Biomedical subjects
Publications and source records attributed to G P Comi.
An Italian multigenerational family with four members affected by an axonal Charcot-Marie-Tooth type 2D (CMT-2D) or distal spinal muscular atrophy (dSMA) phenotype with upper limb predominance, variable age at onset, degree of disability, and autosomal dominant inheritance is reported. A novel heterozygous missense GARS gene mutation (D500N) was identified.
Glycogenosis type III (Cori disease) is an autosomal recessive disorder caused by the deficiency of the glycogen debranching enzyme, encoded by the AGL gene, and existing in six isoforms alternately spliced in a tissue-specific way. Generally, disease onset occurs early on starting from the first year of life, with hepatomegaly, hypoglycemia, hyperlipidemia, increased CK levels, and, in some cases, short stature and slight mental retardation. Frequently, hepatomegaly tends to resolve spontaneously and inexplicably during childhood, when myopathy, often associated with cardiomyopathy, arises. This disease is known to lack almost invariably clear links between the genotype and clinical phenotype. We describe nine new mutations in Italian patients: four nonsense (p.Arg285X, p.Lys422X, p.Arg910X, p.Arg977X), three frameshift (c.442delA, c.753_756delGACA, c.3963delG), and two missense (p.Ala1120Pro, p.Arg524His). Particularly, the nonsense p.Arg285X is linked to an exonic splicing enhancer and it was found to produce two species of transcripts at the same time. Moreover, we discuss a subgroup of subjects carrying c.2681+1G>A, which has proven to be the most frequent mutation among our patients. The previously described c.664+3A>G was also detected in two patients, both homozygous. The present work is yet another confirmation that the individual genetic background plays a pivotal role in influencing the phenotypes, as occurs in other metabolic diseases.
Cell-based therapy may represent a new strategy to treat a vast array of clinical disorders including neurodegenerative diseases. Recent observations indicate that adult somatic stem cells have the capacity to contribute to the regeneration of different tissues, suggesting that differentiative restrictions are not completely irreversible and can be reprogrammed. Cell fusion might account for some changed phenotype of adult cells but it seems to be biologically irrelevant for its extreme rarity. Other experimental evidences are compatible with the hypothesis of wide multipotency of well-defined stem cell populations, but also with transdifferentiation and/or dedifferentiation. Further studies on nuclear reprogramming mechanisms are necessary to fulfil the promise for developing autologous cellular therapies.
BACKGROUND: Caveolin-3 is the muscle-specific protein product of the caveolin gene family and an integral membrane component of caveolae. Mutations in the gene encoding caveolin-3 (CAV3) underlie four distinct disorders of skeletal muscle: the autosomal dominant form of limb-girdle muscular dystrophy type 1C (LGMD-1C), rippling muscle disease (RMD), sporadic and familial forms of hyperCKemia, and distal myopathy. OBJECTIVE: To characterize a multigenerational Italian family affected by an autosomal dominant myopathic disorder and to assess the expression of caveolin-3, dystrophin, dystrophin-associated glycoproteins, and neuronal nitric oxide synthase in the myocardium of an affected patient. METHODS: Clinical analysis involved 15 family members. Skeletal muscle expression of sarcolemmal proteins was evaluated by immunohistochemistry and western blot analysis in three affected individuals. Caveolar structures were analyzed through electron microscopy in muscle biopsies and in one heart biopsy. RESULTS: CAV3 genetic analysis showed a heterozygous 3-bp microdeletion (328-330del) in affected individuals, resulting in the loss of a phenylalanine (Phe97del) in the transmembrane domain. In the skeletal muscle, the mutation was associated with severe caveolin-3 deficiency and caveolar disorganization, whereas the expression of the other analyzed muscle proteins was unaltered. Remarkably, caveolin-3 was expressed in myocardium at a level corresponding to about 60% of that of control individuals and was correctly localized at the myocardial cell membranes, with preservation of cardiac myofiber caveolar structures. Clinical analysis revealed the concomitant presence in this family of the following phenotypes: RMD, LGMD, and hyperCKemia. CONCLUSIONS: Intrafamilial phenotypic heterogeneity is associated with caveolin-3 Phe97 microdeletion. The molecular network interacting with caveolin-3 in skeletal muscle and heart may differ.
