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International audience; Neutrophilic dermatoses (ND) are a group of inflammatory skin conditions characterized by a neutrophilic infiltrate on histopathology with no evidence of infection. ND are classified based upon the localization of neutrophils within the skin and clinical features. Recent findings suggest that ND are due to two main mechanisms: i) a polyclonal hereditary activation of the innate immune system (polygenic or monogenic); or ii) a clonal somatic activation of myeloid cells such as encountered in myelodysplastic syndrome or VEXAS syndrome. ND belong to internal medicine as a great number of patients with ND suffer from an underlying condition (such as hematological malignancy, inflammatory bowel disease, auto-immune and auto-inflammatory diseases). ND are diagnoses of exclusion and physicians should always consider differential diagnoses, particularly skin infections. Here, we review the pathophysiology and classification of the main ND (i.e., subcorneal pustular dermatosis (Sneddon-Wilkinson Disease) and Intercellular IgA dermatoses, aseptic pustulosis of the folds, Sweet syndrome, neutrophilic eccrine hidradenitis, pyoderma gangrenosum, erythema elevatum diutinum, neutrophilic urticarial dermatosis and neutrophilic panniculitis), their clinical and histopathological features, and we highlight the investigations that are useful to identify ND-associated diseases and to exclude the differential diagnoses.

Background: Rare diseases (RDs) affect nearly 3 million people in France and at least 26-30 million people in Europe. These diseases, which represent a major medical concern, are mainly of genetic origin, often chronic, progressive, degenerative, life threatening and disabling, accounting for more than one third of all deaths occurring during infancy. In this context, there are needs for coordinated information on RDs at national /international levels, based on high quality, interoperable and sharable data. The main objective of the RaDiCo (Rare Disease Cohorts) program, coordinated by Inserm, was the development of RD e-cohorts via a national platform. The cohort projects were selected through a national call in 2014. The e-cohorts are supported by an interoperable platform, equivalent to an infrastructure, constructed on the "cloud computing" principle and in compliance with the European General Data Protection Regulation. It is dedicated to allow a continuous monitoring of data quality and consistency, in line with the French Health Data Hub. Results: Depending on cohorts, the objectives are to describe the natural history of the studied RD(s), establish phenotype-genotype correlations, decipher their pathophysiology, assess their societal and medico-economic impact, and/or identify patients eligible for new therapeutic approaches. Inclusion of prevalent and incident cases started at the end of 2016. As of April 2021, 5558 patients have been included within 13 RD e-cohorts covering 67 diseases integrated in 10 European Reference Networks and contributing to the European Joint Program on RDs. Several original results have been obtained in relation with the secondary objectives of the RaDiCo cohorts. They deal with discovery of new disease genes, assessment of treatment management, deciphering the underlying pathophysiological mechanisms, diagnostic approaches, genotype-phenotype relationships, development and validation of questionnaires relative to disease burden, or methodological aspects.Conclusion: RaDiCo currently hosts 13 RD e-cohorts on a sharable and interoperable platform constructed on the “cloud computing” principle. New RD e-cohorts at the European and international levels are targeted.

Introduction La pirfenidone, indiquee dans le traitement de la fibrose pulmonaire idiopathique (FPI), est desormais disponible sous forme de comprimes (nouvelle formulation disponible le 1er avril 2018) ou de gelules. L’objectif de cette etude est de decrire la tolerance, les arrets de traitement et le taux de substitution entre les deux formulations (gelules/comprimes) en France. Methodes Une analyse retrospective de donnees issues de la cohorte RaDiCo-PID (Rare Disease Cohorts — Pneumopathies Interstitielles Diffuses), financee par l’ANR (Agence Nationale de la Recherche) et geree en utilisant l’application REDCap, chez des patients (pts) atteints de FPI ayant recu au moins une dose de pirfenidone (1 juillet 2017–30 juin 2019) a ete realisee. Resultats Parmi les 288 pts traites par la pirfenidone, 162 l’avaient nouvellement initiee sur la periode d’observation. Dans la population des patients traites par la pirfenidone pendant la periode de substitution, apres le 1er avril 2018 (n = 256), les caracteristiques des patients (sexe, âge a l’inclusion/au diagnostic) par type de formulation etaient homogenes. Au total, 44,9 % (IC95 % : 38,8–51,0) des patients sont passes de la gelule au comprime et 4,7 % (IC95 % : 2,10–7,28) du comprime a la gelule. Dans l’ensemble, la duree moyenne du traitement a ete de 21,5 mois (±18,7) et la mediane (Q1 ; Q3) de 16,2 mois [6,3 ; 29,3] avec une dose moyenne de 2106,7 (±460,7) mg/jour. Pres de la moitie des patients (119/256, 46,5 %) ont definitivement arrete le traitement, pour intolerance (81/119 pts, 68,1 %) ou inefficacite (21/119 pts, 10,1 %). Le taux d’arrets de traitement definitifs etait de 36,1 % (IC95 % : 27,5–44,8) dans le groupe « substitution », 45,8 % (IC95 % : 36,9–54,8) dans le groupe « comprimes » et 60,7 % (IC95 % : 51,9–69,5) dans le groupe « gelules uniquement ». Le temps median jusqu’a l’arret de la pirfenidone dans les 3 groupes est presente dans la Fig. 1 . Des evenements indesirables ont ete rapportes chez 112 patients (39 %) dont des evenements indesirables graves (hospitalisations) chez 5 patients (4,9 %). Conclusion Cette etude presente des donnees de vie reelle chez des patients atteints de FPI traites par une seule formulation de la pirfenidone ou ayant eu une substitution entre les deux formulations (gelules ou comprimes). Ces observations suggerent une bonne tolerance de la formulation « comprimes » avec moins d’arrets de traitements et une duree de traitement plus longue par rapport aux gelules.

