Multiplex ligation-dependent probe amplification detection of an unknown large deletion of the CREB-binding protein gene in a patient with Rubinstein-Taybi syndrome

Rubinstein-Taybi syndrome is a rare autosomal dominant congenital disorder characterized by postnatal growth retardation, psychomotor developmental delay, skeletal anomalies, peculiar facial morphology, and tumorigenesis. Mutations in the gene encoding the cAMP response element-binding protein (CREB, also known as CREBBP or CBP) on chromosome 16p13.3 have been identified. In addition, some patients with low intelligence quotients and autistic features bear large deletions. Based on these observations, we used multiplex ligation-dependent probe amplification to search for large deletions affecting the CREBBP gene in a Rubinstein-Taybi syndrome patient. We identified a novel heterozygote deletion removing five exons (exons 17-21), encoding the histone acetyltransferase domain. We propose the use of multiplex ligation-dependent probe amplification as a fast, accurate and cheap test for detecting large deletions in the CREBBP gene in the sub-group of Rubinstein-Taybi syndrome patients with low intelligence quotients and autistic features

Effect of The Gluten-Free Diet on Quality of Life, Gastrointestinal Symptoms and Immune System in Patients with Fibromyalgia and Non-Celiac Wheat Sensitivity. Fibromyalgia and Non-Celiac Wheat Sensitivity.

Fibromyalgia (FM) is a clinical syndrome characterized by chronic pain. FM patients complain hyperalgesia and allodynia and they are frequently affected by Non Celiac Wheat Sensitivity (NCWS), a condition where gastrointestinal and extraintestinal symptoms are triggered by gluten and/or wheat ingestion. The gluten-free diet (GFD) impact was evaluated on fibromyalgia-related and gastrointestinal symptoms, health-related quality of life and immune response of patients with both FM and NCWS in order to detect a possible pathogenetic role of wheat/gluten in the triggering of the inflammatory process. Peripheral blood from 8 FM patients, 10 FM and NCWS patients (FM+NCWS patients), 13 NCWS patients and 14 healthy subjects (HS) was taken before and after 8 weeks of GFD and used for cytofluorimetric analysis. In all FM+NCWS patients after GFD almost all the clinical parameters used to evaluate musculoskeletal and systemic symptoms related to FM were reduced as well as intestinal symptoms present before GFD. Moreover, pro-inflammatory cytokines production (IFN-\u3b3, TNF- \u3b1, IL-17 and IL-22) by T helper (Th) cells of FM+NCWS patients was reduced after GFD. Our findings suggest that gluten/wheat could represent one of the triggering factors of the inflammatory condition playing an important role in the etiopathogenesis of both FM and NCWS

Interleukin-9 Overexpression and Th9 Polarization Characterize the Inflamed Gut, the Synovial Tissue, and the Peripheral Blood of Patients With Psoriatic Arthritis

Objective. To investigate the expression and tis- sue distribution of Th9-related cytokines in patients with psoriatic arthritis (PsA). Methods. Quantitative gene expression analysis of Th1, Th17, and Th9 cytokines was performed in intestinal biopsy samples obtained from patients with PsA, HLA2B272positive patients with ankylosing spondylitis (AS), patients with Crohn’s disease (CD), and healthy controls. Expression and tissue distribu- tion of interleukin-23 (IL-23), IL-17, IL-22, IL-9, and IL-9 receptor (IL-9R) were evaluated by immunohisto- chemistry and confocal microscopy. Flow cytometry was used to study the frequency of Th9 cells among periph- eral blood, lamina propria, and synovial fluid mononuclear cells. The functional relevance of IL-9R expression on epithelial cells was assessed in functional in vitro studies. Th9 cells in synovial tissue from patients with PsA were also studied. Results. Subclinical gut inflammation in PsA patients was characterized by a clear Th17 and Th22, but not Th1, polarized immune response. Unlike AS and CD, a strong and significant up-regulation of IL-9 was observed in PsA gut, especially among infiltrating mononuclear cells, high endothelial venules, and Pan- eth cells. IL-92positive mononuclear cells were demon- strated to be in large part Th9 cells. IL-9 overexpression was accompanied by significant Paneth cell hyperplasia. Paneth cells strongly overexpressed IL-9R, and stimula- tion of epithelial cells, isolated from PsA patients, with IL-9 resulted in overexpression of a-defensin 5 and IL-23p19. Peripheral and synovial expansion of a4b71 Th9 cells was also observed in patients with PsA. Increased expression of IL-9 and IL-9R was also found in synovial tissue. Conclusion. Strong IL-9/Th9 polarization seems to be the predominant immunologic signature in patients in PsA

Comparative multiplex dosage analysis in spinocerebellar ataxia type 2 patients

We developed a new application of comparative multiplex dosage analysis (CMDA) for evaluation of the ataxin 2 gene. Expansions of the triplet CAG can cause spinocerebellar ataxia type 2 (SCA2), a neurodegenerative disease with an autosomal-dominant mode of inheritance. Molecular diagnosis of SCA2 is routinely based on the use of conventional PCR to detect the CAG expansion. However, PCR does not amplify an allele with an expansion of many triplets (>80), which is typically found in infantile and juvenile forms of SCA2, thus leading to false negatives. We propose the analysis of the ATXN2 gene by CMDA to complement existing methods currently used for the detection of large expansions of the CAG repeat. Using CMDA, the presence of any longer mutated allele in a heterozygous patient or fetus would be inferred due to dosage variation of the very frequent normal allele #22. CMDA can be completed in 1 day, at very low cost, and would be a useful tool for prenatal diagnosis and for diagnosis of presymptomatic forms of early-onset SCA2

