[Show abstract][Hide abstract] ABSTRACT: Background
Interstitial 6q deletions, involving the 6q15q25 chromosomal region, are rare events characterized by variable phenotypes and no clear karyotype/phenotype correlation has been determined yet.
We present a child with a 6q21q22.1 deletion, characterized by array-CGH, associated with developmental delay, intellectual disability, microcephaly, facial dysmorphisms, skeletal, muscle, and brain anomalies.
In our patient, the 6q21q22.1 deleted region contains ten genes (TRAF3IP2, FYN, WISP3, TUBE1, LAMA4, MARCKS, HDAC2, HS3ST5, FRK, COL10A1) and two desert gene regions. We discuss here if these genes had some role in determining the phenotype of our patient in order to establish a possible karyotype/phenotype correlation.
Electronic supplementary material
The online version of this article (doi:10.1186/s13039-015-0134-7) contains supplementary material, which is available to authorized users.
[Show abstract][Hide abstract] ABSTRACT: Spinocerebellar ataxia type 28 (SCA28) is a neurodegenerative disease caused by mutations of the mitochondrial protease AFG3L2. The SCA28 mouse model, which is haploinsufficient for Afg3l2, exhibits a progressive decline in motor function and displays dark degeneration of Purkinje cells (PC-DCD) of mitochondrial origin. Here, we determined that mitochondria in cultured Afg3l2-deficient PCs ineffectively buffer evoked Ca2+ peaks, resulting in enhanced cytoplasmic Ca2+ concentrations, which subsequently triggers PC-DCD. This Ca2+-handling defect is the result of negative synergism between mitochondrial depolarization and altered organelle trafficking to PC dendrites in Afg3l2-mutant cells. In SCA28 mice, partial genetic silencing of the metabotropic glutamate receptor mGluR1 decreased Ca2+ influx in PCs and reversed the ataxic phenotype. Moreover, administration of the β-lactam antibiotic ceftriaxone, which promotes synaptic glutamate clearance, thereby reducing Ca2+ influx, improved ataxia-associated phenotypes in SCA28 mice when given either prior to or after symptom onset. Together, the results of this study indicate that ineffective mitochondrial Ca2+ handling in PCs underlies SCA28 pathogenesis and suggest that strategies that lower glutamate stimulation of PCs should be further explored as a potential treatment for SCA28 patients.
[Show abstract][Hide abstract] ABSTRACT: Background
Poland Syndrome (PS) is a rare disorder characterized by hypoplasia/aplasia of the pectoralis major muscle, variably associated with thoracic and upper limb anomalies. Familial recurrence has been reported indicating that PS could have a genetic basis, though the genetic mechanisms underlying PS development are still unknown.
Here we describe a couple of monozygotic (MZ) twin girls, both presenting with Poland Syndrome. They carry a de novo heterozygous 126 Kbp deletion at chromosome 11q12.3 involving 5 genes, four of which, namely HRASLS5, RARRES3, HRASLS2, and PLA2G16, encode proteins that regulate cellular growth, differentiation, and apoptosis, mainly through Ras-mediated signaling pathways.
Phenotype concordance between the monozygotic twin probands provides evidence supporting the genetic control of PS. As genes controlling cell growth and differentiation may be related to morphological defects originating during development, we postulate that the observed chromosome deletion could be causative of the phenotype observed in the twin girls and the deleted genes could play a role in PS development.