Introduction L’urticaire neutrophilique febrile (UNF) est associee a des maladies autoinflammatoires monogeniques (cryopyrinopathies liees a des mutations du gene NLRP3), polygeniques (maladie de Still) ou reactionnelles (syndrome de Schnitzler). Le sequencage a haut debit (NGS) permet de detecter des mutations presentes dans une partie seulement des cellules, au sein d’un organisme (mosaicisme somatique). L’objectif de cette etude a ete d’identifier les bases moleculaires d’une UNF a debut tardif. Materiel et methodes Nous avons etudie deux patients non apparentes, sans antecedents familiaux, atteints d’UNF ayant debute apres l’âge de 45 ans. Le NGS du gene NLRP3 a ete realise sur l’ADN extrait du sang total, des monocytes, neutrophiles, lymphocytes B et T, des cellules epitheliales urinaires et buccales. L’impact fonctionnel des mutations identifiees a ete determine in vitro par l’etude de l’activation de l’inflammasome NLRP3 (la quantification de la formation de specks et le dosage de la secretion d’IL1β). Resultats Les deux patients presentaient des poussees urticariennes ( Figure 1a, b ) survenant tous les 2 a 3 jours, non prurigineuses, non declenchees par le froid. Les poussees etait associees a de la fievre, des arthralgies, un syndrome inflammatoire biologique. Les deux patients ont developpe une surdite de perception. La biopsie cutanee montrait un riche infiltrat dermique de polynucleaires neutrophiles sans vascularite ( Figure 1c, d ). Alors que le sequencage de NLRP3 par methode de Sanger n’etait pas contributif, une mutation somatique, en mosaique, de NLRP3 (une deletion en phase et une mutation faux-sens) a ete identifiee chez chacun des patients par NGS ( Figure 2 ). Les mutations etaient presentes en mosaique dans toutes les populations cellulaires etudiees (entre 5 et 13 % des cellules de chaque sous-type cellulaire - monocytes, lymphocytes, cellules epitheliales...- etaient porteuses de la mutation). Nous avons demontre l’effet gain-de-fonction des deux mutations par l’etude de l’activation de l’inflammasome NLRP3. Alors que les therapeutiques usuelles de l’urticaire chronique (antihistaminiques, immunosuppresseurs) ont echoue, un traitement par anti-IL1 a permis une remission complete. Discussion Chez nos patients, l’absence d’argument en faveur d’une maladie systemique sous-jacente nous a fait evoquer une maladie auto-inflammatoire. L’identification de mutations en mosaique de NLRP3 nous a permis d’introduire un traitement cible. La distribution du mosaicisme somatique dans tous les types cellulaires etudies suggere que l’evenement mutationnel est survenu precocement, malgre un debut tardif de la maladie. La mutation pourrait egalement etre presente dans les gametes des patients et donc theoriquement transmissible a la descendance a l’etat germinal. Conclusion Une UNF sans maladie systemique sous-jacente doit faire rechercher une mutation mosaique de NLRP3 afin de guider la prise en charge therapeutique.

International audience; BACKGROUND: Given the phenotypic similarities between rheumatoid arthritis (RA)-associated interstitial lung disease (ILD) (hereafter, RA-ILD) and idiopathic pulmonary fibrosis, we hypothesized that the strongest risk factor for the development of idiopathic pulmonary fibrosis, the gain-of-function MUC5B promoter variant rs35705950, would also contribute to the risk of ILD among patients with RA. METHODS: Using a discovery population and multiple validation populations, we tested the association of the MUC5B promoter variant rs35705950 in 620 patients with RA-ILD, 614 patients with RA without ILD, and 5448 unaffected controls. RESULTS: Analysis of the discovery population revealed an association of the minor allele of the MUC5B promoter variant with RA-ILD when patients with RA-ILD were compared with unaffected controls (adjusted odds ratio, 3.8; 95% confidence interval [CI], 2.8 to 5.2; P=9.7x10(-17)). The MUC5B promoter variant was also significantly overrepresented among patients with RA-ILD, as compared with unaffected controls, in an analysis of the multiethnic case series (adjusted odds ratio, 5.5; 95% CI, 4.2 to 7.3; P=4.7x10(-35)) and in a combined analysis of the discovery population and the multiethnic case series (adjusted odds ratio, 4.7; 95% CI, 3.9 to 5.8; P=1.3x10(-49)). In addition, the MUC5B promoter variant was associated with an increased risk of ILD among patients with RA (adjusted odds ratio in combined analysis, 3.1; 95% CI, 1.8 to 5.4; P=7.4x10(-5)), particularly among those with evidence of usual interstitial pneumonia on high-resolution computed tomography (adjusted odds ratio in combined analysis, 6.1; 95% CI, 2.9 to 13.1; P=2.5x10(-6)). However, no significant association with the MUC5B promoter variant was observed for the diagnosis of RA alone. CONCLUSIONS: We found that the MUC5B promoter variant was associated with RA-ILD and more specifically associated with evidence of usual interstitial pneumonia on imaging. (Funded by Societe Francaise de Rhumatologie and others.).

BackgroundCryopyrin-associated periodic syndromes (CAPS) consist of a continuum of autoinflammatory diseases caused by a defect in interleukin 1β regulation. Although symptoms may vary widely, the discovery, in 2001, of the gene involved (NLRP3) has dramatically helped diagnosis.ObjectivesTo define the spectrum and prevalence of NLRP3 mutations in France and to delineate initial criteria before molecular analysis.MethodsRetrospective review (2001–9) of genetic analysis data and request forms of patients living in France with an NLRP3 mutation since the set up of CAPS molecular diagnosis by the three French laboratories providing this test (GenMAI network).ResultsOver 800 analyses of this gene have been conducted, identifying 135 cases with an NLRP3 mutation (55 probands; 33 multiplex families); the estimated prevalence in France was equal to 1/360 000. A total of 21 different sequence variants were detected, among which four are common and nine are new mutations.ConclusionsAlthough the number of NLRP3 test requests has doubled over the past 5 years, genetic screening has not contributed to enhanced detection of new index cases each year. There are two possible reasons for this: (i) no clinical prerequisite for genetic diagnosis and (ii) few new large families are now identified (unlike the initial study based on a selection by linkage). A set of initial clinical criteria have been drawn up which it is recommended should be fulfilled before a patient is tested: at least three recurrent bouts, age at disease onset < 20 years and elevated levels of C-reactive protein, especially in individuals with urticaria and moderate fever.

