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Neuroplasticity is a crucial property of the central nervous system to change its activity in response to intrinsic or extrinsic stimuli. This is mainly achieved through the promotion of changes in the epigenome. One of the epi-drivers priming this process is suberoylanilide hydroxamic acid (SAHA or Vorinostat), a pan-histone deacetylase inhibitor that modulates and promotes neuroplasticity in healthy and disease conditions. Knowledge of the specific molecular changes induced by this epidrug is an important area of neuro-epigenetics for the identification of new compounds to treat cognition impairment and/or epilepsy. In this review, we summarize the findings obtained in cellular and animal models of various brain disorders, highlighting the multiple mechanisms activated by SAHA, such as improvement of memory, learning and behavior, and correction of faulty neuronal functioning. Supporting this evidence, in vitro and in vivo data underline how SAHA positively regulates the expression of neuronal genes and microtubule dynamics, induces neurite outgrowth and spine density, and enhances synaptic transmission and potentiation. In particular, we outline studies regarding neurodevelopmental disorders with pharmaco-resistant seizures and/or severe cognitive impairment that to date lack effective drug treatments in which SAHA could ameliorate defective neuroplasticity.

Lysine-specific demethylase 5C (KDM5C) has been identified as an important chromatin remodeling gene, contributing to X-linked neurodevelopmental disorders (NDDs). The KDM5C gene, located in the Xp22 chromosomal region, encodes the H3K4me3-me2 eraser involved in neuronal plasticity and dendritic growth. Here we report 30 individuals carrying 13 novel and one previously identified KDM5C variants. Our cohort includes the first reported case of somatic mosaicism in a male carrying a KDM5C nucleotide substitution, and a dual molecular finding in a female carrying a homozygous truncating FUCA1 alteration together with a de novo KDM5C variant. With the use of next generation sequencing strategies, we detected 1 frameshift, 1 stop codon, 2 splice-site and 10 missense variants, which pathogenic role was carefully investigated by a thorough bioinformatic analysis. The pattern of X-chromosome inactivation was found to have an impact on KDM5C phenotypic expression in females of our cohort. The affected individuals of our case series manifested a neurodevelopmental condition characterized by psychomotor delay, intellectual disability with speech disorders, and behavioral features with particular disturbed sleep pattern; other observed clinical manifestations were short stature, obesity and hypertrichosis. Collectively, these findings expand the current knowledge about the pathogenic mechanisms leading to dysfunction of this important chromatin remodeling gene and contribute to a refinement of the KDM5C phenotypic spectrum., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Glioblastoma multiforme (GBM) is a fatal brain tumor without effective drug treatment. In this study, we highlight, for the first time, the contribution of chromatin remodeling gene Lysine (K)-specific demethylase 5C ( KDM5C ) in GBM via an extensive analysis of clinical, expression, and functional data, integrated with publicly available omic datasets. The expression analysis on GBM samples (N = 37) revealed two informative subtypes, namely KDM5C High and KDM5C Low , displaying higher/lower KDM5C levels compared to the controls. The former subtype displays a strong downregulation of brain-derived neurotrophic factor ( BDNF )-a negative KDM5C target-and a robust overexpression of hypoxia-inducible transcription factor-1A ( HIF1A ) gene, a KDM5C modulator. Additionally, a significant co-expression among the prognostic markers HIF1A , Survivin , and p75 was observed. These results, corroborated by KDM5C overexpression and hypoxia-related functional assays in T98G cells, suggest a role for the HIF1A-KDM5C axis in the hypoxic response in this tumor. Interestingly, fluorescence-guided surgery on GBM sections further revealed higher KDM5C and HIF1A levels in the tumor rim niche compared to the adjacent tumor margin, indicating a regionally restricted hyperactivity of this regulatory axis. Analyzing the TCGA expression and methylation data, we found methylation changes between the subtypes in the genes, accounting for the hypoxia response, stem cell differentiation, and inflammation. High NANOG and IL6 levels highlight a distinctive stem cell-like and proinflammatory signature in the KDM5C High subgroup and GBM niches. Taken together, our results indicate HIF1A-KDM5C as a new, relevant cancer axis in GBM, opening a new, interesting field of investigation based on KDM5C as a potential therapeutic target of the hypoxic microenvironment in GBM.