BMC Medical Genetics 05/2014; 15(1):63. DOI:10.1186/1471-2350-15-63 · 2.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Amyotrophic lateral sclerosis (ALS) is a late-onset fatal neurodegenerative disease reflecting degeneration of upper and lower motoneurons (MNs). The cause of ALS and the mechanisms of neuronal death are still largely obscure, thus impairing the establishment of efficacious therapies. Glutamate (Glu)-mediated excitotoxicity plays a major role in MN degeneration in ALS. We recently demonstrated that the activation of Group I metabotropic Glu autoreceptors, belonging to both type 1 and type 5 receptors (mGluR1 and mGluR5), at glutamatergic spinal cord nerve terminals, produces excessive Glu release in mice over-expressing human superoxide-dismutase carrying the G93A point mutation (SOD1(G93A)), a widely used animal model of human ALS. To establish whether these receptors are implicated in ALS, we generated mice expressing half dosage of mGluR1 in the SOD1(G93A) background (SOD1(G93A)Grm1(crv4/+)), by crossing the SOD1(G93A) mutant mouse with the Grm1(crv4/+) mouse, lacking mGluR1 because of a spontaneous recessive mutation. SOD1(G93A)Grm1(crv4/+) mice showed prolonged survival probability, delayed pathology onset, slower disease progression and improved motor performances compared to SOD1(G93A) mice. These effects were associated to reduction of mGluR5 expression, enhanced number of MNs, decreased astrocyte and microglia activation, normalization of metallothionein and catalase mRNA expression, reduced mitochondrial damage, and decrease of abnormal Glu release in spinal cord of SOD1(G93A)Grm1(crv4/+)compared to SOD1(G93A) mice. These results demonstrate that a lower constitutive level of mGluR1 has a significant positive impact in mice with experimental ALS, thus providing the rationale for future pharmacological approaches to ALS by selectively blocking Group I metabotropic Glu receptors.
Neurobiology of Disease 12/2013; 64. DOI:10.1016/j.nbd.2013.11.006 · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Spinocerebellar ataxia type 1 (SCA1) is a genetic disorder characterized by severe ataxia associated with progressive loss of cerebellar Purkinje cells. The mGlu1 metabotropic glutamate receptor plays a key role in mechanisms of activity-dependent synaptic plasticity in the cerebellum, and its dysfunction is linked to the pathophysiology of motor symptoms associated with SCA1. We used SCA1 heterozygous transgenic mice (Q154/Q2) as a model for testing the hypothesis that drugs that enhance mGlu1 receptor function may be good candidates for the medical treatment of SCA1.
Symptomatic 30-week old SCA1 mice showed reduced mGlu1 receptor mRNA and protein levels in the cerebellum. Interestingly, these mice also showed an intense expression of mGlu5 receptors in cerebellar Purkinje cells, which normally lack these receptors. Systemic treatment of SCA1 mice with the mGlu1 receptor positive allosteric modulator (PAM), Ro0711401 (10 mg/kg, s.c.), caused a prolonged improvement of motor performance on the rotarod and the paw-print tests. A single injection of Ro0711401 improved motor symptoms for several days, and no tolerance developed to the drug. In contrast, the mGlu5 receptor PAM, VU0360172 (10 mg/kg, s.c.), caused only a short-lasting improvement of motor symptoms, whereas the mGlu1 receptor antagonist, JNJ16259685 (2.5 mg/kg, i.p.), further impaired motor performance in SCA1 mice. The prolonged symptomatic benefit caused by Ro0711401 outlasted the time of drug clearance from the cerebellum, and was associated with neuroadaptive changes in the cerebellum, such as a striking reduction of the ectopically expressed mGlu5 receptors in Purkinje cells, increases in levels of total and Ser880-phosphorylated GluA2 subunit of AMPA receptors, and changes in the length of spines in the distal dendrites of Purkinje cells.
These data demonstrate that pharmacological enhancement of mGlu1 receptors causes a robust and sustained motor improvement in SCA1 mice, and lay the groundwork for the development of mGlu1 receptor PAMs as novel "cerebellum-specific", effective, and safe symptomatic drugs for the treatment of SCA1 in humans.
[Show abstract][Hide abstract] ABSTRACT: Background:
GDNF/RET and Endothelin-3 (ET-3)/EDNRB regulate survival, differentiation, migration, and proliferation of neural crest-derived cells. Although several RET and EDNRB signalling mediators have been characterized, most of the genes targeted by these two pathways are still largely unknown. We focused our study on apolipoprotein B (APOB) as a novel target gene of the RET and EDNRB pathways, based on previous data obtained using a Caenorhabditis elegans strain mutant for the homologue of mammalian ECE1.