Una nuova applicazione della Comparative Multiplex Dosage Analysis (CMDA)

La Comparative Multiplex Dosage Analysis (CMDA) \ue8 una tecnica utilizzata per l\u2019analisi di delezioni o duplicazioni di specifiche regioni del genoma (Gable et al. 2003 Hum Mutat 21:379-386). Il metodo si basa su di un\u2019analisi quantitativa delle aree dei picchi di un elettroferogramma ottenuto da elettroforesi capillare condotta su frammenti di DNA amplificati con PCR e marcati con specifici fluorocromi. La presenza di sbilanciamenti \ue8 , in particolare, accertata misurando il rapporto tra le aree dei picchi corrispondenti alla regione da analizzare e una regione del genoma la cui dose \ue8 nota. In studi precedenti noi abbiamo applicato questa tecnica all\u2019analisi di delezioni e duplicazioni di specifici esoni dei geni codificanti per la fenilalanina idrossilasi (PAH) e ubiquitin protein ligase E3A (UBE3A) in pazienti rispettivamente affetti da fenilchetonuria (Cal\uec et al 2010 Exp Mol Med. 42:81-6) e sindrome di Angelman (Cal\uec, et al. 2010 Exp Mol Med. 42:842-8). In questo studio noi mostriamo una nuova applicazione della CMDA per l\u2019analisi del gene ataxina 2 (ATXN2; localizzazione cromosomica: 12q24) nel quale espansioni della tripletta CAG, nell\u2019esone 1 del gene, possono causare l\u2019Atassia Spinocerebellare tipo 2 (SCA2), una malattia neurodegenerativa a trasmissione autosomica-dominante. Individui sani hanno 14-31 ripetizioni della tripletta CAG, mentre gli individui affetti hanno espansioni delle triplette nel range 35-500. Nella SCA2, vi \ue8 inoltre una correlazione inversa tra il numero di ripetizioni CAG e l'et\ue0 di esordio della patologia . La diagnosi molecolare di SCA2 \ue8 basata sull'uso della PCR convenzionale in grado di rilevare l\u2019ampiezza dell\u2019espansione CAG. Tuttavia, il limite di questo test \ue8 che la PCR potrebbe non amplificare un allele con un\u2019espansione di molte triplette, quest\u2019ultime presenti nelle forme di SCA2 a insorgenza infantile e giovanile portando a falsi negativi. In particolare, poich\ue8 l\u2019allele normale di 22 repeats ha una frequenza molto elevata nella popolazione (fino al 90%) individui eterozigoti per un allele normale ( 100 CAG repeats) verrebbero erroneamente diagnosticati come omozigoti per l\u2019allele # 22. L\u2019analisi CMDA permetterebbe di individuare questi falsi omozigoti rivelando la presenza di una singola dose dell\u2019allele normale. Noi proponiamo l\u2019analisi CMDA del gene ATXN2 per complementare metodi correntemente in uso per l\u2019individuazione di ampie espansioni delle triplette CAG quali la Triplet repeat primed PCR (Cagnoli et al., \u2026. e un metodo che utilizza la PCR, le\u2019elettroforesi, il Southern blotting e l\u2019ibridazione molecolare con sonde (CAG)n. L\u2019analisi CMDA pu\uf2 essere condotta in 1 giorno con costi molti contenuti e pu\uf2 rivelarsi particolarmente informativa nella diagnosi prenatale e presintomatica delle forme di SCA2 a insorgenza precoce

Analisi MLPA del gene CREB-binding protein (CREBBP) in un paziente con la sindrome di Rubinstein Taybi

La sindrome di Rubinstein-Taybi \ue8 una rara malattia congenita autosomica dominante caratterizzata da ritardo della crescita postnatale, ritardo dello sviluppo psicomotorio, anomalie scheletriche, peculiare morfologia facciale ed un aumento del rischio oncogeno. La prevalenza alla nascita \ue8 1 su 125.000 nati vivi. La malattia pu\uf2 essere associata a mutazioni nel gene che codifica per la proteina CREB-binding localizzato nella regione cromosomica 16p13.3. Recenti studi hanno dimostrato che pazienti con quoziente intellettivo basso e tratti autistici possono avere grandi delezioni. Sulla base di queste osservazioni, abbiamo usato la Multiplex Ligation-dependent Probe Amplification (MLPA) per ricercare delezioni che interessano il gene CREB-binding in un paziente con sindrome di Rubinstein-Taybi e abbiamo identificato una delezione in eterozigosi che causa la rimozione di cinque esoni (esoni 17-21) codificanti per il dominio istone acetiltransferasi. Noi proponiamo, come primo approccio diagnostico, l'uso del metodo MLPA nel sottogruppo di pazienti con la sindrtome di Rubinstein-Taybi con quoziente di intelligenza basso e fenotipo autistico