Roberto Cusano - Academia.edu (original) (raw)

Papers by Roberto Cusano

Research paper thumbnail of Confirmation of CLIM2/LMX1B interaction by yeast two-hybrid screening and analysis of its involvement in nail-patella syndrome

Nail-patella syndrome (NPS), an autosomal dominant disorder characterized by nail dysplasia, abse... more Nail-patella syndrome (NPS), an autosomal dominant disorder characterized by nail dysplasia, absent or hypoplastic patellae, iliac horns, and often associated with nephropathy and, less frequently, with open angle glaucoma, is caused by mutations in the LMX1B gene. Inter-familial and intra-familial phenotypic variability raises the question whether modifier genes can be identified to explain differences in the expression and severity of clinical features of NPS. Genes encoding proteins that interact with the LMX1B protein are good candidates and, therefore, methods to search for interactions can be used to this purpose. By the yeast two-hybrid screening we detected the CLIM2 gene as a LMX1B interactor, confirming previous reports which described the same interaction by biochemical methods. Sequencing of the CLIM2 coding region in seven NPS cases in which no LMX1B mutation had been found, did not detect any molecular variant in these patients. Moreover, by genotyping a polymorphic dinucleotide repeat close to the CLIM2 gene in affected members of a large Dutch NPS family with high incidence of nephropathy, we were unable to find a correlation between the presence of a specific allele and the expression of nephropathy. In conclusion, although the results of this study could not provide any proof of CLIM2 involvement in the pathogenesis of NPS or in determination of the clinical phenotype, we suggest that the CLIM2 gene can be considered as a good candidate for further studies on normal and disturbed kidney development associated with NPS or other hereditary glomerulopathies.

Research paper thumbnail of Evaluation of the HOX11L1 gene as a candidate for congenital disorders of intestinal innervation

Journal of Medical Genetics, 2000

Research paper thumbnail of Cell-line specific transcription rates of theRET gene and functional domains in its minimal promoter

Gene Function & Disease, 2000

ABSTRACT To gain insight into the RET gene transcriptional regulation we analysed its mRNA expres... more ABSTRACT To gain insight into the RET gene transcriptional regulation we analysed its mRNA expression, transcription rate, and promoter activity in different cell lines, showing that RET transcription is highly cell-line specific. By using a panel of promoter deletion constructs in transient transfection assays we identified the −147/+53 fragment as the main functional element. GMSA experiments indicated binding by Sp1 and a CACCC binding protein to multiple sites within the promoter. Upon deletion analysis Sp1 appeared to be the main positive regulator of promoter activity. The −147/+53 sequence did not reproduce the cell-line specific activity of the endogenous gene in vitro, raising the question of how the regulated pattern of RET transcription is achieved at the molecular level. To address this topic, both the search for far-sited tissue-specific enhancers/silencers and the study of chromatin structure within the RET locus should be envisaged.

Research paper thumbnail of Francalacci.SM

Research paper thumbnail of Prenatal diagnosis of Pelizaeus-Merzbacher disease: detection of proteolipid protein gene duplication by quantitative fluorescent multiplex PCR

Prenatal Diagnosis, 2001

A prenatal diagnosis of Pelizaeus-Merzbacher disease (PMD) resulting from proteolipid protein gen... more A prenatal diagnosis of Pelizaeus-Merzbacher disease (PMD) resulting from proteolipid protein gene (PLP) duplication was performed by a quantitative fluorescent multiplex PCR method. PLP gene copy number was determined in the proband, the pregnant mother, the male fetus and two aunts. Small amounts of genomic DNA extracted from peripheral blood and from chorionic villi were used. The fetus, in common with the proband, was identified as PMD-affected being a carrier of the PLP gene duplication, inherited from the mother, while the two aunts were non-carriers. The data obtained were confirmed by segregation analysis of a PLP-associated dinucleotide-repeat polymorphism amplified by the same multiplex PCR.

