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Summary Signal peptide-CUB-EGF domain-containing protein 3 (SCUBE3) is a member of a small family of multifunctional cell surface-anchored glycoproteins functioning as co-receptors for a variety of growth factors. Here we report that bi-allelic inactivating variants in SCUBE3 have pleiotropic consequences on development and cause a previously unrecognized syndromic disorder. Eighteen affected individuals from nine unrelated families showed a consistent phenotype characterized by reduced growth, skeletal features, distinctive craniofacial appearance, and dental anomalies. In vitro functional validation studies demonstrated a variable impact of disease-causing variants on transcript processing, protein secretion and function, and their dysregulating effect on bone morphogenetic protein (BMP) signaling. We show that SCUBE3 acts as a BMP2/BMP4 co-receptor, recruits the BMP receptor complexes into raft microdomains, and positively modulates signaling possibly by augmenting the specific interactions between BMPs and BMP type I receptors. Scube3−/− mice showed craniofacial and dental defects, reduced body size, and defective endochondral bone growth due to impaired BMP-mediated chondrogenesis and osteogenesis, recapitulating the human disorder. Our findings identify a human disease caused by defective function of a member of the SCUBE family, and link SCUBE3 to processes controlling growth, morphogenesis, and bone and teeth development through modulation of BMP signaling.

Congenital ataxias are nonprogressive neurological disorders characterized by neonatal hypotonia, developmental delay and ataxia, variably associated with intellectual disability and other neurological or extraneurological features. We performed trio-based whole-exome sequencing of 12 families with congenital cerebellar and/or vermis atrophy in parallel with targeted next-generation sequencing of known ataxia genes (CACNA1A, ITPR1, KCNC3, ATP2B3 and GRM1) in 12 additional patients with a similar phenotype. Novel pathological mutations of ITPR1 (inositol 1,4,5-trisphosphate receptor, type 1) were found in seven patients from four families (4/24, ∼16.8%) all localized in the IRBIT (inositol triphosphate receptor binding protein) domain which plays an essential role in the regulation of neuronal plasticity and development. Our study expands the mutational spectrum of ITPR1-related congenital ataxia and indicates that ITPR1 gene screening should be implemented in this subgroup of ataxias.

Wiedemann–Steiner syndrome (WDSTS) is a Mendelian syndromic intellectual disability (ID) condition associated with hypertrichosis cubiti, short stature, and characteristic facies caused by pathogenic variants in the KMT2A gene. Clinical features can be inconclusive in mild and unusual WDSTS presentations with variable ID (mild to severe), facies (typical or not) and other associated malformations (bone, cerebral, renal, cardiac and ophthalmological anomalies). Interpretation and classification of rare KMT2A variants can be challenging. A genome-wide DNA methylation episignature for KMT2A-related syndrome could allow functional classification of variants and provide insights into the pathophysiology of WDSTS. Therefore, we assessed genome-wide DNA methylation profiles in a cohort of 60 patients with clinical diagnosis for WDSTS or Kabuki and identified a unique highly sensitive and specific DNA methylation episignature as a molecular biomarker of WDSTS. WDSTS episignature enabled classification of variants of uncertain significance in the KMT2A gene as well as confirmation of diagnosis in patients with clinical presentation of WDSTS without known genetic variants. The changes in the methylation profile resulting from KMT2A mutations involve global reduction in methylation in various genes, including homeobox gene promoters. These findings provide novel insights into the molecular etiology of WDSTS and explain the broad phenotypic spectrum of the disease.

Biallelic loss-of-function variants in MED23 cause a recessive syndromic intellectual disability condition with or without epilepsy (MRT18). Due to the small number of reported individuals, the clinical phenotype of the disorder has not been fully delineated yet, and the spectrum and frequency of neurologic features have not been fully characterized. Here, we report a 5-year-old girl with compound heterozygous for two additional MED23 variants. Besides global developmental delay, axial hypotonia and peripheral increased muscular tone, absent speech, and generalized tonic seizures, which fit well MRT18, the occurrence of postnatal progressive microcephaly has been here documented. A retrospective assessment of the previously reported clinical data for these subjects confirms the occurrence of postnatal progressive microcephaly as a previously unappreciated feature of the phenotype of MED23-related disorder.