Background: Idiopathic interstitial pneumonia (IIP)s are severe diseases that can occur from neonates to elderly. A genetic cause is identified in around 2% cases in sporadic cases, and up to 20% in familial cases, telomerase genes mutations being the first etiology. We aimed to identify the relevance of a systematic surfactant genetic testing in familial or sporadic early cases (before 50 years-old) of IIP with no telomerase gene mutations. Methods: Patients were recruited through the French national network for rare lung diseases. All the surfactant system genes in which mutations has been involved in IIP were sequenced by Sanger method: genes encoding the surfactant proteins A2, B and C ( SFTPA2 , SFTPB , SFTPC) , and their transporter, the ATP-binding cassette family A member 3 ( ABCA3 ). A signed informed consent and a clinical form were obtained for each patient. Results: A population of 227 patients (203 unrelated families) was included. Forty-two cases (20%) were familial, 89 were children at the time of the diagnosis. A genetic cause was identified for 15 unrelated patients (7.4% of the families): 7 children aged 0 to 1.5 years, and 8 adults aged 28 to 64 years, including 3 familial cases. In children, 4 had a SFTPC mutation, 3 had a homozygous or compound heterozygous ABCA3 mutation. In adults, 2 had a SFTPA2 mutation, 4 a SFTPC mutation and 2 an ABCA3 mutation. In addition, 8 patients had a heterozygous ABCA3 mutation. Discussion: A genetic cause of IIP has been identified in a number of IIP, not only in familial cases (7%), but also in sporadic cases (7%), in children (7.9%) and in adult cases (7%). These results suggest that surfactant testing is of importance in the diagnosis of IIP in children, but also in adults.

Introduction La pneumopathie interstitielle diffuse (PID) est l’une des causes majeure de mortalite chez les patients atteints de polyarthrite rhumatoide (PR). Malgre une prevalence et une mortalite importante, la physiopathologie de la PID associee a la PR (PR-PID) reste meconnue. La PR-PID partage plusieurs caracteristiques phenotypiques avec la fibrose pulmonaire idiopathique (FPI) telles que l’aspect de pneumopathie interstitielle commune sur le scanner et un pronostic tres severe. Enfin, les similitudes entre PR-PID et FPI concernent egalement les facteurs environnementaux (tabac,…) suggerant que ces deux pathologies pourraient egalement partager des facteurs de risques genetiques tels que ceux predisposant a la FPI familiale (FPF). Methodes Nous avons utilise des donnees d’exome obtenue par whole-exome sequencing (WES) de patients atteints de PR-PID provenant de differents centres de rhumatologie et de pneumologie francais. L’ensemble des patients PR-PID+ repondaient aux criteres ACR/EULAR 2010. Le diagnostic de PID etait realise apres analyse de scanner thoracique de haute resolution. Apres une analyse restrictive de 9 genes rapportes comme etant associes aux FPF, nous avons compare le nombre de mutations retrouvees parmi les patients PR-PID au nombre de mutations retrouvees chez des individus temoins. Un burden test a ete effectue pour definir l’exces de mutations chez les patients PR-PID au sein de ces 9 genes d’interet. Resultats Parmi les 101 patients PR-PID inclus, 12 (11,9 %) presentaient des mutations au sein des regions codantes des genes TERT, RTEL1, PARN et SFTPC. Le burden test comparant 81 patients PR-PID a 1010 individus temoins caucasiens europeens a confirme un exces de mutations parmi ces genes chez les patients PR-PID (P = 9,45 × 10−4 ; OR = 3,17 ; IC 95 % 1,53–6,12). Chez les patients PR-PID porteurs de mutations des genes TERT, RTEL1 et PARN, appartenant au complexe telomerase, la taille des telomeres etait significativement plus petite que celle observees chez les temoins (P = 2,87 × 10−2). Conclusion Ces resultats sont en faveur d’un fond genetique commun entre FPI et PR-PID, suggerant une physiopathologie commune entre ces deux pathologies. En cas de confirmation de ces resultats, les avancees therapeutiques realisees dans la prise en charge des FPI pourraient alors beneficier aux patients atteints de PR-PID.

Summary. The effects of emulsome nanosize range lipid particles containing amphotericin B (EAmB) were compared with the reference formulation containing deoxycholate (Fungizone; Bristol-Myers Squibb, Munich, Germany) and with the commercial amphotericin lipid complex preparation (AmBisome; Nexstar, San Dimas, CA, USA). The minimal inhibitory concentrations of Fungizone and EAmB were identical although killing of Candida albicans was delayed when EAmB was used. In a tissue culture model and in mice, the incorporation of AmB into emulsomes resulted in a considerable reduction of toxicity in comparison with Fungizone. For comparison of the in vivo effect of the preparations a mouse model of systemic infection with C. albicans was used. All preparations were able to reduce the fungal burden in the liver and kidneys in comparison with control animals treated with isotonic saline. AmBisome was more efficient in the reduction of the fungal burden of the liver than EAmB and Fungizone, even when applied in a similar dosage of 1 mg kg−1. In the kidneys, EAmB and Fungizone was slightly more effective than AmBisome. Therefore, in our models, the incorporation of AmB into nanosize particles was able to reduce toxicity without loss of efficiency. EAmB may be considered a candidate preparation for the treatment of infections with C. albicans in humans. Zusammenfassung. Ziel der vorliegenden Arbeit war der Vergleich der biologischen Wirksamkeit von in Lipid-Nanopartikeln inkorporiertem Amphotericin B (EAmB) mit der Referenzsubstanz Fungizone, die Amphotericin und Deoxycholat enthalt, sowie mit Amphotericin-Liposomen (AmBisome). Obwohl die Minimalen Hemmkonzentrationen von Fungizone und EAmB identisch waren, war EAmB im Vergleich zu Fungizone verzogert bakterizid wirksam. In einem Zellkulturmodell war EAmB deutlich weniger toxisch als Fungizone. Um die Wirksamkeit der Praparationen in vivo zu untersuchen, wurde das Modell der systemischen Infektion mit Candida albicans verwendet. Alle Praparationen waren imstande, die Pilzmenge in Leber und Nieren im Vergleich zur Kontrollgruppe zu reduzieren, die mit isotonischer Kochsalzlosung behandelt wurde. AmBisome reduzierte die Keimzahlen in der Leber starker als EAmB und Fungizone, auch wenn alle Substanzen in gleicher Dosis (1 mg kg−1) verwendet wurden. Hingegen waren EAmB und Fungizone in den Nieren geringfugig effektiver. In den verwendeten Modellsystemen fuhrte Inkorporierung von AmB in Lipid-Nanopartikel damit zu einer Reduktion der Toxizitat ohne die Effektivitat zu beeintrachtigen. EAmB ist deshalb eine Praparation, die auch zur Anwendung am Menschen in Frage kommt.

