Mitochondrial dysfunction in hereditary optic neuropathies

MITOCHONDRIAL DYSFUNCTION IN HEREDITARY OPTIC NEUROPATHIES Mitochondrial pathologies are a heterogeneous group of clinical manifestations characterized by oxidative phosphorylation impairment. At the beginning of their recognition mitochondrial pathologies were regarded as rare disorders but indeed they are more frequent than originally thought. Due to the unique mitochondria peculiarities mitochondrial pathologies can be caused by mutations in both mitochondrial and nuclear genomes. The poor knowledge of pathologic mechanism of these disorders has not allowed a real development of the “mitochondrial medicine”, that is currently limited to symptoms mitigation. Leber hereditary optic neuropathy (LHON) was the first pathology to be linked to a point mutation in the mtDNA. The mechanism by which point mutations in mitochondrial gene encoding Complex I subunits leads to optic nerve degeneration is still unknown, although is well accepted that other genetic or environmental factors are involved in the modulation of pathology, where a pivotal role is certainly played by oxidative stress. We studied the relationship between the Ala16Val dimorphism in the mitochondrial targeting sequence of nuclear gene SOD2 and the 3460/ND1 LHON mutation. Our results show that, in control population, the heterozygous SOD2 genotype is associated to a higher activity and quantity of MnSOD, particularly with respect to Val homozygotes. Furthermore, we demonstrated that LHON patients harboring at least one Ala allele are characterized by an increased MnSOD activity with respect to relative control population. Since the ATP synthesis rate – severely reduced in LHON patients lymphocytes - is not affected by the SOD2 genotype, we concluded that SOD2 gene could modulate the pathogenicity of LHON mutations through a mechanism associated to an increase of reactive oxygen species production. Autosomal dominant optic atrophy (ADOA) is a pathology linked to mutations in nuclear gene encoding Opa1, a dynamin-related protein localized in the mitochondrial matrix. Although the clinical course is slightly different, the endpoint of ADOA is exactly the same of LHON: optic nerve degeneration with specific involvement of retinal ganglion cells. Opa1 is a relatively new protein, whose major role is the regulation of mitochondrial fusion. Mitochondrial morphology is the results of the equilibrium between two opposite force: fusion and fission, two processes that have to be finely regulated in order to preserve mitochondrial and cellular physiology. We studied fibroblasts deriving from ADOA patients characterized by a new deletion in the GTPase domain of the OPA1 gene. The biochemical characterization of ADOA and control fibroblasts has concerned the evaluation of ATP synthesis rate, mitochondrial membrane potential in different metabolic conditions and the morphological status of mitochondria. Regarding ATP synthesis rate we did not find significant differences between ADOA and control fibroblasts even though a trend toward increased reduction in ADOA samples is observed when fibroblasts are grown in absence of glucose or in the medium containing gramicidin. Furthermore, we found that also in ADOA fibroblasts membrane potential is actively maintained by proton pumping of fully functional respiratory chain complexes. Our results indicate that the mutation found in the pedigree analyzed acts primary impairing the mitochondrial fusion without affecting the energy production, supporting the notion that cell function is tightly linked to mitochondrial morphology. Mitochondrial dysfunctions are acquiring great attention because of their recognized relevance not only in aging but also in age-related pathologies including cancer, cardiovascular disease, type II diabetes, and neurodegenerative disorders. The involvement of mitochondria in such detrimental pathologies that, currently, have become so common enhances the necessity of standardization of therapeutic strategies capable of rescuing the normal mitochondrial function. In order to propose an alternative treatment for energy deficiency-disorders we tested the effect of substrates capable to stimulate the substrate-level phosphorylation on viability and energy availability in different experimental models grown under different metabolic conditions. In fibroblasts, the energy defect was achieved by culturing cells in presence of oligomycin, an inhibitor of ATP synthase complex. NARP cybrids have been used as model of mitochondrial pathology. Cell viability and ATP content have been considered as parameters to assay the capability of exogenous substrate to rescue energy failure. Our results suggest that patients suffering for some forms of ATP synthase deficiency, or characterized by a deficiency in energy production, might benefit from dietary or pharmacological treatment based on supplementation of α-ketoglutarate and aspartate.

