Luigi Donato

Update on Novel CCM Gene Mutations in Patients with Cerebral Cavernous Malformations

Cerebral cavernous malformations (CCMs) are lesions affecting brain microvessels. The pathogenesis is not clearly understood. Conventional classification criterion is based on genetics, and thus, familial and sporadic forms can be distinguished; however, classification of sporadic cases with multiple lesions still remains uncertain. To date, three CCM causative genes have been identified: CCM1/KRIT1, CCM2/MGC4607 and CCM3/PDCD10. In our previous mutation screening, performed in a cohort of 95 Italian patients, with both sporadic and familial cases, we identified several mutations in CCM genes. This study represents further molecular screening in a cohort of 19 Italian patients enrolled by us in the few last years and classified into familial, sporadic and sporadic with multiple lesions cases. Direct sequencing and multiplex ligation-dependent probe amplification (MLPA) analysis were performed to detect point mutations and large genomic rearrangements, respectively. Effects of detected mutations and single-nucleotide polymorphisms (SNPs) were evaluated by an in silico approach and by western blot analysis. A novel nonsense mutation in CCM1 and a novel missense mutation in CCM2 were detected; moreover, several CCM2 gene polymorphisms in sporadic CCM patients were reported. We believe that these data enrich the mutation spectrum of CCM genes, which is useful for genetic counselling to identify both familial and sporadic CCM cases, as early as possible.

miRNAexpression profile of retinal pigment epithelial cells under oxidative stress conditions

Deep analysis of regulative mechanisms of transcription and translation in eukaryotes could improve knowledge of many genetic pathologies such as retinitis pigmentosa (RP). New layers of complexity have recently emerged with the discovery that ‘junk’ DNA is transcribed and, among these, miRNAs have assumed a preponderant role. We compared changes in the expression of miRNAs obtained from whole transcriptome analyses, between two groups of retinal pigment epithelium (RPE) cells, one untreated and the other exposed to the oxidant agent oxidized low‐density lipoprotein (oxLDL), examining four time points (1, 2, 4 and 6 h). We found that 23 miRNAs exhibited altered expression in the treated samples, targeting genes involved in several biochemical pathways, many of them associated to RP for the first time, such as those mediated by insulin receptor signaling and son of sevenless. Moreover, five RP causative genes (KLHL7, RDH11, CERKL, AIPL1 and USH1G) emerged as already validated targets of five altered miRNAs (hsa‐miR‐1307, hsa‐miR‐3064, hsa‐miR‐4709, hsa‐miR‐3615 and hsa‐miR‐637), suggesting a tight connection between induced oxidative stress and RP development and progression. This miRNA expression analysis of oxidative stress‐induced RPE cells has discovered new regulative functions of miRNAs in RP that should lead to the discovery of new ways to regulate the etiopathogenesis of RP.

Detection of Novel Mutation in Ccm3 Causes Familial Cerebral Cavernous Malformations

Cerebral cavernous malformations are vascular lesions that usually involve brain micro-vessels. They can occur both in a sporadic form and familial one. Causes of familial forms are mutations at three loci: CCM1/KRIT1, CCM2/MGC4607, and CCM3/PDCD10. Here, we describe a novel CCM3 missense mutation (c.422T>G) detected in two Greek brothers showing multiple lesions at magnetic resonance imaging; to date, only the youngest is symptomatic. Bioinformatics tools showed this novel variant causes a loss of function in Pdcd10 protein due to its localization in the eighth helix and, particularly, affects Leu141, a highly conserved amino acid. Roles of Pdcd10 in angiogenesis regulation and its association with early development of cerebral cavernous malformations were also considered.