This study focused on the molecular characterization of patients with leukoencephalopathy associated with a specific biochemical defect of mitochondrial respiratory chain complex III, and explores the impact of a distinct magnetic resonance imaging pattern of leukoencephalopathy to detect biallelic mutations in LYRM7 in patients with biochemically unclassified leukoencephalopathy. ‘Targeted resequencing’ of a custom panel including genes coding for mitochondrial proteins was performed in patients with complex III deficiency without a molecular genetic diagnosis. Based on brain magnetic resonance imaging findings in these patients, we selected additional patients from a database of unclassified leukoencephalopathies who were scanned for mutations in LYRM7 by Sanger sequencing. Targeted sequencing revealed homozygous mutations in LYRM7 , encoding mitochondrial LYR motif-containing protein 7, in four patients from three unrelated families who had a leukoencephalopathy and complex III deficiency. Two subjects harboured previously unreported variants predicted to be damaging, while two siblings carried an already reported pathogenic homozygous missense change. Sanger sequencing performed in the second cohort of patients revealed LYRM7 mutations in three additional patients, who were selected on the basis of the magnetic resonance imaging pattern. All patients had a consistent magnetic resonance imaging pattern of progressive signal abnormalities with multifocal small cavitations in the periventricular and deep cerebral white matter. Early motor development was delayed in half of the patients. All patients but one presented with subacute neurological deterioration in infancy or childhood, preceded by a febrile infection, and most patients had repeated episodes of subacute encephalopathy with motor regression, irritability and stupor or coma resulting in major handicap or death. LYRM7 protein was strongly reduced in available samples from patients; decreased complex III holocomplex was observed in fibroblasts from a patient carrying a splice site variant; functional studies in yeast confirmed the pathogenicity of two novel mutations. Mutations in LYRM7 were previously found in a single patient with a severe form of infantile onset encephalopathy. We provide new molecular, clinical, and neuroimaging data allowing us to characterize more accurately the molecular spectrum of LYRM7 mutations highlighting that a distinct and recognizable magnetic resonance imaging pattern is related to mutations in this gene. Inter- and intrafamilial variability exists and we observed one patient who was asymptomatic by the age of 6 years.

Lam et al. characterize a novel hematological/autoinflammatory disorder due to a de novo recurrent missense mutation of CDC42. The authors use in silico, in vitro, and in vivo analyses to correlate the molecular mechanisms altering CDC42 function to the observed phenotype., Hemophagocytic lymphohistiocytosis (HLH) is characterized by immune dysregulation due to inadequate restraint of overactivated immune cells and is associated with a variable clinical spectrum having overlap with more common pathophysiologies. HLH is difficult to diagnose and can be part of inflammatory syndromes. Here, we identify a novel hematological/autoinflammatory condition (NOCARH syndrome) in four unrelated patients with superimposable features, including neonatal-onset cytopenia with dyshematopoiesis, autoinflammation, rash, and HLH. Patients shared the same de novo CDC42 mutation (Chr1:22417990C>T, p.R186C) and altered hematopoietic compartment, immune dysregulation, and inflammation. CDC42 mutations had been associated with syndromic neurodevelopmental disorders. In vitro and in vivo assays documented unique effects of p.R186C on CDC42 localization and function, correlating with the distinctiveness of the trait. Emapalumab was critical to the survival of one patient, who underwent successful bone marrow transplantation. Early recognition of the disorder and establishment of treatment followed by bone marrow transplant are important to survival., Graphical Abstract

