Glu390ArgfsX61 and the IKBKGdel mutations, respectively. Subsequently, by investigating a group of 20 molecularly unsolved IP subjects using a high-density quantitative polymerase chain reaction assay, we have identified seven unique de novo deletions varying from 4.8 to ∼115 kb in length. Each deletion removes partially or completely both IKBKG/NEMO and the overlapping G6PD, thereby uncovering the first deletions disrupting the G6PD gene which were found in patients with IP. Interestingly, the 4.8 kb deletion removes the conserved bidirectional promoterB, shared by the two overlapping IKBKG/NEMO and G6PD genes, leaving intact the alternative IKBKG/NEMO unidirectional promoterA. This promoter, although active in the keratinocytes of the basal dermal layer, is down-regulated during late differentiation. Genomic analysis at the breakpoint sites indicated that other mutational forces, such as non-homologous end joining, Alu-Alu-mediated recombination and replication-based events, might enhance the vulnerability of the IP locus to produce de novo pathological IP alleles.

Ontogenetic profile of innate immune related genes and their tissue-specific expression in brown trout, Salmo trutta (Linnaeus, 1758).

The innate immune system is a fundamental defense weapon of fish, especially during early stages of development when acquired immunity is still far from being completely developed. The present study aims at looking into ontogeny of innate immune system in the brown trout, Salmo trutta, using RT-PCR based approach. Total RNA extracted from unfertilized and fertilized eggs and hatchlings at 0, 1 h and 1, 2, 3, 4, 5, 6, 7 weeks post-fertilization was subjected to RT-PCR using self-designed primers to amplify some innate immune relevant genes (TNF-α, IL-1ß, TGF-ß and lysozyme c-type). The constitutive expression of ß-actin was detected in all developmental stages. IL-1ß and TNF-α transcripts were detected from 4 week post-fertilization onwards, whereas TGF-ß transcript was detected only from 7 week post-fertilization onwards. Lysozyme c-type transcript was detected early from unfertilized egg stage onwards. Similarly, tissues such as muscle, ovary, heart, brain, gill, testis, liver, intestine, spleen, skin, posterior kidney, anterior kidney and blood collected from adult brown trout were subjected to detection of all selected genes by RT-PCR. TNF-α and lysozyme c-type transcripts were expressed in all tissues. IL-1ß and TGF-ß transcripts were expressed in all tissues except for the brain and liver, respectively. Taken together, our results show a spatial-temporal expression of some key innate immune-related genes, improving the basic knowledge of the function of innate immune system at early stage of brown trout.

Genetic and molecular analysis of a new unbalanced X;18 rearrangement: localization of the diminished ovarian reserve disease locus in the distal Xq POF1 region.

BACKGROUND: Diminished ovarian reserve (DOR) is a heterogeneous disorder causing infertility, characterized by a decreased number of oocytes, the genetic cause of which is still unknown. METHODS AND RESULTS: We describe a family with a new unbalanced X;18 translocation der(X) associated with either fully attenuated or DOR phenotype in the same family. Cytogenetics and array comparative genomic hybridization (aCGH) studies have revealed the same partial Xq monosomy and partial 18q trisomy in both the 32-year-old female with DOR and the unaffected mother. The genetic analysis has defined a subtelomeric deletion spanning 13.3 Mb from Xq27.3 to -Xqter, which covers the premature ovarian failure locus 1 (POF1); and a duplication spanning 13.4 Mb, from 18q22.1 to 18qter. From a parental-origin study, we have inferred that the rearranged X chromosome is maternally derived. The Xq27 and 18q22 breakpoint regions fall in a region extremely rich in long interspersed nuclear element, a class of retrotransposons able to trigger mispairing and unusual crossovers. X-inactivation studies reveal a skewing of der(X) both in the mother and the proband. Therefore, the phenotypic expression of der(X) is fully attenuated in the fertile mother and partially attenuated in the DOR daughter. CONCLUSIONS: We report on an unbalanced maternally derived translocation (X;18)(q27;q22) with different intra-familial reproductive performances, ranging from fertility to DOR. Skewed X-inactivation seems to restore the unbalanced genetic make-up, fully silencing the 18q22 trisomy and at least in part the Xq27 monosomy. The chromosomal abnormality observed in this family supports the presence of a DOR susceptibility locus in the distal Xq region and targets the POF1 region for further investigation.

Microdeletion/duplication at the Xq28 IP locus causes a de novo IKBKG/NEMO/IKKgamma exon4_10 deletion in families with Incontinentia Pigmenti.