Molecular and cellular studies of Apob were performed in the murine Neuro2a cells, an in vitro model for studying neural crest-derived cell development, along with a mouse knock-in for the Hirschsprung-associated mutation Ret(C620R). Silencing for Apob and Ret has been performed via shRNA.
GDNF/RET and ET-3/EDNRB cooperated in inducing neuronal differentiation resulting in Apob activation in Neuro2a cell line. Apob expression was downregulated in mouse embryos homozygous for the Ret(C620R) mutation and presenting a severe Hirschsprung phenotype. Ret silencing prevented Apob expression increase. MAPK P38 kinase activation evoked Apob expression via GDNF/RET signalling in Neuro2a cells. A p53-dependent repressor element in Apob promoter resulted in a reduced Apob expression. Silencing of Apob reduced HuD protein expression.
Conclusions & inferences:
Apob is a novel downstream target of the RET/EDNRB pathways with a role in neuronal survival and maintenance, as indicated by its effect on HuD expression. Our data provide a conceptual framework to investigate and establish the role of APOB gene in severe gut dysmotility.
Neurogastroenterology and Motility 08/2012; 24(10):e497-e508. DOI:10.1111/j.1365-2982.2012.01998.x · 3.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The metabotropic glutamate type 1 (mGlu1) and type 5 (mGlu5) receptors, the only members of group I mGlu receptors, are implicated in synaptic plasticity and mechanisms of feedback control of glutamate release. They exhibit nearly complementary distributions throughout the central nervous system, well evident in the cerebellum, where mGlu1 receptor is most intensely expressed while mGlu5 receptor is not. Despite their different distribution, they show a similar subcellular localization and use common transducing pathways. We recently described the Grm1(crv4) mouse with motor coordination deficits and renal anomalies caused by a spontaneous mutation inactivating the mGlu1 receptor. To define the neuropathological mechanisms in these mice, we evaluated expression and function of the mGlu5 receptor in cerebral and cerebellar cortices. Western blot and immunofluorescence analyses showed mGlu5 receptor overexpression. Quantitative reverse transcriptase-polymerase chain reaction results indicated that the up-regulation is already evident at RNA level. Functional studies confirmed an enhanced glutamate release from cortical cerebral and cerebellar synaptosomes when compared with wild-type that is abolished by the mGlu5 receptor-specific inhibitor, 2-methyl-6-(phenylethynyl) pyridine hydrochloride (MPEP). Finally, acute MPEP treatment of Grm1(crv4/crv4) mice induced an evident although incomplete improvement of motor coordination, suggesting that mGlu5 receptors enhanced activity worsens, instead of improving, the motor-coordination defects in the Grm1(crv4/crv4) mice.
[Show abstract][Hide abstract] ABSTRACT: Glutamate-mediated excitotoxicity plays a major role in ALS and reduced astrocytic glutamate transport was suggested as a cause. Based on previous work we have proposed that abnormal release may represent another source of excessive glutamate. In this line, here we studied the modulation of glutamate release in ALS by Group I metabotropic glutamate (mGlu) receptors, that comprise mGlu1 and mGlu5 members. Synaptosomes from the lumbar spinal cord of SOD1/G93A mice, a widely used murine model for human ALS, and controls were used in release, confocal or electron microscopy and Western blot experiments. Concentrations of the mGlu1/5 receptor agonist 3,5-DHPG >0.3 μM stimulated the release of [(3)H]d- aspartate, used to label the releasing pools of glutamate, both in control and SOD1/G93A mice. At variance, ≤0.3 μM 3,5-DHPG increased [(3)H]d-aspartate release in SOD1/G93A mice only. Experiments with selective antagonists indicated the involvement of both mGlu1 and mGlu5 receptors, mGlu5 being preferentially involved in the high potency effects of 3,5-DHPG. High 3,5-DHPG concentrations increased IP3 formation in both mouse strains, whereas low 3,5-DHPG did it in SOD1/G93A mice only. Release experiments confirmed that 3,5-DHPG elicited [(3)H]d-aspartate exocytosis involving intra-terminal Ca(2+) release through IP3-sensitive channels. Confocal microscopy indicated the co-existence of both receptors presynaptically in the same glutamatergic nerve terminal in SOD1/G93A mice. To conclude, activation of mGlu1/5 receptors produced abnormal glutamate release in SOD1/G93A mice, suggesting that these receptors are implicated in ALS and that selective antagonists may be predicted for new therapeutic approaches. This article is part of a Special Issue entitled 'mGluR'.
