Intragenic Polymorphic Markers Research Articles

The cardiac beta-myosin heavy chain gene is not the predominant gene for hypertrophic cardiomyopathy in the Finnish population.

The aim of the study was to screen 36 unrelated patients with hypertrophic cardiomyopathy (HCM; 16 familial and 20 sporadic cases) from a genetically homogeneous area in eastern Finland for variants in the cardiac beta-myosin heavy chain (beta-MHC) and alpha-tropomyosin (alpha-TM) genes. Mutations in the beta-MHC and alpha-TM genes have been reported to be responsible for 30% to 40% and less than 5% of familial HCM cases, respectively. However, most genetic studies have included patients from tertiary care centers and are subject to referral bias. Exons 3-26 and 40 of the beta-MHC gene and the nine exons of the alpha-TM gene were screened with the PCR-SSCP (polymerase chain reaction-single strand conformation polymorphism) method. Linkage analyses between familial HCM locus and two intragenic polymorphic markers (MYO I and MYO II) of the beta-MHC gene were performed in 16 familial HCM kindreds. A previously reported Arg719Trp (arginine converted to tryptophan in codon 719) mutation of the beta-MHC gene was found in one proband and two relatives. In addition, a novel Asn696Ser (asparagine converted to serine in codon 696) substitution was found in one HCM patient. No linkage between familial HCM and the beta-MHC gene was observed in 16 familial kindreds. A previously reported Aspl75Asn (aspartic acid converted to asparagine in codon 175) mutation of the alpha-TM gene was found in four probands and 16 relatives. Mutations in the beta-MHC and alpha-TM genes accounted for 6% and 25% familial HCM cases and 3% and 11% of all cases, respectively. Our results indicate that the beta-MHC gene is not the predominant gene for HCM in the Finnish population, whereas HCM caused by the Aspl75Asn mutation of the a-TM gene is more common than previously reported.

Mapping of human interferon regulatory factor 3 (IRF3) to chromosome 19q13.3–13.4 by an intragenic polymorphic marker

We have identified a highly polymorphic intragenic marker composed of two dinucleotide repeats in an intron of the human interferon regulatory factor (IRF3) gene. This polymorphic marker has allowed us to map IRF3 to the boundary of 19q13.3-13.4 between the polymorphic markers D19S604 and D19S206 close to KLK1. An intron is present in a homologous position in the mouse Irf3 gene, but lacks the dinucleotide repeats present in the human intron. Syntenic conservation between this region of 19q13.3-13.4 maps Irf3 to either mouse chromosome 7 or 17.

Molecular analysis of the FHIT gene in human prostate cancer.

A variety of studies suggest that the FHIT gene, which encompasses the fragile site at 3p14.2, is a candidate tumor suppressor gene in several forms of human cancer. To determine whether the FHIT gene is altered in prostate carcinomas, we examined 15 prostate tumors, four normal prostate tissue specimens and RNA from a pool of 62 normal human prostate tissues (Clontech) for the integrity of FHIT transcripts, using a robust single-stage PCR and a nested PCR method. In each case a major FHIT-specific full-length product was observed. Additional aberrantly sized products, which were more numerous in the nested PCR strategy, were present at a far lower level than the full-length transcripts in 14 of 15 tumors, three of four normal human prostate tissues and in the pooled normal prostate RNA. Sequence analysis revealed that these aberrant products corresponded to alternatively spliced FHIT transcripts, which were neither more numerous nor more prominent in the tumors than in the normal prostate specimens. Deletion at the FHIT locus was also evaluated by using three intragenic polymorphic markers (D13S1481, D3S1300, and D3S1234). Allelic loss was observed in two tumors, but these genomic alterations did not correspond to the aberrant FHIT transcripts. DNA analysis, furthermore, suggested that the tumor heterogeneity was not a likely explanation for presence of normal and alternatively spliced FHIT transcripts in the prostate tumors. In conclusion, we detected neither frequent loss of heterozygosity nor tumor specific transcripts of the FHIT gene in human prostate cancer.

Abnormalities of fragile histidine triad genomic and complementary DNAs in cervical cancer: association with human papillomavirus type.

Chromosome 3p14.2 contains FRA3B, the most active chromosome breakage site in the human genome. The fragile histidine triad (FHIT) gene, a putative tumor suppressor gene, overlaps FRA3B. Human papillomavirus (HPV), a known cofactor in cervical carcinogenesis, can integrate into FRA3B. We examined abnormalities in FHIT and its RNA transcripts in cervical cancer cell lines and tumors. We also investigated the relationship between loss of heterozygosity (LOH) in FHIT/FRA3B and the presence of oncogenic HPV types. Eleven cell lines, 40 tumors (20 fresh and 20 archival), and 10 normal cervical epithelia were examined. Two intragenic polymorphic markers (D3S1300 and D3S4103) and the polymerase chain reaction (PCR) were used to examine FHIT LOH. Reverse transcription-PCR (RT-PCR) analysis and single-strand conformation polymorphism analysis of RT-PCR products were used to characterize FHIT transcripts. Oncogenic HPV types were identified by PCR, using general and type-specific primers. All normal epithelia, 19 of 20 fresh tumors and nine of 11 cell lines expressed wild-type and, occasionally, exon 8-deleted FHIT transcripts. Additional aberrant FHIT transcripts were seen in nine of 20 fresh tumors and in seven of 11 cell lines. DNA sequencing of the aberrant transcripts revealed a variety of insertions and deletions but no point mutations. Three cell lines also had homozygous FHIT deletions. Oncogenic HPV types (i.e., 16, 18, 31, and 33) were detected in 18 of 20 archival tumors, and, in these tumors, LOH within FHIT was identified in nine of 16 informative cases. HPV 16 was found to be associated with LOH in the FHIT/FRA3B region (P = .041). FHIT/FRA3B is frequently altered in cervical cancer, demonstrating LOH, occasional homozygous deletions, and frequent aberrant transcripts not found in normal epithelia. However, the presence of wild-type transcripts and the lack of protein-altering point mutations raise questions about FHIT's function as a classic tumor suppressor gene in cervical tissue.

