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NEMO/IKK? is the regulatory subunit of the I?B Kinase (IKK) complex, required for the activation of the NF-?B pathway, which is involved in a variety of key processes, including immunity, inflammation, differentiation, and cell survival. Termination of NF-?B activity on specific -?B responsive genes, which is crucial for the resolution of inflammatory responses, can be achieved by direct degradation of the chromatin-bound NF-?B subunit RelA/p65, a process mediated by a protein complex that contains Copper Metabolism Murr1 Domain 1 (COMMD1). In this study, we identify COMMD7, another member of the COMMDs protein family, as a novel NEMO-interacting protein. We show that COMMD7 exerts an inhibitory effect on NF-?B activation upon TNF? stimulation. COMMD7 interacts with COMMD1 and together they cooperate to down-regulate NF-?B activity. Accordingly, termination of TNF?-induced NF-?B activity on the -?B responsive gene, Icam1, is defective in cells silenced for COMMD7 expression. Furthermore, this impairment is not greatly increased when we silence the expression of both COMMD7 and COMMD1 indicating that the two proteins participate in the same pathway of termination of TNF?-induced NF-?B activity. Importantly, we have demonstrated that COMMD7's binding to NEMO does not interfere with the binding to the IKKs, and that the disruption of the IKK complex through the use of the NBP competitor impairs the termination of NF-?B activity. We propose that an intact IKK complex is required for the termination of NF-?B-dependent transcription and that COMMD7 acts as a scaffold in the IKK-mediated NF-?B termination.

X-linked Intellectual Disability (XLID) is a group of genetically heterogeneous disorders caused by mutations in genes on the X chromosome. Disease mutations in ~10% of X chromosome genes are implicated in causing XLID disorders in ~50% of known and suspected XLID disorders. Mutations in Lysine-specific demethylase 5C (KDM5C) gene, located at Xp11.22, have been reported as an important cause of both syndromic and non-syndromic X-linked intellectual disability (XLID) in males. KDM5C is a chromatin remodelling regulator with histone demethylase activity for di- and trimethylated histone 3 lysine 4 (H3K4me2 and H3K4me3), acting as transcriptional repressor during brain development and neuronal maturation. With Regulatory Element-1-Silencing Transcription factor (REST), a critical regulator of the spatio-temporal transition of neural progenitors to neurons, KDM5C co-occupies the promoters of a subset of REST target genes. We recently identified a disease path, linking functionally KDM5C to another XLID/Epilepsy gene, encoding the homeotic transcription factor ARX, whose mutations impair severely KDM5C transcript regulation.Expanding our study, we analysed two additional XLID proteins that also bind the KDM5C promoter. Our findings open new perspectives towards the exploitation of rational strategies to treat the growing group of ID and cognition diseases that are caused by chromatin and/or transcriptional defects.

Alterazioni del gene responsabile dell'Incontinentia pigmenti possono provocare, in età scolare, difficoltà nella lettura e nel calcolo. Lo suggerisce, per la prima volta, uno studio dell'Igb-Cnr di Napoli, in collaborazione con l'Irccs-Fondazione Santa Lucia di Roma. I risultati sono stati pubblicati su Plos One

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 NF-kB signaling. In more than 80% of cases, IP is due to recurrent or non-recurrent 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 micro-indel (c.436_471delinsT, p.Val146X) arising through a DNA-replication-repair Fork-Stalling-and-Template-Switching (FoSTeS) and Microhomology-Mediated-End-Joining (MMEJ) 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), while 7/21 predict a partial loss of NEMO/IKKgamma activity (2 splicing and 5 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