Non-classical congenital adrenal hyperplasia (NC-CAH) includes a group of genetic disorders due to a broad class of CYP21A2 variants identifying a disease-causing ‘C’ genotype. The heterozygous carriers of CYP21 mutations are at increased risk of developing clinically evident hyperandrogenism, even though clinical and laboratory characteristics are still underestimated. With the aim of obtaining a more accurate delineation of the phenotype of heterozygous carrier of CAH, we analyzed clinical, biochemical and molecular characteristics in a cohort of Sicilian subjects. Fifty-seven females with biallelic and monoallelic CYP21A2 variants classifying NC-CAH (24) and heterozygous carriers of CAH (33), respectively were selected. Forty-four females age-matched healthy controls were also enrolled and genotyped for CYP21A2. Clinical, hormonal and genetic data were collected. CYP21A2 monoallelic mutations, defining the heterozygous carriers state, were identified in subjects with clinical features including hirsutism, oligomenorrhoea, overweight and a PCO-like phenotype, particularly occurring in the age of adolescence. Consistently, levels of 17OHP and cortisol were found to be significantly different from NC-CAH. Overall, some clinical and laboratory findings including oligomenorrhea and 17OHP/cortisol ratio were observed as independent markers associated with carriers of CAH. Here we report a high prevalence of late-onset signs of polycystic ovary syndrome (PCOS) and hyperandrogenism in heterozygous carriers. The 17OHP/cortisol ratio may be a predictive tool to identify the carriers of CAH, even though specific cut-off values have not yet been identified.

Background Heterozygous mutations in OPA1 are a common cause of autosomal dominant optic atrophy, sometimes associated with extra-ocular manifestations. Few cases harboring compound heterozygous OPA1 mutations have been described manifesting complex neurodegenerative disorders in addition to optic atrophy. Results We report here three patients: one boy showing an early-onset mitochondrial disorder with hypotonia, ataxia and neuropathy that was severely progressive, leading to early death because of multiorgan failure; two unrelated sporadic girls manifesting a spastic ataxic syndrome associated with peripheral neuropathy and, only in one, optic atrophy. Using a targeted resequencing of 132 genes associated with mitochondrial disorders, in two probands we found compound heterozygous mutations in OPA1: in the first a 5 nucleotide deletion, causing a frameshift and insertion of a premature stop codon (p.Ser64Asnfs*7), and a missense change (p.Ile437Met), which has recently been reported to have clinical impact; in the second, a novel missense change (p.Val988Phe) co-occurred with the p.Ile437Met substitution. In the third patient a homozygous mutation, c.1180G > A (p.Ala394Thr) in OPA1 was detected by a trio-based whole exome sequencing approach. One of the patients presented also variants in mitochondrial DNA that may have contributed to the peculiar phenotype. The deleterious effect of the identified missense changes was experimentally validated in yeast model. OPA1 level was reduced in available patients’ biological samples, and a clearly fragmented mitochondrial network was observed in patients’ fibroblasts. Conclusions This report provides evidence that bi-allelic OPA1 mutations may lead to complex and severe multi-system recessive mitochondrial disorders, where optic atrophy might not represent the main feature. Electronic supplementary material The online version of this article (doi:10.1186/s13023-017-0641-1) contains supplementary material, which is available to authorized users.

Deafness is caused by a variety of facts, genetic and environmental. Regarding the acquired causes, deafness can be the consequence of prenatal infections, acoustic or cerebral trauma, and the use of ototoxic drugs. Deafness can be the only manifestation (nonsyndromic forms) or it may occur together with other phenotypic findings (syndromic forms). The majority of nonsyndromicdeafness has a genetic basis [Van Camp et al., 1997]. In recent years, deafness and hearing loss have assumed a clinical importance in the study of congenital disorders [Morton et al., 1991]. The clinical interest for hearing loss is supported by the social impact that this disorder has; if not treated, delays in the development of language and learning skills will occur [Yoshinaga-Itano et al., 1998]. In the absence of newborn screening, hearing loss might not be noticed by parents, teachers or paediatricians until the child begins to have difficulties at speaking and learning, sometimes as late as age 2 or 3 years. In genetic nonsyndromic hearing loss (NSHL) the inheritance pattern is autosomal recessive (80%), autosomal dominant (17%), X-linked (2–3%), and mitochondrial (

