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P Fortina

Publications and source records attributed to P Fortina.

At least 19 recordsLinked to original sources
PubMed1995

Infundibulopelvic stenosis, multicystic kidney, and calyectasis in a kindred: clinical observations and genetic analysis.

Congenital obstructive anomalies of the urinary tract usually occur sporadically. We describe inheritance in a three-generation kindred of a spectrum of kidney anomalies consistent with an autosomal-dominant mode of transmission, with incomplete penetrance, calyectasis (maternal grandmother), infundibulopelvic stenosis (uncle), and multicystic kidney (male proband, age 4 years). The proband's mother, father and half sister had normal renal imaging studies. Inheritance of informative polymorphic markers (3'-HVR, GGG1, GGG9, SM-7, KG8, and CW3) mapping close to the adult polycystic kidney disease type 1 (PKD-1) and tuberous sclerosis (TSC-2) loci on chromosome 16p was evaluated by Southern blot studies and by PCR-based, fluorescent genotyping for linkage to phenotype. The 3 affected individuals, as well as the unaffected mother (obligate carrier) and unaffected half-sister, inherit a common chromosome haplotype linked to the PKD1 locus. Our findings support the hypothesis that these anomalies may be part of a spectrum of obstructive renal dysplasia which are inherited as a simple Mendelian trait exhibiting an autosomal-dominant mode of transmission with variable expression and incomplete penetrance.

Adult↗
PubMed1995

Nucleic acid detection using non-radioactive labelling methods.

Nucleic acid probe-based assays are now widely used in genetic research, human identification, forensics and in a broad spectrum of clinical assays in the fields of microbiology, haematology/oncology and virology. Labelled probes are used in a variety of assay formats including dot-blots, Southern blots (DNA target), Northern blots (RNA target), Western blots (protein target), in situ hybridization, plaque or colony screening and immobilized arrays on silicon or glass surfaces. Traditionally, the probes used in these assays have a radioactive 32phosphorous label that has a short shelf-life, is dangerous, has high disposal costs and, when labelled to high specific-activity, may be unstable. Extensive efforts to develop alternative labelling techniques have resulted in colorimetric, chemiluminescent and fluorescent assay formats. This review summarizes the properties desired in a probe, describes the advantages and disadvantages of the different non-radioactive labelling strategies, and illustrates examples of probe-based assays in which detection is facilitated by imaging samples using a general purpose fluorescence scanner.

Colorimetry↗
PubMed1995

Fluorescence-based DNA minisequence analysis for detection of known single-base changes in genomic DNA.

We describe a rapid, automated method for direct detection of known single-base changes in genomic DNA. Fluorescence-based DNA minisequence analysis is employed in a template-dependent reaction which involves a single nucleotide extension of an oligonucleotide primer by the correct fluorescently-tagged dideoxynucleotide chain terminator. Detection following electrophoresis on denaturing acrylamide gels is facilitated by alkaline phosphatase treatment of reaction products after extension followed by isopropanol precipitation of the dye-tagged, single-base-extended primer to remove unincorporated deoxynucleotides. Fluorescence analysis of the incorporated dye tag reveals the identity of the template nucleotide immediately 3' to the primer site. This technique does not require radioactivity or biotinylated PCR product, relies on the incorporation of a single dideoxynucleotide terminator to extend the primer by one nucleotide and takes advantage of the sensitivity of fluorescent terminators developed for automated DNA sequence analysis. As a demonstration, we have applied the assay to human genomic DNA for detection of the sickle mutation in the beta-globin gene, and have also examined feasibility for simultaneous delineation using a multiplex-like strategy in a single gel-lane of some of the most common beta-thalassemia mutations in the Mediterranean basin.

Anemia, Sickle Cell↗
PubMed1995

Angiotensin converting enzyme gene deletion allele is independently and strongly associated with coronary atherosclerosis and myocardial infarction.

