Simonetta Sangiorgi

Mitochondrial DNA haplogroup K is associated with a lower risk of Parkinson's disease in Italians

It has been proposed that European mitochondrial DNA (mtDNA) haplogroups J and K, and their shared 10398G single-nucleotide polymorphism (SNP) in the ND3 gene, are protective from Parkinsons disease (PD). We evaluated the distribution of the different mtDNA haplogroups in a large cohort of 620 Italian patients with adult-onset (>50, <65 years of age) idiopathic PD vs two groups of ethnic-matched controls. Neither the frequencies of haplogroup J nor that of 10398G were significantly different. However, the frequency of haplogroup K was significantly lower in PD. Stratification by sex and age indicated that the difference in the distribution of haplogroup K was more prominent in >50year old males. In spite of the common 10398G SNP, haplogroups J and K belong to widely diverging mitochondrial clades, a consideration that may explain the different results obtained for the two haplogroups in our cohorts. Our study suggests that haplogroup K might confer a lower risk for PD in Italians, corroborating the idea that the mitochondrial oxidative phosphorylation pathway is involved in the susceptibility to idiopathic PD.

Leber's hereditary optic neuropathy Biochemical effect of 11778/ND4 and 3460/ND1 mutations and correlation with the mitochondrial genotype

To clarify the bioenergetic relevance of mtDNA mutations in Lebers hereditary optic neuropathy (LHON), we investigated affected individuals and healthy carriers from six Italian LHON families harboring the 11778/ ND4 and the 3460/ND1 mtDNA mutations. The enzymatic activities of mitochondrial complex I and its sensitivity to the potent inhibitors rotenone and rolliniastatin-2 were studied in mitochondrial particles from platelets, in correlation with mtDNA analysis of platelets and leukocytes. In platelets homoplasmic for mutant mtDNA, both 11778/ND4 and 3460/ND1 mutations induced resistance to rotenone and the 3460/ND1 mutation also provoked a marked decrease in the specific activity of complex I. Individuals heteroplasmic in platelets for either mutation showed normal biochemical features, indicating functional complementation of wild-type mtDNA. There was no correlation between the clinical status and mtDNA homoheteroplasmy in platelets, but the biochemical features correlated with the mitochondrial genotype of platelets. In some cases, the degree of mtDNA heteroplasmy differed in platelets and leukocytes from the same individual with a prevalence of wild-type mtDNA in the platelets. These results imply that biochemical studies on mitochondrial diseases should always be integrated with mtDNA analysis of the same tissue investigated and also suggest that the mtDNA analysis on the leukocyte fraction, as usually performed in LHON, does not necessarily reflect the mutant genotype level of other tissues. The differential tissue heteroplasmy may be more relevant than previously thought in determining disease penetrance.

Functional alterations of the mitochondrially encoded ND4 subunit associated with Leber's hereditary optic neuropathy

Lebers hereditary optic neuropathy (LHON) is a maternally inherited disease associated with point mutations in mitochondrial DNA. The most frequent of these mutations is the G‐to‐A substitution at nucleotide position 11,778 which changes an evolutionarily conserved arginine with a histidine at position 340 in subunit ND4 of NADH: ubiquinone reductase (respiratory complex I). We report that this amino acid substitution alters the affinity of complex I for the ubiquinone substrate and induces resistance towards its potent inhibitor rotenone in mitochondria of LHON patients. Such changes could reflect a substantial loss in the energy conserving function of NADH: ubiquinone reductase and thus explain the pathological effect of the ND4/11,778 mutation.

Abnormal platelet mitochondrial function in patients affected by migraine with and without aura

To investigate energy metabolism in migraine, we determined platelet mitochondrial enzyme activities in 40 patients with migraine with aura and in 40 patients with migraine without aura during attack-free intervals and in 24 healthy control subjects. NADH-dehydrogenase, citrate synthase and cytochrome-c-oxidase activities in both patient groups were significantly lower than in controls (p < 0.01), while NADH-cytochrome-c-reductase activity was reduced only in migraine with aura (p < 0.01). No alteration in succinate-dehydrogenase was observed. Monoamine-oxidase activity differed between sexes (p < 0.05) but within each sex group no difference was observed between patients and controls. We hypothesize that the defect in mitochondrial enzymes observed indicates a systemic impairment of mitochondrial function in migraine patients.

Testing Models for Genetic Determination in Migraine

We collected two clinically matched samples of patients, one sample affected by migraine with aura the other by migraine without aura, to investigate the genetic determination of these conditions. A maternal and X-linked transmission for both these diseases was considered unlikely after pedigree analysis. Classical segregation analysis indicated a likely autosomal recessive kind of transmission for both. Reduced penetrance and the h2 values, however, imply the presence of additional genetic and/or environmental factors controlling the phenotypic expression of migraine.

Evidence of polymorphic CYP2C19 involvement in the human metabolism of N-desmethylclobazam.