[Show abstract][Hide abstract] ABSTRACT: Nephrin is an immunoglobulin-like adhesion molecule first discovered as a major component of the podocyte slit diaphragm, where its integrity is essential to the function of the glomerular filtration barrier. Outside the kidney, nephrin has been shown in other restricted locations, most notably in the central nervous system (CNS) of embryonic and newborn rodents. With the aim of better characterizing nephrin expression and its role in the CNS of adult rodents, we studied its expression pattern and possible binding partners in CNS tissues and cultured neuronal cells and compared these data to those obtained in control renal tissues and podocyte cell cultures. Our results show that, besides a number of locations already found in embryos and newborns, endogenous nephrin in adult rodent CNS extends to the pons and corpus callosum and is expressed by granule cells and Purkinje cells of the cerebellum, with a characteristic alternating expression pattern. In primary neuronal cells we find nephrin expression close to synaptic proteins and demonstrate that nephrin co-immunoprecipitates with Fyn kinase, glutamate receptors and the scaffolding molecule PSD95, an assembly that is reminiscent of those made by synaptic adhesion molecules. This role seems to be confirmed by our findings of impaired maturation and reduced glutamate exocytosis occurring in Neuro2A cells upon nephrin silencing. Of note, we disclose that the very same nephrin interactions occur in renal glomeruli and cultured podocytes, supporting our hypothesis that podocytes organize and use similar molecular intercellular signalling modules to those used by neuronal cells.
The Journal of Pathology 09/2011; 225(1):118-28. DOI:10.1002/path.2923 · 7.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Eight-month old WAG/Rij rats, which developed spontaneous occurring absence seizures, showed a reduced function of mGlu1 metabotropic glutamate receptors in the thalamus, as assessed by in vivo measurements of DHPG-stimulated polyphosphoinositide hydrolysis, in the presence of the mGlu5 antagonist MPEP as compared to age-matched non-epileptic control rats. These symptomatic 8-month old WAG/Rij rats also showed lower levels of thalamic mGlu1α receptors than age-matched controls and 2-month old (pre-symptomatic) WAG/Rij rats, as detected by immunoblotting. Immunohistochemical and in situ hybridization analysis indicated that the reduced expression of mGlu1 receptors found in symptomatic WAG/Rij rats was confined to an area of the thalamus that excluded the ventroposterolateral nucleus. No mGlu1 receptor mRNA was detected in the reticular thalamic nucleus. Pharmacological manipulation of mGlu1 receptors had a strong impact on absence seizures in WAG/Rij rats. Systemic treatment with the mGlu1 receptor enhancer SYN119, corresponding to compound RO0711401, reduced spontaneous spike and wave discharges spike-wave discharges (SWDs) in epileptic rats. Subcutaneous doses of 10 mg/kg of SYN119 only reduced the incidence of SWDs, whereas higher doses (30 mg/kg) also reduced the mean duration of SWDs. In contrast, treatment with the non-competitive mGlu1 receptor antagonist, JNJ16259685 (2.5 and 5 mg/kg, i.p.) increased the incidence of SWDs. These data suggest that absence epilepsy might be associated with a reduction of mGlu1 receptors in the thalamus, and that compounds that amplify the activity of mGlu1 receptors might be developed as novel anti-absence drugs. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.
