G. Casari - Profile on Academia.edu (original) (raw)

Papers by G. Casari

EMBO Molecular Medicine, 2016

Migraine is a common disabling brain disorder. A subtype of migraine with aura (familial hemipleg... more Migraine is a common disabling brain disorder. A subtype of migraine with aura (familial hemiplegic migraine type 2: FHM2) is caused by loss-of-function mutations in 2 Na(+),K(+) ATPase (2 NKA), an isoform almost exclusively expressed in astrocytes in adult brain. Cortical spreading depression (CSD), the phenomenon that underlies migraine aura and activates migraine headache mechanisms, is facilitated in heterozygous FHM2-knockin mice with reduced expression of 2 NKA The mechanisms underlying an increased susceptibility to CSD in FHM2 are unknown. Here, we show reduced rates of glutamate and K(+) clearance by cortical astrocytes during neuronal activity and reduced density of GLT-1a glutamate transporters in cortical perisynaptic astrocytic processes in heterozygous FHM2knockin mice, demonstrating key physiological roles of 2 NKA and supporting tight coupling with GLT-1a. Using ceftriaxone treatment of FHM2 mutants and partial inhibition of glutamate transporters in wild-type mice, we obtain evidence that defective glutamate clearance can account for most of the facilitation of CSD initiation in FHM2-knockin mice, pointing to excessive glutamatergic transmission as a key mechanism underlying the vulnerability to CSD ignition in migraine.

Biochemical Society Transactions, 1997

American journal of human genetics, 1996

A hereditary defect of the distal tubule accounts for the clinical features of Gitelman syndrome ... more A hereditary defect of the distal tubule accounts for the clinical features of Gitelman syndrome (GS), an autosomal recessive disease characterized by hypokalemia, hypomagnesemia, metabolic alkalosis, and hypocalciuria. Recently, we cloned the cDNA coding for the human Na-Cl thiazide-sensitive cotransporter (TSC; also known as ¿NCCT¿ or ¿SLC12A3¿) as a possible candidate for GS, and Simon et al., independently, described mutations in patients with GS. Now, we show 12 additional mutations consistent with a loss of function of the Na-Cl cotransporter in GS. Two missense replacements, R209W and P349L, are common to both studies and could represent ancient mutations. The other mutations include three deletions, two insertions, and six missense mutations. When all mutations from both studies are considered, missense mutations seem to be more frequently localized within the intracellular domains of the molecule, rather than in transmembrane or extracellular domains. One family, previously...

The locus for a novel syndromic form of neuronal intestinal pseudoobstruction maps to Xq28

American journal of human genetics, 1996

The neuronal type of primary chronic idiopathic intestinal pseudoobstruction (CIIP) results from ... more The neuronal type of primary chronic idiopathic intestinal pseudoobstruction (CIIP) results from the developmental failure of enteric neurons to migrate or differentiate correctly. This leads to intestinal motility disorders, which are characterized by symptoms and signs of bowel obstruction in the absence of a mechanical obstacle. Most of these conditions are congenital, and among them some are inherited. One syndromic condition characterized by intestinal pseudoobstruction with morphological abnormalities of the argyrophil neurons in the myenteric plexus, associated with short small bowel, malrotation, and pyloric hypertrophy, has been previously described. We have studied a family affected by this disorder, in which the disease appeared to segregate as an X-linked recessive trait. In order to map the CIIP locus in this family, we performed linkage analysis in 26 family members by use of highly polymorphic microsatellite markers from the X chromosome. One of these markers, DXYS154...

Novel rat calpastatin mRNA variants

Biochemistry and molecular biology international, 1993

Rat calpastatin cDNAs obtained by RT-PCR method were isolated and directly sequenced; this allowe... more Rat calpastatin cDNAs obtained by RT-PCR method were isolated and directly sequenced; this allowed the identification of two new variants both characterized by a 23 amino acids deletion at the end of the unique N-terminal domain L, increasing the number of functional isoforms. The deletion shows high homology to the amino acid sequence coded by exon 8 of human calpastatin gene and properly conserved splicing consensus sequences, suggesting exon skipping in domain L. Furthermore, three point mutations scattered along the coding region were found.

Towards the identification of (a) gene(s) for autosomal dominant medullary cystic kidney disease

Journal of nephrology

Medullary cystic kidney disease (MCKD) belongs with nephronophthisis (NPH) in a group of inherite... more Medullary cystic kidney disease (MCKD) belongs with nephronophthisis (NPH) in a group of inherited tubulo-interstitial nephritis, which has been referred to as the NPH-MCKD complex. Although MCKD and NPH share morphological features, they differ in several respects. The most common variant is recessive juvenile NPH, with onset in childhood and leading to end-stage renal disease (ESRD) within the 2nd decade of life; the most frequent extrarenal involvement is tapeto-retinal degeneration. MCKD is a dominant condition recognized in later life and leading to ESRD at the age of 50 years; hyperuricemia and gout can be associated features. The first sign of MCKD is polyuria; later, the clinical findings relate to renal insufficiency. Originally, NPH and MCKD were considered separate entities. Subsequently, it has been suggested that the two diseases were a single disorder due to the clinico-pathological identity. This unifying conception was later refuted due to the identification of MCKD ...