OBJECTIVE: To better understand the still unknown pathologic mechanism involved in the accumulation of multiple mtDNA deletions in stable tissues. METHODS: A large-scale screening of mtDNA molecules from skeletal muscle was performed in 14 patients with progressive external ophthalmoplegia (PEO) and 2 patients with mitochondrial neurogastrointestinal encephalomyopathy carrying mutations on ANT1, C10ORF2 or POLG1, and TP genes. RESULTS: Patients with at least one mutation in the exonuclease domain of POLG1 showed the highest frequency of individually rare point mutations only in the mtDNA control region; in addition, high levels, in terms of frequency and heteroplasmy, of recurrent mutations (A189G, T408A, and T414G) and alterations affecting the (HT)D310 region were detectable in many of the patients. Two homozygous POLG1 mutations, within the exonuclease domain, were able to induce an increased mutational burden also in fibroblasts from patients with PEO. CONCLUSIONS: Specific POLG1 mutations directly affect the integrity of the mtDNA by reducing its proof-reading exonuclease activity, resulting in the accumulation of heteroplasmic levels of both randomly rare and recurrent point mutations in the skeletal muscle tissue and fibroblasts.
We have identified a heteroplasmic G to A mutation at position 12,183 of the mitochondrial transfer RNA Histidine (tRNA(His)) gene in three related patients. These phenotypes varied according to mutation heteroplasmy: one had severe pigmentary retinopathy, neurosensorial deafness, testicular dysfunction, muscle hypotrophy, and ataxia; the other two had only retinal and inner ear involvement. The mutation is in a highly conserved region of the T(psi)C stem of the tRNA(His) gene and may alter secondary structure formation. This is the first described pathogenic, maternally inherited mutation of the mitochondrial tRNA(His) gene.
Limb girdle muscular dystrophy (LGMD) type 2B and distal Miyoshi myopathy (MM) are caused by mutations in a recently discovered mammalian gene coding for a skeletal muscle protein called dysferlin. The protein is normally expressed at the skeletal muscle level and absent or reduced in affected patients. We selected a clinically heterogeneous population of Italian myopathic patients with clinical evidence of myopathy and/or hyperCKemia, EMG myopathic pattern, and no alterations of the dystrophin-sarcoglycan complex. Calpain, merosin, emerin and caveolin were also tested and found normal in all patients. Dysferlin immunohistochemical and Western blot analyses allowed us to identify six patients with dysferlin deficiency: one with distal myopathy, four with limb girdle myopathy and one with hyperCKemia. No apoptosis was found in any of the six muscle specimens, although expression of the pro-apoptotic Fas antigen was mildly increased in two cases. Inflammatory reactions were present in two of the six cases, but we found no evidence of immune-mediated processes.
We analyzed dystrophin alternative splicing events in a large number of Becker muscular dystrophy (BMD) affected individuals presenting major hot-spot deletions. Evidence is shown that altered splicing patterns in these patients do not directly result from the gene defect but probably derive from modifications in trans- rather than cis-acting factors. Several potential CUG-binding protein 2 (CUG-BP2) binding sites were found to be located in the dystrophin gene region encompassing exons 43-60 and CUG-BP2 transcript analysis indicated that not only expression levels are increased in dystrophic muscles but also that different CUG-BP2 isoforms are expressed. The possibility that CUG-BP2 might have a role in dystrophin splicing regulation is discussed.
Total or partial lack of glycogen debranching enzyme (GDE or AGL, amylo-1,6-glucosidase, 4-alpha-glucanotransferase) is responsible for Glycogen Storage Disease type III (GSDIII), a rare autosomal recessive disorder of glycogen metabolism. The clinical and biochemical features of GSDIII subjects are quite heterogeneous, and this mirrors the genotype-phenotype heterogeneity among patients. In this paper, we report the molecular characterisation of five unrelated subjects, four Italian and one Tunisian. The following new mutations are described and confirm the genetic heterogeneity of this disease: p.R864X, p.R428K, c.3911 insA, p.G1087R and c.3512_3549dup+c.3512_3519del. The functional relevance of these mutations is discussed on the basis of the recently acquired knowledge about the boundaries and structures of the two catalytic domains.
Two muscle dystrophin transcripts and proteins were detected in a 17-year-old boy with a persistently elevated serum creatine kinase level. A decreased amount of full-length dystrophin and a 360 kDa polypeptide lacking the COOH-terminus were detectable in the patient's muscle biopsy; accordingly, transcript analysis revealed the expression of a wild type messenger RNA together with a shorter frameshifted one. No genomic DNA mutation was found and the presence of a somatic mosaicism was excluded. This dystrophinopathy may be caused by a novel dystrophin gene transcriptional defect, namely aberrant intraexonic splicing.
Dysferlin, the protein product of the dysferlin gene (DYSF), has been shown to have a role in calcium-induced membrane fusion and repair. Dysferlin is absent or drastically reduced in patients with the following autosomal recessive disorders: limb-girdle muscular dystrophy type 2B (LGMD-2B), Miyoshi myopathy (MM) and distal anterior compartment myopathy. To date, less than 45 mutations have been described in DYSF and a wide inter- and intra-familial variation in clinical phenotype has been associated with the same mutation. This observation underlines the relevance of any new report describing genotype/phenotype correlations in dysferlinopathic patient and families. Here we present the results of clinical, biochemical and genetic analysis performed on one MM and three LGMD Italian families. By screening the entire coding region of DYSF, we identified three novel mutations (two missense substitutions and one frame shift microdeletion). The possible existence of a founder effect for the Arg959Trp mutation in the Italian population is discussed.
