Agnese Brega

Mitochondrial DNA polymorphisms in Italy I. Population data from Sardinia and Rome

The polymorphisms of human mitochondrial DNA were studied in about 150 Sardinians from Cagliari and 100 other Italians living in Rome, using total blood cell DNA and the following restriction enzymes: HpaI, BamHI, HaeII, MspI, AvaII and Hinc II.

The shortness of Pygmies is associated with severe under-expression of the growth hormone receptor.

The stature is a highly heritable trait controlled by genetic and environmental factors. The African Pygmies represent a paradigmatic example of non-disease-related idiopathic short stature (ISS), showing a similar endocrine profile of Caucasian individuals with ISS. Pygmy children show normal anthropometric and endocrine parameters until puberty, while adult Pygmies show normal baseline and post-stimulation serum growth hormone (GH) levels but low values of baseline serum GH-binding protein (GHBP) and insulin-like growth factor-I (IGF-I). This discrepancy suggests a defective response to GH occurring in adulthood since Pygmies lack both the pubertal serum IGF-I surge and the growth spurt. However, sequencing of the key genes of the GH-IGF-I axis failed to identify Pygmy specific variants or haplotypes. We therefore aimed at assessing whether the quantitative gene expression profile of two key genes of the GH-IGF-I axis, GH and GHR, was also similar in low-stature and normal stature populations. We showed that the GH gene expression is 1.8-fold reduced and the GH receptor (GHR) gene expression is 8-fold reduced in adult Pygmies in comparison with sympatric adult Bantu, and that this reduction is not associated with sequence variants of the GHR gene. The marked decrease of the GHR expression in Pygmies is associated with reduced serum levels of the IGF-I and GHBP. Our results, documenting a markedly reduced GHR gene expression in adult Pygmies, could contribute to elucidate the mechanisms involved in ISS in Caucasoid subjects.

Barth syndrome associated with compound hemizygosity and heterozygosity of the TAZ and LDB3 genes.

Barth syndrome is an X‐linked recessive disorder caused by the tafazzin (TAZ) gene mutations and includes dilated cardiomyopathy (DCM) with left ventricular non‐compaction, neutropenia, skeletal myopathy, abnormal mitochondria and 3‐methylglutaconic aciduria. Dilated cardiomyopathy with left ventricular non‐compaction transmitted as an autosomal dominant condition has also been associated with LIM domain‐binding 3 (LDB3) gene defects. We describe a family in which the 12‐year‐old proband had left ventricular non‐compaction and DCM. His mother had five miscarriages, two other sons who died in infancy, and a healthy son and daughter. The proband showed left ventricular non‐compaction–DCM, skeletal myopathy, recurrent oral aphthous ulcers and cyclic neutropenia. The DCM progressively improved with age; medical therapy was discontinued at 5 years of age. At present, left ventricular function is normal and arrhythmias are absent. Magnetic resonance imaging documented left ventricular non‐compaction. However, oral aphthous ulcers and cyclic neutropenia have recurred. In the proband we identified two novel mutations, one of maternal origin in the TAZ gene (p.[Glu202ValfsX15]) and one of paternal origin in the LDB3 gene (p.[Thr350Ile]). The mother, brother and father are healthy; although the latter two show prominent left ventricle trabeculation without dysfunction. Expression studies of TAZ and LDB3 genes were conducted in family members and controls. In the proband, brother and father, LDB3 expression was similar to control cases. TAZ and LDB3 expression progressively declined with age in control both blood and myocardial samples. However, an endomyocardial biopsy performed in the proband at 6 months of age, showed significantly lower TAZ and LDB3 expression than in age‐matched myocardial controls. We believe that the clinical, genetic and expression data support the hypothesis that tafazzins are essential during fetal and early post‐natal life.

The mitochondrial DNA mutation T12297C affects a highly conserved nucleotide of tRNA(Leu(CUN)) and is associated with dilated cardiomyopathy.