OBJECTIVE: To investigate the association of the three angiotensin converting enzyme (ACE) genotypes, DD, ID, and II, with the occurrence or absence of coronary atherosclerosis and with myocardial infarction and hypertension. DESIGN: Cohort analysis study. SETTING: North-Italy reference centre. SUBJECTS: 388 white Italian patients (281 males; mean age 60.7 (SD 12.5) years) with proven coronary atherosclerosis (n = 255) or with angiographically normal coronary arteries (n = 133). A further group of 290 healthy blood donors was tested for allele frequency comparison. INTERVENTIONS: ACE/ID polymorphism was analysed with polymerase chain reaction on DNA from white blood cells. MAIN OUTCOME MEASURES: Coronary atherosclerosis, myocardial infarction, hypertension. RESULTS: The D and I allele frequencies were respectively 0.63 and 0.37 in the overall healthy blood donor group and 0.66 and 0.34 in the overall study group. In the latter, univariate analysis showed (1) that coronary atherosclerosis (255 patients) was associated with the deletion allele, with an odds ratio (OR) of 5.78 for DD/II, P < 0.001, and 2.39 for ID/II, P = 0.006; and (2) that myocardial infarction (154 patients) was associated with the DD genotype (OR DD/II = 2.56, P = 0.007), but not with the ID genotype (OR DD/II = 1.96, P = 0.056). Finally, hypertension proved to be unrelated with the ACE genotype. The distribution between the three genotypes of known risk factors for coronary artery disease was similar. Logistic regression modelling, performed to test the association of the selected risk factors simultaneously with coronary atherosclerosis and myocardial infarction, showed that the deletion allele (whether DD or ID) was the strongest risk factor for atherosclerosis, and that the D allele was significantly associated with the risk of infarction (although to a lesser extent than with coronary atherosclerosis). CONCLUSION: ACE deletion polymorphism is strongly and independently associated with coronary atherosclerosis and, to a lesser extent, with myocardial infarction. As such, the results are analogous to what has already been reported in French white, Japanese, and Welsh coronary patients.

Adolescent↗
PubMed1994

Interaction of rare illegitimate recombination event and a poly A addition site mutation resulting in a severe form of alpha thalassemia.

The clinical diversity of thalassemia depends on interaction of diverse genetic defects. We have characterized a severe form of alpha thalassemia caused by coinheritance of a rare alpha-globin gene deletion and a nondeletional defect in a southern Italian family. The proband, a 7-year-old girl, exhibited an abnormal hemoglobin electrophoresis pattern with hemoglobin H and hemoglobin Barts, indicating inheritance of H and hemoglobin Barts, indicating inheritance of a severe form of alpha thalassemia. Southern blot analysis of DNA showed normal as well as aberrant alpha-globin gene fragments indicating heterozygosity for a deletional form of alpha thalassemia in the proband and her mother. The coinheritance of a nondeletional form of alpha thalassemia (alpha alpha T) was suspected because of the severity of the proband's phenotype and the presence of normal alpha-globin gene fragments in the father. Selective polymerase chain reaction of the paternal alpha 1- and alpha 2-globin genes in the proband followed by DNA sequence analysis showed an AATAAA to AATGAA mutation in the polyadenylation signal sequence of the alpha 2-globin gene. Genomic DNA mapping and sequence analysis of a unique polymerase chain reaction product generated across the deletion breakpoint of the maternal allele showed a 5,201-bp deletion extending from 870 nucleotides 5' of the alpha 2-globin gene to nucleotide +519 in the alpha 1-globin gene. This deletion is similar to that previously suggested by blotting studies in a Greek family (Pressley et al, Nucleic Acids Res 8:4889, 1980) and removes the entire alpha 2-globin gene and a portion of the 5' end of the alpha 1-globin gene. Sequence characterization of the resultant aberrant truncated alpha 1-globin gene from the proband showed a 27 nucleotide duplication corresponding to the 3' end of the alpha-globin gene IVS-2 region separated by the insertion of a tetranucleotide (GGTT), suggesting that this deletion is caused by an illegitimate recombination event.

Base Sequence↗
PubMed1994

Rapid detection of medium chain acyl-CoA dehydrogenase gene mutations by non-radioactive, single strand conformation polymorphism minigels.

Medium chain acyl-CoA dehydrogenase (MCAD) deficiency is a common inherited metabolic disorder affecting fatty acid beta oxidation. Identification of carriers is important since the disease can be fatal and is readily treatable once diagnosed. Twelve molecular defects have been identified in the MCAD gene; however, a single highly prevalent mutation, A985G, accounts for > 90% of mutant alleles in the white population. In order to facilitate the molecular diagnosis of MCAD deficiency, oligonucleotide primers were designed to amplify the exon regions encompassing the 12 mutations enzymatically, and PCR products were then screened with a single strand conformation polymorphism (SSCP) based method. Minigels were used allowing much faster run times, and silver staining was used after gel electrophoresis to eliminate the need for radioisotopic labelling strategies. Our non-radioactive, minigel SSCP approach showed that normals can be readily distinguished from heterozygotes and homozygotes for all three of the 12 known MCAD mutations which were detected in our sampling of 48 persons. In addition, each band pattern is characteristic for a specific mutation, including those mapping in the same PCR product like A985G and T1124C. When necessary, the molecular defect was confirmed using either restriction enzyme digestion of PCR products or by direct DNA sequence analysis or both. This rapid, non-radioactive approach can become routine for molecular diagnosis of MCAD deficiency and other genetic disorders.