Background. Several mutations in CYP21 locus cause 21-Hydroxylase Deficiency (21-OHD). The most common mutations are widespread among the different geographic areas and their frequencies have been also reported to differ among certain populations. Aim. To obtain a large view on the frequencies of the most common mutations in the CYP21 locus, in Sicily, in Mediterranean and in other major geographic areas in the worldwide. Subjects and Methods. 308 unrelated CYP21A2 alleles leading 21-OHD in Sicily were genetically typed and compared with other series previously reported in Sicily and in surrounding regions. An analysis of the frequencies of the different geographic areas was also carried out. CYP21A2 typing was carried out using PCR-RFLP, for the detection of the CYP21A2 deletion, while sequencing analysis was performed to evaluate all the missense/non-sense mutations. Results: our study revealed that p.V281L (44.4%), I2splice (21.6%) and p.P30L (11.2%) were very frequent alleles, del8bp (0.4%) was found very rarely in Sicily and a novel mutation leading to nonclassical phenotype, p.L198F, was also discovered in this population. Allele frequencies were found to be significantly different from previously observed frequencies in Sicily. In addition, here we present the most significant frequency modifications among different geographic areas in the worldwide. Conclusions: as the distribution of the disease CYP21A2 alleles is heterogeneous around the world, the knowledge of the relative distributions allows a better management of 21-OHD for foetuses and newborns in different geographic areas.

We have previously described the development of a scaffold/matrix attachment region (S/MAR) episomal vector system for in vivo application and demonstrated its utility to sustain transgene expression in the mouse liver for at least 6 months following a single administration. Subsequently, we observed that transgene expression is sustained for the lifetime of the animal. The level of expression, however, does drop appreciably over time. We hypothesised that by eliminating the bacterial components in our vectors, we could improve their performance since bacterial sequences have been shown to be responsible for the immunotoxicity of the vector and the silencing of its expression when applied in vivo. We describe here the development of a minimally sized S/MAR vector, which is devoid of extraneous bacterial sequences. This minicircle vector comprises an expression cassette and an S/MAR moiety, providing higher and more sustained transgene expression for several months in the absence of selection, both in vitro and in vivo. In contrast to the expression of our original S/MAR plasmid vector, the novel S/MAR minicircle vectors mediate increased transgene expression, which becomes sustained at about twice the levels observed immediately after administration. These promising results demonstrate the utility of minimally sized S/MAR vectors for persistent, atoxic gene expression.

Background: Excessive androgen production, suspected in women when acne is accompanied by hirsutism and menstrual irregularities, may be due to congenital adrenal hyperplasia. This inherited disorder of cortisol biosynthesis is caused in more than 90–95% of all cases by 21-hydroxylase deficiency (21-OHD). The steroid 21-hydroxylase gene (CYP21) has a high degree of variability. Objective: This study was conducted to evaluate CYP21 gene mutations in a selected group of women with papulopustular and comedonal acne refractory to treatment, irregular menses and hirsutism. Methods: 30 out of 61 women enrolled underwent pelvic ultrasound examination and hormonal screening. In 9 patients with a polycystic ovary and hormonal pattern of adrenal hyperandrogenism a significant elevation of adrenocorticotropic hormone (ACTH) stimulated 17-hydroxyprogesterone was detected. These women positive in the ACTH stimulation test were submitted to CYP21 gene analysis. Results: Genetic testing revealed several different point mutations and demonstrated that a cohort of patients resistant to acne therapy can be carriers or affected by non-classical 21-OHD (late onset). Conclusion: Persistent acne can be the unique presenting sign of non-classical 21-OHD. Evaluation of CYP21 gene mutations may identify female carriers or patients for genetic counselling.

A barrier limiting the use of nonviral vectors for gene therapy is related to the short duration of transgene expression in vivo. Development, evaluation, and optimisation of a long term transgene expression using a non viral vector is currently the primary aim in our research field. Recently, we have demonstrate that a nonviral episomal plasmid (pDNA) vector combined with a scaffold/matrix attachment region (S/MAR) is able to sustain long-term expression in murine liver for at least six months following hydrodynamic injection. However, plasmids contain sequences, which are essential for propagation in bacteria but are unnecessary for expression in mammalian cells. These bacterial components are responsible for the epigenetic silencing of the vector and toxicity when applied in vivo.