Polyalanine expansion mutations of Aristaless-related homeobox gene (ARX) cause a spectrum of X-chromosome phenotypes with Intellectual Disabilily (ID) and various forms of malignant paediatric epilepsy. We have established that ARX regulates and binds a CNE element in the 5' region of the lysine (K)-specific demethylase 5C (KDM5C), a known XLID gene involved in chromatin remodeling and neuronal maturation. By in vitro studies, we have analyzed five Polyalanine mutants establishing a decreased trans-activating activity and a reduced, but not abolished, binding to the KDM5C regulatory region. By quantitative RT-PCR, we have showed in murine Arx-KO ES cells and neurospheres a dramatic downregulation of the Kdm5C mRNA levels that leads to a decrease of the KDM5C protein. In Arx KO GABA-oriented model, which presents severe abnormalities in dendrite formation and GABAergic maturation, we have found a KDM5C reduction in coupling with a global increase of H3K4me3 signalling, potentially due to a compromised KDM5C activity. Since H3K4me3 is the hallmark of open chromatin, ARX-dependent KDM5C defects could compromise cyclical rounds of methylation-demethylation and consequently the chromatin remodelling. Starting from these data, we are now able to break up a unique ARX-dependent epigenetic road involved in epileptogenesis, suggesting that the molecular pathogenesis of ARX Polyalanine mutations may be in part caused by aberrant histone demethylation as a result of KDM5C defect. As chromatin modifications are reversible, it is possible that epigenetic drugs could compensate KDM5C-H3K4me3 deregulation opening further studies to cure or ameliorate ARX Polyalanine-related epilepsy.

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-?, IL-1?, TGF-? and lysozyme c-type). The constitutive expression of ?-actin was detected in all developmental stages. IL-1? and TNF-? transcripts were detected from 4 week post-fertilization onwards, whereas TGF-? 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-? and lysozyme c-type transcripts were expressed in all tissues. IL-1? and TGF-? 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.

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.

Malattie ereditarie diverse, uno stesso gene responsabile. Arriva dalla ricerca di base una nuova scoperta che chiarisce il meccanismo di ricombinazione del gene Ikbkg/Nemo, già noto per essere coinvolto in una rara patologia ereditaria chiamata Incontinentia Pigmenti, e che ora risulta implicato anche in alcune forme di ritardo mentale associate al cromosoma X. Lo hanno dimostrato un gruppo di ricercatrici dell'Istituto di genetica e biofisica (Igb) del Cnr di Napoli, composto da Matilde Valeria Ursini, Maria Giuseppina Miano e Francesca Fusco, con uno studio pubblicato sulla prestigiosa rivista della Società americana di genetica umana, l''American Journal of Human Genetics'.

Diminished Ovarian Reserve (DOR) is a heterogeneous disorder belonging to the gonad failure spectrum, characterized by low numbers of remaining oocytes in the ovaries, usually accompanied by high follicle stimulating hormone (FSH) level. Such diminished ovarian reserve is thought to reflect both a decreased number of eggs and a decrease in egg quality. DOR aetiology factors are multiple and different, such as genetic factors, aging, autoimmune disorders, adrenal gland impairment, iatrogenic, e.g., due to radiation or chemotherapy. At present the exact genetic cause of DOR is still unknown, although DOR with a severe reduction of reproductive performance was reported with a significant high incidence (44.4%) in females carrying X chromosome mosaicisms (45,X/46,XX; 46,XX/47,XXX) without balanced autosomal rearrangements. We report the first unbalanced translocation with segmental loss and gain of genomic fragments associated with DOR. Array CGH and FISH studies revealed a partial Xq monosomy and a partial 18q trisomy resulting from an unbalanced t(X;18) translocation. Real time experiments showed a Xq27 monosomy starting at position 141.2 Mb and a 18q22 duplication starting at position 64.5 Mb. Sequence inspection revealed the presence of a genomic context of LINE-1 (L1) clusters, at both the Xq27 and the 18q22 breakpoints. This structure might increase the likelihood of mispairing, unusual crossover and therefore, gain or loss of genomic materials. To further characterize the outcome of DOR rearrangement, the status of X chromosome inactivation was investigated. Androgen Receptor assay and late-replication DNA analysis revealed a preferential inactivation of rearranged X:18 chromosome, therefore completely skewed inactivation and late replication might mask the outcome of the 18 trisomy as well as of the X monosomy. Our patient has only DOR and no other unusual clinical features. This finding is in agreement with other reports that demonstrated that in X:autosome unbalanced translocation, inactivation signals of X-inactivation centre spread across the X:autosome boundary therefore, resulting in various attenuated phenotypes. Our data include DOR condition among the gonad failure spectrum of diseases linked to X-chromosome abnormalities, and confirm the feature of the Xq27-28 as a region of high genome instability. The hypothesis that the DOR phenotype occurs as a result of faulty meiotic pairing and recombination will be discussed.