Acyl-CoA Dehydrogenase↗
PubMed1994

Glanzmann thrombasthenia secondary to a Gly273-->Asp mutation adjacent to the first calcium-binding domain of platelet glycoprotein IIb.

We studied the defect responsible for Glanzmann thrombasthenia in a patient whose platelets expressed < 5% of the normal amount of GPIIb-IIIa. Genetic and biochemical evidence indicated that the patient's GPIIIa genes were normal. However, DNA analysis revealed the patient homozygous for a G818-->A substitution in her GPIIb genes, resulting in a Gly273-->Asp substitution adjacent to the first GPIIb calcium-binding domain. To determine how this mutation impaired GPIIb-IIIa expression, recombinant GPIIb containing the mutation was coexpressed with GPIIIa in COS-1 cells. The GPIIb mutant formed stable GPIIb-IIIa heterodimers that were not immunoprecipitated by either of two heterodimer-specific monoclonal antibodies, indicating that the mutation disrupted the epitopes for these antibodies. Moreover, the GPIIb in the heterodimers was not cleaved into heavy and light chains, indicating that the heterodimers were not transported from the endoplasmic reticulum to the Golgi complex where GPIIb cleavage occurs, nor were the mutant heterodimers expressed on the cell surface. These studies demonstrate that a Gly273-->Asp mutation in GPIIb does not prevent the assembly of GPIIb-IIIa heterodimers, but alters the conformation of these heterodimers sufficiently to impair their intracellular transport. The impaired GPIIb-IIIa transport is responsible for the thrombasthenia in this patient.

Adenosine Diphosphate↗
PubMed1994

Cytologically balanced t(2;20) in a two-generation family with alagille syndrome: cytogenetic and molecular studies.

Alagille syndrome is a clinically defined, dominantly inherited disorder affecting the liver, heart, face, eye, and vertebrae. Alagille syndrome has previously been localized to the short arm of chromosome 20, on the basis of reports of a small number of patients with chromosomal deletions of 20p. We undertook a cytogenetic study of patients with Alagille syndrome and identified a family in which a cytologically balanced translocation between chromosomes 2 and 20, 46,XX/XY, t(2;20)(q21.3;p12), is segregating concordantly with the disease. The breakpoint on chromosome 20p in this t(2;20) is consistent with the shortest region of overlap demonstrated in the reported deletion patients. This is the first report of a translocation associated with 20p and Alagille syndrome, and this rearrangement confirms the location of the Alagille disease gene at 20p12. We have established a somatic cell hybrid from a lymphoblastoid cell line from one of the affected individuals that contains the derivative chromosome 20 (20qter-->p12::2q21.3-->qter) but not the derivative chromosome 2, the normal chromosome 2, or the normal chromosome 20. Southern blot and PCR analysis of probes and sequences from 20p have been studied to define the location of the translocation breakpoint. Our results show that the breakpoint lies distal to D20S61 and D20S56 within band 20p12.

Alagille Syndrome↗
PubMed1993

Duchenne/Becker muscular dystrophy carrier detection using quantitative PCR and fluorescence-based strategies.

Dystrophin gene deletions account for up to 68% of all Duchenne (DMD) and Becker (BMD) muscular dystrophy mutations. In affected males, these deletions can be detected easily using multiplex PCR tests which monitor for exon presence. In addition, quantitative dosage screening can discriminate female carriers. We previously analyzed multiplex PCR products by gel electrophoresis and quantitation of fluorescently labeled primers with the Gene Scanner in order to test carrier status. These multiplex PCR protocols detect DMD gene deletions adequately, but require up to 18 pairs of fluorochrome-labeled primers. We previously described two alternative fluorescent labeling strategies, each with approximately 1,000-fold greater sensitivity than ethidium bromide staining, which can be used to quantify the products of multiplex PCR. The first method uses the DNA intercalating thiazole orange dye TOTO-1 to stain PCR products after 20 cycles. In the second method, fluorescein-12,2'-dUTP is incorporated into products during PCR as a fluorescent tag for subsequent quantitative dosage studies. Both methods label all multiplexed exons including the 506 bp exon 48 fragment that is difficult to detect and quantify by standard ethidium bromide staining. Using this approach, we determined DMD/BMD carrier status in 24 unrelated families using a fluorescent fragment analyzer. Analysis of fluorochrome-labeled PCR products facilitates quantitative multiplex PCR for gene-dosage analysis.