Bioinformatic Analysis of a “Functional Cluster” Probably Related to Retinitis Pigmentosa

RESEARCH ARTICLE Bioinformatic Analysis of a “Functional Cluster” Probably Related to Retinitis Pigmentosa Luigi Donato and Lucia Denaro Department of Biomedical and Dental Sciences and Morphofunctional Images, Division of Medical Biotechnologies and Preventive Medicine, University of Messina, Messina, Italy Department of vanguard medicine and therapies, biomolecular strategies and neuroscience, Section of NeuroscienceApplied Molecular Genetics and Predictive Medicine, I. E. ME. S. T., Palermo, Italy Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Messina, Italy

Stargardt Phenotype Associated With Two ELOVL4 Promoter Variants and ELOVL4 Downregulation: New Possible Perspective to Etiopathogenesis?

Purpose Stargardt disease (STGD) is the most common form of inherited juvenile macular degeneration. It is inherited as autosomal recessive trait (STGD1), although STGD3 and STGD4 are inherited as autosomal dominant inheritance pattern. STGD3 is caused by mutations in the elongation of very long-chain fatty acids-like 4 (ELOVL4) gene encoding for a very long-chain fatty acid elongase. Mutations lead to a truncated Elovl4, lacking of a dilysine motif necessary for retention of transmembrane proteins in the endoplasmic reticulum. STGD occurs due to altered synthesis of very long-chain polyunsaturated fatty acids (VLC-PUFA). Our work investigates the role of two variants in the ELOVL4 gene promoter region, c.-236 C>T (rs240307) and c.-90 G>C (rs62407622), identified in a patient with STGD in transconfiguration. Methods Their effects on ELOVL4 expression were examined by Dual-Luciferase Reporter assay. Results rs62407622 and rs240307 variants caused 14% and 18% of expression reduction, respectively, compared with wild-type promoter. A very strong decreased gene expression was caused by coexistence of both variants. Conclusions A highly reduced activity of the ELOVL4 promoter was registered due to combination of two variants. Decrease of ELOVL4 enzymatic activity could lead to a deficiency of VLC-PUFA, essential components for rods function and longevity, which are among the parameters involved in the etiopathogenesis of STGD.

Corrigendum to: miRNA expression profile of retinal pigment epithelial cells under oxidative stress conditions

[This corrects the article DOI: 10.1002/2211-5463.12360.].

Relevance of CCM gene polymorphisms for clinical management of sporadic cerebral cavernous malformations

Cerebral cavernous malformations (CCMs) are clusters of capillaries in the brain that may cause focal deficits or seizures in affected patients. They occur in both sporadic and inherited autosomal dominant form. Germline mutations in CCM1, CCM2 and CCM3 were identified in familial cases. Over the past 13years we performed sequencing and MLPA of the CCM genes in all sporadic and familial CCM cases coming from some hospital clinics of Neurology and Neurosurgery of Messina and other Italian cities. Our results showed that CCM sporadic patients, negative for previously reported CCM gene causative mutations, always carried known CCM polymorphisms. Previously, we reported polymorphisms in CCM2 gene associated with an increase in risk for CCM. Here, we undertook a case-control study to investigate the possible association of others polymorphisms (c.485+65 C/G, c.989+63 C/G, c.1980 A/G in CCM1 gene, c.472+127 C/T in CCM2 and c.150 G/A in CCM3) with CCMs. The five polymorphisms were characterized in 64 sporadic patients and in 90 healthy controls by ASO-PCR. Statistically significant differences in frequencies between patients and controls were found for c.485+65C/G, c.1980 A/G and c.472+127C/T polymorphisms. For c.485+65C/G polymorphism, a higher frequency of mutated allele (G) was found in patients group (9%) than in controls (2%) (p=0.0041); for c.1980 A/G polymorphism, we found a frequency of mutated allele (G) higher in the control group (25%) compared to that of patients (8%) (p=0.0396). Same trend was observed for c.472+127C/T polymorphism (T allele frequency=34% and 6% in control group and patients, respectively; p=0.0001). Polymorphisms c.485+65C/G, c.1980 A/G and c.472+127C/T were associated with an increased risk of CCM as indicated by odds ratio values. Furthermore, c.1980 A/G and c.472+127C/T polymorphisms were associated with less severe CCM symptomatology. Identification of these polymorphisms in CCM sporadic patient may represent a useful tool for clinicians to determine prognosis, scheduled periodic checks and appropriate treatment strategy.