Familial Mediterranean fever (FMF) is an autosomal recessively inherited disease affecting patients of the Mediterranean basin. FMF is characterized by recurrent episodes of fever accompanied with topical signs of inflammation. Some patients can develop a renal amyloidosis associated (AA) amyloidosis. The administration of colchicine is an effective preventive treatment of both the attacks and amyloidosis. The FMF gene (MEFV) was cloned and missense mutations were found to be responsible for the disease. We investigated a large series of 303 unselected and unrelated patients of various ethnic backgrounds with a clinical suspicion of FMF to confirm or invalidate the diagnosis of FMF and to determine the spectrum of MEFV mutations. Molecular analysis focused on all the most frequent mutations identified so far, and an exhaustive analysis of exon 10, containing the mutational hotspot, was performed through DNA sequencing. Sixty-two percent of Sephardic, North African Arabs, Armenian and Turkish patients were either homozygous or compound heterozygous for MEFV mutations. In other populations surrounding the Mediterranean Sea such as Greek, Italian, Portuguese, Kurdish and Lebanese populations, mutations were also found. In general, patients without Mediterranean origin had no mutations in the MEFV gene. Two new mis-sense mutations were identified in exon 10 of the MEFV gene: the S675N in an Italian patient and the M680L in a French patient without any known at-risk ethnic ancestry.

Le syndrome d'insensibilite a l'hormone de croissance (GHIS) est associe a de nombreuses mutations differentes du gene du recepteur de la GH et a une anomalie genetique nouvellement decrite du gene de l'IGF I. Nous avons etudie les caracteristiques phenotypiques et biochimiques de 82 patients presentant un GHIS, d'âge moyen 8,25 ans, vivant dans 23 pays. Le score DS (deviation standard) de la taille moyenne etait de -6,09 et le score DS de la proteine liante de l'IGF (IGFBP-3) de-7,99. Vingt-trois pour cent etaient GHBP-positifs (> 10 %). Le score DS de la taille moyenne chez les patients GHBP-negatifs etait de - 6,5 et celui des patients GHBP-positifs de -4,9 (p < 0,001). Quinze mutations differentes du gene du recepteur de la GH ont ete identifiees chez 27 patients. Tous presentaient des anomalies homozygotes, a l'exception d'un patient chez lequel une anomalie heterozygote composee a ete identifiee. Aucune relation n'a ete observee entre le type de mutation ou l'exon du gene du recepteur de la GH implique et la taille ou le score DS de l'IGFBP-3. Un nouveau phenotype du a une deletion partielle du gene de l'IGF I associee a une insensibilite a la GH est decrit. Ce patient, un garcon âge de 15 ans, avait presente un retard de croissance intra-uterin severe, une stagnation postnatale de la croissance, une surdite neurosensorielle et un leger retard mental. La GH etait elevee avec des taux indetectables d'IGF I et des taux normaux d'IGFBP-3. Aucune reponse au traitement par la GH exogene n'a ete observee. Une deletion partielle impliquant les exons 4 et 5 du gene de l'IGF I a ete identifiee. L'insensibilite a la GH est associee a une large variation de la severite des phenotypes cliniques et biochimiques. Cette variation ne peut s'expliquer clairement par des anomalies du gene du recepteur de la GH. Une deletion du gene de l'IGF I doit etre envisagee lorsqu'il existe une insensibilite a la GH associee a un retard de croissance intra-uterin severe.

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Patients with growth hormone releasing hormone receptor (GHRHR) mutations exhibit pronounced dwarfism and are phenotypically and biochemically indistinguishable from other forms of isolated growth hormone deficiency (IGHD). We presented here two siblings with clinical findings of IGHD due to a nonsense mutation in the GHRHR gene who reached their target height in spite of late GH treatment. Two female siblings were admitted to our clinic with severe short stature at the age of 13.8 (patient 1) and 14.8 years (patient 2). On admission, height in patient 1 was 107 cm (−8.6 SD) and 117 cm (−6.7 SD) in patient 2. Bone age was delayed in both patients (6 years and 9 years). Clinical and biochemical analyses revealed a diagnosis of complete IGHD (peak GH levels on stimulation test was 0.06 ng/mL in patient 1 and 0.16 ng/mL in patient 2). Patients were given recombinant human GH treatment. Genetic analysis of the GH and GHRHR genes revealed that both patientscarried the GHRHR gene mutation p.Glu72X (c.214 G>T) in exon 3 in homozygous (or hemizygous) state. After seven years of GH treatment, the patients reached a final height appropriate for their target height. Final height was 151 cm (−1.5 SD) in patient 1 and 153 cm (−1.2 SD) in patient 2. In conclusion, genetic analysis is indicated in IGHD patients with severe growth failure and a positive family history. In spite of the very late diagnosis in these two patients who presented with severe growth deficit due to homozygous loss−of−function mutations in GHRHR, their final heights reached the target height. Conflict of interest:None declared.