Deoxyuracil Nucleotides↗
PubMed1993

Fluorescent approaches to diagnosis of Lesch-Nyhan syndrome and quantitative analysis of carrier status.

Lesch-Nyhan syndrome is an X-linked recessive disorder caused by molecular defects within the HPRT gene. Deletional forms of this syndrome, most of which are inherited, account for 15% of the cases. In addition, a large percentage of cases are due to de novo point mutations. We have used complementary fluorescence-based PCR assays to analyse disease-causing mutations in three unrelated families: (1) inheritance of dye-labelled PCR products of linked polymorphic loci mapping within and flanking the HPRT gene; (2) dye-labelled exon dosage analysis and (3) automated fluorescence-based DNA sequence analysis. Our results using fluorescent, dye-tagged PCR products show that inheritance of two polymorphic small tandem repeats, HPRTB [AGAT]n, mapping within intron 3 of the HPRT gene, and the CA-repeat at DXS294 can be used to establish linkage to the disease. In addition, we modified a previously described PCR protocol to use fluorescent dye-labelled oligoprimers and an ABI Gene Scanner in order to rapidly quantitate deletional forms of Lesch-Nyhan syndrome. Quantitative PCR analysis of individual exons followed by dosage analysis confirmed a deletion encompassing exon 9. A similar approach was used to confirm a previously described HPRT gene duplication involving exons 2 and 3. In this analysis, we co-amplified the HPRTB [AGAT]n and HUMARA [AGC]n repeats and confirmed increased exon dosage in carriers for the duplication. DNA sequence analysis remains the method of choice for delineating new disease-causing mutations, most of which are non-deletional forms of Lesch-Nyhan syndrome. We have also used a cycle-sequencing strategy employing dye-labelled dideoxy terminators and a laser-activated, fluorescence-emission DNA sequencer in order to define carrier status in 10 family members at risk for Lesch-Nyhan syndrome due to a splice donor mutation in intron 7. Our DNA sequence analyses corroborate small tandem repeat (STR) inheritance patterns in this family. Multiple fluorescence-based strategies should facilitate rapid diagnosis of the various Lesch-Nyhan disease-causing mutations.

Adolescent↗
PubMed1992

Non-radioactive detection of the most common mutations in the cystic fibrosis transmembrane conductance regulator gene by multiplex allele-specific polymerase chain reaction.

A rapid, simple, nonradioactive method for detection of four common mutations causing cystic fibrosis (CF) has been developed combining multiplexing with allele-specific polymerase chain reaction amplification. This approach (MASPCR) provides an easy assay for direct genotyping of normal and mutant CF alleles in homozygotes and heterozygotes. The strategy involves multiplex PCR of exons 10, 11, and 21 within the cystic fibrosis transmembrane conductance regulator (CFTR) gene in a single reaction containing three common oligoprimers and either the four normal or four mutant oligos corresponding to the delta F508, G551D, G542X, and N1303K mutations. Primers are chosen so that the size of the four PCR products differ, thereby facilitating detection on agarose gels following amplification in the same reaction. Patient samples are primed with either four normal or four mutant oligo mixtures, and PCR products run in parallel on gels to detect band presence or absence. This approach provides a simple and potentially automated method for cost-effective population screening.

Alleles↗
PubMed1992

Fluorescence-based, multiplex allele-specific PCR (MASPCR) detection of the delta F508 deletion in the cystic fibrosis transmembrane conductance regulator (CFTR) gene.

Cystic fibrosis (CF) is a common genetic disorder in Caucasians, and in some populations 70% of cases are associated with a 3 base pair (bp) deletion (delta F508) in the CFTR gene. We have implemented a fluorescence-based, multiplex allele-specific polymerase chain reaction (MASPCR) assay for deletion of the delta F508 mutation. Different allele-specific fluorescently-tagged primers are used in the PCR reaction to distinguish between normal and delta F508 alleles. Fluorescent PCR products are then visualized in a single lane on an agarose gel following electrophoresis combined with real-time multicolour fluorescence detection. The approach simplifies diagnosis of the most common mutation in the CFTR gene, and holds promise for a multiplex allele-specific, fluorescence-tagged gene amplification strategy for detection of additional CF mutations which may result in more cost-effective testing without increasing the risk of missed or erroneous diagnoses.

Alleles↗
PubMed1992

Detection of the most common mutations causing beta-thalassemia in Mediterraneans using a multiplex amplification refractory mutation system (MARMS).