Trombofilia ereditaria e complicanze gravidiche nella popolazione siciliana. È ormai noto che la trombofilia congenita rappresen- ti un fattore di rischio per la gravidanza. Può infatti es- sere causa di abortività ricorrente, perdita fetale (MEF), ritardo di crescita fetale (IUGR) e altri problemi nel mantenimento della gravidanza (preeclampsia e di- stacco di placenta). Nel presente studio è stata indaga- ta la prevalenza dei fattori trombofilici nella popolazio- ne siciliana e la possibile relazione fra essi ed un even- to trombotico uteroplacentare in gravidanza. Sono sta- ti comparati due gruppi di gestanti con anamnesi posi- tiva per presenza di almeno un fattore trombofilico e storia di insuccessi riproduttivi e/o complicanze gravi- diche: gruppo di studio (120 donne) trattato con LMWH 0.3 mL associata all’ASA 50 mg; gruppo con- trollo (44 donne) non trattato. L’analisi dei genotipi associati al fenomeno compli- canze gravidiche ha evidenziato come, fra i fattori trombofilici, il polimorfismo PAI-1 4G/4G sia preva- lente nella popolazione siciliana e che esso, da solo o in associazione con altri difetti genetici, sia in grado di au- mentare il rischio di complicanze ostetriche del 50%. Come emerso già da altri lavori pubblicati in letteratu- ra, anche nel nostro studio si è avuta la conferma che il trattamento con un'anticoagulante come l'eparina a basso peso molecolare (LMWH) è utile nel ridurre le complicazioni.

La sindrome di Crigler-Najjar (CNS) è una malattia molto rara (prevalenza 1/1.000.000 nati) a trasmissione autosomica recessiva, caratterizzata da un incremento cronico e grave di bilirubina sierica indiretta (non coniugata), dovuta alla parziale (tipo 2) o completa (tipo 1) assenza funzionale dell’enzima epatico glucoronosil transferasi UGT1. La malattia si manifesta in età neonatale con un ittero precoce e intenso, dovuto alla presenza di bilirubina non coniugata e l'esame fisico è nella norma; in epoca di sviluppo gli effetti clinici della CNS tipo 1 sono letali in quanto l’eccesso di bilirubina porta inevitabilmente ad una encefalopatia essendo tale pigmento fortemente liposolubile. Le mutazioni ad oggi descritte causano l’assenza di sintesi enzimatica o la sintesi di un prodotto aberrante non funzionale. La CNS tipo 2, in particolare, è caratterizzata dalla presenza di un’attività enzimatica residua ed è causata da mutazioni autosomiche sia dominanti, a penetranza incompleta, che recessive nel gene UGT1A1. La prova clinica del fenobarbital consente di distinguere le due forme (tipo I e tipo II) mentre il test genetico è il metodo più specifico per la corretta diagnosi. Il gene UGT1A1 è situato in posizione telomerica nel cromosoma 2 (2q37.1) e si estende per 110Kb. Il gene è costituito da 4 esoni consecutivi (ex2-ex5) e da un gruppo di 10 varianti dell’esone 1 posti all’estremità 5’. La selezione per splicing alternativo dell’esone 1 è finemente regolato dal promotore ed è responsabile della sintesi delle varie isoforme dell’enzima (Fig. 1). La proteina enzimatica (precursore UDP-glucuronosyltransferase 1-1) consta di 533 aminoacidi. Le mutazioni missense che correlano con tale patologia sono circa 79 e tale numero è in continuo aggiornamento presso il database internazionale (sito http://www.hgmd.cf.ac.uk/ac/index.php). Qui presentiamo due casi di Crigler Najjar tipo II dovuti a due nuove mutazioni del gene UGT1A1 e vogliamo sottolineare che l’eterogeneità genomica presso il nostro territorio suggerisce una valutazione completa del gene UGT1A1 per una corretta diagnosi di Crigler Najjar ed una valutazione funzionale delle nuove mutazioni eventualmente riscontrate.