Molecular epidemiology of the long QT syndrome

The Journal of Neuroscience, 2009

Paraplegin and AFG3L2 are ubiquitous nuclear-encoded mitochondrial proteins that form hetero-olig... more Paraplegin and AFG3L2 are ubiquitous nuclear-encoded mitochondrial proteins that form hetero-oligomeric paraplegin-AFG3L2 and homo-oligomeric AFG3L2 complexes in the inner mitochondrial membrane, namedm-AAA proteases. These complexes ensure protein quality control in the inner membrane, jointly with a chaperone-like activity on the respiratory chain complexes. Despite coassembling in the same complex, mutations of either paraplegin or AFG3L2 cause two different neurodegenerative disorders. Indeed, mutations of paraplegin are responsible for a recessive form of hereditary spastic paraplegia, whereas mutations of AFG3L2 have been recently associated to a dominant form of spinocerebellar ataxia (SCA28). In this work, we report that the mouse model haploinsufficient forAfg3l2recapitulates important pathophysiological features of the human disease, thus representing the first SCA28 model. Furthermore, we propose a pathogenetic mechanism in which respiratory chain dysfunction and increase...

Pediatric Nephrology, 2000

Limited phenotypic variability has been reported in patients with Bartter syndrome type I, with m... more Limited phenotypic variability has been reported in patients with Bartter syndrome type I, with mutations in the Na-K-2Cl cotransporter gene (BSC). The diagnosis of this hereditary renal tubular disorder is usually made in the antenatal-neonatal period, due to the presence of polyhydramnios, premature delivery, hypokalemia, metabolic alkalosis, hypercalciuria, and nephro-calcinosis. Among nine children with hypercalciuria and nephrocalcinosis, we identified new mutations consistent with a loss of function of the mutant allele of the BSC gene in five. Three of the five cases with BSC gene mutations were unusual due to the absence of hypokalemia and metabolic alkalosis in the first years of life. The diagnosis of incomplete distal renal tubular acidosis was considered before molecular evaluation. Three additional patients with hypokalemia and hypercalciuria, but without nephrocalcinosis in the first two and with metabolic acidosis instead of alkalosis in the third, were studied. Two demonstrated the same missense mutation A555T in the BSC gene as one patient of the previous group, suggesting a single common ancestor. The third patient presented with severe hypernatremia and hyperchloremia for about 2 months, and a diagnosis of nephrogenic diabetes insipidus was hypothesized until the diagnosis of Bartter syndrome type I was established by molecular evaluation. We conclude that in some patients with Bartter syndrome type I, hypokalemia and/or metabolic alkalosis may be absent in the first years of life and persistent metabolic acidosis or hypernatremia and hyperchloremia may also be present. Molecular evaluation can definitely establish the diagnosis of atypical cases of this complex hereditary tubular disorder, which, in our experience, may exhibit phenotypic variability.

Human Molecular Genetics, 2003

The disease complex medullary cystic disease/familial juvenile hyperuricemic nephropathy (MCKD/FJ... more The disease complex medullary cystic disease/familial juvenile hyperuricemic nephropathy (MCKD/FJHN) is characterized by alteration of urinary concentrating ability, frequent hyperuricemia, tubulo-interstitial fibrosis, cysts at the cortico-medullary junction and renal failure. MCKD/FJHN is caused by mutations of the gene encoding uromodulin, the most abundant protein in urine. Here, we describe new missense mutations in three families with MCKD/FJHN and demonstrate allelism with a glomerulocystic kidney disease (GCKD) variant, showing association of cyst dilatation and collapse of glomeruli with some clinical features similar to MCKD/FJHN as hyperuricemia and impairment of urine concentrating ability. Furthermore, we provide the first functional characterization of uromodulin mutations. The four newly identified mutants were characterized by immunofluorescence and FACS analysis on transfected cells. These experiments showed that all uromodulin mutations cause a delay in protein export to the plasma membrane due to a longer retention time in the endoplasmic reticulum. Immunohistochemistry on GCKD and MCKD/FJHN kidney biopsies revealed dense intracellular accumulation of uromodulin in tubular epithelia of the thick ascending limb of Henle's loop. Electron microscopy demonstrated accumulation of dense fibrillar material within the endoplasmic reticulum. Consistently, patient urines show a severe reduction of excreted uromodulin. The maturation impairment is consistent with the clinical findings and suggests a pathogenetic mechanism leading to these kidney diseases.

A New Benign Adult Familial Myoclonic Epilepsy (BAFME) Pedigree Suggesting Linkage to Chromosome 2p11.1-q12.2

Epilepsia, 2004

Benign adult familial myoclonic epilepsy (BAFME) is an autosomal dominant condition characterized... more Benign adult familial myoclonic epilepsy (BAFME) is an autosomal dominant condition characterized by cortical tremor and generalized seizures, mapped on chromosome 8q24 by Japanese authors. Recently the same phenotype also was reported in European families, with linkage on chromosome 2. We present a new family with suggestion of linkage to chromosome 2p11.1-2q12.2 (lod score value, 1.55). This observation would confirm that BAFME is a worldwide, genetically heterogeneous condition, probably with Japanese families linked to 8q24 and European families to 2p11.1-q12.2.

Diabetes, 2003

Sodium-lithium countertransport (SLC) is an ouabain-insensitive exchange of Na for Li found in th... more Sodium-lithium countertransport (SLC) is an ouabain-insensitive exchange of Na for Li found in the erythrocyte membrane of several mammalian species. Although increased SLC activity is presently the most consistent intermediate phenotype of essential hypertension and diabetic nephropathy in humans, the gene responsible for this membrane transport has not been identified. Because of functional similarities, SLC was suggested to represent an in vitro mode of operation of the Na-H exchanger (NHE). This hypothesis, however, has been long hampered by the total insensitivity of SLC to amiloride, which is an intrinsic inhibitor of the first isoform of NHE, the only NHE isoform detected in human erythrocytes. We describe here the identification in human reticulocytes and erythrocytes of an alternative splicing of NHE lacking the amiloride binding site. Transfection experiments with this spliced variant restore amiloride-insensitive, phloretin-sensitive SLC activity. Expression of both regul...