Optimized gene transfer into human primary leukemic T cell with NOD-SCID/leukemia-initiating cell activity

The innate immune system, which corresponds to the first line of defense against microorganisms, brings into play cell surface and intracellular sensors that detect pathogen ligands and danger signals. Among them, NOD-like receptors (NLRs) are intracellular proteins involved in inflammatory signaling pathways. NLRs are part of multiprotein complexes, called inflammasomes, which usually bring into play a NLR, an adaptor protein called ASC, and the pro-inflammatory caspase 1 protein. The activation of inflammasome by different stimuli triggers the proteolytic cleavage of pro-caspase 1 into active caspase 1, which, in turn, converts pro-interleukin 1β (pro-IL1β) into the mature IL1β. IL1β plays a crucial role in systemic inflammation due to its ability to induce the expression of a large panel of pro-inflammatory genes and to act on various target organs. Mutations in NLR genes are responsible for several autoinflammatory and/or autoimmune disorders. For example, mutations in NLRP3, which are responsible for three Mendelian autoinflammatory disorders called cryopyrinopathies, lead to inflammasome autoactivation. Peripheral blood mononuclear cells from patients carrying NLRP3 mutations secrete high levels of IL1β; in many patients presenting with autoinflammatory disorders, blocking IL1 activity by anti-IL1 therapy significantly improves their manifestations. The mechanisms leading to IL1β hypersecretion in other autoinflammatory disorders remain to be identified, as is the case for the role of each inflammasome in vivo. Better knowledge in this field should also contribute to the development of new anti-inflammatory treatments.

We have sequenced the deletion borders of the muscle mitochondrial DNA from 24 patients with heteroplasmic deletions. The length of these deletions varies from 2.310 bp to 8.476 bp and spans from position 5.786 to 15.925 of the human mitochondrial genome preserving the heavy chain and light chain origins of replication. 12 cases are common deletions identical to the mutation already described by other workers and characterized by 13 bp repeats at the deletion boundaries, one of these repeats being retained during the deletion process. The other cases (10 out of 12) have shown deletions which have not been previously described. All these deletions are located in the H strand DNA region which is potentially single stranded during mitochondrial DNA replication. In two cases, the retained Adenosine from repeat closed to the heavy strand origin of replication would indicate slippage mispairing. Furthermore in one patient two mt DNA molecules have been cloned and their sequences showed the difference of four nucleotides in the breakpoint of the deletion, possibly dued to slippage mispairing. Taken together our results suggest that deletions occur either by slippage mispairing or by internal recombination at the direct repeat level. They also suggest that different mechanisms account for the deletions since similarly located deletions may display different motives at the boundaries including the absence of any direct repeat.

The primary ciliary dyskinesias (PCD) are rare diseases characterised by infection of the airways due to impaired muco-ciliary clearance. Half the patients have situs inversus making up Kartagener's syndrome.Primary cilia play a role in development. In the adult ciliated cells occur mainly in the airways and the genital tract. The axoneme, the internal structure of the cilia, is made up of a central pair of microtubules surrounded by peripheral doublets carrying the inner and outer dynein arms. These multiprotein complexes are composed of chains of dynein whose ATPase activity is the basis of ciliary movement. Structural and functional abnormalities of the respiratory ciliated cells are the cause of PCD, diseases that are heterogeneous at both the genetic and ultrastructural levels.There are more than two hundred axonemal proteins. The synthesis and assembly of these proteins are controlled by transcription factors and intraflagellar transport molecules respectively. The genes that code for these proteins are as numerous as candidate genes for PCD.To date only two dynein genes, DNA11 and DNAH5, have been implicated and only in individuals suffering from PCD with absence of outer dynein arms.

The effects of emulsome nanosize range lipid particles containing amphotericin B (EAmB) were compared with the reference formulation containing deoxycholate (Fungizone; Bristol-Myers Squibb, Munich, Germany) and with the commercial amphotericin lipid complex preparation (AmBisome; Nexstar, San Dimas, CA, USA). The minimal inhibitory concentrations of Fungizone and EAmB were identical although killing of Candida albicans was delayed when EAmB was used. In a tissue culture model and in mice, the incorporation of AmB into emulsomes resulted in a considerable reduction of toxicity in comparison with Fungizone. For comparison of the in vivo effect of the preparations a mouse model of systemic infection with C. albicans was used. All preparations were able to reduce the fungal burden in the liver and kidneys in comparison with control animals treated with isotonic saline. AmBisome was more efficient in the reduction of the fungal burden of the liver than EAmB and Fungizone, even when applied in a similar dosage of 1 mg kg(-1). In the kidneys, EAmB and Fungizone was slightly more effective than AmBisome. Therefore, in our models, the incorporation of AmB into nanosize particles was able to reduce toxicity without loss of efficiency. EAmB may be considered a candidate preparation for the treatment of infections with C. albicans in humans.

We studied the NF1 gene in 93 unrelated patients with neurofibromatosis type1, focusing the analysis on four exons that contain the highest number of possible mutations occurring at CpG sites. We used denaturing gradient gel electrophoresis to analyse exons 16, 28, 29 and 49, which contain 45 (25%) of the 183 possible mutations that could occur at the 120 CpG dinucleotides of the coding sequence. Six different mutations were identified, five of which are novel: two truncating mutations, W1810X and 5448insG, located in exon29; two splice defects leading to exon29 skipping, 5206-2AG and 5546GA; and one missense mutation, L844F, located in exon16. The already described R1748X mutation located in exon29 was found in two unrelated patients. The 5546GA and R1748X mutations are located at CpG sites, whereas the W1810X involves a CpNpG site. Four novel polymorphisms, which may be helpful for family studies, were also identified. Overall, all but one mutations were found in exon29, a result which suggests that all the CpG sites of the NF1 coding sequence do not have the same mutability, and that exon29, the most CpG-rich exon, contains mutational hotspots associated with NF1.