The authors report preliminary findings on the potential contribution of CYP2C19 isoenzyme to the human metabolism of N-desmethylclobazam (N-CLB), the main active metabolite of clobazam (CLB), a benzodiazepine frequently used as add-on therapy in patients with refractory epilepsy. Two children on CLB treatment showing extremely high plasma concentration/dose ratio (C/D) of N-CLB and metabolite/parent drug ratio (N-CLB/CLB), suggestive of a putative poor metabolizer (PM) phenotype, were tested for CYP2C19 polymorphisms. Eleven epileptic patients on stable CLB therapy were included for reference values of CLB and N-CLB metabolic variables and tested for possible CYP2C19 polymorphisms. Detection of the CYP2C19*2, CYP2C19*3, and CYP2C19*4 mutations was performed in the genomic DNA by PCR amplification and enzyme digestion procedures. In the two presumed CYP2C19 PM patients, the N-CLB/CLB ratio was 10- to 27-fold higher than matched median values of the control epileptic patients. According to CYP2C19 genotyping, one patient was homozygous for CYP2C19*2, while the second presented only one copy of the same mutation, a genotype also found in three control patients. These observations provide further indirect in vivo evidence of CYP2C19 isoenzyme involvement in the metabolism of the CLB main metabolite. According to genotyping, subjects carrying one or two copies of the defective CYP2C19*2 allele might develop markedly elevated steady-state plasma concentrations of N-CLB and be at higher risk of adverse effects.

Unusual side-effects due to clobazam: A case report with genetic study of CYP2C19

We describe the case of a 10-year-old girl with two epileptic seizures and subcontinuous spike-waves during sleep, who presented unusual side-effects related to clobazam (CLB) monotherapy. High plasma levels of N-desmethyl-clobazam (N-CLB), the major metabolite of CLB were detected. The patient and her parents underwent molecular analysis of the CYP2C19 gene, which may be implicated in the metabolism of this drug. Our patient presents one copy of the most common mutation (CYP2C19*2) affecting the activity of the isoenzyme and probably another rare or private mutation. CLB and N-CLB plasma level dosages and molecular analysis may be useful when a poor metabolic condition is suspected.

ADH SYSTEM AND GENETIC BACKGROUND: INTERACTION WITH WING LENGTH IN DROSOPHILA MELANOGASTER

in order to test the adaptive value of Adh genotypes in connection with the genetic background, we crossed two unrelated strains of Drosophila melanogaster, Canton and Oregon; the former, homozygous for AdhS, exhibited shorter wing length and higher reproductive fitness, the latter, homozygous for AdhF, exhibited longer wing length and lower reproductive fitness in standard conditions of rearing. The F2 progenies appear to be in equilibrium at the enzyme locus; nevertheless the Adh genotypes are not randomly distributed throughout wing length classes, pointing to a linkage disequilibrium between the enzyme locus and wing length polygenes. Moreover the preferential combinations enjoy an adaptive advantage; in fact Adh F homozygotes and heterozygotes show a peak of reproductive fitness and frequency in connection with longer wing, while AdhS do so in connection with shorter wing. Such a dissimilar behaviour of Adh genotypes may be due to coadaptation to different genetic backgrounds, namely the original Canton strain in the case of AdhS and new genie combinations of heterotic value in the case ofAdh F and AdhF/S. The authors conclude that each allozyme at a locus can be related to a different fitness system, and that the adaptive value of isozyme genotypes cannot be measured on the assumption of equivalent dependence on genetic background.

Mapping of the Emery-Dreifuss gene through reconstruction of crossover points in two Italian pedigrees.

SummaryTwo unrelated pedigrees, which show recurrence of Emery-Dreifuss muscular dystrophy (EDMD) in three generations, have been studied using 13 X-linked DNA polymorphisms and somatic cell hybrids to establish the phase of the corresponding alleles in some obligate carriers. The reconstruction of cross-over points on the X chromosomes carrying the EDMD gene excludes from mapping most regions of the X chromosome except for the terminal portion of Xq. Pooled linkage data from the two pedigrees confirm the linkage previously reported with locus DXS15. A cross-over in a carrier female suggests that the EDMD gene is probably located distally to DXS15. In addition the recombinant meioses from one of the two pedigrees suggest the following order for some Xq polymorphic loci: DXS1 (DXYS1-DXS178) DXS42 (F9-DXS15).

Definitive localization of Becker muscular dystrophy in Xp by linkage to a cluster of DNA polymorphisms (DXS43 and DXS9).

SummaryA study of linkage between Becker muscular dystrophy and four X chromosome-specific DNA polymorphisms in 17 kindreds has indicated that this gene is located in Xp, as already anticipated by single pedigree analysis. In particular the DXS43 and DXS9 loci, identified by probes D2 and RC8, respectively, are closely linked to each other and are both located at approximately 15 cM from the Becker locus. These linkage data, together with the previously established linkage between Becker and the DXS7 locus identified by probe L 1.28, indicate that the Becker gene is located in the same region where Duchenne has been mapped and also yield information about relative genetic distances among different DNA polymorphisms of the X chromosome.