Brain, 2001

We describe a pedigree in which eight individuals presented with a non-progressive disorder with ... more We describe a pedigree in which eight individuals presented with a non-progressive disorder with onset between the ages of 12 and 50 years. It was characterized by predominantly distal, semi-continuous rhythmic myoclonus (all patients), generalized tonic-clonic seizures (all patients) and complex partial seizures (three patients). Most individuals had rarely suffered seizures and had a normal cognitive level, but three individuals with intractable seizures had mild mental retardation. The pattern of inheritance was autosomal dominant with high penetrance. We defined this disorder as autosomal dominant cortical myoclonus and epilepsy (ADCME). All patients had frontotemporal as well as generalized interictal EEG abnormalities. A neurophysiological study of the myoclonus suggested a cortical origin. Backaveraging of the data generated a series of waves with a frequency that mirrored the frequency of EMG bursts. Frequency analysis identified significant peaks with coherence between EMG and EEG, which were recorded over the contralateral rolandic area in five patients. The frequency of coherence was 8-25 Hz and phase spectra confirmed that EEG activity preceded EMG activity by 8-15 ms. In two individuals there was also significant coherence between the ipsilateral EEG and EMG, consistent with the transcallosal spread of myoclonic © Oxford University Press 2001 activity. The C-reflex at rest was enhanced and somatosensory and visual evoked potentials were of high amplitude. The resting motor threshold intensity to transcranial magnetic stimulation was significantly reduced (38%; SD ⍨ 7; P ⍧ 0.01) and the post-motor evoked potential silent period (101 ms; SEM ⍨ 10) was significantly shortened compared with the controls (137 ms; SEM ⍨ 18). These clinical and neurophysiological characteristics suggest diffuse cortical hyperexcitability and high propensity for intra-hemispheric and inter-hemispheric cortical spread, as well as rhythmic myoclonic activity. Genome-wide linkage analysis identified a critical region spanning 12.4 cM between markers D2S2161 and D2S1897 in 2p11.1-q12.2, with a maximum two-point LOD score of 3.46 at θ 0.0 for marker D2S2175. Multipoint LOD score values, reaching 3.74 around D2S2175, localize the ADCME gene to the centromeric region of chromosome 2. The exclusion of the locus for familial adult myoclonic epilepsy on chromosome 8q23.3-q24 from linkage to our family and the new localization of the responsible gene to chromosome 2cen, together with the different phenotype, define a new epilepsy syndrome. We hypothesize that the responsible gene causes cortical hyperexcitability that is widespread but particularly involves the frontotemporal circuits.

Archives of Neurology, 2004

Background: Spinocerebellar ataxia type 17 is an autosomal dominant cerebellar ataxia caused by a... more Background: Spinocerebellar ataxia type 17 is an autosomal dominant cerebellar ataxia caused by a CAG repeat expansion in the TATA box-binding protein gene. Ataxia is typically the first sign whereas behavioral symptoms occur later. Objective: To characterize the unusual phenotypic expression of a large spinocerebellar ataxia type 17 kindred. Design: Clinical, neuropathological, and molecular genetic characterization of a 4-generation family with 16 affected patients. Results: Behavioral symptoms and frontal impairment dominated the early stages preceding ataxia, rigidity, and dystonic movements. Neuropathological examination showed cortical, subcortical, and cerebellar atrophy. Pur-(REPRINTED) ARCH

American Journal of Medical Genetics, 2001

Nonspeci®c X-linked mental retardation is a nonprogressive, genetically heterogeneous condition t... more Nonspeci®c X-linked mental retardation is a nonprogressive, genetically heterogeneous condition that affects cognitive function in the absence of other distinctive clinical manifestations. We report here linkage data on a large Pakistani family affected by a form of X-linked nonspeci®c mental retardation. X chromosome genotyping of family members and linkage analysis allowed the identi®cation of a new disease locus, MRX53. The de®ned critical region spans approximately 15 cM between DXS1210 and DXS1047 in Xq22.2±26. A LOD score value of 3.34 at no recombination was obtained with markers DXS1072 and DXS8081. ß 2001 Wiley-Liss, Inc.

Human Molecular Genetics, 2012

The mitochondrial protein AFG3L2 forms homo-oligomeric and hetero-oligomeric complexes with parap... more The mitochondrial protein AFG3L2 forms homo-oligomeric and hetero-oligomeric complexes with paraplegin in the inner mitochondrial membrane, named m-AAA proteases. These complexes are in charge of quality control of misfolded proteins and participate in the regulation of OPA1 proteolytic cleavage, required for mitochondrial fusion. Mutations in AFG3L2 cause spinocerebellar ataxia type 28 and a complex neurodegenerative syndrome of childhood. In this study, we demonstrated that the loss of AFG3L2 in mouse embryonic fibroblasts (MEFs) reduces mitochondrial Ca 21 uptake capacity. This defect is neither a consequence of global alteration in cellular Ca 21 homeostasis nor of the reduced driving force for Ca 21 internalization within mitochondria, since cytosolic Ca 21 transients and mitochondrial membrane potential remain unaffected. Moreover, experiments in permeabilized cells revealed unaltered mitochondrial Ca 21 uptake speed in Afg3l2 2/2 cells, indicating the presence of functional Ca 21 uptake machinery. Our results show that the defective Ca 21 handling in Afg3l2 2/2 cells is caused by fragmentation of the mitochondrial network, secondary to respiratory dysfunction and the consequent processing of OPA1. This leaves a number of mitochondria devoid of connections to the ER and thus without Ca 21 elevations, hampering the proper Ca 21 diffusion along the mitochondrial network. The recovery of mitochondrial fragmentation in Afg3l2 2/2 MEFs by overexpression of OPA1 rescues the impaired mitochondrial Ca 21 buffering, but fails to restore respiration. By linking mitochondrial morphology and Ca 21 homeostasis, these findings shed new light in the molecular mechanisms underlining neurodegeneration caused by AFG3L2 mutations.