Research paper thumbnail of Autosomal dominant aplasia cutis congenita: report of a large Italian family and no hint for candidate chromosomal regions

Archives of Dermatological Research, 1999

We studied a three-generation pedigree in which seven individuals were affected by aplasia cutis ... more We studied a three-generation pedigree in which seven individuals were affected by aplasia cutis congenita, a rare disorder characterized by the congenital absence of the epidermis, dermis and subcutaneous tissue of the vertex or occipital region. Accurate clinical and formal genetic analysis suggested that this family was affected by the autosomal dominant form of the disease, a hereditary condition

Research paper thumbnail of Low-Pass DNA Sequencing of 1200 Sardinians Reconstructs European Y-Chromosome Phylogeny

Research paper thumbnail of Immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX): report of the first prenatal mutation testing

Research paper thumbnail of Weak linkage at 4p16 to predisposition for human neuroblastoma

Research paper thumbnail of Clinical and genetic heterogeneity in autosomal recessive nemaline myopathy

Neuromuscular Disorders, 1999

Research paper thumbnail of Pseudoxanthoma elasticum: Point mutations in the ABCC6 gene and a large deletion including also ABCC1 and MYH11

Research paper thumbnail of Novel MC1R variants in Ligurian melanoma patients and controls

Research paper thumbnail of Molecular characterization of Italian nevoid basal cell carcinoma syndrome patients

Research paper thumbnail of Natural transmission of USP9Y gene mutations: a new perspective on the role of AZFa genes in male fertility

Human Molecular Genetics, 2006

Research paper thumbnail of Epstein syndrome: another renal disorder with mutations in the nonmuscle myosin heavy chain 9 gene

Human Genetics, 2002

Epstein syndrome (EPTS) is an autosomal dominant disease characterized by nephritis, mild hearing... more Epstein syndrome (EPTS) is an autosomal dominant disease characterized by nephritis, mild hearing loss, and thrombocytopenia with giant platelets. Renal and hearing abnormalities are indistinguishable from those observed in Fechtner syndrome (FTNS), an Alport-like variant. EPTS macrothrombocytopenia is similar to that described in FTNS, May-Hegglin anomaly (MHA), and Sebastian syndrome (SBS), three disorders caused by mutations in the nonmuscle heavy chain myosin IIA ( MYH9). Unlike FTNS, MHA, and SBS, EPTS does not show inclusion bodies in the leukocytes. The clinical features of EPTS and the chromosomal localization of the respective gene in the same region as MYH9 suggest that this disorder is allelic with the other giant platelet disorders. We identified a MYH9 missense mutation in two EPTS familial cases. In both families, an R702H substitution was found, probably inducing conformational changes to the myosin head. A different amino acid substitution at the same codon (R702C) has been previously identified in FTNS. On the basis of predictions from molecular modeling of the X-ray crystallographic structure of chick smooth muscle myosin, the mutated thiol reactive group of R702C may lead to intermolecular disulfide bridges, with the consequent formation of the inclusions typical of FTNS. On the contrary, the R702H mutation does not allow the protein to aggregate and thus to generate "Döhle-like" bodies, which are indeed absent in EPTS. In conclusion, our results extend the allelic heterogeneity of MYH9 mutations to another clinical syndrome and contribute to the clarification of the pathogenesis of the various inherited giant platelet disorders.

Research paper thumbnail of Gene dosage of the spermidine/spermine N1-acetyltransferase (SSAT) gene with putrescine accumulation in a patient with a Xp21.1p22.12 duplication and keratosis follicularis spinulosa decalvans (KFSD)