X-linked lissencephaly with abnormal genitalia (XLAG) and developmental epileptic encephalopathy-1 (DEE1) are caused by mutations in the Aristaless-related homeobox (ARX) gene, which encodes a transcription factor responsible for brain development. It has been unknown whether the phenotypically diverse XLAG and DEE1 phenotypes may converge on shared pathways. To address this question, a label-free quantitative proteomic approach was applied to the neonatal brain of Arx knockout (ArxKO/Y) and knock-in polyalanine (Arx(GCG)7/Y) mice that are respectively models for XLAG and DEE1. Gene ontology and protein-protein interaction analysis revealed that cytoskeleton, protein synthesis and splicing control are deregulated in an allelic-dependent manner. Decreased α-tubulin content was observed both in Arx mice and Arx/alr-1(KO) Caenorhabditis elegans ,and a disorganized neurite network in murine primary neurons was consistent with an allelic-dependent secondary tubulinopathy. As distinct features of Arx(GCG)7/Y mice, we detected eIF4A2 overexpression and translational suppression in cortex and primary neurons. Allelic-dependent differences were also established in alternative splicing (AS) regulated by PUF60 and SAM68. Abnormal AS repertoires in Neurexin-1, a gene encoding multiple pre-synaptic organizers implicated in synaptic remodelling, were detected in Arx/alr-1(KO) animals and in Arx(GCG)7/Y epileptogenic brain areas and depolarized cortical neurons. Consistent with a conserved role of ARX in modulating AS, we propose that the allelic-dependent secondary synaptopathy results from an aberrant Neurexin-1 repertoire. Overall, our data reveal alterations mirroring the overlapping and variant effects caused by null and polyalanine expanded mutations in ARX. The identification of these effects can aid in the design of pathway-guided therapy for ARX endophenotypes and NDDs with overlapping comorbidities., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The X-linked gene encoding aristaless-related homeobox ( ARX ) is a bi-functional transcription factor capable of activating or repressing gene transcription, whose mutations have been found in a wide spectrum of neurodevelopmental disorders (NDDs); these include cortical malformations, paediatric epilepsy, intellectual disability (ID) and autism. In addition to point mutations, duplications of the ARX locus have been detected in male patients with ID. These rearrangements include telencephalon ultraconserved enhancers, whose structural alterations can interfere with the control of ARX expression in the developing brain. Here, we review the structural features of 15 gain copy-number variants (CNVs) of the ARX locus found in patients presenting wide-ranging phenotypic variations including ID, speech delay, hypotonia and psychiatric abnormalities. We also report on a further novel Xp21.3 duplication detected in a male patient with moderate ID and carrying a fully duplicated copy of the ARX locus and the ultraconserved enhancers. As consequences of this rearrangement, the patient-derived lymphoblastoid cell line shows abnormal activity of the ARX-KDM5C-SYN1 regulatory axis. Moreover, the three-dimensional (3D) structure of the Arx locus, both in mouse embryonic stem cells and cortical neurons, provides new insight for the functional consequences of ARX duplications. Finally, by comparing the clinical features of the 16 CNVs affecting the ARX locus, we conclude that-depending on the involvement of tissue-specific enhancers-the ARX duplications are ID-associated risk CNVs with variable expressivity and penetrance.