A rapid, simple, cost-effective, non-radioactive method for detection of the most common mutations causing beta-thalassemia in Mediterranean people has been developed by combining multiplexing with the amplification refractory system. This approach, the multiplex amplification refractory mutation system (MARMS), provides an easy assay for direct detection of normal and mutant beta-globin genes in homozygotes and heterozygotes. The strategy involves multiplex PCR of four of the five regions of interest within the beta-globin gene in a single reaction containing a common oligoprimer and either the normal or mutant oligonucleotides corresponding to IVS-1 nucleotide 1 or IVS-1 nucleotide 6, IVS-1 nucleotide 110, codon 39, and IVS-2 nucleotide 1 regions. Primers are chosen so that the sizes of the four PCR products differ, thereby facilitating detection on agarose gels following amplification. Patient samples are primed with either four normal or four mutant oligonucleotide mixtures and the common oligoprimer, and PCR products run in parallel on gels to detect band presence or absence. This approach simplifies mutation detection and shows promise for automation employing fluorescent-tagged primers.

Alleles↗
PubMed1991

A newly-characterized alpha-thalassaemia-1 deletion removes the entire alpha-like globin gene cluster in an Italian family.

We describe a new deletional form of alpha thalassaemia which encompasses the entire alpha-like globin gene cluster in a 15-year-old boy of Southern Italian descent. The deletion removes approximately 31 kb, the 5'-end point is located approximately 4 kb upstream of the xi gene, while the 3'-end point maps between the alpha 1- and theta 1-globin genes. The interaction of this deletion with the common-alpha 3.7 form gives origin to a classical form of haemoglobin (Hb) H disease in the propositus of this study. Deletional forms of xi alpha-thalassaemia are uncommon in the Mediterranean basin; as for other unusual xi alpha-thalassaemia forms, heterozygotes for this mutation may escape detection in population surveys based on zeta and alpha probes.

Adolescent↗
PubMed1991

A greater than 200 kb deletion removing the entire beta-like globin gene cluster in a family of Irish descent.

We describe a new deletional form of gamma delta beta-thalassemia segregating in two generations of a family of Irish descent. Affected family members present with a beta-thalassemia minor phenotype, normal Hb A2 and Hb F levels. Genomic blotting analyses on DNA from affected family members show heterozygosity for a large deletion beginning at least 15 kb upstream of the 5' endpoint of the gamma delta beta-thalassemia-1 deletion, extending through the entire beta-like globin gene cluster, and continuing for at least 10 kb beyond the 3' endpoint of the deletion associated with the Spanish form of delta beta 0-thalassemia. This deletion is among the largest described so far, and removes at least 205 kb encompassing the entire beta-like globin gene cluster on chromosome 11.

Adult↗
PubMed1989

RFLPs of the phenylalanine hydroxylase gene in the Italian population.

Different mutations of the phenylalanine hydroxylase (PAH) gene leading to phenylketonuria (PKU) have been described associated with specific haplotypes in several European countries. In order to investigate the distribution of DNA haplotypes in Italy, restriction fragment length polymorphism (RFLP) analysis of the PAH gene was performed in nine Italian PKU patients from eight unrelated families, and in the available relatives. The analysis of eight polymorphic sites revealed haplotypes 1 and 6 in association with PKU. This pattern appears to differ from those reported for other European populations. The majority of the 14 PKU subjects studied showed compound heterozygosity for different haplotypes, as observed for other European series. RFLP analysis at the PAH locus allowed us to offer the possibility of prenatal diagnosis to six of the studied families. One prenatal diagnosis was performed and a normal fetus was diagnosed.

Alleles↗
PubMed1988

A large deletion encompassing the entire alpha-like globin gene cluster in a family of northern European extraction.

We describe a new deletional form of alpha thalassemia segregating in three generations of a family of northern European origin. A full-term female girl had hypochromic, microcytic anemia since early infancy associated with delayed language development, slow growth and weight gain. Hematologic studies suggested the presence of alpha thalassemia. Gene-blotting studies showed no abnormal alpha-like globin gene fragments; however, studies of inheritance of informative polymorphic restriction fragments using zeta, alpha and 3'-alpha-hypervariable region (3'-HVR) probes showed evidence for an extensive deletion encompassing the entire alpha-like globin gene cluster. The 3' breakpoint of this deletion maps beyond the 3'-HVR, a region implicated as a hot spot for the generation of other large deletional events within the alpha-like cluster. The 5' breakpoint maps at least 10 kilobases (kb) 5' to the zeta-globin gene. The minimum size estimate for this deletion is greater than 47 kilobases.

Child, Preschool↗