Human Genetics, 2002

Keratosis follicularis spinulosa decalvans (KFSD) or Siemens-1 syndrome is a rare X-linked diseas... more Keratosis follicularis spinulosa decalvans (KFSD) or Siemens-1 syndrome is a rare X-linked disease of unknown etiology affecting the skin and the eye. Although most affected families are compatible with X-linked inheritance, KFSD appears to be clinically and genetically heterogeneous. So far, the gene has been mapped to Xp22.13p22.2 in two extended KFSD families. Analysis of additional recombination events in the first Dutch pedigree located the gene to an interval covering approximately 1 Mb between markers DXS7163 and DXS7593/DXS7105, whereas haplotype reconstruction in the second German family positioned the gene outside the previously identified region, proximal to marker DXS274. We report here the molecular characterization of an Xp21.1p22.12 duplication present in a patient affected with dosage-sensitive sex reversal (DSS) and KFSD. The duplicated region includes both the DAX1 gene (previously demonstrated to be responsible for DSS) and the KFSD interval, in which the gene encoding spermidine/spermine N(1)-acetyltransferase ( SSAT) is located. This enzyme catalyzes the N(1)-acetylation of spermidine and spermine and, by the successive activity of polyamine oxidase, the spermine can be converted to spermidine and the spermidine to putrescine. Overexpression of the SSAT enzyme in a mouse model results in putrescine accumulation and a phenotype with skin and hair abnormalities reminiscent of human KFSD. Analysis of polyamine metabolism in the cells of the patient indicated that the levels of metabolites such as putrescine, spermidine and spermine were consistent with the overexpression of the SSAT gene as in the murine model. Thus, we propose that overexpression of SSAT and the consequent putrescine accumulation are involved in the KFSD phenotype, at least in our propositus.

Research paper thumbnail of Exclusion of the Sonic Hedgehog gene as responsible for Currarino syndrome and anorectal malformations with sacral hypodevelopment

Human Genetics, 1999

Anorectal malformations (ARMs) are common congenital anomalies that account for 1:4 digestive mal... more Anorectal malformations (ARMs) are common congenital anomalies that account for 1:4 digestive malformations. ARM patients show different degrees of sacral hypodevelopment while the hemisacrum is characteristic of the Currarino syndrome (CS). Cases of CS present an association of ARM, hemisacrum and presacral mass. A gene responsible for CS has recently been mapped in 7q36. Among the genes localized in this critical region, sonic hedgehog (SHH) was thought to represent a candidate gene for CS as well as for ARM with different levels of sacral hypodevelopment according to its role in the differentiation of midline mesoderm. By linkage analysis we confirmed the critical region in one large family with recurrence of CS. In addition, the screening of SHH in 7 CS and in 15 sporadic ARM patients with sacral hypodevelopment allowed us to exclude its role in the pathogenesis of these disorders.

Research paper thumbnail of Contribution of arylsulfatase A mutations located on the same allele to enzyme activity reduction and metachromatic leukodystrophy severity

Research paper thumbnail of The First Intron of the Human Osteopontin Gene Contains a C/EBP-Beta-Responsive Enhancer

Gene Expression, 2003

The osteopontin (OPN) protein is found expressed at high level in several processes including fib... more The osteopontin (OPN) protein is found expressed at high level in several processes including fibrotic evolution of organ injuries, tumorigenesis, and immune response. The molecular mechanisms that underly overexpression, especially at the transcriptional level, have been only partially clarified. Therefore, this study was undertaken in search for additional DNA elements in the regulatory regions of the OPN gene and cognate transcription factors. Our results on the region upstream of the transcription start site confirmed that essential elements are located within the first 100 bp. Analysis of the sequence that includes the first untranslated exon and first intron revealed that it could enhance the promoter activity. Experiments of transfection of constructs containing different fragments of this sequence showed that most of the enhancer activity was confined in the terminal 30-bp tract of the first intron, although it was not functioning in a myofibroblast cell line. DNA/protein binding assays and cotransfection experiments showed that the C/EBP-beta transcription factor was able to bind a recognition sequence in this 30-bp segment. We found a bi-allelic sequence polymorphism at +245 in the first intron, which did not show a significant functional effect, but is a useful tool for future association studies.