Dysregulation of transcriptional pathways is observed in multiple forms of neurodevelopmental disorders (NDDs), such as intellectual disability (ID), epilepsy and autism spectrum disorder (ASD). We previously demonstrated that the NDD genes encoding lysine-specific demethylase 5C ( KDM5C ) and its transcriptional regulators Aristaless related-homeobox (ARX), PHD Finger Protein 8 (PHF8) and Zinc Finger Protein 711 ( ZNF711 ) are functionally connected. Here, we show their relation to each other with respect to the expression levels in human and mouse datasets and in vivo mouse analysis indicating that the coexpression of these syntenic X-chromosomal genes is temporally regulated in brain areas and cellular sub-types. In co-immunoprecipitation assays, we found that the homeotic transcription factor ARX interacts with the histone demethylase PHF8, indicating that this transcriptional axis is highly intersected. Furthermore, the functional impact of pathogenic mutations of ARX , KDM5C , PHF8 and ZNF711 was tested in lymphoblastoid cell lines (LCLs) derived from children with varying levels of syndromic ID establishing the direct correlation between defects in the KDM5C-H3K4me3 pathway and ID severity. These findings reveal novel insights into epigenetic processes underpinning NDD pathogenesis and provide new avenues for assessing developmental timing and critical windows for potential treatments.

Background: The application of single-cell RNA sequencing (scRNASeq) represents a unique approach to identify hundreds to millions of cells in mammalian cortical multilayers at different stages of embryogenesis. ScRNASeq technology applied to neurological studies requires the use of fresh starting materials because standard cryopreservation methods do not guarantee high viability of cortical primary cells derived from dissected brain areas., New Method: Here we set up and validate an innovative strategy to perform scRNASeq studies in cryopreserved primary cortical cells isolated from E15.5 mouse embryo. In order to freeze cortical primary cells, we have employed Neurostore, a medium able to guarantee high viability and cell composition of embryonic cortex after thawing., Comparison With Existing Methods: We showed for the first time the possibility to run scRNASeq experiments on primary cortical cells in an off-line set-up, ensuring cellular integrity and diversity., Results: By trypan blue assay and flow cytometry analysis, we found that Neurostore-cryopreserved cortical cells showed approximately 95 % of viability. Satisfactory RNA recovery and cDNA libraries were achieved. Transcriptome sequencing of 35,763 cryoconserved single cells yielded a robust data-set, identifying 25 cell clusters in three biological samples. Prevalence of peculiar neural populations before and after the cryopreservation-resuscitation procedure was verified by marker gene expression and immunofluorescence analysis., Conclusions: Our findings support the evidence that frozen primary cortical cells can be successfully employed in scRNASeq experiments allowing an unprecedented flexibility in experimental procedures, such as sample preparation and subsequent processing steps performed in different locations., (Copyright © 2020 Consiglio nazionale delle ricerche CNR Italy. Published by Elsevier B.V. All rights reserved.)

Detecting the ultrastructure of brain tissue in human archaeological remains is a rare event that can offer unique insights into the structure of the ancient central nervous system (CNS). Yet ancient brains reported in the literature show only poor preservation of neuronal structures. Using scanning electron microscopy (SEM) and advanced image processing tools, we describe the direct visualization of neuronal tissue in vitrified brain and spinal cord remains which we discovered in a male victim of the AD 79 eruption in Herculaneum. We show exceptionally well preserved ancient neurons from different regions of the human CNS at unprecedented resolution. This tissue typically consists of organic matter, as detected using energy-dispersive X-ray spectroscopy. By means of a self-developed neural image processing network, we also show specific details of the neuronal nanomorphology, like the typical myelin periodicity evidenced in the brain axons. The perfect state of preservation of these structures is due to the unique process of vitrification which occurred at Herculaneum. The discovery of proteins whose genes are expressed in the different region of the human adult brain further agree with the neuronal origin of the unusual archaeological find. The conversion of human tissue into glass is the result of sudden exposure to scorching volcanic ash and the concomitant rapid drop in temperature. The eruptive-induced process of natural vitrification, locking the cellular structure of the CNS, allowed us to study possibly the best known example in archaeology of extraordinarily well-preserved human neuronal tissue from the brain and spinal cord., Competing Interests: The authors have declared that no competing interests exist.