Riabilitazione protesica ed implanto-protesica di pazienti in età evolutiva affetti da Displasia Ectodermica

Introduction. Ectodermal Dysplasias are a heterogeneous group of inherited disorders characterized by dysplasia of tissues of ectodermal origin (hair, nails, teeth, skins and glands). Clinically, it may be divided into two broad categories: the X-linked hypoidrotic form and the hidrotic form. Hypohidrotic Ectodermal Dysplasia (H.E.D) is characterized by the triad oligo-anodontia, hypotricosis, hypo-anhydrosis (Christ-Siemens-Tourane syndrome). The incidence of HED is about 1/100,000. Mutation in the actodysplasin-A (EDA) and ectodysplasin-A receptor (EDAR) genes are responsible for X-linked and autosomal HED. The clinical features include sparse, fine hair, missing or conical-shaped teeth, decreased sweat and mucous glands, hypoplastic skin, and heat intolerance with exercise or increased ambient temperature. Complete or partial anodontia and malformation of teeth are the most frequent dental findings. Incisors and canines are often conical-shaped while primarily second molars, if present, are mostly affected by taurodontism. Treatment is supportive and includes protection from heat exposure, early prosthetic rehabilitation, skin, hair ear, nose and nail care, and genetic counseling for family planning. The diagnosis of HED in the neonatal and early infancy period may be difficult since sparse hair and absent teeth are normal finding at this age. In childhood the diagnosis is more easily made on the basis of history and clinical examination. Dental abnormalities are the most common complaint. Prosthetic rehabilitation has been recommended as an essential part of the management of HED because is important from functional, esthetic, and psychological standpoint. A team approach that includes input from a pediatric dentist, an orthodontist, a prosthodontist, and an oral and maxillofacial surgeon is necessary for a successful outcome. Conventional prosthodontic rehabilitation in young patient is often difficult because of the anatomical abnormalities of existing teeth and alveolar ridges. The conical shaped teeth and “knife-edge” alveolar ridges result in poor retention and instability of dentures. Moreover, denture must permit jaws expansion and a correct pattern of growth. Materials and Methods. Complete removable dentures were provided to allow for normal physiological development and a corrected masticatory function. Initial maxillary and mandibular impressions were made with smallest stock trays and irreversible hydrocolloid and then final impressions ware made with light-bodied polysulfide rubber base impression material. A base of autopolymerizing resin was constructed and a wax rim was added to the base. The patient’s vertical dimension of occlusion was established by assessing phonetic and esthetic criteria. Preliminary occlusal relations were recorded, and the mandibular cast was mounted on the articulator. Acrylic resin teeth specific for children dentures were selected and mounted. The dentures were tried in and, after proper adjustments, were inserted. The patients were monitored clinically every month to fit prostheses. Cephalometric radiographs were taken every 6 month with the prostheses in place in order to evaluate correct pattern of growth. Cephalometric measurements were realized and used to evaluate the effect of rehabilitation on craniofacial growth. Cephalometric measurements of sound patients were compared with ED patients. After two month expander screws (three-way screw in the upper denture and two-way the lower one)were inserted in each denture in order to permit the expansion of the denture and the jaws growth. Where conical teeth were present, composite crown were realized and luted to improve the esthetic and phonesis. In order to improve retention the placement of endosseous implants was carried out. TC 3D Accuitomo was performed and a resin model of mandibular bone of the patient was realized. At the age of 11 years two implants were inserted into anterior mandible in a child with anodontia. Despite a remarkable multi-dimensional atrophy of the mandibular alveolar process, the insertion of two tapered screw implants (SAMO Smiler, diameter 3.8, length 10 mm). After a submerged healing period of two-three month, the implants were exposed. Implants were connected with an expansion guide that permits mandibular growth and prosthetic retention. The amount of mandibular growth was also evaluate dusing the expansion guide. Results. Early oral rehabilitation improve oral function, phonesis and esthetic, reducing social impairment. Treated patients showed normal cephalometric measurement. Early rehabilitation is able to prevent the prognatissm of the mandibula . The number of teeth was significantly related to several changes in craniofacial morphology. Discussion. In the present study the 5,3% of ED patients showed hypodontia, the l’89,4% di oligodontia, and the 5,3% di anodontia. The cephalometric analysis supports that ED patients showed midface hypoplasia. ED groups showed an increased pogonion to nasion measurement than sound patients, indicative of class III tendency. The present study demonstrated that number of teeth was significantly correlated with deviation of cephalometric measurements from normality. Oligoanodontia is responsible for changing of cephalometric measuraments also on sagittal plane with a class III tendency. Maxillary jaw showed a retrused position related to the presence of hypodontia.