Brain, 2003

Restless legs syndrome (RLS) is a common neurological disorder characterized by an irresistible d... more Restless legs syndrome (RLS) is a common neurological disorder characterized by an irresistible desire to move the extremities associated with paraesthesia/dysaesthesia. These symptoms occur predominantly at rest and worsen at night, resulting in nocturnal insomnia and chronic sleep deprivation. In this paper, we show sig-ni®cant evidence of linkage to a new locus for RLS on chromosome 14q13-21 region in a 30-member, threegeneration Italian family affected by RLS and periodic leg movements in sleep (PLMS). This is the second RLS locus identi®ed so far and the ®rst consistent with an autosomal dominant inheritance pattern. The new RLS critical region spans 9.1 cM, between markers D14S70 and D14S1068. The maximum two-point log of odds ratio score value, of 3.23 at q = 0.0, was obtained for marker D14S288. The accurate clinical evaluation of RLS-affected, as well as unaffected, family members allowed for the con®guring of RLS as a phenotypic spectrum ranging from PLMS to RLS. Motor component, both while awake and during sleep, was an important aspect of the phenotype in the family analysed. The complementary clinical and genetic studies on multiplex families are likely to be of the utmost importance in unfolding the complete expressivity of RLS phenotype spectrum.

Molecular and Cellular Biology, 2006

The m -AAA protease, an ATP-dependent proteolytic complex in the mitochondrial inner membrane, co... more The m -AAA protease, an ATP-dependent proteolytic complex in the mitochondrial inner membrane, controls protein quality and regulates ribosome assembly, thus exerting essential housekeeping functions within mitochondria. Mutations in the m -AAA protease subunit paraplegin cause axonal degeneration in hereditary spastic paraplegia (HSP), but the basis for the unexpected tissue specificity is not understood. Paraplegin assembles with homologous Afg3l2 subunits into hetero-oligomeric complexes which can substitute for yeast m -AAA proteases, demonstrating functional conservation. The function of a third paralogue, Afg3l1 expressed in mouse, is unknown. Here, we analyze the assembly of paraplegin into m -AAA complexes and monitor consequences of paraplegin deficiency in HSP fibroblasts and in a mouse model for HSP. Our findings reveal variability in the assembly of m -AAA proteases in mitochondria in different tissues. Homo-oligomeric Afg3l1 and Afg3l2 complexes and hetero-oligomeric as...

BMC Medical Genetics, 2008

Background Mutations in the SPG4 gene (spastin) and in the SPG3A gene (atlastin) account for the ... more Background Mutations in the SPG4 gene (spastin) and in the SPG3A gene (atlastin) account for the majority of 'pure' autosomal dominant form of hereditary spastic paraplegia (HSP). Recently, mutations in the REEP1 gene were identified to cause autosomal dominant HSP type SPG31. The purpose of this study was to determine the prevalence of REEP1 mutations in a cohort of 162 unrelated Caucasian index patients with 'pure' HSP and a positive family history (at least two persons per family presented symptoms). Methods 162 patients were screened for mutations by, both, DHPLC and direct sequencing. Results Ten mutations were identified in the REEP1 gene, these included eight novel mutations comprising small insertions/deletions causing frame shifts and subsequently premature stop codons, one nonsense mutation and one splice site mutation as well as two missense mutations. Both missense mutations and the splice site mutation were not identified in 170 control subjects. Conclus...

Nucleic Acids Research, 2005

An increasing number of eukaryotic and prokaryotic genes are being found to have natural antisens... more An increasing number of eukaryotic and prokaryotic genes are being found to have natural antisense transcripts (NATs). There is also growing evidence to suggest that antisense transcription could play a key role in many human diseases. Consequently, there have been several recent attempts to set up computational procedures aimed at identifying novel NATs. Our group has developed the AntiHunter program for the identification of expressed sequence tag (EST) antisense transcripts from BLAST output. In order to perform an analysis, the program requires a genomic sequence plus an associated list of transcript names and coordinates of the genomic region. After masking the repeated regions, the program carries out a BLASTN search of this sequence in the selected EST database, reporting via email the EST entries that reveal an antisense transcript according to the user-supplied list. Here, we present the newly developed version 2.0 of the AntiHunter tool. Several improvements have been added to this version of the program in order to increase its ability to detect a larger number of antisense ESTs. As a result, AntiHunter can now detect, on average, .45% more antisense ESTs with little or no increase in the percentage of the false positives. We also raised the maximum query size to 3 Mb (previously 1 Mb). Moreover, we found that a reasonable trade-off between the program search sensitivity and the maximum allowed size of the input-query sequence could be obtained by querying the database with the MEGABLAST program, rather than by using the BLAST one. We now offer this new opportunity to users, i.e. if choosing the MEGABLAST option, users can input a query sequence up to 30 Mb long, thus considerably improving the possibility to analyze longer query regions.