Unstable repeat disorders comprise a variable group of incurable human neurological and neuromuscular diseases caused by an increase in the copy number of tandem repeats located in various regions of their resident genes. It has become clear that dense DNA methylation in hyperexpanded non-coding repeats induces transcriptional silencing and, subsequently, insufficient protein synthesis. However, the ramifications of this paradigm reveal a far more profound role in disease pathogenesis. This review will summarize the significant progress made in a subset of non-coding repeat diseases demonstrating the role of dense landscapes of 5-methylcytosine (5mC) as a common disease modifier. However, the emerging findings suggest context-dependent models of 5mC-mediated silencing with distinct effects of excessive DNA methylation. An in-depth understanding of the molecular mechanisms underlying this peculiar group of human diseases constitutes a prerequisite that could help to discover novel pathogenic repeat loci, as well as to determine potential therapeutic targets. In this regard, we report on a brief description of advanced strategies in DNA methylation profiling for the identification of unstable Guanine-Cytosine (GC)-rich regions and on promising examples of molecular targeted therapies for Fragile X disease (FXS) and Friedrich ataxia (FRDA) that could pave the way for the application of this technique in other hypermethylated expansion disorders., Competing Interests: Graphical Abstract: DNA methylation-induced transcriptional silencing in unstable repeat diseases. Three specific epigenetic mechanisms have been identified, depending on the location of the expanded repeat in the promoter or in the 5’ regulatory region (a), intronic region (b) and 3’ regulatory region (c) of the resident gene. Abbreviations: 5’ untranslated region (5’UTR); 3’ untranslated region (3’UTR).

A disproportional large number of neurodevelopmental disorders (NDDs) is caused by variants in genes encoding transcription factors and chromatin modifiers. However, the functional interactions between the corresponding proteins are only partly known. Here, we show that KDM5C, encoding a H3K4 demethylase, is at the intersection of transcriptional axes under the control of three regulatory proteins ARX, ZNF711 and PHF8. Interestingly, mutations in all four genes (KDM5C, ARX, ZNF711 and PHF8) are associated with X-linked NDDs comprising intellectual disability as a core feature. in vitro analysis of the KDM5C promoter revealed that ARX and ZNF711 function as antagonist transcription factors that activate KDM5C expression and compete for the recruitment of PHF8. Functional analysis of mutations in these genes showed a correlation between phenotype severity and the reduction in KDM5C transcriptional activity. The KDM5C decrease was associated with a lack of repression of downstream target genes Scn2a, Syn1 and Bdnf in the embryonic brain of Arx-null mice. Aiming to correct the faulty expression of KDM5C, we studied the effect of the FDA-approved histone deacetylase inhibitor suberanilohydroxamic acid (SAHA). In Arx-KO murine ES-derived neurons, SAHA was able to rescue KDM5C depletion, recover H3K4me3 signalling and improve neuronal differentiation. Indeed, in ARX/alr-1-deficient Caenorhabditis elegans animals, SAHA was shown to counteract the defective KDM5C/rbr-2-H3K4me3 signalling, recover abnormal behavioural phenotype and ameliorate neuronal maturation. Overall, our studies indicate that KDM5C is a conserved and druggable effector molecule across a number of NDDs for whom the use of SAHA may be considered a potential therapeutic strategy., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

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., (© 2013 WILEY PERIODICALS, INC.)

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., (Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

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.

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.

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.

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.

