Guidelines for the genetic diagnosis of hereditary recurrent fevers.

Hereditary recurrent fevers (HRFs) are a group of monogenic autoinflammatory diseases characterised by recurrent bouts of fever and serosal inflammation that are caused by pathogenic variants in genes important for the regulation of innate immunity. Discovery of the molecular defects responsible for these diseases has initiated genetic diagnostics in many countries around the world, including the Middle East, Europe, USA, Japan and Australia. However, diverse testing methods and reporting practices are employed and there is a clear need for consensus guidelines for HRF genetic testing. Draft guidelines were prepared based on current practice deduced from previous HRF external quality assurance schemes and data from the literature. The draft document was disseminated through the European Molecular Genetics Quality Network for broader consultation and amendment. A workshop was held in Bruges (Belgium) on 18 and 19 September 2011 to ratify the draft and obtain a final consensus document. An agreed set of best practice guidelines was proposed for genetic diagnostic testing of HRFs, for reporting the genetic results and for defining their clinical significance.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome (APECED) due to AIRET16M mutation in a consanguineous Greek girl.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome (APECED) or autoimmune polyendocrine syndrome type 1 (APS-1) is a rare autosomal recessive disease caused by mutations of the AutoImmune REgulator (AIRE) gene, an important mediator of tolerance to self-antigens. It is characterized by two out of three major components: chronic mucocutaneous candidiasis, hypoparathyroidism and Addison's disease. We present an 11-year-old girl suffering from recurrent episodes of mucocutaneous candidiasis and onychomycosis from 1 to 6 years of age, and transient alopecia at the age of 4 years. Hypoparathyroidism and dental enamel hypoplasia were diagnosed at 8 years. Autoantibodies to thyroid and adrenal glands were not detected and all other endocrine functions have remained normal. Genetic analysis revealed that the patient was homozygous for the mutation T16M in exon 1 of the AIRE gene (p.T16M, c.47C>T). This is the first APECED case reported for carrying this mutation in homozygous form. Parents were third cousins and heterozygous carriers of this mutation.

De novo duplication of MECP2 in a girl with mental retardation and no obvious dysmorphic features.

Loss-of-function mutations of MECP2 are responsible for Rett syndrome (RTT), an X-linked neurodevelopmental disorder affecting mainly girls. The availability of MECP2 testing has led to the identification of such mutations in girls with atypical RTT features and the recognition of milder forms. Furthermore, duplication of the entire gene has recently been described in boys with mental retardation and recurrent infections. We describe a girl with a heterozygous de novo MECP2 duplication. The patient, at the age of 19, has mental retardation with no autistic features. She is friendly but gets frequently anxious. She has neither dysmorphic features nor malformations. Her motor development was delayed with walking at 20 months. Speech is fluid with good pronunciation but is simple and repetitive. Diagnosis was made after single-strand conformation analysis (SSCA) and multiplex ligation-dependent probe amplification (MLPA) analysis of MECP2. Array comparative genomic hybridization (aCGH) analysis showed a duplication of 29 kb including MECP2 and part of IRAK1. Fluorescent in situ hybridization (FISH) has revealed that the duplicated region is inserted near the telomere of the short arm of chromosome 10. X-chromosome inactivation in leukocyte DNA was not skewed. We conclude that it is likely that this MECP2 duplication is responsible for the mental retardation in this patient. This case broadens the phenotypic spectrum of MECP2 abnormalities with consequent implication in diagnosis and genetic counselling of girls with non-syndromic mental retardation.

Molecular analysis of F8 in Lebanese haemophilia A patients: novel mutations and phenotype-genotype correlation.

Haemophilia A (HA) is an X-linked recessive hereditary bleeding disorder affecting one in 5000 men, resulting from mutations in the F8 gene. Our objective was to identify the spectrum of mutations of the F8 gene in Lebanese patients, and to perform genotype/phenotype correlations. A group of 79 HA patients from 55 unrelated families was studied. Patients were screened for intron 22 and intron 1 inversion using PCR. In the absence of mutations in both introns, a dHPLC screening followed by a DNA sequencing of all coding regions was performed. When patients presented novel mutations, 150 control chromosomes were tested to exclude common polymorphisms. Large deletions were confirmed by MLPA technique. The mRNA was specifically studied whenever a splice site mutation was detected. In addition, studies of the putative biochemical function and FVIII 3D structures were conducted. Thirty-four mutations were identified in this study of which 21 were novel: 11 missense, two nonsense, two splice sites, five small deletions and one large deletion. Inhibitor found in three over 75 patients correlated with large deletion, intron 22 inversion, and nonsense mutations. We were able to identify all causative mutations in those HA patients. This knowledge represents a huge step for genetic counselling.