Studies of OPA1 pathogenic mechanisms in Dominant Optic Atrophy and novel protein function in mitochondrial DNA stability

The mitochondrion is an essential cytoplasmic organelle that provides most of the energy necessary for eukaryotic cell physiology. Mitochondrial structure and functions are maintained by proteins of both mitochondrial and nuclear origin. These organelles are organized in an extended network that dynamically fuses and divides. Mitochondrial morphology results from the equilibrium between fusion and fission processes, controlled by a family of “mitochondria-shaping” proteins. It is becoming clear that defects in mitochondrial dynamics can impair mitochondrial respiration, morphology and motility, leading to apoptotic cell death in vitro and more or less severe neurodegenerative disorders in vivo in humans. Mutations in OPA1, a nuclear encoded mitochondrial protein, cause autosomal Dominant Optic Atrophy (DOA), a heterogeneous blinding disease characterized by retinal ganglion cell degeneration leading to optic neuropathy (Delettre et al., 2000; Alexander et al., 2000). OPA1 is a mitochondrial dynamin-related guanosine triphosphatase (GTPase) protein involved in mitochondrial network dynamics, cytochrome c storage and apoptosis. This protein is anchored or associated on the inner mitochondrial membrane facing the intermembrane space. Eight OPA1 isoforms resulting from alternative splicing combinations of exon 4, 4b and 5b have been described (Delettre et al., 2001). These variants greatly vary among diverse organs and the presence of specific isoforms has been associated with various mitochondrial functions. The different spliced exons encode domains included in the amino-terminal region and contribute to determine OPA1 functions (Olichon et al., 2006). It has been shown that exon 4, that is conserved throughout evolution, confers functions to OPA1 involved in maintenance of the mitochondrial membrane potential and in the fusion of the network. Conversely, exon 4b and exon 5b, which are vertebrate specific, are involved in regulation of cytochrome c release from mitochondria, and activation of apoptosis, a process restricted to vertebrates (Olichon et al., 2007). While Mgm1p has been identified thanks to its role in mtDNA maintenance, it is only recently that OPA1 has been linked to mtDNA stability. Missense mutations in OPA1 cause accumulation of multiple deletions in skeletal muscle. The syndrome associated to these mutations (DOA-1 plus) is complex, consisting of a combination of dominant optic atrophy, progressive external ophtalmoplegia, peripheral neuropathy, ataxia and deafness (Amati- Bonneau et al., 2008; Hudson et al., 2008). OPA1 is the fifth gene associated with mtDNA “breakage syndrome” together with ANT1, PolG1-2 and TYMP (Spinazzola et al., 2009). In this thesis we show for the first time that specific OPA1 isoforms associated to exon 4b are important for mtDNA stability, by anchoring the nucleoids to the inner mitochondrial membrane. Our results clearly demonstrate that OPA1 isoforms including exon 4b are intimately associated to the maintenance of the mitochondrial genome, as their silencing leads to mtDNA depletion. The mechanism leading to mtDNA loss is associated with replication inhibition in cells where exon 4b containing isoforms were down-regulated. Furthermore silencing of exon 4b associated isoforms is responsible for alteration in mtDNA-nucleoids distribution in the mitochondrial network. In this study it was evidenced that OPA1 exon 4b isoform is cleaved to provide a 10kd peptide embedded in the inner membrane by a second transmembrane domain, that seems to be crucial for mitochondrial genome maintenance and does correspond to the second transmembrane domain of the yeasts orthologue encoded by MGM1 or Msp1, which is also mandatory for this process (Diot et al., 2009; Herlan et al., 2003). Furthermore in this thesis we show that the NT-OPA1-exon 4b peptide co-immuno-precipitates with mtDNA and specifically