Recent evidence suggests that cells from bone marrow can acquire neuroectodermal phenotypes in cell culture or after transplantation in animal models and in the human brain. However, isolation of the bone marrow cell subpopulation with neuronal differentiation potential remains a challenge. To isolate and expand neural progenitors from whole murine bone marrow, bone marrow was obtained from hind limb bone of C57BL6 mice and plated in culture with neuronal medium with basic fibroblast growth factor and epidermal growth factor. After 5-7 days in culture, cellular spheres similar to brain neurospheres appeared either floating or attached to culture dishes. These spheres were collected, dissociated, and expanded. The bone marrow-derived spheres were positive for nestin as assessed by immunocytochemistry and by reverse transcriptase polymerase chain reaction. Thy-1- and Sca-1-positive bone marrow cells selected by magnetic cell sorting resulted in a higher yield of nestin-positive spheres. After exposure to neuronal differentiative medium retinoic acid with and without Sonic hedgehog, cells positive for neuronal markers tubulin III (TuJ-1) and neurofilament (NF) were detected. The mRNA profile of these cells included the expression of TuJ-1, neuronal-specific enolase (NSE), and NF-light chain. To evaluate the in vivo behavior of these cells, spheres derived from bone marrow-derived cells of transgenic green fluorescent protein (GFP) mice were transplanted into newborn mouse brain. Two months later, the mouse neural cortex contained a minor proportion of GFP(+) cells co-expressing neuronal markers (TuJ-1, NF, MAP-2, NeuN). Although cell fusion phenomena with the host cells could not be ruled out, bone marrow-derived neurosphere transplantation could be a strategy for cellular mediated gene therapy.
Stem cell transplantation through cell replacement or as vector for gene delivery is a potential strategy for the treatment of neurodegenerative diseases. Several studies have reported the transdifferentiation of different somatic stem cells into neurons in vitro or after transplantation into animal models. This observation has pointed out the perspective of using an ethical and accessible cell source to "replace" damaged neurons or provide support to brain tissue. However, recent findings such as the cell fusion phenomenon have raised some doubts about the real existence of somatic stem cell plasticity. In this review, we will discuss current evidence and controversial issues about the neuroneogenesis from various sources of somatic cells focusing on the techniques of isolation, expansion in vitro as well as the inductive factors that lead to transdifferentiation in order to identify the factors peculiar to this process. The morphological, immunochemical, and physiological criteria to correctly judge whether the neuronal transdifferentation occurred are critically presented. We will also discuss the transplantation experiments that were done in view of a possible clinical therapeutic application. Animal models of stroke, spinal cord and brain trauma have improved with Mesenchymal Stem Cells or Bone Marrow transplantation. This improvement does not seem to depend on the replacement of the lost neurons but may be due to increased expression levels of neurotrophic factors, thus suggesting a beneficial effect of somatic cells regardless of transdifferentiation. Critical understanding of available data on the mechanisms governing the cell fate reprogramming is a necessary achievement toward an effective cell therapy.
There is now evidence that bone marrow (BM) can generate cells expressing neuronal antigens in adult mouse brain. In the present study, we examined the spinal cord and dorsal root ganglia (DRG) of adult mice 3 months after BM cell transplantation from transgenic donor mice expressing the enhanced green fluorescent protein (GFP). To determine whether GFP(+) cells acquire neuroectodermal phenotypes, we tested, by immunocytochemistry followed by confocal analysis, the coexpression of the astrocytic marker glial fibrillary acidic protein (GFAP) and the neuronal markers NeuN, neurofilament (NF), and class III beta-tubulin (TuJ1). Rare GFP(+) cells coexpressing TuJ1, NF, and NeuN were found both in spinal cord and in sensory ganglia. These cells have small dimensions and short cytoplasmic processes, probably reflecting an immature phenotype. Double GFP and GFAP positivity was found only in spinal cord. To determine whether cell fusion with endogenous cells occurred, we investigated the nuclear content of cells coexpressing GFP and neuronal or astrocytic markers, demonstrating that these cells have only one nucleus and a DNA ploidy that it is not different from that of surrounding neurons and astrocytes. Large numbers of GFP(+) cells are also positively stained for F4/80, a microglial-recognizing antibody, and present a characteristic microglial-like morphology both in spinal cord and, with a higher frequency, in sensory ganglia. These data support a potential role for BM-derived stem cells in spinal cord neuroneogenesis. They also confirm that the microglial compartment within the CNS and in DRG undergoes a relatively fast turnover, with the contribution of hematopoietic stem cells. Both these findings might prove useful for the development of treatments for spinal cord neurodegenerative and acquired disorders.