Background: Cognitive impairments are heterogeneous conditions, and it is estimated that 10% may be caused by a defect of mental function genes on the X chromosome. One of those genes is Aristaless related homeobox (ARX) encoding a polyA-rich homeobox transcription factor essential for cerebral patterning and its mutations cause different neurologic disorders. We reported on the clinical and genetic analysis of an Italian family with X-linked mental retardation (XLMR) and intra-familial heterogeneity, and provided insight into its molecular defect., Methods: We carried out on linkage-candidate gene studies in a new MRX family (MRX87). All coding regions and exon-intron boundaries of ARX gene were analysed by direct sequencing., Results: MRX87 patients had moderate to profound cognition impairment and a combination of minor congenital anomalies. The disease locus, MRX87, was mapped between DXS7104 and DXS1214, placing it in Xp22-p21 interval, a hot spot region for mental handicap. An in frame duplication of 24 bp (ARXdup24) in the second polyAlanine tract (polyA_II) in ARX was identified., Conclusion: Our study underlines the role of ARXdup24 as a critical mutational site causing mental retardation linked to Xp22. Phenotypic heterogeneity of MRX87 patients represents a new observation relevant to the functional consequences of polyAlanine expansions enriching the puzzling complexity of ARXdup24-linked diseases.

Background: The association between premature ovarian failure (POF) and the FMR1 repeat number (41> CGGn< 200) has been widely investigated. Current findings suggest that the risk estimation for POF can be calculated in the offspring of women with pre-mutated FMR1 alleles., Case Presentation: We describe the coexistence in a large Italian kindred of Fragile X syndrome and familial POF in females with ovarian dysfunctions who carried normal or expanded FMR1 alleles. Genetic analysis of the FMR1 gene in over three generations of females revealed that six carried pre-mutated alleles (61-200), of which two were also affected by POF. However a young woman, who presented a severe ovarian failure with early onset, carried normal FMR1 alleles (<40). The coexistence within the same family of two dysfunctional ovarian conditions, one FMR1-related and one not FMR1-related, suggests that the complexity of familial POF conditions is larger than expected., Conclusion: Our case study represents a helpful observation and will provide familial cases with heterogeneous etiology that could be further studied when candidate genes in addition to the FMR1 premutation will be available.

NEMO/IKKgamma gene, which is responsible of two allelic diseases in human, EDA-ID and IP, encodes for a protein with a central regulatory role in the activation of the NF-kB pathway. We here provide insights into the molecular mechanism governing NEMO/IKKgamma expression. We mapped 4 distinctive NEMO/IKKgamma transcription start sites each corresponding to an alternative first exon, controlled by two conserved promoters. A distal promoter, named promoter A, located 10 kb upstream of the coding region and a proximal promoter, promoter B, with strong bi-directional activity driving also the transcription of G6PD gene in the opposite direction. The promoter B is housekeeping, it is embedded in a CpG island, required for proper expression and it is down-regulated by methylation. The promoter A is active in cells of hepatic origin and it directs transcription of the main NEMO/IKKgamma 5' UTR alternative transcript in liver, which starts at a tissue-specific site. Qualitative and quantitative expression analysis revealed that each NEMO/IKKgamma 5' UTR alternative transcript has different expression profiles indicating that the control of NEMO/IKKgamma expression is mediated through tissue-specific transcription initiation sites and multiple regulatory regions.

Background: Deletions of Xq chromosome are reported for a number of familial conditions exhibiting premature ovarian failure (POF) and early menopause (EM)., Methods and Results: We describe the inheritance of an interstitial deletion of the long arm of the X chromosome associated with either POF or EM in the same family. Cytogenetic studies and heterozygosity mapping by quantitative fluorescent PCR revealed a 46,X,del(X)(q26.2-q28) karyotype in a POF female, in her EM mother, and also in her aborted fetus with severe cardiopathy. Applying a microsatellite approach, we have narrowed the extension of an identical interstitial deletion located between DXS1187 and DXS1073. These data, in line with other mapped deletions, single out the proximal Xq28 as the region most frequently involved in ovarian failure. We also propose that other factors may influence the phenotypic effect of this alteration. Indeed, skewed X inactivation has been ascertained in EM and POF to be associated with different X haplotypes., Conclusion: Our analysis indicates that Xq26.2-q28 deletion is responsible for gonad dysgenesis in a family with EM/POF. The dissimilar deletion penetrance may be due to epigenetic modifications of other X genes that can contribute to human reproduction, highlighting that ovarian failure should be considered as a multifactorial disease.