interacts with two major components of the mitochondrial nucleoids: the polymerase gamma and Tfam. Thus, from these experiments the conclusion is that NT-OPA1- exon 4b peptide contributes to the nucleoid anchoring in the inner mitochondrial membrane, a process that is required for the initiation of mtDNA replication and for the distribution of nucleoids along the network. These data provide new crucial insights in understanding the mechanism involved in maintenance of mtDNA integrity, because they clearly demonstrate that, besides genes implicated in mtDNA replications (i.e. polymerase gamma, Tfam, twinkle and genes involved in the nucleotide pool metabolism), OPA1 and mitochondrial membrane dynamics play also an important role. Noticeably, the effect on mtDNA is different depending on the specific OPA1 isoforms down-regulated, suggesting the involvement of two different combined mechanisms. Over two hundred OPA1 mutations, spread throughout the coding region of the gene, have been described to date, including substitutions, deletions or insertions. Some mutations are predicted to generate a truncated protein inducing haploinsufficiency, whereas the missense nucleotide substitutions result in aminoacidic changes which affect conserved positions of the OPA1 protein. So far, the functional consequences of OPA1 mutations in cells from DOA patients are poorly understood. Phosphorus MR spectroscopy in patients with the c.2708delTTAG deletion revealed a defect in oxidative phosphorylation in muscles (Lodi et al., 2004). An energetic impairment has been also show in fibroblasts with the severe OPA1 R445H mutation (Amati-Bonneau et al., 2005). It has been previously reported by our group that OPA1 mutations leading to haploinsufficiency are associated in fibroblasts to an oxidative phosphorylation dysfunction, mainly involving the respiratory complex I (Zanna et al., 2008). In this study we have evaluated the energetic efficiency of a panel of skin fibroblasts derived from DOA patients, five fibroblast cell lines with OPA1 mutations causing haploinsufficiency (DOA-H) and two cell lines bearing mis-sense aminoacidic substitutions (DOA-AA), and compared with control fibroblasts. Although both types of DOA fibroblasts maintained a similar ATP content when incubated in a glucose-free medium, i.e. when forced to utilize the oxidative phosphorylation only to produce ATP, the mitochondrial ATP synthesis through complex I, measured in digitonin-permeabilized cells, was significantly reduced in cells with OPA1 haploinsufficiency only, whereas it was similar to controls in cells with the missense substitutions. Furthermore, evaluation of the mitochondrial membrane potential (DYm) in the two fibroblast lines DOA-AA and in two DOA-H fibroblasts, namely those bearing the c.2819-2A>C mutation and the c.2708delTTAG microdeletion, revealed an anomalous depolarizing response to oligomycin in DOA-H cell lines only. This finding clearly supports the hypothesis that these mutations cause a significant alteration in the respiratory chain function, which can be unmasked only when the operation of the ATP synthase is prevented. Noticeably, oligomycin-induced depolarization in these cells was almost completely prevented by preincubation with cyclosporin A, a well known inhibitor of the permeability transition pore (PTP). This results is very important because it suggests for the first time that the voltage threshold for PTP opening is altered in DOA-H fibroblasts. Although this issue has not yet been addressed in the present study, several are the mechanisms that have been proposed to lead to PTP deregulation, including in particular increased reactive oxygen species production and alteration of Ca2+ homeostasis, whose role in DOA fibroblasts PTP opening is currently under investigation. Identification of the mechanisms leading to altered threshold for PTP regulation will help our understanding of the pathophysiology of DOA, but also provide a strategy for therapeutic intervention.