[Show abstract][Hide abstract] ABSTRACT: The metabotropic glutamate (mGlu) receptor 1 (GRM1) has been shown to play an important role in neuronal cells by triggering, through calcium release from intracellular stores, various signaling pathways that finally modulate neuron excitability, synaptic plasticity, and mechanisms of feedback regulation of neurotransmitter release. Herein, we show that Grm1 is expressed in glomerular podocytes and that a glomerular phenotype is exhibited by Grm1(crv4) mice carrying a spontaneous recessive inactivating mutation of the gene. Homozygous Grm1(crv4/crv4) and, to a lesser extent, heterozygous mice show albuminuria, podocyte foot process effacement, and reduced levels of nephrin and other proteins known to contribute to the maintenance of podocyte cell structure. Overall, the present data extend the role of mGlu1 receptor to the glomerular filtration barrier. The regulatory action of mGlu1 receptor in dendritic spine morphology and in the control of glutamate release is well acknowledged in neuronal cells. Analogously, we speculate that mGlu1 receptor may regulate foot process morphology and intercellular signaling in the podocyte.
American Journal Of Pathology 03/2011; 178(3):1257-69. DOI:10.1016/j.ajpath.2010.11.050 · 4.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This paper presents a top-down strategy to detect features in genomic sequences. The strategy's core is to exploit dictionary-based compression algorithms and analyse the content of the automatically generated dictionary. We classify the different over-represented segments and in the case study we correlate them to experimentally identified or theoretically forecasted biological features. A large spectrum analysis reveals that the only feature co-located with the a priori extracted segments is the torsional flexibility of DNA, while non-B DNA configurations are anti-localized and other features are mostly independent of the extracted sequences. This analysis unravels complex relationships between the linguistic structures investigated under our approach and some known biological features.
[Show abstract][Hide abstract] ABSTRACT: Human immunodeficiency virus-1 (HIV-1)-encoded transactivator of transcription (Tat) potentiated the depolarization-evoked exocytosis of [(3)H]D-aspartate ([(3)H]D-ASP) from human neocortical terminals. The metabotropic glutamate (mGlu) 1 receptor antagonist 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt) prevented this effect, whereas the mGlu5 receptor antagonist 2-methyl-6-(phenylethynyl) pyridine hydrochloride (MPEP) was ineffective. Western blot analysis showed that human neocortex synaptosomes possess mGlu1 and mGlu5 receptors. Tat potentiated the K(+)-evoked release of [(3)H]D-ASP or of endogenous glutamate from mouse neocortical synaptosomes in a CPCCOEt-sensitive and MPEP-insensitive manner. Deletion of mGlu1 receptors (crv4/crv4 mice) or mGlu5 receptors (mGlu5(-/-)mouse) silenced Tat effects. Tat enhanced inositol 1,4,5-trisphosphate production in human and mouse neocortical synaptosomes, consistent with the involvement of group I mGlu receptors. Tat inhibited the K(+)-evoked release of [(3)H]gamma-aminobutyric acid ([(3)H]GABA) from human synaptosomes and that of endogenous GABA or [(3)H]GABA from mouse nerve terminals; the inhibition was insensitive to CPCCOEt or MPEP. Tat-induced effects were retained by Tat(37-72) but not by Tat(48-85). In mouse neocortical slices, Tat facilitated the K(+)- and the veratridine-induced release of [(3)H]D-ASP in a CPCCOEt-sensitive manner and was ineffective in crv4/crv4 mouse slices. These observations are relevant to the comprehension of the pathophysiological effects of Tat in central nervous system and may suggest new potential therapeutic approaches to the cure of HIV-1-associated dementia.