EMBO Molecular Medicine, 2016

Migraine is a common disabling brain disorder. A subtype of migraine with aura (familial hemipleg... more Migraine is a common disabling brain disorder. A subtype of migraine with aura (familial hemiplegic migraine type 2: FHM2) is caused by loss-of-function mutations in 2 Na(+),K(+) ATPase (2 NKA), an isoform almost exclusively expressed in astrocytes in adult brain. Cortical spreading depression (CSD), the phenomenon that underlies migraine aura and activates migraine headache mechanisms, is facilitated in heterozygous FHM2-knockin mice with reduced expression of 2 NKA The mechanisms underlying an increased susceptibility to CSD in FHM2 are unknown. Here, we show reduced rates of glutamate and K(+) clearance by cortical astrocytes during neuronal activity and reduced density of GLT-1a glutamate transporters in cortical perisynaptic astrocytic processes in heterozygous FHM2knockin mice, demonstrating key physiological roles of 2 NKA and supporting tight coupling with GLT-1a. Using ceftriaxone treatment of FHM2 mutants and partial inhibition of glutamate transporters in wild-type mice, we obtain evidence that defective glutamate clearance can account for most of the facilitation of CSD initiation in FHM2-knockin mice, pointing to excessive glutamatergic transmission as a key mechanism underlying the vulnerability to CSD ignition in migraine.

Biochemical Society Transactions, 1997

American journal of human genetics, 1996

A hereditary defect of the distal tubule accounts for the clinical features of Gitelman syndrome ... more A hereditary defect of the distal tubule accounts for the clinical features of Gitelman syndrome (GS), an autosomal recessive disease characterized by hypokalemia, hypomagnesemia, metabolic alkalosis, and hypocalciuria. Recently, we cloned the cDNA coding for the human Na-Cl thiazide-sensitive cotransporter (TSC; also known as ¿NCCT¿ or ¿SLC12A3¿) as a possible candidate for GS, and Simon et al., independently, described mutations in patients with GS. Now, we show 12 additional mutations consistent with a loss of function of the Na-Cl cotransporter in GS. Two missense replacements, R209W and P349L, are common to both studies and could represent ancient mutations. The other mutations include three deletions, two insertions, and six missense mutations. When all mutations from both studies are considered, missense mutations seem to be more frequently localized within the intracellular domains of the molecule, rather than in transmembrane or extracellular domains. One family, previously...

The locus for a novel syndromic form of neuronal intestinal pseudoobstruction maps to Xq28

American journal of human genetics, 1996

The neuronal type of primary chronic idiopathic intestinal pseudoobstruction (CIIP) results from ... more The neuronal type of primary chronic idiopathic intestinal pseudoobstruction (CIIP) results from the developmental failure of enteric neurons to migrate or differentiate correctly. This leads to intestinal motility disorders, which are characterized by symptoms and signs of bowel obstruction in the absence of a mechanical obstacle. Most of these conditions are congenital, and among them some are inherited. One syndromic condition characterized by intestinal pseudoobstruction with morphological abnormalities of the argyrophil neurons in the myenteric plexus, associated with short small bowel, malrotation, and pyloric hypertrophy, has been previously described. We have studied a family affected by this disorder, in which the disease appeared to segregate as an X-linked recessive trait. In order to map the CIIP locus in this family, we performed linkage analysis in 26 family members by use of highly polymorphic microsatellite markers from the X chromosome. One of these markers, DXYS154...

Novel rat calpastatin mRNA variants

Biochemistry and molecular biology international, 1993

Rat calpastatin cDNAs obtained by RT-PCR method were isolated and directly sequenced; this allowe... more Rat calpastatin cDNAs obtained by RT-PCR method were isolated and directly sequenced; this allowed the identification of two new variants both characterized by a 23 amino acids deletion at the end of the unique N-terminal domain L, increasing the number of functional isoforms. The deletion shows high homology to the amino acid sequence coded by exon 8 of human calpastatin gene and properly conserved splicing consensus sequences, suggesting exon skipping in domain L. Furthermore, three point mutations scattered along the coding region were found.

Towards the identification of (a) gene(s) for autosomal dominant medullary cystic kidney disease

Journal of nephrology

Medullary cystic kidney disease (MCKD) belongs with nephronophthisis (NPH) in a group of inherite... more Medullary cystic kidney disease (MCKD) belongs with nephronophthisis (NPH) in a group of inherited tubulo-interstitial nephritis, which has been referred to as the NPH-MCKD complex. Although MCKD and NPH share morphological features, they differ in several respects. The most common variant is recessive juvenile NPH, with onset in childhood and leading to end-stage renal disease (ESRD) within the 2nd decade of life; the most frequent extrarenal involvement is tapeto-retinal degeneration. MCKD is a dominant condition recognized in later life and leading to ESRD at the age of 50 years; hyperuricemia and gout can be associated features. The first sign of MCKD is polyuria; later, the clinical findings relate to renal insufficiency. Originally, NPH and MCKD were considered separate entities. Subsequently, it has been suggested that the two diseases were a single disorder due to the clinico-pathological identity. This unifying conception was later refuted due to the identification of MCKD ...