Research paper thumbnail of Germline MLH1 and MSH2 mutations in Italian pancreatic cancer patients with suspected Lynch syndrome

Research paper thumbnail of Confirmation of CLIM2/LMX1B interaction by yeast two-hybrid screening and analysis of its involvement in nail-patella syndrome

Nail-patella syndrome (NPS), an autosomal dominant disorder characterized by nail dysplasia, abse... more Nail-patella syndrome (NPS), an autosomal dominant disorder characterized by nail dysplasia, absent or hypoplastic patellae, iliac horns, and often associated with nephropathy and, less frequently, with open angle glaucoma, is caused by mutations in the LMX1B gene. Inter-familial and intra-familial phenotypic variability raises the question whether modifier genes can be identified to explain differences in the expression and severity of clinical features of NPS. Genes encoding proteins that interact with the LMX1B protein are good candidates and, therefore, methods to search for interactions can be used to this purpose. By the yeast two-hybrid screening we detected the CLIM2 gene as a LMX1B interactor, confirming previous reports which described the same interaction by biochemical methods. Sequencing of the CLIM2 coding region in seven NPS cases in which no LMX1B mutation had been found, did not detect any molecular variant in these patients. Moreover, by genotyping a polymorphic dinucleotide repeat close to the CLIM2 gene in affected members of a large Dutch NPS family with high incidence of nephropathy, we were unable to find a correlation between the presence of a specific allele and the expression of nephropathy. In conclusion, although the results of this study could not provide any proof of CLIM2 involvement in the pathogenesis of NPS or in determination of the clinical phenotype, we suggest that the CLIM2 gene can be considered as a good candidate for further studies on normal and disturbed kidney development associated with NPS or other hereditary glomerulopathies.

Research paper thumbnail of Evaluation of the HOX11L1 gene as a candidate for congenital disorders of intestinal innervation

Journal of Medical Genetics, 2000

Research paper thumbnail of Cell-line specific transcription rates of theRET gene and functional domains in its minimal promoter

Gene Function & Disease, 2000

ABSTRACT To gain insight into the RET gene transcriptional regulation we analysed its mRNA expres... more ABSTRACT To gain insight into the RET gene transcriptional regulation we analysed its mRNA expression, transcription rate, and promoter activity in different cell lines, showing that RET transcription is highly cell-line specific. By using a panel of promoter deletion constructs in transient transfection assays we identified the −147/+53 fragment as the main functional element. GMSA experiments indicated binding by Sp1 and a CACCC binding protein to multiple sites within the promoter. Upon deletion analysis Sp1 appeared to be the main positive regulator of promoter activity. The −147/+53 sequence did not reproduce the cell-line specific activity of the endogenous gene in vitro, raising the question of how the regulated pattern of RET transcription is achieved at the molecular level. To address this topic, both the search for far-sited tissue-specific enhancers/silencers and the study of chromatin structure within the RET locus should be envisaged.

Research paper thumbnail of Francalacci.SM

Research paper thumbnail of Prenatal diagnosis of Pelizaeus-Merzbacher disease: detection of proteolipid protein gene duplication by quantitative fluorescent multiplex PCR

Prenatal Diagnosis, 2001

A prenatal diagnosis of Pelizaeus-Merzbacher disease (PMD) resulting from proteolipid protein gen... more A prenatal diagnosis of Pelizaeus-Merzbacher disease (PMD) resulting from proteolipid protein gene (PLP) duplication was performed by a quantitative fluorescent multiplex PCR method. PLP gene copy number was determined in the proband, the pregnant mother, the male fetus and two aunts. Small amounts of genomic DNA extracted from peripheral blood and from chorionic villi were used. The fetus, in common with the proband, was identified as PMD-affected being a carrier of the PLP gene duplication, inherited from the mother, while the two aunts were non-carriers. The data obtained were confirmed by segregation analysis of a PLP-associated dinucleotide-repeat polymorphism amplified by the same multiplex PCR.