[Show abstract][Hide abstract] ABSTRACT: The 22q11.2 region is a hotspot for chromosomal rearrangements mediated by LCR22A-D low-copy repeats. Sequence motifs and homology-driven mechanisms have been suggested to mediate rearrangements. Nevertheless, recent evidence has emphasized the role of functional properties in genome instability, suggesting that replication timing transition regions could be peculiarly prone to genetic damage. In this work, we show that an early-late replication-transition zone is localised within LCR22A, the shared proximal endpoint of the majority of deletions and duplications of 22q11.2 region. Transition zone is characterized by asynchronous replication and by a DNA flexibility peak, features which are relevant for double-strand breaks and rearrangements at fragile sites. This and other flexibility peaks, associated with less relevant replication anomalies, are present in clusters inside LCR22A, B and D. All of them are composed of modules of AT-rich sequences, DNA satellites, and a HIV-1 integration site; moreover, they have coincidental position with boundaries of duplicons inside segmental duplications and with breakpoints of recurrent translocations. Noteworthy, flexibility peaks also lay at breakpoints of translocation partner chromosomes, three of which, 1p21.2, 8q24.13 and 11q23.3, have been positioned inside known common fragile sites. In many cases peaks are associated with potential matrix attachment regions (MARs). We propose that, similarly to fragile sites, replication perturbation and flexibility peaks may mediate strand breakage and rearrangements. Consistently with this view we show that the replication timing transition zone detected inside LCR22A is susceptible to replicative stress by aphidicolin, known inducer of fragile sites. These findings emphasize the significance of mutagenic exposure for the constitutional syndrome origin.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 02/2010; 686(1-2):74-83. DOI:10.1016/j.mrfmmm.2010.01.021 · 3.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The metabotropic glutamate (mGlu) 1 receptor, coded by the GRM1 gene, is involved in synaptic activities, learning and neuroprotection. Eleven different mouse Grm1 mutations, either induced or spontaneously occurring, have been reported, including one from our group. All the mutations result in a complex phenotype with ataxia and intention tremor in mice. Moreover, autoantibodies against mGlu1 receptor have been associated with paraneoplastic cerebellar ataxia in humans. In spite of the large clinical and genetic heterogeneity displayed by the inherited forms of cerebellar ataxia, forms remain with a yet unknown molecular definition. With the evidence coming out from mouse models and from paraneoplastic ataxia, it seems that GRM1 represents a good candidate gene for early-onset ataxia forms, though no GRM1 mutations have thus far been looked for. The aim of this study was to investigate the possible involvement of GRM1 in early-onset or familial forms of ataxia. We searched for gene mutations in a panel of patients with early-onset ataxia as yet molecularly undefined. No causative mutations were found, though we detected synonymous variants in the exons and changes in flanking intronic sequences which are unlikely to alter correct splicing upon bioinformatics prediction. As for other known forms of inherited ataxias, absence of mutations in GRM1 seems to suggest a relatively low frequency in cerebellar ataxias.
Journal of Neurology 11/2009; 257(4):598-602. DOI:10.1007/s00415-009-5380-3 · 3.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The effects of mGlu1 and mGlu5 receptor activation on the depolarization-evoked release of [3H]d-aspartate ([3H]D-ASP) from mouse cortical synaptosomes were investigated. The mGlu1/5 receptor agonist 3,5-DHPG (0.1-100microM) potentiated the K+(12mM)-evoked [3H]D-ASP overflow. The potentiation occurred in a concentration-dependent manner showing a biphasic pattern. The agonist potentiated [3H]D-ASP exocytosis when applied at 0.3microM; the efficacy of 3,5-DHPG then rapidly declined and reappeared at 30-100microM. The fall of efficacy of agonist at intermediate concentration may be consistent with 3,5-DHPG-induced receptor desensitization. Facilitation of [3H]D-ASP exocytosis caused by 0.3microM 3,5-DHPG was prevented by the selective mGlu5 receptor antagonist MPEP, but was insensitive to the selective mGlu1 receptor antagonist CPCCOEt. In contrast, CPCCOEt prevented the potentiation by 50microM 3,5-DHPG, while MPEP had minimal effect. Unexpectedly, LY 367385 antagonized both the 3,5-DHPG-induced effects. A total of 0.3microM 3,5-DHPG failed to facilitate the K+-evoked [3H]D-ASP overflow from mGlu5 receptor knockout (mGlu5-/-) cortical synaptosomes, but not from nerve terminals prepared from the cortex of animals lacking the mGlu1 receptors, the crv4/crv4 mice. On the contrary, 50microM 3,5-DHPG failed to affect the [3H]D-ASP exocytosis from cortical synaptosomes obtained from crv4/crv4 and mGlu5-/-mice. Western blot analyses in subsynaptic fractions support the existence of both mGlu1 and mGlu5 autoreceptors located presynaptically, while immunocytochemistry revealed their presence at glutamatergic terminals. We propose that mGlu1 and mGlu5 autoreceptors exist on mouse glutamatergic cortical terminals; mGlu5 receptors may represent the "high affinity" binding sites for 3,5-DHPG, while mGlu1 autoreceptors represent the "low affinity" binding sites.