Familial Mediterranean fever (FMF) is an autosomal recessively inherited disease affecting patients of the Mediterranean basin. FMF is characterized by recurrent episodes of fever accompanied with topical signs of inflammation. Some patients can develop a renal amyloidosis associated (AA) amyloidosis. The administration of colchicine is an effective preventive treatment of both the attacks and amyloidosis. The FMF gene (MEFV) was cloned and missense mutations were found to be responsible for the disease. We investigated a large series of 303 unselected and unrelated patients of various ethnic backgrounds with a clinical suspicion of FMF to confirm or invalidate the diagnosis of FMF and to determine the spectrum of MEFV mutations. Molecular analysis focused on all the most frequent mutations identified so far, and an exhaustive analysis of exon 10, containing the mutational hotspot, was performed through DNA sequencing. Sixty-two percent of Sephardic, North African Arabs, Armenian and Turkish patients were either homozygous or compound heterozygous for MEFV mutations. In other populations surrounding the Mediterranean Sea such as Greek, Italian, Portuguese, Kurdish and Lebanese populations, mutations were also found. In general, patients without Mediterranean origin had no mutations in the MEFV gene. Two new mis-sense mutations were identified in exon 10 of the MEFV gene: the S675N in an Italian patient and the M680L in a French patient without any known at-risk ethnic ancestry.

The diagnosis of familial Mediterranean fever (FMF) has until recently been based on clinical signs alone. Discovery of the MEFV gene has enabled a molecular approach to diagnosis, which is already well established for diagnosing typical clinical forms of FMF. We evaluated the utility of this molecular approach in a large series of patients with various clinical presentations and ethnic origins. We looked for mutations in the MEFV gene in 303 unselected consecutive patients with a variable (from high to low) clinical suspicion of FMF. Two mutations were found in 133 patients (44%). In 22 patients (7%), the clinical diagnosis of FMF was unlikely according to the Tel Hashomer clinical criteria. Our results suggest that the spectrum of FMF-associated signs is broader than previously believed. Wider indications for genotyping should lead to more frequent diagnosis of FMF.

Prop-1 is a newly isolated pituitary-specific paired-like homeodomain transcription factor whose cDNA sequence is well known in mouse. To study its involvement in human combined pituitary hormone deficiency (CPHD), we have isolated the human cDNA ortholog and determined the exon/intron organization and chromosomal localization of the human gene. A Prop-1 defect was characterized in three CPHD families. One missense mutation (R73C) involves a residue conserved in 95% of the more than 400 homeodomain proteins so far identified; in vitro splicing assays demonstrated the functional importance of the second defect, whereas the remaining mutation is a frameshift. Given the disease phenotype documented in the patients, these data, which will facilitate molecular investigations in other patients, demonstrate the crucial role of Prop-1 in the proper development of somatotrophs, lactotrophs, thyreotrophs and gonadotrophs.

GH secretion and release are complex phenomena depending on activation of several genes, including those encoding GH, GHRH, and its receptor (GHRH-R). The GH gene, which is the most extensively analyzed sequence in patients with familial GH deficiency (GHD), represents the main known target of mutations. To test the involvement of the GHRH-R gene in this disease phenotype, we investigated one candidate Tamoulean family originating from Sri Lanka. Two brothers, with extremely short stature (-4 SD) and no dysmorphy, were diagnosed as having complete GHD, unresponsive to exogenous GHRH and associated with PRL levels within the lower normal range. Magnetic resonance imaging examination showed anterior pituitary hypoplasia with a normal pituitary stalk. Both patients increased their growth rate while under GH therapy. Molecular investigations revealed a homozygous GHRH-R gene mutation that introduces a stop codon at residue 72. This mutation, which predicts a severely truncated receptor lacking the seven membrane spanning domains, is identical to that recently reported in one Indian Moslem family, raising the possibility of a founder effect. There was no clear evidence for height reduction in the three heterozygous individuals studied. This observation, which underlines the phenotypic criteria associated with a loss of GHRH-R function, raises the question of the frequency of GHRH-R abnormalities among GHD patients.

Previously, we reported the identification of a new human GH receptor (hGHR) messenger RNA species that encodes a smaller hGHR isoform, termed hGHRtr. Its messenger RNA is expressed in several human tissues and predicts a severely truncated GHR protein that lacks 97.5% of the intracellular domain. Because these two hGHR isoforms, which display similar binding affinity, are coexpressed in several tissues, they may reside side by side and, therefore, interrelate. To further characterize the biological properties of hGHRtr in comparison with hGHR, we generated Chinese hamster ovary (CHO) cell lines stably expressing each of these hGHR isoforms. Cross-linking of [125I]hGH to CHO/hGHRtr cells revealed a majored specific complex with apparent Mr of approximately 100 kDa, which would indicate the hGHRtr to be in molecular mass form of about 80 kDa. When compared with CHO/hGHR, CHO/hGHRtr cells secreted higher amounts of soluble GH-binding protein (GHBP). In contrast to CHO/hGHR cells, CHO/hGHRtr cells did not exhibit any GH-induced receptor down-regulation, and internalization was markedly reduced. Analysis of the constitutive turnover of cellular hGHR and soluble GHBP showed that incubation of CHO/hGHR cells with cycloheximide caused parallel disappearance of hGHR and GHBP. This contrasted with the stability of GHRtr, which showed no decline after cycloheximide treatment for up to 4 h, suggesting that the bulk GHRtr and GHBP may be derived from preformed proteins. Thus, in contrast to hGHR, hGHRtr is fixed at the cell membrane; it undergoes minimal internalization, no down-regulation by hGH, no constitutive turnover for as long as 4 h, but increased capacity to generate a soluble GHBP. Because hGHRtr failed to undergo ligand-induced internalization, the source of the continuous, undisturbed GHBP released into the medium may be from an intracellular storage pool. The relative abundance of these two hGHR isoforms, through regulation of splicing, could be of critical importance in modulating the biological effects of GH.