Incontinentia Pigmenti (IP) is an X-linked genodermatosis that is lethal for males and present in females with abnormal skin pigmentation and high variable clinical signs, including retinal detachment, anodontia, alopecia, nail dystrophy and nervous system defects. The NF-kappaB essential modulator (NEMO) gene, responsible for IP, encodes the regulatory subunit of the IkappaB kinase (IKK) complex required for nuclear factor kappaB (NF-kappaB) activation. We analyzed the NEMO gene in 122 IP patients and identified mutations in 83 (36 familiar and 47 sporadic cases). The recurrent NEMO exon 4-10 deletion that is the major cause of the disease was present in 73 females (59.8%). In addition 10 point alterations (8.2% of females) were identified: three frameshift, three nonsense, three missense and one in-frame deletion of a single amino acid. We measured the effects of these NEMO point-mutations on NF-kappaB signaling in nemo(-/-) deficient murine pre-B cells. A mutation in the N-terminal domain, required for IKK assembly, reduced but did not abolish NF-kappaB activation following lipopolysaccharide stimulation. Mutations that disrupt the C-terminal domain, required for the recruitment of upstream factors, showed lower or no NF-kappaB activation. A phenotype score based on clinical features of our IP patients was applied for summarizing disease severity. The score did not correlate with mutation type or domain affected indicating that other factors influence the severity of IP. Such a factor is likely to be X-inactivation. Indeed, 64% of our patients have extremely skewed X-inactivation pattern (>/=80 : 20). Overall IP pathogenesis thus depends on a combination of X-inactivation and protein domain that recruit upstream factors and activate NF-kappaB.

X-linked nonspecific mental retardation (MRX) accounts for approximately 25% of mental retardation in males. A number of MRX loci have been mapped on the X chromosome, reflecting the complexity of gene action in central nervous system (CNS) specification and function. Eleven MRX genes have been identified, but many other causative loci remain to be refined to the single gene level. In 21 MRX families, the causative gene is located in the pericentromeric region; and we report here the identification by linkage analysis of a further such locus, MRX81. The new MRX locus was identified by two- and multi-point parametric analysis carried out on a large Italian family. Tight linkage of MRX81 to DNA markers ALAS2, DXS991, and DXS7132 was observed with a maximum LOD score of 3.43. Haplotype construction delineates an MRX81 critical region of 8 cM, the smallest MRX pericentromeric interval so far described, between DXS1039 and DXS1216, and placing it in Xp11.2-Xq12. So far, automated sequencing of two candidates in the region, the MRX gene oligophrenin (OPHN1) and the brain-specific ephrinB1 (EFNB1) gene, in DNA from affected males excluded their candidacy for MRX81, suggesting a novel disease gene., (Copyright 2003 Wiley-Liss, Inc.)

Membrane-associated guanylate kinase (MAGUK) proteins are cell-cell contact organizing molecules that mediate targeting, clustering and anchoring of proteins at synapses and other cell junctions. MAGUK proteins may contain multiple protein-protein interaction motifs including PDZ, SH3 and guanylate kinase (GuK) domains. In this study, we performed a detailed analysis of the expression pattern of MPP4, a recently described member of the MAGUK protein family. We confirmed that this gene is highly expressed in retina, and demonstrate that it is also present, at lower levels, in brain. We identified a new retina specific isoform encoding a predicted protein lacking 71 amino acids. This protein region contains a newly identified L27 domain, another module playing a role in protein-protein interaction. By RNA in situ hybridization, Mpp4 expression was found to be localized to photoreceptor cells in postnatal retina. The MPP4 gene is localized to chromosome 2, in band 2q31-33, where a locus for autosomal recessive retinitis pigmentosa (RP26) has been mapped. Mutation analysis of the entire open reading frame of the MPP4 gene in a RP26 family revealed no pathologic mutations. In addition, we did not identify mutations in a panel of 300 unrelated patients with retinitis pigmentosa.