When normal development and growth of the calvarial sutures is disrupted, craniosynostosis (premature calvarial suture fusion) may result. Classical craniosynostosis syndromes are autosomal dominant traits and include Apert, Pfeiffer, Crouzon, Jackson-Weiss, and Saethre-Chotzen syndromes. In these conditions, there is premature fusion of skull bones leading to an abnormal head shape, ocular hypertelorism with proptosis, and midface hypoplasia. It is known that mutations in the fibroblast growth factor receptors 1, 2, and 3 cause craniosynostosis. We report on a child with a clinically diagnosed Pfeiffer syndrome that shows the missense point mutation Q289P in exon 8 of the FGFR2 gene. This is a mutation not previously described in the Pfeiffer syndrome but reported in the Crouzon, Jackson-Weiss, and Saethre-Chotzen syndromes. In this paper, we propose the concept that these disorders may represent one genetic condition with phenotypic variability.

Un vettore ideale dovrebbe consentire l'espressione di un transgene senza alcuna limitazione di sicurezza e di riproducibilità. Qui riportiamo lo sviluppo di un nuovo vettore NON-virale basato su DNA episomale plasmidico (pDNA) che sembra soddisfare al pieno le caratteristiche del vettore ideale nel tessuto epatico. Questo pDNA deriva dalla combinazione tra un promotore epatospecifico (AAT promoter) posto a monte del transgene e un elemento S-MAR (Scafold/Matrix Attachment) posto a valle, mentre il reporter è gene della luciferasi. L'applicazione nel tessuto epatico è stata effettuata mediante iniezione ad alta pressione per via vena caudale del modello murino (hydrodynamic delivery). L'espressione a lungo termine è stata dimostrata attraverso diverse metodiche in vivo ed in vitro. In vivo, mediante l'utilizzo del Xenogen in vivo bio-imaging ai tempi "

L’Emofilia A, la più comune causa di disordine emorragico nell’uomo, è causata da una grande varietà di mutazioni genetiche nel gene F8 con un ampio range di quadri clinici definiti: forma severa (FaVIIIC G al nucleotide -2 nell’introne 16 (IVS16 -2). Tale mutazione porta verosimilmente ad una alterazione del sito di splicing e non essendo presente nei due genitori è da ritenersi una mutazione de novo.

Per una migliore comprensione patogenetica del ritardo mentale (RM) in combinazione o meno con le manifestazioni malformative, i tests di laboratorio sono di notevole importanza. La metodica CGH Array ha migliorato la sensibilità nella valutazione dei punti di rottura nelle delezioni cromosomiche. Qui presentiamo un caso di delezione 15q26.2-15qter in un bambina con medie note dismorfiche esterne (come la clinodattilia), interne (doppio distretto renale a destra) ed alcune alterazioni del linguaggio. L'analisi GCH Microarray è stata effettuata estraendo da linfociti di sangue periferico (Puregene DNA isolation kit, Gentra) e marcando (Cy5) il DNA del probando e realizzando l'Array CGH (Agilent human genome CGH Microarray Kit 4 x 44K, capacità risolutiva: 200Kb). La metodica FISH è stata realizzata mediante il multiprobe-T (Cytocell, UK) in accordo con le raccomandazioni della casa costruttrice. La comparazione genomica tra il DNA del probando ed il DNA di controllo (Human DNA, Promega), opportunamente marcato (Cy3), ha evidenziato la presenza di una monosomia parziale di circa 5,55 Mb del braccio lungo del cromosoma 15 (posizioni: da 94.783.205 a 100.338.915). La valutazione dei break points in tale monosomia ci consente di valutare i geni potenzialmente coinvolti nella patogenesi del RM. Particolare rilievo hanno i geni IGFR1, MEF2A e CHSY1 le cui alterazioni sembrano essere legate a più processi di sviluppo somatico e del SNC .