Phenotypic heterogeneity of dopa-responsive dystonia in monozygotic twins.

The clinical expression of dopa-responsive dystonia (DRD) was found to be different in a pair of affected monozygotic twins. An earlier onset was associated with a more disabling course of disease. Whereas monozygosity was genetically proven, the search for pathogenic mutations in the GTP-cyclohydrolase-1 gene was negative. The contribution of environmental factors appeared minimal. Intrafamilial variability of DRD phenotype may be related to yet unknown non-Mendelian epigenetic or proteomic factors.

N-acetylcysteine augments adenovirus-mediated gene expression in human endothelial cells by enhancing transgene transcription and virus entry.

BACKGROUND: It has previously been shown that oxidants reduce the efficiency of adenoviral transduction in human umbilical vein endothelial cells (HUVECs). In this study, the effect of the antioxidant N-acetylcysteine (NAC) in adenovirus-mediated gene transfer has been investigated. METHODS: HUVECs were pretreated or not with NAC, and infected with E1E3-deleted adenovirus (Ad) containing the LacZ gene expressed from the RSV-LTR promoter/enhancer in the presence and absence of NAC. Transgene expression was assessed at the protein level (histochemical staining, measurement of beta-Gal activity, and western blot), mRNA level (real-time RT-PCR) and gene level (nuclear run on) 24 h and 48 h after infection. Adenoviral DNA was quantitated by real-time PCR, and cell surface expression of Coxsackie/adenovirus receptors (CAR) was determined by FACS analysis. RESULTS: Pretreatment of cells with NAC prior to Ad infection enhanced beta-Gal activity by two-fold due to an increase in viral DNA, which was related to increased CAR expression. When NAC was present only during the post-infection period, a five-fold increase in beta-Gal activity and LacZ gene transcriptional activity was observed. When NAC was present during both the pretreatment and the post-infection period, beta-Gal activity was further enhanced, by 15-fold. Augmentation of beta-Gal activity was paralleled by an increase in beta-Gal protein and mRNA levels. NAC did not affect the half-life of LacZ mRNA. CONCLUSION: Pretreatment with NAC prior to Ad infection enhances virus entry, while treatment with NAC post-infection increases transgene transcription. This strategy permits the use of lower adenoviral loads and thus might be helpful for gene therapy of vascular diseases.

A case of (X;15) translocation diagnosed as a paracentric inversion of Xp: diagnostic revision with FISH.

In 1990 we reported the case of a 17 years old girl with growth retardation, overweight and primary amenorrhea, presenting a de novo chromosomal rearrangement cytogenetically characterized as a paracentric inversion of the short arm of X chromosome. The FISH analyses that were recently performed, revealed that in fact our patient presented a case of unbalanced translocation, 46,X, t(X;15)(p11.2; q15).

Effects of first generation E1E3-deleted and second generation E1E3E4-deleted/modified adenovirus vectors on human endothelial cell death.

Adenoviral vectors are promising tools for pulmonary vascular gene transfer. In first generation vectors, the viral E4 region is preserved (E4+ Ad), but E4 is deleted in second generation vectors (E4- Ad). These vectors were compared for their toxicity in human endothelial cells in terms of apoptosis and necrosis. Infection with E4+ Ad vectors reduced whereas E4- Ad vectors enhanced apoptosis under normal culture conditions. Furthermore, E4+ Ad protected against apoptosis induced by growth factor deprivation, while E4- Ad enhanced apoptosis triggered by ceramide. Ad vectors containing different E4 open reading frames, alone or in different combinations, showed similar effects to E4- Ad, leaving the viral genes that might be responsible for reducing apoptosis unidentified at the present time. As previously observed with E4+ Ad devoid of transgene, E4+ Ad carrying beta-galactosidase or green fluorescent protein under the control of either the RSV or CMV promoter also reduced apoptosis triggered by growth factor deprivation. In contrast, E4+ Ad containing a CFTR expression cassette did not reduce apoptosis, and E4- Ad with CFFR showed increased toxicity. We conclude that Ad vectors may have important effects on the control of apoptosis in transfected cells, depending on the residual expression of viral genes. This effect can be complicated by the action of transgene expression on cell survival.