Sindrome di Buschke-Ollendorff: clinica e genetica

Buschke Ollendorff syndrome (BOS) is a rare autosomal dominant genodermatosis, with high penetrance and variable expressivity, characterized by the association of connective tissue naevi and osteopoikilosis. Both cutaneous and osseous manifestations are usually asymptomatic. The disease is caused by a loss-of-function mutation in the gene LEMD3, that is located on chromosome 12q13. Differential diagnosis mainly includes pseudoxantoma elasticum, morphea, lipoid proteinosis, papular elastorrhexis, juvenile elastoma, papular mucinosis. The 2 cases of BOS here reported are an example of segmental type 2 autosomal dominant genodermatosis, that is due to the loss of heterozygosity occurring at an early developmental stage in a heterozygous patient, causing a segmental homozygosity. Such patients usually have pronounced segmental lesions in the first years of life and later develop disseminated symmetrical lesions.

Pathogenic mechanisms in mitochondrial optic neuropathies

Leber’s hereditary optic neuropathy (LHON) and Autosomal Dominant Optic Atrophy (ADOA) are the two most common inherited optic neuropathies and both are the result of mitochondrial dysfunctions. Despite the primary mutations causing these disorders are different, being an mtDNA mutation in subunits of complex I in LHON and defects in the nuclear gene encoding the mitochondrial protein OPA1 in ADOA, both pathologies share some peculiar features, such a variable penetrance and tissue-specificity of the pathological processes. Probably, one of the most interesting and unclear aspect of LHON is the variable penetrance. This phenomenon is common in LHON families, most of them being homoplasmic mutant. Inter-family variability of penetrance may be caused by nuclear or mitochondrial ‘secondary’ genetic determinants or other predisposing triggering factors. We identified a compensatory mechanism in LHON patients, able to distinguish affected individuals from unaffected mutation carriers. In fact, carrier individuals resulted more efficient than affected subjects in increasing the mitochondrial biogenesis to compensate for the energetic defect. Thus, the activation of the mitochondrial biogenesis may be a crucial factor in modulating penetrance, determining the fate of subjects harbouring LHON mutations. Furthermore, mtDNA content can be used as a molecular biomarker which, for the first time, clearly differentiates LHON affected from LHON carrier individuals, providing a valid mechanism that may be exploited for development of therapeutic strategies. Although the mitochondrial biogenesis gained a relevant role in LHON pathogenesis, we failed to identify a genetic modifying factor for the variable penetrance in a set of candidate genes involved in the regulation of this process. A more systematic high-throughput approach will be necessary to select the genetic variants responsible for the different efficiency in activating mitochondrial biogenesis. A genetic modifying factor was instead identified in the MnSOD gene. The SNP Ala16Val in this gene seems to modulate LHON penetrance, since the Ala allele in this position significantly predisposes to be affected. Thus, we propose that high MnSOD activity in mitochondria of LHON subjects may produce an overload of H2O2 for the antioxidant machinery, leading to release from mitochondria of this radical and promoting a severe cell damage and death ADOA is due to mutation in the OPA1 gene in the large majority of cases. The causative nuclear defects in the remaining families with DOA have not been identified yet, but a small number of families have been mapped to other chromosomal loci (OPA3, OPA4, OPA5, OPA7, OPA8). Recently, a form of DOA and premature cataract (ADOAC) has been associated to pathogenic mutations of the OPA3 gene, encoding a mitochondrial protein. In the last year OPA3 has been investigated by two different groups, but a clear function for this protein and the pathogenic mechanism leading to ADOAC are still unclear. Our study on OPA3 provides new information about the pattern of expression of the two isoforms OPA3V1 and OPA3V2, and, moreover, suggests that OPA3 may have a different function in mitochondria from OPA1, the major site for ADOA mutations. In fact, based on our results, we propose that OPA3 is not involved in the mitochondrial fusion process, but, on the contrary, it may regulate mitochondrial fission. Furthermore, at difference from OPA1, we excluded a role for OPA3 in mtDNA maintenance and we failed to identify a direct interaction between OPA3 and OPA1. Considering the results from overexpression and silencing of OPA3, we can conclude that the overexpression has more drastic consequences on the cells than silencing, suggesting that OPA3 may cause optic atrophy via a gain-of-function mechanism. These data provide a new starting point for future investigations aimed at identifying the exact function of OPA3 and the pathogenic mechanism causing ADOAC.

Osteogenesi imperfetta e Dentinogenesi imperfetta Tipo I: correlazioni cliniche e istologiche