Molecular epidemiology of the long QT syndrome

The Journal of Neuroscience, 2009

Paraplegin and AFG3L2 are ubiquitous nuclear-encoded mitochondrial proteins that form hetero-olig... more Paraplegin and AFG3L2 are ubiquitous nuclear-encoded mitochondrial proteins that form hetero-oligomeric paraplegin-AFG3L2 and homo-oligomeric AFG3L2 complexes in the inner mitochondrial membrane, namedm-AAA proteases. These complexes ensure protein quality control in the inner membrane, jointly with a chaperone-like activity on the respiratory chain complexes. Despite coassembling in the same complex, mutations of either paraplegin or AFG3L2 cause two different neurodegenerative disorders. Indeed, mutations of paraplegin are responsible for a recessive form of hereditary spastic paraplegia, whereas mutations of AFG3L2 have been recently associated to a dominant form of spinocerebellar ataxia (SCA28). In this work, we report that the mouse model haploinsufficient forAfg3l2recapitulates important pathophysiological features of the human disease, thus representing the first SCA28 model. Furthermore, we propose a pathogenetic mechanism in which respiratory chain dysfunction and increase...

Pediatric Nephrology, 2000

Limited phenotypic variability has been reported in patients with Bartter syndrome type I, with m... more Limited phenotypic variability has been reported in patients with Bartter syndrome type I, with mutations in the Na-K-2Cl cotransporter gene (BSC). The diagnosis of this hereditary renal tubular disorder is usually made in the antenatal-neonatal period, due to the presence of polyhydramnios, premature delivery, hypokalemia, metabolic alkalosis, hypercalciuria, and nephro-calcinosis. Among nine children with hypercalciuria and nephrocalcinosis, we identified new mutations consistent with a loss of function of the mutant allele of the BSC gene in five. Three of the five cases with BSC gene mutations were unusual due to the absence of hypokalemia and metabolic alkalosis in the first years of life. The diagnosis of incomplete distal renal tubular acidosis was considered before molecular evaluation. Three additional patients with hypokalemia and hypercalciuria, but without nephrocalcinosis in the first two and with metabolic acidosis instead of alkalosis in the third, were studied. Two demonstrated the same missense mutation A555T in the BSC gene as one patient of the previous group, suggesting a single common ancestor. The third patient presented with severe hypernatremia and hyperchloremia for about 2 months, and a diagnosis of nephrogenic diabetes insipidus was hypothesized until the diagnosis of Bartter syndrome type I was established by molecular evaluation. We conclude that in some patients with Bartter syndrome type I, hypokalemia and/or metabolic alkalosis may be absent in the first years of life and persistent metabolic acidosis or hypernatremia and hyperchloremia may also be present. Molecular evaluation can definitely establish the diagnosis of atypical cases of this complex hereditary tubular disorder, which, in our experience, may exhibit phenotypic variability.

Human Molecular Genetics, 2003

The disease complex medullary cystic disease/familial juvenile hyperuricemic nephropathy (MCKD/FJ... more The disease complex medullary cystic disease/familial juvenile hyperuricemic nephropathy (MCKD/FJHN) is characterized by alteration of urinary concentrating ability, frequent hyperuricemia, tubulo-interstitial fibrosis, cysts at the cortico-medullary junction and renal failure. MCKD/FJHN is caused by mutations of the gene encoding uromodulin, the most abundant protein in urine. Here, we describe new missense mutations in three families with MCKD/FJHN and demonstrate allelism with a glomerulocystic kidney disease (GCKD) variant, showing association of cyst dilatation and collapse of glomeruli with some clinical features similar to MCKD/FJHN as hyperuricemia and impairment of urine concentrating ability. Furthermore, we provide the first functional characterization of uromodulin mutations. The four newly identified mutants were characterized by immunofluorescence and FACS analysis on transfected cells. These experiments showed that all uromodulin mutations cause a delay in protein export to the plasma membrane due to a longer retention time in the endoplasmic reticulum. Immunohistochemistry on GCKD and MCKD/FJHN kidney biopsies revealed dense intracellular accumulation of uromodulin in tubular epithelia of the thick ascending limb of Henle's loop. Electron microscopy demonstrated accumulation of dense fibrillar material within the endoplasmic reticulum. Consistently, patient urines show a severe reduction of excreted uromodulin. The maturation impairment is consistent with the clinical findings and suggests a pathogenetic mechanism leading to these kidney diseases.

A New Benign Adult Familial Myoclonic Epilepsy (BAFME) Pedigree Suggesting Linkage to Chromosome 2p11.1-q12.2

Epilepsia, 2004

Benign adult familial myoclonic epilepsy (BAFME) is an autosomal dominant condition characterized... more Benign adult familial myoclonic epilepsy (BAFME) is an autosomal dominant condition characterized by cortical tremor and generalized seizures, mapped on chromosome 8q24 by Japanese authors. Recently the same phenotype also was reported in European families, with linkage on chromosome 2. We present a new family with suggestion of linkage to chromosome 2p11.1-2q12.2 (lod score value, 1.55). This observation would confirm that BAFME is a worldwide, genetically heterogeneous condition, probably with Japanese families linked to 8q24 and European families to 2p11.1-q12.2.

Diabetes, 2003

Sodium-lithium countertransport (SLC) is an ouabain-insensitive exchange of Na for Li found in th... more Sodium-lithium countertransport (SLC) is an ouabain-insensitive exchange of Na for Li found in the erythrocyte membrane of several mammalian species. Although increased SLC activity is presently the most consistent intermediate phenotype of essential hypertension and diabetic nephropathy in humans, the gene responsible for this membrane transport has not been identified. Because of functional similarities, SLC was suggested to represent an in vitro mode of operation of the Na-H exchanger (NHE). This hypothesis, however, has been long hampered by the total insensitivity of SLC to amiloride, which is an intrinsic inhibitor of the first isoform of NHE, the only NHE isoform detected in human erythrocytes. We describe here the identification in human reticulocytes and erythrocytes of an alternative splicing of NHE lacking the amiloride binding site. Transfection experiments with this spliced variant restore amiloride-insensitive, phloretin-sensitive SLC activity. Expression of both regul...