Research paper thumbnail of Autosomal dominant aplasia cutis congenita: report of a large Italian family and no hint for candidate chromosomal regions

Archives of Dermatological Research, 1999

We studied a three-generation pedigree in which seven individuals were affected by aplasia cutis ... more We studied a three-generation pedigree in which seven individuals were affected by aplasia cutis congenita, a rare disorder characterized by the congenital absence of the epidermis, dermis and subcutaneous tissue of the vertex or occipital region. Accurate clinical and formal genetic analysis suggested that this family was affected by the autosomal dominant form of the disease, a hereditary condition

Research paper thumbnail of Low-Pass DNA Sequencing of 1200 Sardinians Reconstructs European Y-Chromosome Phylogeny

Research paper thumbnail of Immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX): report of the first prenatal mutation testing

Research paper thumbnail of Weak linkage at 4p16 to predisposition for human neuroblastoma

Research paper thumbnail of Clinical and genetic heterogeneity in autosomal recessive nemaline myopathy

Neuromuscular Disorders, 1999

Research paper thumbnail of Pseudoxanthoma elasticum: Point mutations in the ABCC6 gene and a large deletion including also ABCC1 and MYH11

Research paper thumbnail of Novel MC1R variants in Ligurian melanoma patients and controls

Research paper thumbnail of Molecular characterization of Italian nevoid basal cell carcinoma syndrome patients

Research paper thumbnail of Natural transmission of USP9Y gene mutations: a new perspective on the role of AZFa genes in male fertility

Human Molecular Genetics, 2006

Research paper thumbnail of Epstein syndrome: another renal disorder with mutations in the nonmuscle myosin heavy chain 9 gene

Human Genetics, 2002

Epstein syndrome (EPTS) is an autosomal dominant disease characterized by nephritis, mild hearing... more Epstein syndrome (EPTS) is an autosomal dominant disease characterized by nephritis, mild hearing loss, and thrombocytopenia with giant platelets. Renal and hearing abnormalities are indistinguishable from those observed in Fechtner syndrome (FTNS), an Alport-like variant. EPTS macrothrombocytopenia is similar to that described in FTNS, May-Hegglin anomaly (MHA), and Sebastian syndrome (SBS), three disorders caused by mutations in the nonmuscle heavy chain myosin IIA ( MYH9). Unlike FTNS, MHA, and SBS, EPTS does not show inclusion bodies in the leukocytes. The clinical features of EPTS and the chromosomal localization of the respective gene in the same region as MYH9 suggest that this disorder is allelic with the other giant platelet disorders. We identified a MYH9 missense mutation in two EPTS familial cases. In both families, an R702H substitution was found, probably inducing conformational changes to the myosin head. A different amino acid substitution at the same codon (R702C) has been previously identified in FTNS. On the basis of predictions from molecular modeling of the X-ray crystallographic structure of chick smooth muscle myosin, the mutated thiol reactive group of R702C may lead to intermolecular disulfide bridges, with the consequent formation of the inclusions typical of FTNS. On the contrary, the R702H mutation does not allow the protein to aggregate and thus to generate "Döhle-like" bodies, which are indeed absent in EPTS. In conclusion, our results extend the allelic heterogeneity of MYH9 mutations to another clinical syndrome and contribute to the clarification of the pathogenesis of the various inherited giant platelet disorders.

Research paper thumbnail of Gene dosage of the spermidine/spermine N1-acetyltransferase (SSAT) gene with putrescine accumulation in a patient with a Xp21.1p22.12 duplication and keratosis follicularis spinulosa decalvans (KFSD)