Osteogenesis imperffecta (OI) is a heterogeneous group of heritable connetive tissue diseases, quantity and/or qualitative defect in type 1 collagen syntesis; sometimes and in some types it can be associated to dentinogenesis imperfecta (DI), a hereditary disorder in dentin formation that comprises a group of autosomal dominant genetic conditions characterized by abnormal dentine structure affecting either the primary or both the primary and secondary dentitions. Aim: the aim of this study was to assess the correlation between OI and DI from both a clinical and histological point of view, clarifying the structural and ultrastructural changes. Eighteen children (&-15 years aged) with diagnosis of OI were examined for dental alterations referable to DI; for each patient, the OI type (I, III, IV) was recorded. Extracted or normally exfolied teeth were subjected to a histological examination.Results: a total of eleven patients had abnormal discolourations referable to DI: five patients were affected by OI type I, three by OI III, and three patients by OI type IV. The discolourations, yellow/brown or oplaescent grey, could not be related to the different types of OI. Histological exam of primary teeth showed severe pathological change in dentin, structured into four diffeent layers. A collagen defect due to odontoblast dysfunction was theorized to be on the base of the histological changes. Conclusions: there is no correlation between the type of OI and the type of discolouration. The underlying dentinal defect seems to be related to an odontoblast dysfunction.

Caratteristiche ortopediche-ortodontiche in pazienti affetti da Sindrome di Nooan

La sindrome di Noonan (SN) è una patologia a trasmissione autosomica dominante caratterizzata da bassa statura, difetti cardiaci congeniti, dismorfia facciale. In letteratura sono stati pubblicati pochi case reports riguardanti le condizioni orali-facciali in pazienti affetti da SN. Obiettivo. Individuare patologie di pertinenza ortopedico-ortodontica caratteristiche della sindrome utilizzando un campione di pazienti con diagnosi di SN. Metodi. Un gruppo di 10 pazienti affetti da SN è stato sottoposto a esame obiettivo extraorale ed intraorale, ortopantomografia, teleradiografia latero-laterale, impronte delle arcate dentarie. Le misurazioni sulle TLL sono state effettuate sulla base dell'analisi MBT; i valori palatali provengono dai modelli di studio dell’arcata superiore. È stata utilizzato il test t-Student per mettere a confronto il gruppo di studio e il gruppo di controllo riguardo le misure cefalometriche e i valori palatali. Risultati. Nel gruppo di studio sono state rilevate anomalie di numero (un dente deciduo soprannumerario e una agenesia di un dente permanente). Il test t-Student rivela differenze statisticamente significative per 7 variabili cefalometriche su 13 e per 2 variabili palatali. Conclusioni. Basandosi su questo studio è possibile concludere che i pazienti con SN mostrano II classe scheletrica di tipo mandibolare, crescita iperdivergente, tendenza al morso aperto scheletrico, palatoversione degli incisivi superiori, palato stretto. Questi risultati possono fornire informazioni utili sia per la diagnosi di SN sia per la pianificazione del corretto trattamento ortodontico.

Down Syndrome: Neuropsychological phenotype and mitochondrial DNA

Introduction. Down Syndrome (DS) is the most known autosomal trisomy, due to the presence in three copies of chromosome 21. Many studies were designed to identify phenotypic and clinical consequences related to the triple gene dosage. However, the general conclusion is a senescent phenotype; in particular, the most features of physiological aging, such as skin and hair changes, vision and hearing impairments, thyroid dysfunction, Alzheimer-like dementia, congenital heart defects, gastrointestinal malformations, immune system changes, appear in DS earlier than in normal age-matched subjects. The only established risk factor for the DS is advanced maternal age, responsible for changes in the meiosis of oocytes, in particular the meiotic nondisjunction of chromosome 21. In this process mitochondria play an important role since mitochondrial dysfunction, due to a variety of extrinsic and intrinsic influences, can profoundly influence the level of ATP generation in oocytes, required for a correct chromosomal segregation. Aim. The aim of this study is to investigate an integrated set of molecular genetic parameters (sequencing of complete mtDNA, heteroplasmy of the mtDNA control region, genotypes of APOE gene) in order to identify a possible association with the early neurocognitive decline observed in DS. Results. MtDNA point mutations do not accumulate with age in our study sample and do not correlate with early neurocognitive decline of DS subjects. It seems that D-loop heteroplasmy is largely not inherited and tends to accumulate somatically. Furthermore, in our study sample no association of cognitive impairment and ApoE genotype is found. Conclusions. Overall, our data cast some doubts on the involvement of these mutations in the decline of cognitive functions observed in DS.