NF-?B activity is regulated by interaction with inhibitory proteins (I?B). The canonical p65/p50 heterodimer is bound to I?B? that inhibits the NF-?B activity by masking its NLS domain and so blocking NF-?B ability to bind DNA. NF-?B-I?B? complex is continuously shuttling between nucleus and cytoplasm. Its nuclear export rate exceeds its import rate and thus the complex is generally cytoplasmic. NF-kB signalling studies during oocyte maturation have been limited. The upregulated I?B? expression has been observed in murine oocyte aging suggesting that NF-?B pathway deregulation may be related to oocyte competence. In male gamete development, NF-?B is activated in pachitene stage and in the following germinal differentiation steps triggering specialized genes expression. We analysed I?B? transcript levels in meiotic arrested bovine oocytes at different stages of maturation (diplotene of prophase I and metaphase II) and in early embryogenesis. We included three expressed oocyte genes: BMP15, GDF9, SPIN1. We examined ZAR1, specific oocyte maturation marker, whose transcript generally underwent degradation during meiosis. We observed a significant reduction of the I?B? mRNA during meiotic maturation. On the contrary, we observed that the amount of I?B? protein increased during the transition from immature to mature oocytes, while it was significantly decreased in embryos. Further analysis will be performed to establish how I?B? is regulated during developmental competence and if it is involved in human diseases such as premature aging and infertility.

Human chromosome Xq28 is a gene-rich region, which lies within the critical linkage interval for many human diseases and characterized by an unusual degree of genomic instability. Here we report the structural/functional analysis of a new Xq28 gene FUNDC2 that encodes for a small mitochondrial protein with unknown function. The gene maps approximately 4,1 kb centromeric to F8 and 6,5 kb telomeric to MTCP1. Comparative and chromosomal mapping indicates that FUNDC2 has duplicated to specific locations near the pericentromeric region of human chromosomes highlighting a phenomenon for paralogous genome evolution common to other Xq28 genes. FUNDC2 has ubiquitously expression in human embryos and adult tissues with high level in the heart and skeletal muscle. Its predicted protein shows high conservation from Archeabacteria to Human suggesting that it could be a member of a new family of functionally related genes. Its 189-amino-acid sequence appears rich of hydrophobic residues and lacks a signal-sequence for protein sorting or target membrane. In HeLa and COS cells, confocal immuno-fluorescence studies co-localized FUNDC2 protein to the mitochondria stained by anti-HSP60 antibody and pECFP-mito. We also performed subcellular fractionation by differential centrifugation and alkali treatment establishing that FUNDC2 is located in the mitochondria inner membrane. Based on a high-throughput yeast 2-hybrid screen, we found that Drosophila FUNDC2 orthologue interacts with mtacp1 (mitochondrial acyl carrier protein 1. Its human counterpart is NDUFAB1, a subunit of the mitochondrial respiratory Complex I that is the entry point for electrons donated from NADH. Mutations in the structural building blocks of Complex I have been detected in 40% of patients with Complex I deficiencies. Further investigation of FUNDC2 will contribute to understanding of its function as well as provide a new candidate for mitochondrial pathogenesis including those associated to Complex I deficiency.

NF-kB is a transcription factor involved in the regulation of gamete maturation, a complex process, which concludes meiosis. This process that sustains genome reprogramming and embryo development is associated to a specific expression pattern. NF-kB activity is primarily regulated by interaction with inhibitory proteins (IkB). The best-studied NF-kB-IkB interaction is IkBa-p50/RelA that inhibits the NF-kB activity by masking its NLS domain and so blocking the ability of NF-kB to bind DNA. NF-kB-IkBa complex is continuously shuttling between the nucleus and the cytoplasm, but its nuclear export rate exceeds its import rate and thus the complex is generally cytoplasmic. An upregulation of IkBa expression has been observed in association with murine oocyte aging suggesting that a deregulation of NF-kB pathway may be related to oocyte competence. However, NF-kB signalling studies during oocyte maturation have been limited. In male gamete development, NF-kB is activated in a stage-specific manner. It appears at pachitene and remains activated in the following germinal differentiation steps triggering specialized genes expression. We report on the study of IkBa in bovine oocytes at different meiotic stages that are transcriptionally quiescent: diplotene of prophase I and metaphase II. During this time, a subset of maternal transcript undergoes post-transcriptional regulation and is stored protected; when the protein is required, dormant transcripts can undergo polyadenylation and translation, and rapid degradation. Immature oocytes arrested in diplotene were obtained from bovine cumulus ovary and in vitro meiosis progression were performed with FSH and estradiol hormones. Using Real-TimePCR, we examined IkBa transcript levels in immature and mature oocyte pools. We included four expressed oocyte genes that are BMP15, GDF9, SPIN1, FMR1. We examined also ZAR1, as a specific oocyte maturation marker, whose transcript generally underwent degradation during meiosis. Our data showed a reduction of IkBa transcript during in vitro nuclear maturation suggesting that oocyte competence could be regulated through NF-kB pathway. Western-blot analysis in immature and mature oocytes is currently carrying out to correlate transcript and protein levels. Further analysis will be performed to establish how IkBa is regulated during developmental competence and if it is involved in human diseases such as premature aging and infertility.