[Show abstract][Hide abstract] ABSTRACT: Positional cloning is the approach of choice for the identification of genetic mutations underlying the pathological development of diseases with simple Mendelian inheritance. It consists of different consecutive steps, starting with recruitment of patients and DNA collection, that are critical to the overall process. A genetic analysis of the enrolled patients and their families is performed, based on genetic recombination frequencies generated by meiotic cross-overs and on genome-wide molecular studies, to define a critical DNA region of interest. This analysis culminates in a statistical estimate of the probability that disease features may segregate in the families independently or in association with specific molecular markers located in known regions. In this latter case, a marker can be defined as being linked to the disease manifestations. The genetic markers define an interval that is a function of their recombination frequencies with the disease, in which the disease gene is localised. The identification and characterisation of chromosome abnormalities as translocations, deletions and duplications by classical cytogenetic methods or by the newly developed microarray-based comparative genomic hybridisation (array CGH) technique may define extensions and borders of the genomic regions involved. The step following the definition of a critical genomic region is the identification of candidate genes that is based on the analysis of available databases from genome browsers. Positional cloning culminates in the identification of the causative gene mutation, and the definition of its functional role in the pathogenesis of the disorder, by the use of cell-based or animal-based experiments. More often, positional cloning ends with the generation of mice with homologous mutations reproducing the human clinical phenotype. Altogether, positional cloning has represented a fundamental step in the research on genetic renal disorders, leading to the definition of several disease mechanisms and allowing a proper diagnostic approach to many conditions.
[Show abstract][Hide abstract] ABSTRACT: Germline RET mutations are responsible for different inherited disorders: Hirschsprung disease (congenital aganglionic megacolon), caused by loss of function mutations, familial medullary thyroid carcinoma and multiple endocrine neoplasia type 2, caused by gain of function mutations. Intriguingly, some RET mutations, including C620R, are associated with both types of diseases. To investigate the dual role of such RET mutations, a mouse model with a targeted mutation ret(C620R) was generated. ret(C620R/C620R) offspring die during the first postnatal day, and show kidney agenesis and intestinal aganglionosis. Decreased outgrowth of the Ret-positive cells was observed in ret(C620R/C620R) neuronal cell cultures, which is suggestive of an impaired migration, proliferation or survival of the Ret-expressing cells. Electronmicroscopy revealed the absence of membrane-bound Ret in ret(C620R/C620R) cells as compared to ret(+/+) and ret(+/C620R) cells. On the other hand, aged ret(+/C620R) mice develop precancerous lesions in the adrenal gland or in the thyroid. Our results suggest that the ret(C620R) mutation has a loss of function effect in homozygotes and exhibits a dominant gain of function effect with low penetrance causing hyperplasia in heterozygotes.
International Journal of Cancer 07/2007; 121(2):292-300. DOI:10.1002/ijc.22378 · 5.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We describe a novel spontaneous autosomal recessive mutation, cervelet-4 (crv4), which arose in a BALB/c strain. Mice homozygous for the mutation exhibit principally a reduced body size, a congenital neurological phenotype characterized by ataxic gait and intention tremor, with no gross anomalies observed in brain or cerebellum, and skeletal anomalies. Using linkage analysis, we mapped the crv4 locus to the proximal region of chromosome 10, at the location of the Grm1 gene. Genetic complementation crosses between crv4 and Grm1 KO mice confirmed that crv4 is a new allele of Grm1. Molecular analysis of the Grm1 gene in mutant mice revealed the insertion of a 190-bp LTR fragment in intron 4. Our results also indicated that the presence of the LTR fragment caused the disruption of the Grm1 normal splicing process and complete absence of the wild-type protein. crv4 is an interesting model to extend the study of Grm1 function and the pathological effects of Grm1 deficiency in vivo.
International Journal of Molecular Medicine 11/2006; 18(4):593-600. DOI:10.3892/ijmm.18.4.593 · 2.09 Impact Factor