GH insensitivity syndrome (GHIS) is associated with many different mutations of the GH receptor (GHR) gene. We examined the phenotypic and biochemical features in 82 GHIS patients from 23 countries, each fulfilling diagnostic criteria of GHIS. There were 45 males and 37 females [mean age, 8.25 yr; mean height, -6.09 SD score, and mean insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3), -7.99 SD score]. Sixty-three were GH-binding protein (GHBP) negative; 19 were GHBP positive (10% binding). The mean height in GHBP-negative subjects was -6.5 SD score, and that in GHBP-positive patients was -4.9 SD score (P =0.001). Clinical and biochemical heterogeneity was demonstrated by the wide range of height (-2.2 to -10.4 SD score) and IGFBP-3 (-1.4 to -14.7 SD score) values, which were positively correlated (r2 = 0.45; P =0.001). This contrasted with the lack of correlation between mean parental height SD score and height SD score (r2 = 0.01). Fifteen different GH receptor gene mutations were identified in 27 patients. All had homozygous defects, except 1 who had a compound heterozygous defect. The mutations were 5 nonsense, 2 frame shift, 4 splice, 4 missense, and 1 compound heterozygote. There was no relationship between mutation type or exon of the GHR gene involved and height or IGFBP-3 SD score. In conclusion, GHIS is associated with wide variation in the severity of clinical and biochemical phenotypes. This variation cannot clearly be accounted for by defects in the GHR gene. Other genetic and/or environmental factors must, therefore, contribute to phenotype in GHIS.

The pharmacokinetics of dexanabinol (HU-211), a synthetic, nonpsychotropic cannabinoid with neuroprotectant action, was evaluated in a phase I clinical trial. The compound was administered at doses of 48 mg, 100 mg, and 200 mg as short i.v. infusions in a Cremophor-ethanol vehicle diluted with saline. All administrations were well-tolerated and no compound-related side-effects were observed. Plasma concentrations of dexanabinol were quantitated using a GC/MS/MS technique which provided a limit of quantitation of 100 pg/ml. The elimination of dexanabinol was best fitted to a 3-compartment model with a rapid distribution half-life (5 min), an intermediate phase half-life of approximately 90 min, and a slow terminal elimination half-life (approximately 9 h). The pharmacokinetics were linear over the evaluated dose range. The plasma clearance of the drug was high (1,700 ml/min) and the volume of distribution approximately 15 l/kg. These data are similar to those reported for naturally occurring cannabinoids such as delta 9-tetrahydrocannabinol and cannabidiol.

The GH receptor (GHR) is a member of the cytokine receptor superfamily; GH binding protein is the solubilized extracellular domain of the GHR. Abnormalities in the GHR produce an autosomal recessive form of GH resistance, the Laron syndrome, characterized by growth failure and the clinical appearance of severe GH deficiency despite elevated circulating GH levels. In 13 unrelated patients with undetectable levels of GH binding protein, we characterized nine novel mutations in the GHR gene. These molecular defects comprise three nonsense mutations (Q65X, W80X, and W157X), one frameshift (36delC), two splice defects (G--A at 70 + 1, C--T at 723), and three missense mutations (C38S, S40L, and W50R) located in the extracellular domain of the receptor, and thus would be expected to interfere with GH binding activity. These results further confirm the broad heterogeneity of mutations underlying this rare GH resistance syndrome.

Laron syndrome (LS) is a hereditary form of GH resistance due to molecular defects in the GH receptor (GHR). Most of the identified mutations are located in the extracellular domain of the receptor, resulting in a lack of serum GHBP in the majority of LS patients. We present an LS patient with supranormal levels of serum GHBP, in addition to 35 of her relatives. The proband is a 3.5 year-old Druse girl with severe short stature (height SDS -5.1), high GH (250 micrograms/l), low IGF-I (2.7 nmol/l) and IGFBP-3 (410 micrograms/l), both unresponsive to exogenous GH. The binding capacity of the serum GHBP was 22 nM (adult reference serum, 0.7 nM), with an affinity constant Ka = 1.9 x 10(9) M-1 comparable to that of normal sera (Ka = 1.7-2.1 x 10(9) M-1). The apparent MW of the GHBP was approximately 60-80 kDa, similar to that of control sera. In the proband's sister, parents, grandparents and uncles, extremely high GHBP values were observed (43.0 +/- 4.8 RSB, n = 10) compared with normal adults (0.81 +/- 0.06 RSB) (p0.001). The remaining subjects had normal or moderately elevated GHBP levels. Serum GH in adults with high GHBP was significantly elevated above control values (6.0 +/- 0.9 micrograms/l vs 0.76 +/- 0.13 microgram/l, p0.001). Serum IGF-I and IGFBP-3 levels were normal in all the subjects, with the exception of an aunt (IGF-I 3.9 nmol/l) and the proband's sister (IGFBP-3 460 micrograms/l). All the subjects' heights were within the normal range. Analysis of the GHR gene performed in the proband revealed an as yet undescribed homozygous intronic point mutation. It consists of a G--T substitution at nucleotide 785-1 preceding exon 8, a sequence that encodes the transmembrane domain. This mutation, which destroys the invariant dinucleotide of the splice acceptor site, is expected to alter GHR mRNA splicing and to be responsible for skipping exon 8. The resulting truncated protein that retains GH binding activity is probably no longer anchored in the cell membrane, affecting signal transmission in the homozygous patient and causing high GHBP levels in the heterozygous relatives.

Desmopressin acetate (DDAVP) is used parenterally and intranasally in the control of several diseases. Oral administration of DDAVP, while most desirable, is not practical presently due to low bioavailability. The objective of the present study was to explore the feasibility for employing oil-in-water MucoAdhesive SubMicron Emulsion (MA-SME), a novel mucoadhesive vehicle with polymer-coated droplets, for enhanced oral delivery of DDAVP.We used a modified pharmacopeal method, based on measurement of the antidiuretic activity, for the assessment of oral delivery of DDAVP in rats. DDAVP formulated in two MA-SME preparations, in non-mucoadhesive SME (plain-SME), in saline and in other control solutions was administered orally to rats via a stomach tube at a dose of 0.5 units/kg. At various times following DDAVP administration, water was given via a stomach tube. Excretion times for 30% and 60% of the total water load were measured.Excretion times for DDAVP in MA-SME formulations were always longer (up to 2-fold) than those following DDAVP in saline. By contrast, excretion times for DDAVP in plain-SME and in non-SME Carbopol (a Mucoadhesive polymer) solution were virtually identical to those for DDAVP in saline.Formulations of MA-SME were shown to generate substantial enhancement (up to 12-fold) of the rat oral bioavailability of DDAVP with regard to simple saline solution of the drug. From the results it is also evident that MA-SME, but not plain-SME or non-SME Carbopol solution, is responsible for the enhancement of oral delivery of DDAVP in rats.