Anomalie delle funzioni cognitive costituiscono un gruppo eterogeneo di neuropatologie congenite. Il 10% delle forme genetiche di Ritardo Mentale (MR) è causato da mutazioni in geni X-linked. Di questi, ARX rappresenta un sito hot spot per MR. Esso mappa in Xp22 e codifica un fattore trascrizionale omeotico, essenziale per lo sviluppo neuronale. Abbiamo eseguito un'analisi clinico-genetica di una famiglia italiana con Ritardo Mentale X-linked. I maschi affetti mostrano un'elevata eterogeneità intra-familiare con handicap mentale e piccole alterazioni neurologiche. L'analisi molecolare di ARX ha evidenziato la segregazione di una duplicazione di 24 bp dell'esone 2 (ARXdup24) che determina un'espansione nel secondo tratto di poli-Alanine. Mediante analisi in silico della struttura secondaria, ARXdup24 sembra incrementare l'idrofobicità del polyA_II alterandone la struttura ad ? elica. Attraverso uno studio di genomica comparativa, è stata analizzata la storia evolutiva di ARX. Applicando il metodo Ka/Ks, emerge che ARX ha subito una pressione selettiva positiva nei Roditori. Riguardo ai quattro tratti di polyA che caratterizzano ARX, essi appaiono in tempi differenti durante la speciazione. In particolare, poliA_I e poliA_II, siti per mutazioni MR, compaiono a partire dai Mammiferi placentali. Ciò suggerisce un'evoluzione di ARX per aggiunta di domini e/o funzioni pleiotropiche parallelamente all'acquisizione di espressioni cognitive, peculiari dei Mammiferi superiori.

Decline aging-oocyte competence and premature ovarian failure (POF) are common factors in human female infertility. In both conditions, follicular senescence and ovarian dysfunctions occur at different speed. Understanding how an immature oocyte transforms into an egg during oocyte maturation is critical for knowledge of fertility and decline-oocyte aging. This process guides the achievement of the final competence essential to fertilization and zygote division, through evolutionary conserved nuclear and cytoplasmatic events. The maturation of mammalian oocyte requires a co-ordinated programme of gene expression events that itself regulates the development of ovarian follicles. Starting from selected genes involved in POF disorders and oocyte aging, we are carrying out a project directed to characterize the expression level of bovine ortologous genes during oocyte maturation. The cow constitutes the best surrogate model for studying reproductive human disorders given the great similarity between the two reproduction systems (such as reproduction cycle, ovulation rate, ovarian maturation, gamete biology, etc.). We assayed systematically the expression level of each transcript in bovine oocytes at different stage of maturation (immature-MI stage and mature-MII stage) coming from young (12 months old) and older animals (13 years old). Oocyte pools (20-100) were collected by puncturing follicles from ovaries of cows and RNA extraction methods were carried out using high recovery rate methods. This presentation showed our preliminary results on those transcripts expressed during meiosis I and II. We believe that they may represent a good starting point to investigate on the presence of specific mRNAs in the former maturation stages and assay how they vary during decline- competence in aging oocyte.

