Matteo Bertelli

Mutations and polymorphisms of the CLCN2 gene in idiopathic epilepsy

The authors analyzed the CLCN2 chloride channel gene in 112 probands with familial epilepsy, detecting 18 common polymorphisms. Two brothers with generalized epilepsy and their asymptomatic father, and a father and son with focal epilepsy carried variants of possible functional significance that were not found in 192 controls. The authors conclude that CLCN2 mutations may be a rare cause of familial epilepsy. Further studies are needed to test if polymorphisms in this gene are associated with epilepsy.

Neurological Disorders of Purine and Pyrimidine Metabolism

Purines and pyrimidines, regarded for a long time only as building blocks for nucleic acid synthesis and intermediates in the transfer of metabolic energy, gained increasing attention since genetically determined aberrations in their metabolism were associated clinically with various degrees of mental retardation and/or unexpected and often devastating neurological dysfunction. In most instances the molecular mechanisms underlying neurological symptoms remain undefined. This suggests that nucleotides and nucleosides play fundamental but still unknown roles in the development and function of several organs, in particular central nervous system. Alterations of purine and pyrimidine metabolism affecting brain function are spread along both synthesis (PRPS, ADSL, ATIC, HPRT, UMPS, dGK, TK), and breakdown pathways (5NT, ADA, PNP, GCH, DPD, DHPA, TP, UP), sometimes also involving pyridine metabolism. Explanations for the pathogenesis of disorders may include both cellular and mitochondrial damage: e.g. deficiency of the purine salvage enzymes hypoxanthine-guanine phosphoribosyltransferase and deoxyguanosine kinase are associated to the most severe pathologies, the former due to an unexplained adverse effect exerted on the development and/or differentiation of dopaminergic neurons, the latter due to impairment of mitochondrial functions. This review gathers the presently known inborn errors of purine and pyrimidine metabolism that manifest neurological syndromes, reporting and commenting on the available hypothesis on the possible link between specific enzymatic alterations and brain damage. Such connection is often not obvious, and though investigated for many years, the molecular basis of most dysfunctions of central nervous system associated to purine and pyrimidine metabolism disorders are still unexplained.

Molecular basis of hypoxanthine-guanine phosphoribosyltransferase deficiency in Italian Lesch-Nyhan patients: Identification of nine novel mutations

Summary: Lesch-Nyhan syndrome (LSN, McKusick 300322) is an X-linked genetic disease due, in its typical form, to the complete absence of hypoxanthine-guanine phosphoribosyltransferase (HPRT, EC 2.4.2.8) enzyme activity. It is characterized by hyperuricaemia, leading to gout and kidney stones, accompanied by severe neurological dysfunction with self-injurious behaviour, choreoathetosis and spasticity. Based on a worldwide birth incidence estimate of about 1:380000, one or two new cases are expected every year in Italy. We performed biochemical and molecular genetic studies on 28 Italian patients from 25 families who are likely to represent most living individuals with the syndrome in the country. They all had absent HPRT activity and a typical LNS phenotype. Genetic analysis identified 24 HPRT mutations, 9 of which had not been previously reported: 74C>G (P25R), IVS2+1G>C, 194-195delTC, 329-332delCAAC insTCTs, IVS9-1G>A, 506insC, IVS8-1G>C, 606G>T (L202F), 418G>C (G140R). No mutation hotspots were identified. Only two mutations were found in more than one family, indicating the lack of any major mutation causing LNS in Italy. Three mutations arose de novo, two in the probands mother, one in the maternal grandmother. The virtual complete absence of HPRT activity was related to deletions, nonsense, or missense mutations leading to nonconservative amino acid changes.

Multimodal Approach to Monitoring and Investigating Cone Structure and Function in an Inherited Macular Dystrophy

PURPOSE To examine a female subject, her father, and a brother harboring a missense mutation of the retinitis pigmentosa 1-like 1 (RP1L1) gene, over 2 years of follow-up. DESIGN Observational case series. METHODS setting: Fondazione G.B. Bietti IRCCS, Rome, Italy. STUDY POPULATION RP1L1 family members and controls. MAIN OUTCOME MEASURES Images of the cone mosaic acquired with an adaptive optics retinal camera, spectral-domain optical coherence tomography (SD OCT), and full-field and multifocal electroretinography (mfERG). RESULTS In the proband, best-corrected visual acuity (≤0.7 logMAR) was stable in both eyes during follow-up, though analysis of adaptive optics images showed decreased cone density in the central 9 degrees from the fovea with respect to controls (P < .05) and cone density loss in the parafoveal area (2 degrees; <12%-16%) during follow-up. Texture analysis of SD OCT images identified abnormalities of the ellipsoid zone in the central 7 degrees, while mfERG response amplitudes were reduced only in the central 5 degrees relative to controls. In the probands father, who had 0.0 logMAR visual acuity, significant cone loss was found in the central 7 degrees from the fovea (P < .05); abnormal SD OCT and mfERG values with respect to controls were found in corresponding retinal areas. No defects in the cone structure and function were found in the probands brother, who had 0.0 logMAR visual acuity. CONCLUSIONS Occult macular dystrophy was diagnosed based on genetic and multimodal ophthalmic findings. The quantitative assessment of photoreceptor survival or loss, based on analysis of adaptive optics retinal images, was valuable to monitor disease progression at a cellular level.