Human Genetics, 2002

Keratosis follicularis spinulosa decalvans (KFSD) or Siemens-1 syndrome is a rare X-linked diseas... more Keratosis follicularis spinulosa decalvans (KFSD) or Siemens-1 syndrome is a rare X-linked disease of unknown etiology affecting the skin and the eye. Although most affected families are compatible with X-linked inheritance, KFSD appears to be clinically and genetically heterogeneous. So far, the gene has been mapped to Xp22.13p22.2 in two extended KFSD families. Analysis of additional recombination events in the first Dutch pedigree located the gene to an interval covering approximately 1 Mb between markers DXS7163 and DXS7593/DXS7105, whereas haplotype reconstruction in the second German family positioned the gene outside the previously identified region, proximal to marker DXS274. We report here the molecular characterization of an Xp21.1p22.12 duplication present in a patient affected with dosage-sensitive sex reversal (DSS) and KFSD. The duplicated region includes both the DAX1 gene (previously demonstrated to be responsible for DSS) and the KFSD interval, in which the gene encoding spermidine/spermine N(1)-acetyltransferase ( SSAT) is located. This enzyme catalyzes the N(1)-acetylation of spermidine and spermine and, by the successive activity of polyamine oxidase, the spermine can be converted to spermidine and the spermidine to putrescine. Overexpression of the SSAT enzyme in a mouse model results in putrescine accumulation and a phenotype with skin and hair abnormalities reminiscent of human KFSD. Analysis of polyamine metabolism in the cells of the patient indicated that the levels of metabolites such as putrescine, spermidine and spermine were consistent with the overexpression of the SSAT gene as in the murine model. Thus, we propose that overexpression of SSAT and the consequent putrescine accumulation are involved in the KFSD phenotype, at least in our propositus.

Research paper thumbnail of Exclusion of the Sonic Hedgehog gene as responsible for Currarino syndrome and anorectal malformations with sacral hypodevelopment

Human Genetics, 1999

Anorectal malformations (ARMs) are common congenital anomalies that account for 1:4 digestive mal... more Anorectal malformations (ARMs) are common congenital anomalies that account for 1:4 digestive malformations. ARM patients show different degrees of sacral hypodevelopment while the hemisacrum is characteristic of the Currarino syndrome (CS). Cases of CS present an association of ARM, hemisacrum and presacral mass. A gene responsible for CS has recently been mapped in 7q36. Among the genes localized in this critical region, sonic hedgehog (SHH) was thought to represent a candidate gene for CS as well as for ARM with different levels of sacral hypodevelopment according to its role in the differentiation of midline mesoderm. By linkage analysis we confirmed the critical region in one large family with recurrence of CS. In addition, the screening of SHH in 7 CS and in 15 sporadic ARM patients with sacral hypodevelopment allowed us to exclude its role in the pathogenesis of these disorders.

Research paper thumbnail of Contribution of arylsulfatase A mutations located on the same allele to enzyme activity reduction and metachromatic leukodystrophy severity

Research paper thumbnail of The First Intron of the Human Osteopontin Gene Contains a C/EBP-Beta-Responsive Enhancer

Gene Expression, 2003

The osteopontin (OPN) protein is found expressed at high level in several processes including fib... more The osteopontin (OPN) protein is found expressed at high level in several processes including fibrotic evolution of organ injuries, tumorigenesis, and immune response. The molecular mechanisms that underly overexpression, especially at the transcriptional level, have been only partially clarified. Therefore, this study was undertaken in search for additional DNA elements in the regulatory regions of the OPN gene and cognate transcription factors. Our results on the region upstream of the transcription start site confirmed that essential elements are located within the first 100 bp. Analysis of the sequence that includes the first untranslated exon and first intron revealed that it could enhance the promoter activity. Experiments of transfection of constructs containing different fragments of this sequence showed that most of the enhancer activity was confined in the terminal 30-bp tract of the first intron, although it was not functioning in a myofibroblast cell line. DNA/protein binding assays and cotransfection experiments showed that the C/EBP-beta transcription factor was able to bind a recognition sequence in this 30-bp segment. We found a bi-allelic sequence polymorphism at +245 in the first intron, which did not show a significant functional effect, but is a useful tool for future association studies.

Research paper thumbnail of Germline MLH1 and MSH2 mutations in Italian pancreatic cancer patients with suspected Lynch syndrome