The Incontinentia Pigmenti (IP) locus contains the IKBKG/NEMO/IKKgamma gene and its truncated pseudogene copy, IKBKGP/deltaNEMO. The major genetic defect in IP is a heterozygous exon4_10 IKBKG deletion (IKBKGdel) caused by a recombination between two consecutive MER67B repeats. We analyzed 91 IP females carrying the IKBKGdel, 59 of whom carrying de novo mutations (65%). In eight parents, we found two recurrent nonpathological variants of IP locus, which were also present as rare polymorphism in control population: the IKBKGPdel, corresponding to the exon4_10 deletion in the pseudogene, and the MER67Bdup, that replicates the exon4_10 region downstream of the normal IKBKG gene. Using quantitative DNA analysis and microsatellite mapping, we established that both variants might promote the generation of the pathological IKBKGdel. Indeed, in family IP-516, the exon4_10 deletion was repositioned in the same allele from the pseudogene to the gene, whereas in family IP-688, the MER67Bdup generated the pathological IKBKGdel by recombination between two direct nonadjacent MER67Bs. Moreover, we found an instance of somatic recombination in a MER67Bdup variant, creating the IKBKGdel in an IP male. Our data suggest that the IP locus undergoes recombination producing recurrent variants that might be "at risk" of generating de novo IKBKGdel by NAHR during either meiotic or mitotic division.

Alterations of the IKBKG locus and diseases: an update and a report of 13 novel mutations.

Mutations in the inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase gamma (IKBKG), also called nuclear factor-kappaB (NF-kB) essential modulator (NEMO), gene are the most common single cause of incontinentia pigmenti (IP) in females and anhydrotic ectodermal dysplasia with immunodeficiency (EDA-ID) in males. The IKBKG gene, located in the Xq28 chromosomal region, encodes for the regulatory subunit of the inhibitor of kappaB (IkB) kinase (IKK) complex required for the activation of the NF-kB pathway. Therefore, the remarkably heterogeneous and often severe clinical presentation reported in IP is due to the pleiotropic role of this signaling transcription pathway. A recurrent exon 4_10 genomic rearrangement in the IKBKG gene accounts for 60 to 80% of IP-causing mutations. Besides the IKBKG rearrangement found in IP females (which is lethal in males), a total of 69 different small mutations (missense, frameshift, nonsense, and splice-site mutations) have been reported, including 13 novel ones in this work. The updated distribution of all the IP- and EDA-ID-causing mutations along the IKBKG gene highlights a secondary hotspot mutation in exon 10, which contains only 11% of the protein. Furthermore, familial inheritance analysis revealed an unexpectedly high incidence of sporadic cases (>65%). The sum of the observations can aid both in determining the molecular basis of IP and EDA-ID allelic diseases, and in genetic counseling in affected families.

EDA-ID and IP, two faces of the same coin: how the same IKBKG/NEMO mutation affecting the NF-κB pathway can cause immunodeficiency and/or inflammation.

Anhidrotic Ectodermal Dysplasia with ImmunoDeficiency (EDA-ID, OMIM 300291) and Incontinentia Pigmenti (IP, OMIM 308300) are two rare diseases, caused by mutations of the IKBKG/NEMO gene. The protein NEMO/IKKγ is essential for the NF-κB activation pathway, involved in a variety of physiological and cellular processes, such as immunity, inflammation, cell proliferation, and survival. A wide spectrum of IKBKG/NEMO mutations have been identified so far, and, on the basis of their effect on NF-κB activation, they are considered hypomorphic or amorphic (loss of function) mutations. IKBKG/NEMO hypomorphic mutations, reducing but not abolishing NF-κB activation, have been identified in EDA-ID and IP patients. Instead, the amorphic mutations, abolishing NF-κB activation by complete IKBKG/NEMO gene silencing, cause only IP. Here, we present an overview of IKBKG/NEMO mutations in EDA-ID and IP patients and describe similarities and differences between the clinical/immunophenotypic and genetic aspects, highlighting any T and B lymphocyte defect, and paying particular attention to the cellular and molecular defects that underlie the pathogenesis of both diseases.

Antioxidant Potential of the Polyherbal Formulation "ImmuPlus": A Nutritional Supplement for Horses.

In order to counteract harmful effects of oxidative stress due to pathological conditions or physical exercise, horses are often administered dietary supplements having supposed high antioxidant activities. The aim of the present study was to identify the in vitro antioxidant potential of "ImmuPlus", a polyherbal formulation (Global Herbs LTD, Chichester, West Sussex, Great Britain), containing three medicinal plants (Withania somnifera, Tinospora cordifolia, and Emblica officinalis), known in Ayurveda for their use in human disease treatment. Extracts obtained by different solvents (water, methanol, ethanol, acetone, and hexane) were tested for total antioxidant capacity, total reducing power, scavenging activity against DPPH radical, and total polyphenol and flavonoid contents. Our results showed that, except as regards hexane, all the used solvents are able to extract compounds having high antioxidant activity, even when compared to ascorbic acid. Regression analysis showed significant correlations between antioxidant properties and polyphenol/flavonoid contents, indicating the latter, known for their beneficial effects on health of human and animal beings, as major components responsible for the strong antioxidant capacities. Moreover, obtained results suggest the effective role of the polyherbal mixture as good source of antioxidants in horses.

Incontinentia pigmenti: report on data from 2000 to 2013.

We report here on the building-up of a database of information related to 386 cases of Incontinentia Pigmenti collected in a thirteen-year activity (2000-2013) at our centre of expertise. The database has been constructed on the basis of a continuous collection of patients (27.6/year), the majority diagnosed as sporadic cases (75.6%). This activity has generated a rich source of information for future research studies by integrating molecular/clinical data with scientific knowledge. We describe the content, architecture and future utility of this collection of data on IP to offer comprehensive anonymous information to the international scientific community.