An ideal gene therapy vector should enable persistent transgene expression without limitations of safety and reproducibility. Here we report the development of a non-viral episomal plasmid DNA (pDNA) vector that appears to fulfil these criteria. This pDNA vector combines a scaffold/matrix attachment region (S/MAR) with a human liver-specific promoter (alpha1-antitrypsin (AAT)) in such a way that long-term expression is enabled in murine liver following hydrodynamic injection. Long-term expression is demonstrated by monitoring the longitudinal luciferase expression profile for up to 6 months by means of in situ bioluminescent imaging. All relevant control pDNA constructs expressing luciferase are unable to sustain significant transgene expression beyond 1 week post-administration. We establish that this shutdown of expression is due to promoter methylation. In contrast, the S/MAR element appears to inhibit methylation of the AAT promoter thereby preventing transgene silencing. Although this vector appears to be maintained as an episome throughout, we have no evidence for its establishment as a replicating entity. We conclude that the combination of a mammalian, tissue-specific promoter with the S/MAR element is sufficient to drive long-term episomal pDNA expression of genes in vivo.

La deficienza enzimatica di G6PDH è uno dei più comuni disordini nella popolazione siciliana in quanto più di 400 milioni di persone ne sono affette. Al fine di valutare la reale prevalenza dei casi nel nostro territorio presentiamo i dati di uno studio di genotipizzazione del locus G6PD (Xq28). 349 soggetti Siciliani di sesso maschile affetti da deficienza di G6PDH sono stati tipizzati secondo varie metodiche. Gli approcci di laboratorio sono: RFLPs (NlaIII, BclI, PstI e BspHI), PCR-Reverse Dot Blot (RDB) ed il sequenziamento diretto del gene. Le prime metodiche sono utili per definire le mutazioni già descritte e comunque le più comuni; il sequenziamento diretto è determinante per la valutazione delle nuove varianti genotipiche. Oltre alla coorte di campioni è stata tipizzata anche una coorte di controllo costituita da 104 individui maschi Siciliani da almeno tre generazioni e clinicamente asintomatici. I dati ottenuti consentono di definire la prevalenza delle mutazioni più frequenti del gene G6PD (G6PD*Siattle G844C, 37%; G6PD*Mediterranea C563T, 32%; G6PD*AG202A / A376G, 18%; G6PD*Santamaria A376G / A542T, 9%). Inoltre, grazie al sequenziamento genico diretto è stato possibile identificare una nuova variante G6PD*Palermo R257M (C769A / G770A Esone 7). Lo studio dei genotipi G6PD in Sicilia acquisisce notevole importanza in quanto anche questi sono stati oggetto di selezione naturale positiva in tempi endemici di Malaria.

An ideal gene therapy vector should enable persistent transgene expression without limitations of safety and reproducibility. Here we report the development of a non-viral episomal plasmid DNA (pDNA) vector that appears to fulfil these criteria. This pDNA vector combines a scaffold/matrix attachment region (S/MAR) with a human liver-specific promoter (a1-antitrypsin (AAT)) in such a way that long-term expression is enabled in murine liver following hydrodynamic injection. Long-term expression is demonstrated by monitoring the longitudinal luciferase expression profile for up to 6 months by means of in situ bioluminescent imaging. We conclude that the combination of a mammalian, tissue-specific promoter with the S/MAR element is sufficient to drive longterm episomal pDNA expression of genes in vivo.

Myhre syndrome is a developmental disorder characterized by reduced growth, generalized muscular hypertrophy, facial dysmorphism, deafness, cognitive deficits, joint stiffness, and skeletal anomalies. Here, by performing exome sequencing of a single affected individual and coupling the results to a hypothesis-driven filtering strategy, we establish that heterozygous mutations in SMAD4, which encodes for a transducer mediating transforming growth factor β and bone morphogenetic protein signaling branches, underlie this rare Mendelian trait. Two recurrent de novo SMAD4 mutations were identified in eight unrelated subjects. Both mutations were missense changes altering Ile500 within the evolutionary conserved MAD homology 2 domain, a well known mutational hot spot in malignancies. Structural analyses suggest that the substituted residues are likely to perturb the binding properties of the mutant protein to signaling partners. Although SMAD4 has been established as a tumor suppressor gene somatically mutated in pancreatic, gastrointestinal, and skin cancers, and germline loss-of-function lesions and deletions of this gene have been documented to cause disorders that predispose individuals to gastrointestinal cancer and vascular dysplasias, the present report identifies a previously unrecognized class of mutations in the gene with profound impact on development and growth.