A novel RLBP1 gene geographical area-related mutation present in a young patient with retinitis punctata albescens

BackgroundAutosomal recessive forms of retinitis punctata albescens (RPA) have been described. RPA is characterized by progressive retinal degeneration due to alteration in visual cycle and consequent deposit of photopigments in retinal pigment epithelium. Five loci have been linked to RPA onset. Among these, the retinaldehyde-binding protein 1 gene, RLBP1, is the most frequently involved and several founder mutations were reported. We report results of a genetic molecular investigation performed on a large Sicilian family in which appears a young woman with RPA.ResultsThe proband is in homozygous condition for a novel RLBP1 single-pair deletion, and her healthy parents, both heterozygous, are not consanguineous. Thenovelc.398delC (p.P133Qfs*258) involves the exon 6 and leads to a premature stop codon, resulting in a truncated protein entirely missing of CRAL-TRIO lipid-binding domain.Pedigree analysis showed other non-consanguineous relatives heterozygous for the same mutation in the family. Extension of mutation research in the native town of the proband revealed its presence also in healthy subjects, in a heterozygous condition.ConclusionsA novel RLBP1 truncating mutation was detected in a young girl affected by RPA. Although her parents are not consanguineous, the mutation was observed in a homozygous condition. Being them native of the same small Sicilian town of Fiumedinisi, the hypothesis of a geographical area-related mutation was assessed and confirmed.

First case of Currarino Syndrome and Trimethylaminuria: two rare diseases for a complex clinical presentation.

Currarino syndrome (CS; Online Mendelian Inheritance in Man [OMIM] no. 176450), also known as Currarino triad, is a rare congenital condition characterized by three main anomalies: partial sacral agenesis, presacral mass and anorectal malformation (ARM). Sacral agenesis usually does not involve the first sacral vertebra but is found in the S2–S5 vertebrae, taking a typical shape of a sickle on X-ray examination. The presacral mass can be anterior myelomeningocele (approximately 60%), teratoma (25%), neurenteric cyst, or hamartoma. These lesions are usually detectable on magnetic resonance imaging (MRI). Although a presacral mass is often benign, cases of malignant transformation and meningitis have been reported. Anorectal stenosis, rectal duplication, anal atresia, anteriorly placed anus and fistulae are, instead, the most common ARM; more rarely, urogenital malformations have also been described. The triad can be incomplete, with only one or two anomalies, but sacral agenesis is usually present, although it may be asymptomatic. About one-third of patients are symptomatic. Chronic constipation is the main clinical manifestation from early childhood onwards and it can also be temporary. Among patients, 39% manifest a severe phenotype, 28.3% show only one anomaly, in 28% malformations are detected only by X-ray examination and 4% are asymptomatic. To date, the incidence of CS remains not precisely established but seems to range between 0.001% and 0.009%. Women are more frequently affected. A familial form has been described as an inherited autosomal dominant pattern with incomplete penetrance and variable expression. Almost all familial cases and about 30% of sporadic ones are caused by mutations of the homeobox gene HLXB9 (7q36), now renamed motor neuron and pancreas homeobox 1 (MNX1; GenBank: NG_013212.1). MNX1 encodes for the nuclear homeobox HB9 (HLXB9) protein, acting as a transcription factor for genes involved in organ development. MNX1 exon 1 is a mutational hotspot, with the consequent alteration of DNA-binding specificity and the reduced nuclear translocation of the mutated protein. Homeobox protein HB9 contains a homeodomain, preceded by a highly conserved region of 82 amino acids (159–241) and a region of polyalanine that expands from residue 121 to 134 in exon 1. The polyalanine tract is coded by polymorphic GCCN repeats that may range from GCC8 to GCC13. 13