Brain, 2001

We describe a pedigree in which eight individuals presented with a non-progressive disorder with ... more We describe a pedigree in which eight individuals presented with a non-progressive disorder with onset between the ages of 12 and 50 years. It was characterized by predominantly distal, semi-continuous rhythmic myoclonus (all patients), generalized tonic-clonic seizures (all patients) and complex partial seizures (three patients). Most individuals had rarely suffered seizures and had a normal cognitive level, but three individuals with intractable seizures had mild mental retardation. The pattern of inheritance was autosomal dominant with high penetrance. We defined this disorder as autosomal dominant cortical myoclonus and epilepsy (ADCME). All patients had frontotemporal as well as generalized interictal EEG abnormalities. A neurophysiological study of the myoclonus suggested a cortical origin. Backaveraging of the data generated a series of waves with a frequency that mirrored the frequency of EMG bursts. Frequency analysis identified significant peaks with coherence between EMG and EEG, which were recorded over the contralateral rolandic area in five patients. The frequency of coherence was 8-25 Hz and phase spectra confirmed that EEG activity preceded EMG activity by 8-15 ms. In two individuals there was also significant coherence between the ipsilateral EEG and EMG, consistent with the transcallosal spread of myoclonic © Oxford University Press 2001 activity. The C-reflex at rest was enhanced and somatosensory and visual evoked potentials were of high amplitude. The resting motor threshold intensity to transcranial magnetic stimulation was significantly reduced (38%; SD ⍨ 7; P ⍧ 0.01) and the post-motor evoked potential silent period (101 ms; SEM ⍨ 10) was significantly shortened compared with the controls (137 ms; SEM ⍨ 18). These clinical and neurophysiological characteristics suggest diffuse cortical hyperexcitability and high propensity for intra-hemispheric and inter-hemispheric cortical spread, as well as rhythmic myoclonic activity. Genome-wide linkage analysis identified a critical region spanning 12.4 cM between markers D2S2161 and D2S1897 in 2p11.1-q12.2, with a maximum two-point LOD score of 3.46 at θ 0.0 for marker D2S2175. Multipoint LOD score values, reaching 3.74 around D2S2175, localize the ADCME gene to the centromeric region of chromosome 2. The exclusion of the locus for familial adult myoclonic epilepsy on chromosome 8q23.3-q24 from linkage to our family and the new localization of the responsible gene to chromosome 2cen, together with the different phenotype, define a new epilepsy syndrome. We hypothesize that the responsible gene causes cortical hyperexcitability that is widespread but particularly involves the frontotemporal circuits.

Archives of Neurology, 2004

Background: Spinocerebellar ataxia type 17 is an autosomal dominant cerebellar ataxia caused by a... more Background: Spinocerebellar ataxia type 17 is an autosomal dominant cerebellar ataxia caused by a CAG repeat expansion in the TATA box-binding protein gene. Ataxia is typically the first sign whereas behavioral symptoms occur later. Objective: To characterize the unusual phenotypic expression of a large spinocerebellar ataxia type 17 kindred. Design: Clinical, neuropathological, and molecular genetic characterization of a 4-generation family with 16 affected patients. Results: Behavioral symptoms and frontal impairment dominated the early stages preceding ataxia, rigidity, and dystonic movements. Neuropathological examination showed cortical, subcortical, and cerebellar atrophy. Pur-(REPRINTED) ARCH

American Journal of Medical Genetics, 2001

Nonspeci®c X-linked mental retardation is a nonprogressive, genetically heterogeneous condition t... more Nonspeci®c X-linked mental retardation is a nonprogressive, genetically heterogeneous condition that affects cognitive function in the absence of other distinctive clinical manifestations. We report here linkage data on a large Pakistani family affected by a form of X-linked nonspeci®c mental retardation. X chromosome genotyping of family members and linkage analysis allowed the identi®cation of a new disease locus, MRX53. The de®ned critical region spans approximately 15 cM between DXS1210 and DXS1047 in Xq22.2±26. A LOD score value of 3.34 at no recombination was obtained with markers DXS1072 and DXS8081. ß 2001 Wiley-Liss, Inc.

Human Molecular Genetics, 2012

The mitochondrial protein AFG3L2 forms homo-oligomeric and hetero-oligomeric complexes with parap... more The mitochondrial protein AFG3L2 forms homo-oligomeric and hetero-oligomeric complexes with paraplegin in the inner mitochondrial membrane, named m-AAA proteases. These complexes are in charge of quality control of misfolded proteins and participate in the regulation of OPA1 proteolytic cleavage, required for mitochondrial fusion. Mutations in AFG3L2 cause spinocerebellar ataxia type 28 and a complex neurodegenerative syndrome of childhood. In this study, we demonstrated that the loss of AFG3L2 in mouse embryonic fibroblasts (MEFs) reduces mitochondrial Ca 21 uptake capacity. This defect is neither a consequence of global alteration in cellular Ca 21 homeostasis nor of the reduced driving force for Ca 21 internalization within mitochondria, since cytosolic Ca 21 transients and mitochondrial membrane potential remain unaffected. Moreover, experiments in permeabilized cells revealed unaltered mitochondrial Ca 21 uptake speed in Afg3l2 2/2 cells, indicating the presence of functional Ca 21 uptake machinery. Our results show that the defective Ca 21 handling in Afg3l2 2/2 cells is caused by fragmentation of the mitochondrial network, secondary to respiratory dysfunction and the consequent processing of OPA1. This leaves a number of mitochondria devoid of connections to the ER and thus without Ca 21 elevations, hampering the proper Ca 21 diffusion along the mitochondrial network. The recovery of mitochondrial fragmentation in Afg3l2 2/2 MEFs by overexpression of OPA1 rescues the impaired mitochondrial Ca 21 buffering, but fails to restore respiration. By linking mitochondrial morphology and Ca 21 homeostasis, these findings shed new light in the molecular mechanisms underlining neurodegeneration caused by AFG3L2 mutations.