Mitochondrial DNA (mtDNA) mutations have been causally linked with cardiomyopathies, both dilated (DCM) and hypertrophic. We identified the T12297C mutation in the mtDNA-tRNALeu(CUN) of a 36-year-old male patient diagnosed with DCM. The mutation was heteroplasmic, with high amount (88%) of mutant DNA in the myocardium, and was absent in normal (n=120) and disease (n=150) controls. It affects a highly conserved nucleotide (adjacent to the anticodon triplet) that allows the phospho-ribose backbone to turn and form the loop. The potential pathological role of T12297C mutation is further supported by its recent identification in another unrelated Italian family with DCM associated with endocardial fibroelastosis. In the variable loop of the same tRNA, our patient also carried the A12308G transition that is debated as pathological mutation or neutral polymorphism in progressive external ophthalmoplegia: the two defects could exert a synergistic effect on the tRNA structure and function. The endomyocardial biopsy study showed abnormal ring-like mitochondria and occasional cytochrome c oxydase negative myocytes. Overall, the heteroplasmy, the highly conserved position of the mutated nucleotide, the absence of the mutation in large series of diseased and normal controls, and the cardiac mitochondrial changes support a causative link of the mutation with the disease.

Ultrastructural definition of apoptosis in heart failure.

Cardiac myocytes die through apoptosis, oncosis, and autophagy. Apoptosis affects single cells and is morphologically characterized by nuclear fragmentation with generation of apoptotic bodies that can be seen either within dying cells or free in the interstitial spaces. Dead myocytes are removed by macrophages through phagocytosis without triggering inflammation. The circulating markers of myocyte necrosis are not increased by apoptosis. The morphologic changes of the induction and early execution phases are seen at electron microscopy while late fragmentation is visible on both light and electron microscopy. Immunoelectron microscopy provides combined functional and structural information showing cytochrome c immuno-labelling release from mitochondria, TUNEL labelling of apoptotic nuclei, annexin V translocation in the outer plasma cell layer. Oncosis is characterized by specific morphologic features that may coexist with apoptosis, especially in ischemic myocardium. Autophagy is a defense process that is associated with significant myocardial damage and necrosis when removal of the lysosomal content is impaired. Morphological features of apoptosis, oncosis, and autophagocytosis may coexist at the same time. Although dead myocytes showing characteristics of autophagy and apoptosis are rarely observed in human decompensated hearts, autophagic vacuoles, and early apoptotic changes may be seen more often in morphologically viable myocytes. Such features may occur in failing hearts of both ischemic and non-ischemic etiology. The shared mode of cardiac myocyte death in failing human hearts of different etiologies suggests that preservation of myocyte integrity may be possible by similar therapeutic strategies.

Mitochondrial DNA polymorphisms in Italy II. Molecular analysis of new and rare morphs from Sardinia and Rome

A molecular analysis of morphs found in a previous survey of mtDNA restriction enzyme polymorphisms in Italy revealed that different site changes can give similar patterns and that the same mutation can yield variant morphs for apparently unrelated enzymes.

Mitochondrial DNA polymorphisms in Italy. III: Population data from Sicily: a possible quantitation of maternal African Ancestry

mtDNA polymorphisms were studied in a sample of 90 individuals of the Sicilian population using six restriction enzymes: HpaI, BamHI, HaeII, MspI, AvaII and HincII.

Mitochondrial DNA polymorphisms among Hindus: A comparison with the Tharus of Nepal

The polymorphisms of mitochondrial DNA for the restriction enzymes Hpal, Bamill, Haell, Mspl, Avail and Hindi were studied in a sample of 79 Hindus, 45 from New Delhi (India) and 34 from Terai (Nepal), both to characterize another Caucasian population and to investigate some possible Hindu component in the genetic structure of the Tharus, a Nepalese population, the anthropological position of which is still disputed.