New approach in the diagnosis and therapy of hyperphenylalaninemia

Background. Phenylketonuria is the most prevalent inborn error of aminoacid metabolism. Is an autosomal recessive disorder. It results from mutations in the phenylalanine hydroxilase (PAH) gene. Phenotypes can vary from mild hyperphenylalaninemia to a severe phenylketonuria wich, if untreated, results in severe mental retardation. Thanks to neonatal screening programmes, early detection and promp dietetic intervention (phenylalanine restricted diet lifelong) has allowed to avoid neurocognitive complications. Recently, a new therapy is become widely used: the oral supplementation with the PAH cofactor (BH4), wich can alleviate the diet burden. Genotype-phenotype correlation is a reliable tool to predict metabolic phenotype in order to establish a better tailored diet and to assess the potential responsiveness to BH4 therapy. Aim Molecular analysis of the PAH gene, evaluation of genotype-phenotype correlation and prediction of BH4 responsiveness in a group of HPA patients living in Emilia Romagna. Patients and methods. We studied 48 patients affected by PAH deficiency in regular follow-up to our Metabolic Centre. We performed the molecular analysis of these patients using genomic DNA extracted from peripheral blood samples Results. We obtained a full genotipic characterization of 46 patients. We found 87 mutant alleles and 35 different mutations, being the most frequent IVS10-11 G>A (19.3%), R261Q (9.1%), R158Q (9.1%), R408Q (6.8%) and A403V (5.7%), including 2 new ones (L287, N223Y) ever described previously. Notably, we found 15 mutations already identified in BH4-responsive patients, according to the literature. We found 42 different genotipic combinations, most of them in single patients and involving a BH4-responsive mutation. Conclusion. BH4 responsiveness is shown by a consistent number of PAH deficient hyperphenylalaninemic patients. This treatment, combined with a less restricted diet or as monotherapy, can reduce nutritional complications and improve the quality of life of these patients.

The KIT gene in familial mastocytosis

Familial cutaneous mastocytosis is an exceptional condition of unknown etiology. In this study we report the largest series of patients with familial cutaneous mastocytosis without other manifestations (18 affected subjects from seven unrelated families), and we investigate the role of germ-line KIT mutations in the pathogenesis of the disease. The mean age at onset was 5.4 years (range from birth to 22 years), and the clinical behavior was variable over a mean follow up period of 15.1 years (range 2-36): improvement in seven, stability in eight and worsening in the remaining three patients. The pattern of inheritance was compatible with an autosomal dominant trait with incomplete penetrance; a female preponderance (14 females vs 4 males, ratio 3.5:1) was noted; among the six women who have been pregnant at least once, three experienced important clinical changes during pregnancy. No germ-line mutation was found in the exons 10, 11, and 17 of the KIT proto-oncogene, which are the most commonly mutated exons in sporadic mastocytosis. However, in the majority of affected subjects we found the Met541Leu polymorphic variant of the KIT gene, which seems to confer a growth advantage to mast cells in vitro. This observation further suggests that the Met541Leu may be a predisposing factor of cutaneous mastocytosis, although it seems to be neither necessary nor sufficient for the development of the disease.

Comparing genetic and linguistic diversity in African populations with a focus on the Khoisan of southern Africa

The interaction between disciplines in the study of human population history is of primary importance, profiting from the biological and cultural characteristics of humankind. In fact, data from genetics, linguistics, archaeology and cultural anthropology can be combined to allow for a broader research perspective. This multidisciplinary approach is here applied to the study of the prehistory of sub-Saharan African populations: in this continent, where Homo sapiens originally started his evolution and diversification, the understanding of the patterns of human variation has a crucial relevance. For this dissertation, molecular data is interpreted and complemented with a major contribution from linguistics: linguistic data are compared to the genetic data and the research questions are contextualized within a linguistic perspective. In the four articles proposed, we analyze Y chromosome SNPs and STRs profiles and full mtDNA genomes on a representative number of samples to investigate key questions of African human variability. Some of these questions address i) the amount of genetic variation on a continental scale and the effects of the widespread migration of Bantu speakers, ii) the extent of ancient population structure, which has been lost in present day populations, iii) the colonization of the southern edge of the continent together with the degree of population contact/replacement, and iv) the prehistory of the diverse Khoisan ethnolinguistic groups, who were traditionally understudied despite representing one of the most ancient divergences of modern human phylogeny. Our results uncover a deep level of genetic structure within the continent and a multilayered pattern of contact between populations. These case studies represent a valuable contribution to the debate on our prehistory and open up further research threads.