Late onset MLD with normal nerve conduction associated with two novel missense mutations in the ASA gene

Metachromatic leukodystrophy (MLD) rarely has its clinical onset in young adults, with a combination of cognitive and behavioural symptoms and peripheral neuropathy. Here we present an exceptional case with very late onset at 42 years of age and no clinical or neurophysiological sign of peripheral neuropathy. Molecular analysis revealed compound heterozygosity for two novel missense mutations affecting conserved residues in the arylsulphatase A (ASA) sulphatase and carboxyterminal domains, resulting in an 89% loss of enzymatic activity. This case indicates that MLD needs to be considered in the differential diagnosis of very late onset white matter diseases, even if not accompanied by peripheral nerve involvement.

Gene expression and mRNA editing of serotonin receptor 2C in brains of HPRT gene knock-out mice, an animal model of Lesch-Nyhan disease.

Lesch-Nyhan disease (LND), a genetic disorder associated with motor and psychiatric disturbance and self-injurious behaviour (SIB) is caused by a complete deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT). The connection between enzyme deficiency and neurological involvement is still unclear. Evidence exists for a role of basal ganglia dysfunction with decreased dopamine and excess serotonin striatal content. In this study, we investigate the role of serotonin receptor 2C (HTR2C) in the brains of HPRT gene knock-out mice, a model of LND. HTR2C expression is analyzed by real-time polymerase chain reaction (PCR) using SYBR-green detection methods. The percentage of edited HTR2C mRNA was determined by direct sequencing of amplification products of the region containing the editing sites. We found a 55% increase in the expression of HTR2C gene but no significant difference in mRNA editing levels between knock-out and control mice. The above alteration found in HPRT-deficient mice is similar to those found in other animal models used to study aggressive and self-injurious behaviour.

Novel mutations in the arylsulfatase A gene in eight Italian families with metachromatic leukodystrophy.

Metachromatic leukodystrophy (MLD) is an autosomal recessive lysosomal storage disease caused by arylsulfatase A (ARSA) deficiency. We analysed the ARSA gene in eight unrelated Italian families with different clinical variants of MLD and identified three novel mutations: two Ser406Gly, (Glu329Ter) associated with late infantile MLD and one (Leu52Pro) with juvenile MLD. Only one family carried a pseudodeficiency allele (Asn350Ser). The IVS2+1G>A mutation occurred in four families. We also identified three polymorphisms, all in heterozygosis: Thr391Ser was present in five families, Trp193Cys in four families, and Ala210Ala in one family. We could identify 100% of the alleles causing MLD in the families, involving 12 different mutations, resulting in improved prognosis and genetic counselling.

Bilateral Symmetry of Visual Function Loss in Cone-Rod Dystrophies.

PURPOSE To investigate bilateral symmetry of visual impairment in cone-rod dystrophy (CRD) patients and understand the feasibility of clinical trial designs treating one eye and using the untreated eye as an internal control. METHODS This was a retrospective study of visual function loss measures in 436 CRD patients followed at the Ophthalmology Department of the Catholic University in Rome. Clinical measures considered were best-corrected visual acuity, focal macular cone electroretinogram (fERG), and Ganzfeld cone-mediated and rod-mediated electroretinograms. Interocular agreement in each of these clinical indexes was assessed by t- and Wilcoxon tests for paired samples, structural (Deming) regression analysis, and intraclass correlation. Baseline and follow-up measures were analyzed. A separate analysis was performed on the subset of 61 CRD patients carrying likely disease-causing mutations in the ABCA4 gene. RESULTS Statistical tests show a very high degree of bilateral symmetry in the extent and progression of visual impairment in the fellow eyes of CRD patients. CONCLUSIONS These data contribute to a better understanding of CRDs and support the feasibility of clinical trial designs involving unilateral eye treatment with the use of fellow eye as internal control.

Physical exercise to prevent multifactorial diseases: a warning written in our genes?

The human genome has about forty thousand genes and has been subject to strong selective pressure in various periods and different geographical areas. Various evidence sustains the hypothesis that the widespread nature of multifactor diseases is partly due to modified life-styles (reduced physical activity, over-eating) and partly to genetic inheritance selected over thousands of years for life-styles that persisted almost up to the start of the twentieth century. Physical activity could therefore be a natural remedy for recovering part of the imbalance caused by modern life-styles in bodies “born to run” and fed parsimoniously. In order to prescribe it as a cure, more research is necessary into the effects of physical activity on the body at molecular level and into individual predispositions to be used in targeted prevention programmes.

NAD metabolism in HPRT-deficient mice

The activity of hypoxanthine-guanine phosphoribosyltransferase (HPRT) is virtually absent in Lesch-Nyhan disease (LND), an X-linked genetic disorder characterized by uric acid accumulation and neurodevelopmental dysfunction. The biochemical basis for the neurological and behavioral abnormalities have not yet been completely explained. Prior studies of cells from affected patients have shown abnormalities of NAD metabolism. In the current studies, NAD metabolism was evaluated in HPRT gene knock-out mice. NAD content and the activities of the enzymes required for synthesis and breakdown of this coenzyme were investigated in blood, brain and liver of HPRT- and control mice. NAD concentration and enzyme activities were found to be significantly increased in liver, but not in brain or blood of the HPRT- mice. These results demonstrate that changes in NAD metabolism occur in response to HPRT deficiency depending on both species and tissue type.