L'identificazione di geni associati a ritardo mentale (MR) costituisce uno strumento fondamentale nello studio delle basi molecolari della plasticità neuronale. Questa patologia dello sviluppo del sistema nervoso centrale (SNC) altera gravemente le capacità di apprendimento e memoria. Tra le forme genetiche, quelle legate al cromosoma X (MRX) costituiscono circa il 30% sottolineando lo straordinario contributo di questo cromosoma allo sviluppo delle funzioni cognitive nell'Uomo. Numerosi sono i geni e i loci MRX finora identificati, con regioni hot spot nelle bande Xp22, Xper e Xq28. Riportiamo qui l'analisi genetica di una famiglia affetta da una severa forma di MR non sindromico con segregazione X-linked e il mappaggio del locus corrispondente. Mediante X chromosome wide-screening abbiamo stabilito un aplotipo malattia tra i marcatori DXS987 e DXS1214. L'analisi statistica parametrica a due punti ha stabilito un valore di lodscore massimo di 2.43 per DXS207. Sulla base di questi dati, abbiamo posizionato la regione critica tra Xp22-Xp21.3, corrispondente ad uno degli intervalli hot spot per mutazioni MRX. Tre sono i geni malattia già identificati che mappano nel locus stabilito: RSK2 codificante una serina/treonina chinasi che regola la struttura della cromatina, ARX che codifica un fattore di trascrizione della famiglia homeobox, e infine IL1RAPL codificante un recettore per la segnalazione cellulare di NF-kB nel SNC. Attualmente è in corso la ricerca di mutazioni puntiformi in ognuno di essi mediante sequenziamento automatico diretto e analisi di segregazione. Abbiamo inoltre verificato lo stato di metilazione del cromosoma X allo scopo di stabilire nelle femmine portatrici l'esistenza di un'associazione preferenziale tra aplotipo malattia e livello di inattivazione. I dati a nostra disposizione escludono l'esistenza di una metilazione preferenziale del cromosoma X mutato, suggerendo il coinvolgimento di un gene non necessario alla sopravvivenza cellulare.

Il ritardo mentale X linked non sindromico (MRX) ha una frequenza di 1/600 individui nella popolazione umana. E' una patologia del SNC causata da una alterazione nello sviluppo delle capacità cognitive, spesso causato da mutazioni in geni che regolano la plasticità neuronale. Peculiarità di questa malattia è l'elevata eterogeneità genetica. Finora sono stati identificati fino a 16 geni MRX, e come suggerito dall'elevata densità di loci malattia, altri rimangono ancora sconosciuti, soprattutto nella regione pericentromerica del cromosoma X (Xper). Nel nostro laboratorio, mediante studi di linkage genetico parametrico, abbiamo identificato il locus MRX81. Esso mappa nella regione Xper, tra i marcatori ricombinanti DXS1039 e DXS1216 in p11.2 e q12 rispettivamente, indicando la presenza del gene malattia in una regione critica di 8 cM. Lo scopo del presente studio è stato restringere i confini del locus MRX81 e selezionare accuratamente i geni candidati da sottoporre a ricerca di mutazioni. Abbiamo analizzato nuovi marcatori polimorfici distribuiti ogni 1-2 cM nell'intervallo MRX81 precedentemente definito. La genotipizzazione in automatico effettuata su 24 individui della famiglia MRX81 ha permesso l'identificazione di un nuovo aplotipo associato alla malattia per i marcatori DXS1199-ALAS2-DXS8032-DXS991-DXS7132. L'analisi di linkage ha fornito un massimo multipoint LOD score di 3.77 nella regione da ALAS2 a DXS991. Dai nuovi dati di linkage abbiamo potuto restringere la ricerca del gene MRX81 da un intervallo di 8 cM a 3 cM e quindi costruire una mappa fisica della nuova regione critica. E' in corso l'analisi di geni e trascritti che mappano al suo interno, selezionati sulla base del coinvolgimento in altre forme MRX, della loro espressione in ippocampo e/o del loro coinvolgimento in funzioni cellulari correlate alla regolazione della plasticità.

Incontinentia Pigmenti (IP) is an X-linked genodermatosis, lethal for males and presents in females with abnormal skin pigmentation and high variable clinical signs. NEMO gene, responsible for IP, encodes the regulatory subunit of the IKK complex for NF-kB activation. We analyzed NEMO in 122 IP patients from EC and identified mutations in 83 (36 familiar and 47 sporadic cases). However, NEMO mutations isolated in this study account for the majority but not all clinical cases. The recurrent NEMO 4-10 deletion that is the major cause of the disease was present in 73 females. In addition 10 point alterations were identified. A phenotype scoring system was used for correlation between the mutation type and the clinical IP presentation. To assess genotype-phenotype correlation in IP patients we listed features that are typical for IP but not consistent in all patients and could therefore reflect the severity of the syndrome. For calculating the score we included only defects relative to nervous system, eyes, teeth, hair and nail, that are most frequently, but not always, observed in IP patients. If a genotype-phenotype correlation does exist, females who carry loss-of-function mutations would be expected to have the most severe phenotype. In our study, however, this is not the case: 50 patients with the same exon 4-10 NEMO deletion have severity score ranging from 1 to 8. In addition, we found that mutations preserving some activity (i.e. same point mutations) show an atypical phenotype characterized by involvement of much more tissues than classical IP phenotype. Thus, the detected mutation spectrum do not allow genotypephenotype correlation, leading to the hypothesis that other factors may modulate the IP pathogenesis. 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-kB.