Conservation genetics of European wildcat (Felis silvestris silvestris): a wide and integrating analysis protocol for admixture inferences and population structure

Introgression of domestic cat genes into European wildcat (Felis silvestris silvestris) populations and reduction of wildcats’ range in Europe, leaded by habitat loss and fragmentation, are considered two of the main conservation problems for this endangered feline. This thesis addressed the questions related with the artificial hybridization and populations’ fragmentation, using a conservation genetics perspective. We combined the use of highly polymorphic loci, Bayesian statistical inferences and landscape analyses tools to investigate the origin of the geographic-genetic substructure of European wildcats (Felis silvestris silvestris) in Italy and Europe. The genetic variability of microsatellites evidenced that European wildcat populations currently distributed in Italy differentiated in, and expanded from two distinct glacial refuges during the Last Glacial Maximum. The genetic and geographic substructure detected between the eastern and western sides of the Apennine ridge, resulted by adaptation to specific ecological conditions of the Mediterranean habitats. European wildcat populations in Europe are strongly structured into 5 geographic-genetic macro clusters corresponding to: the Italian peninsular & Sicily; Balkans & north-eastern Italy; Germany eastern; central Europe; and Iberian Peninsula. Central European population might have differentiated in the extra-Mediterranean Würm ice age refuge areas (Northern Alps, Carpathians, and the Bulgarian mountain systems), while the divergence among and within the southern European populations might have resulted by the Pleistocene bio geographical framework of Europe, with three southern refugia localized in the Balkans, Italian Peninsula and Iberia Peninsula. We further combined the use of most informative autosomal SNPs with uniparental markers (mtDNA and Y-linked) for accurately detecting parental genotypes and levels of introgressive hybridization between European wild and domestic cats. A total of 11 hybrids were identified. The presence of domestic mitochondrial haplotypes shared with some wild individuals led us to hypnotize the possibility that ancient introgressive events might have occurred and that further investigation should be recommended.

Rationale for an adjunctive therapy with fenofibrate in pharmacoresistant nocturnal frontal lobe epilepsy (NFLE).

Nocturnal Frontal Lobe Epilepsy (NFLE) is characterized by onset during infancy or childhood with persistence in adulthood, family history of similar nocturnal episodes simulating non-REM parasomnias (sleep terrors or sleepwalking), general absence of morphological substrates, often by normal interictal electroencephalographical recordings (EEGs) during wakefulness. A family history of epilepsy may be present with Mendelian autosomal dominant inheritance has been described in some families. Recent studies indicate the involvement of neuronal nicotinic acetylcholine receptors (nAChRs) in the molecular mechanisms of NFLE. Mutations in the genes encoding for the α4 (CHRNA4) and ß2 (CHRNB2) subunits of the nAChR induce changes in the biophysical properties of nAChR, resulting generally in a “gain of function”. Preclinical studies report that activation of a nuclear receptor called type peroxisome proliferator-activated receptor (PPAR-α) by endogenous molecules or by medications (e.g. fenofibrate) reduces the activity of the nAChR and, therefore, may decrease the frequency of seizures. Thus, we hypothesize that negative modulation of nAChRs might represent a therapeutic strategy to be explored for pharmacological treatment of this form of epilepsy, which only partially responds to conventional antiepileptic drugs. In fact, carbamazepine, the current medication for NFLE, abolishes the seizures only in one third of the patients. The aim of the project is: 1)_to verify the clinical efficacy of adjunctive therapy with fenofibrate in pharmacoresistant NFLE and ADNFLE patients; focousing on the analysis of the polysomnographic action of the PPAR- agonist (fenofibrate). 2)_to demonstrate the subtended mechanism of efficacy by means of electrophysiological and behavioral experiments in an animal model of the disease: particularly, transgenic mice carrying the mutation in the nAChR 4 subunit (Chrna4S252F) homologous to that found in the humans. Given that a PPAR-α agonist, FENOFIBRATE, already clinically utilized for lipid metabolism disorders, provides a promising therapeutic avenue in the treatment of NFLE\ADNFLE.