Bone marrow (BM) transplantation in mice suggests the existence of pluripotent cells able to differentiate into skeletal muscle tissue, although sustained myofiber reconstitution has not yet been achieved. We investigated the myogenic potential of mouse BM cells and evaluated whether a BM fraction enriched for cells expressing skeletal muscle markers would ameliorate muscle repair, when compared to whole BM, into the dystrophic mdx mouse. We demonstrate that cells expressing striated-muscle-specific proteins are already present in the BM independently from experimentally forced myogenic conversion. We observed the presence of both markers of early myogenic program such as Pax3, Myf5, MyoD, desmin, and late myogenesis such as myosin heavy chain and alpha-sarcomeric actin. These myogenic cells are more represented in the early nonadherent BM fraction, which generates clones able to fully differentiate into myotubes. Transplantation in mdx mice by intravenous injection of whole BM and a tenfold BM myogenic enriched fraction resulted in BM reconstitution and limited dystrophin restoration. Taken together, these data show that a fraction of BM cells have a definite potential for differentiation along the skeletal muscle pathway and can be recruited by muscle repair mechanisms. They also indicate that factors limiting the degree of muscle recruitment and the host stem cell competition should be assessed in order to evaluate the usefulness of BM-derived myogenic cells into the context of cell-mediated gene therapy of inherited muscle diseases.
Deficiency of amylo-1,6-glucosidase, 4-alpha-glucanotransferase enzyme (AGL or glycogen debrancher enzyme) is responsible for glycogen storage disease type III, a rare autosomal recessive disorder of glycogen metabolism. The AGL gene is located on chromosome 1p21, and contains 35 exons translated in a monomeric protein product. The disease has recognized clinical and biochemical heterogeneity, reflecting the genotype-phenotype heterogeneity among different subjects. The clinical manifestations of GSD III are represented by hepatomegaly, hypoglycemia, hyperlipidemia, short stature and, in a number of subjects, cardiomyopathy and myopathy. In this article, we discuss the genotypic-phenotypic heterogeneity of GSD III by the molecular characterization of mutations responsible for the disease on a collection of 18 independent alleles from the Mediterranean area. We identified by heteroduplex band shift, DNA direct sequencing, and restriction analysis, seven novel mutations (four nonsense point-mutations: R34X, S530X, R1218X, W1398X; two microinsertions: 1072insT and 4724insAA; and one bp deletion: 676DeltaG), together with two new cases carrying a IVS21 + 1 G --> A splicing site mutation previously described in Italian patients. Altogether, 15 alleles were characterized. The correlation between type of mutation and clinical severity was studied in six patients in whom both mutated alleles were detected. Our data confirm the extreme genetic heterogeneity of this disease, thus precluding a strategy of mutation finding based on screening of recurrent common mutations.
We have analysed splicing patterns in the human dystrophin gene region encoding the rod and cysteine-rich domains in normal skeletal muscle, brain and heart tissues. Sixteen novel alternative transcripts were identified, the majority of them being present in all three tissues. Tissue-specific variants were also identified, suggesting a functional role of transcriptional diversity. Transcript analysis in dystrophinopathic autoptic and bioptic specimens revealed that pre-mRNAs secondary structure formation and relative strength of exon/exon association play little or no role in directing alternative splicing events. This analysis also showed that independent deletion events leading to the loss of the same exons may be associated with transcriptional variability.
Deficiency of amylo-1,6-glucosidase, 4-alpha-glucanotransferase enzyme (AGL or glycogen debranching enzyme) is causative of Glycogen Storage Disease type III, a rare autosomal recessive disorder of glycogen metabolism. The disease has been demonstrated to show clinical and biochemical heterogeneity, reflecting the genotype-phenotype heterogeneity among different subjects. The aim of this study was the molecular characterisation of eight unrelated patients from an ethnically heterogeneous population (six Italians, one from India and another one from Tunisia). We describe six novel mutations responsible for the disease (C234R, R675W, 2547delG, T38A, W1327X, IVS6 +3 A>G) and the presence in two Italian subjects of a splice variant (IVS21(+1) G>A) already described elsewhere. This last one is confirmed to be the most frequent mutation among the Italian patients come to our observation, accounting for 28% of 21 patients. One subject was found to be a compound heterozygous. Our data confirm the substantial genetic heterogeneity of this disease. Consequently, the strategy of mutation finding based on screening of recurrent common mutations is limited, as far as regards Italian GSD III patients, to check for the presence of IVS21(+1) G>A.