The cytogenetic region q28 of the human X chromosome has great health relevance in Medical Genetics because it contains several disease loci whose causative genes are still unknown. In particular, two hereditary diseases of the central nervous system (CNS) map in the distal Xq28, X-linked polymicrogyria (PMGX) and a form of non specific X mental retardation (MRX72). The difficulty to isolate new Xq28 disease genes is remarkable because of the paucity of polymorphic markers available for linkage study. Thus, we have initiated a systematic search of new short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs) in 1.6 Mb of distal Xq28, between G6PD gene and the telomeric repeat TTAGGG. Starting from the nucleotide sequence, performed in our laboratory, we have selected new microsatellites. Sequence analysis of the genomic clones RP11-211L10, RP11-196H18, RP11-273L24, RP11-405N23 and RP11-53A12 was performed by CENSOR software looking for DNA repeated elements. Specific primer pairs were designed for each potential STRs and used in QF-PCR reactions. DNA pools from males of 40 CEPH families were genotyped to establish the number of alleles and their frequencies in the Caucasian population. Based on this strategy, we are able to establish the index of heterozigosity and consequently the power of each new Xq28 marker to be used in linkage studies. We found significant evidences of the presence of several new STRs that we are currently using to narrow down the MRX72 locus. Furter study are in progress to construct haplotypes of distal Xq28 to evaluate linkage disiquilibrium.

Familial incontinentia pigmenti (IP) is a rare X-linked dominant disorder that affects ectodermal tissues. Over 90% of IP carrier females have a recurrent genomic deletion of exons 4-10 of the NEMO (IKBKG-IKKgamma) gene, which encodes a regulatory component of the IkB kinase complex, required to activate the NF-kB pathway. In IP, mutations in NEMOlead to the complete loss of NF-kB activation creating a susceptibility to cellular apoptosis in response to TNF-alpha. This condition is lethal for males during embryogenesis while females, who are mosaic as a result of X-inactivation, can survive. Recently, a second nonfunctional copy of the gene, DeltaNEMO, was identified, opposite in direction to NEMO in a 35.5-kb duplicated sequence tract. PCR-based detection of the NEMO deletion is diagnostic for IP disease. However, we present instances in which ex 4-10 DeltaNEMO pseudogene deletion occurs in unaffected parents of two females with clinically characteristic IP. These were missed by the currently standard PCR-based method, but can be easily discriminated by a new PCR-based test reported here that permits unambiguous molecular diagnosis and proper familial genetic counseling for IP.

Severe combined immunodeficiency (SCID) is a heterogeneous group of disorders characterized by defect of T- and B-cell immunity. In many cases of autosomal recessive SCID, thus far described, the molecular alteration involves genes encoding for molecules that participate in the signal transduction. We report on a patient affected by a combined immunodeficiency, characterized by severe T-cell functional impairment, in spite of a close to normal number of circulating mature type T and B cells. NK cells were absent. Associated with the immunodeficiency, this patient also showed short stature characterized by very low growth velocity, delayed bone age and absence of increase of the plasma levels of Insulin growth factor-I (IGF-I) after growth hormone (GH) in vivo stimulation indicating peripheral hyporesponsiveness to GH. Evaluation of the protein tyrosine phosphorylation events occurring following either T-cell receptor (TCR) or GH receptor (GHR) triggering revealed striking abnormalities. No molecular alteration of GHR gene was found, thus suggesting the presence of postreceptorial blockage. Mutational screening and expression analysis failed to reveal any molecular alteration of JAK2 and STAT 5 A/B genes thus ruling out the involvement of these genes in the pathogenesis of this form of SCID. Mutational analysis of IL2Rgamma chain gene revealed the presence of a L183S missense mutation, thus indicating an atypical and a more complex clinical presentation of this X-linked form of SCID. At our knowledge, this is the first report on the GH hyporesponsiveness in this disease.