Brain, 2003

Restless legs syndrome (RLS) is a common neurological disorder characterized by an irresistible d... more Restless legs syndrome (RLS) is a common neurological disorder characterized by an irresistible desire to move the extremities associated with paraesthesia/dysaesthesia. These symptoms occur predominantly at rest and worsen at night, resulting in nocturnal insomnia and chronic sleep deprivation. In this paper, we show sig-ni®cant evidence of linkage to a new locus for RLS on chromosome 14q13-21 region in a 30-member, threegeneration Italian family affected by RLS and periodic leg movements in sleep (PLMS). This is the second RLS locus identi®ed so far and the ®rst consistent with an autosomal dominant inheritance pattern. The new RLS critical region spans 9.1 cM, between markers D14S70 and D14S1068. The maximum two-point log of odds ratio score value, of 3.23 at q = 0.0, was obtained for marker D14S288. The accurate clinical evaluation of RLS-affected, as well as unaffected, family members allowed for the con®guring of RLS as a phenotypic spectrum ranging from PLMS to RLS. Motor component, both while awake and during sleep, was an important aspect of the phenotype in the family analysed. The complementary clinical and genetic studies on multiplex families are likely to be of the utmost importance in unfolding the complete expressivity of RLS phenotype spectrum.

Molecular and Cellular Biology, 2006

The m -AAA protease, an ATP-dependent proteolytic complex in the mitochondrial inner membrane, co... more The m -AAA protease, an ATP-dependent proteolytic complex in the mitochondrial inner membrane, controls protein quality and regulates ribosome assembly, thus exerting essential housekeeping functions within mitochondria. Mutations in the m -AAA protease subunit paraplegin cause axonal degeneration in hereditary spastic paraplegia (HSP), but the basis for the unexpected tissue specificity is not understood. Paraplegin assembles with homologous Afg3l2 subunits into hetero-oligomeric complexes which can substitute for yeast m -AAA proteases, demonstrating functional conservation. The function of a third paralogue, Afg3l1 expressed in mouse, is unknown. Here, we analyze the assembly of paraplegin into m -AAA complexes and monitor consequences of paraplegin deficiency in HSP fibroblasts and in a mouse model for HSP. Our findings reveal variability in the assembly of m -AAA proteases in mitochondria in different tissues. Homo-oligomeric Afg3l1 and Afg3l2 complexes and hetero-oligomeric as...

BMC Medical Genetics, 2008

Background Mutations in the SPG4 gene (spastin) and in the SPG3A gene (atlastin) account for the ... more Background Mutations in the SPG4 gene (spastin) and in the SPG3A gene (atlastin) account for the majority of 'pure' autosomal dominant form of hereditary spastic paraplegia (HSP). Recently, mutations in the REEP1 gene were identified to cause autosomal dominant HSP type SPG31. The purpose of this study was to determine the prevalence of REEP1 mutations in a cohort of 162 unrelated Caucasian index patients with 'pure' HSP and a positive family history (at least two persons per family presented symptoms). Methods 162 patients were screened for mutations by, both, DHPLC and direct sequencing. Results Ten mutations were identified in the REEP1 gene, these included eight novel mutations comprising small insertions/deletions causing frame shifts and subsequently premature stop codons, one nonsense mutation and one splice site mutation as well as two missense mutations. Both missense mutations and the splice site mutation were not identified in 170 control subjects. Conclus...

Nucleic Acids Research, 2005

An increasing number of eukaryotic and prokaryotic genes are being found to have natural antisens... more An increasing number of eukaryotic and prokaryotic genes are being found to have natural antisense transcripts (NATs). There is also growing evidence to suggest that antisense transcription could play a key role in many human diseases. Consequently, there have been several recent attempts to set up computational procedures aimed at identifying novel NATs. Our group has developed the AntiHunter program for the identification of expressed sequence tag (EST) antisense transcripts from BLAST output. In order to perform an analysis, the program requires a genomic sequence plus an associated list of transcript names and coordinates of the genomic region. After masking the repeated regions, the program carries out a BLASTN search of this sequence in the selected EST database, reporting via email the EST entries that reveal an antisense transcript according to the user-supplied list. Here, we present the newly developed version 2.0 of the AntiHunter tool. Several improvements have been added to this version of the program in order to increase its ability to detect a larger number of antisense ESTs. As a result, AntiHunter can now detect, on average, .45% more antisense ESTs with little or no increase in the percentage of the false positives. We also raised the maximum query size to 3 Mb (previously 1 Mb). Moreover, we found that a reasonable trade-off between the program search sensitivity and the maximum allowed size of the input-query sequence could be obtained by querying the database with the MEGABLAST program, rather than by using the BLAST one. We now offer this new opportunity to users, i.e. if choosing the MEGABLAST option, users can input a query sequence up to 30 Mb long, thus considerably improving the possibility to analyze longer query regions.