Leprechaunism is characterized by severe intrauterine growth retardation, elfin-like face, relatively large hands, feet and genitalia and abnormal skin with hypertrichosis, acanthosis nigricans and low subcutaneous fat. The insulin receptors have multiple defects.A boy was born after cesarean section at the 35th week of gestation because of intrauterine growth retardation: weight: 930 g; height: 36 cm; head circumference: 27 cm. He had trigonocephaly, coarse features and hyperkeratosis. Ultrasonography confirmed the presence of a ventricular septal defect detected during pregnancy. Hyperglucosemia (3 g/l) was associated with insulinemia above 350 mU/l; his C-peptide concentration was above 20 ng/ml. The patient was given intravenous insulin, up to 2,500 U/kg/d. He died at the age of 95 days, weighing 1500 g, with persistent hyperglucosemia and cholestasis. Postmortem examination showed adrenal and thymus hypoplasia and hyperplasia of pancreatic islet cells. Molecular biology studies showed that this patient was heterozygotic for two mutations, one in exon 20 inherited from his father, the other in exon 18 inherited from his mother; both mutations are associated with tyrosine-kinase activity of the insulin receptor. These results will be used for antenatal diagnosis in any future pregnancy.Molecular biology can indicate specific defects in the insulin receptor. It may also allow antenatal diagnosis in some families.

Fusariosis, a rare infectious disease of the immunocompromised host, is relatively resistant to amphotericin B (AmB) or other antifungal agents. We describe a 5-year follow-up of a 40 year old woman with T-type acute lymphoblastic leukemia who following chemotherapy developed prolonged high fever, chills, night sweats, and severe weakness. Liver function tests were impaired and abdominal computerized tomography (CT) showed multiple lesions in the liver and abnormal structure of the spleen. A laparotomy revealed multiple granulomas containing Fusarium sp. in the liver, and the spleen was heavily infiltrated by the same fungus. The patient failed to respond to the conventional AmB dosage form (Fungizone) even after a total dose of 3.0 g was given, and developed significant renal impairment. AmB was complexed (in a mole ratio of 1:16) with a mixture of the phospholipids dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol (mixed in 7:3 mole ratio). The resulting drug complex, AmB-PLC, was then administered (1-4 mg/kg/day, total dose 4.2 g) and subsequently the patient was cured of all symptoms of fusariosis. There were only mild side effects and no nephrotoxicity was evident. On the contrary, marked improvement of the renal function tests occurred during AmB-PLC treatment. Eight months later, she developed a spinal lesion with dense consistency in L5 and S1, and after receiving another course of AmB-PLC (3.1 g) she recovered completely. In a 2 year follow-up period the patient had no further relapse of the fungal disease. Subsequent chemotherapy given for relapse of the leukemia was followed by a new fungal infection, which was treated with AmB-cholesteryl sulfate complex (Amphocil).(ABSTRACT TRUNCATED AT 250 WORDS)

A deficiency in the activity of uroporphyrinogen decarboxylase (URO-D), the fifth enzyme of the haem biosynthetic pathway, is found in two hereditary diseases, familial porphyria cutanea tarda (PCT) and hepatoerythropoietic porphyria (HEP). Little is known about the genetic relationship between those two diseases and it has been postulated that HEP is the homozygous form of PCT. A URO-D cDNA was cloned from an HEP patient and the comparison between the mutant and the wild-type sequences showed a single base difference within the coding sequence leading to the replacement of a glutamic acid by a lysine at codon 167 of the mutant protein. This replacement produced a protein which is rapidly degraded in the presence of cell lysate. On the basis of hybridization of synthetic oligomers to amplified genomic DNA, we demonstrated that this patient is homozygous for this single base mutation. In order to look for any relationship between HEP and PCT, we tested six unrelated patients with familial PCT and could not detect the codon 167 mutation in any of them. These results indicate an heterogeneity in the mutations responsible for the PCT and HEP phenotypes.

The polymerase chain reaction (PCR) allows the detection of minimal amounts of nucleic sequences and has been successfully used to test for the chronic myeloid leukemia-specific bcr/abl transcripts. We studied blood samples from 17 patients who had undergone allogeneic bone marrow transplantation for CML, using a modified polymerase chain reaction-based assay for the detection of leukemic mRNA. This nested PCR technique was found to be highly sensitive, detecting the chimeric bcr/abl transcript in 16 of 17 patients including several long-term survivors. Cytogenetic techniques failed to detect Ph mitoses. The clinical significance of the persisting bcr/abl transcript for long periods following BMT is poorly understood and remains to be elucidated by further studies.

Many mutations in the LDL receptor (LDLR) gene have now been identified mostly as gross gene rearrangements, however they only represent a weak percentage of all deleterious gene mutations causing Familial Hypercholesterolemia (FH). This discrepancy may be related to the difficulties in characterizing point or small defective mutations. In a three-generation family with Familial Hypercholesterolemia, one specific haplotype constructed with 12 intragenic restriction fragment length polymorphisms (RFLP) cosegregated with the disease, while in the consanguineous propositus there was homozygosity for this haplotype. By polymerase chain reaction (PCR) amplification followed by direct sequencing there was unequivocal evidence for a double dose of a unique mutation, (namely a duplication of 4 bases in exon 17), while there was a single dose in heterozygote relatives. We consequently screened a population selected under clinical and geographical criteria for this mutation by PCR and allele specific oligonucleotides (ASO) hybridization. None of the 158 type IIa individuals tested carried the same mutation. Herein, is a rapid combined genetic and molecular approach to characterize and evaluate the frequency of LDL Receptor gene mutations causing Familial Hypercholesterolemia, towards targeted prevention and therapy.