Prenatal and presymptomatic diagnosis of Marfan syndrome using fluorescence PCR and an automated sequencer.

The Marfan syndrome (MFS) is an autosomal dominant heritable connective tissue disorder characterized by variable and pleiotropic manifestations primarily in the skeletal, ocular, and cardiovascular systems (). Molecular defects in fibrillin-1, a 350 kDa glycoprotein, encoded by the FBN1 gene located on chromosome 15 are now well documented in individuals and families with MFS (see ref. for a review). The variability in phenotypic expression of MFS is significant not only between families, but also within a family, while the effective treatment of patients with MFS relies heavily on early and accurate diagnosis. It, therefore, seems likely that prenatal and presymptomatic molecular genetic analysis would prove a valuable adjunct to clinical diagnosis for purposes of risk assessment (, , ). Mutations have been found along the entire 10 kb coding region of fibrillin and in most of its 65 exons. In addition, most families appear to have unique mutations. Therefore, the routine use of mutation screening for prenatal and presymptomatic diagnosis is impractical in this disorder. We have used intragenic polymorphic markers in FBN-1 () to perform linkage analyses in MFS families. We have adapted a “one-step” or “two-step” fluorescence PCR and automated sequencer to accurately, reproducibly, and rapidly perform prenatal or presymptomatic diagnosis of MFS in informative families.

Resistance to thyrotropin (TSH) in three families is not associated with mutations in the TSH receptor or TSH.

Resistance to TSH (RTSH) is a recently described syndrome of reduced sensitivity to TSH that manifests as euthyroid hyperthyrotropinemia. It is usually identified at birth during routine neonatal screening for congenital hypothyroidism. In less than 2 yr, 13 subjects with RTSH belonging to 8 families have been reported, and all were shown to harbor mutations in the TSH receptor (TSHR) gene. We now report the occurrence of RTSH in 3 unrelated families. Contrary to previous reports, the inheritance of RTSH in 2 of the families was dominant rather than recessive and was not associated with abnormalities in the TSHR gene. Abnormalities in the TSHR gene were excluded by sequencing all coding sequences, exon/intron junctions, and the promoter region of the gene. Furthermore, the involvement of the TSHR in the manifestation of the RTSH phenotype was excluded in 2 families by linkage analysis using intragenic polymorphic markers. We excluded defects in the TSH beta-subunit by sequencing its gene and by showing that the circulating TSH in affected subjects from all families had normal bioactivity. Also, no abnormalities were found in the Gs alpha gene of one family analyzed by GC-clamped denaturing gradient gel electrophoresis. This study shows that RTSH may be a manifestation of several different genetic defects that requires the exploration of other candidate genes involved in the TSH-TSHR-Gs alpha cascade and genes participating in its regulation.

Allelic imbalance at the beta-catenin gene (CTNNB1 at 3p22-21.3) in various human tumor types

beta-catenin is a multifunctional protein: it plays a central role in the cell-cell adhesive junctions, and participates in transduction of the morphogenic Wingless/Wnt-signal. Upon detailed analysis of the human beta-catenin gene, an intragenic polymorphic microsatellite marker could be identified. This marker shows 62% heterozygosity and was used in a study of eleven different tumor types. A high level of beta-catenin allelic imbalance was observed for small cell lung carcinoma, squamous cell lung carcinoma and cervix carcinoma. Other microsatellite markers on 3p24-21 could demonstrate frequent but not invariable codeletion of flanking chromosomal loci. This intragenic polymorphic marker will allow selection of tumor types and tumor samples possibly bearing recessive mutations in the remaining allele of the beta-catenin gene.

Enhanced apoptotic cell death of renal epithelial cells in mice lacking transcription factor AP-2beta.

Expression of AP-2 transcription factors has been detected previously in embryonic renal tissues. We show here that AP-2beta -/- mice complete embryonic development and die at postnatal days 1 and 2 because of polycystic kidney disease. Analyses of kidney development revealed that induction of epithelial conversion, mesenchyme condensation, and further glomerular and tubular differentiation occur normally in AP-2beta-deficient mice. At the end of embryonic development expression of bcl-X(L), bcl-w, and bcl-2 is down-regulated in parallel to massive apoptotic death of collecting duct and distal tubular epithelia. Addressing the molecular mechanism we show that transfection of AP-2 into cell lines in vitro strongly suppresses c-myc-induced apoptosis pointing to a function of AP-2 in programming cell survival during embryogenesis. The position of the human AP-2beta gene was identified at chromosome 6p12-p21.1, within a region that has been mapped for autosomal recessive polycystic kidney disease (ARPKD). Sequence analyses of ARPKD patients and linkage analyses using intragenic polymorphic markers indicate that the AP-2beta gene is located in close proximity to but distinct from the ARPKD gene.

Malignant-Hyperthermia Susceptibility Is Associated with a Mutation of the a1-Subunit of the Human Dihydropyridine-Sensitive L-Type Voltage-Dependent Calcium-Channel Receptor in Skeletal Muscle

Malignant hyperthermia susceptibility (MHS) is characterized by genetic heterogeneity. However, except for the MHS1 locus, which corresponds to the skeletal muscle ryanodine receptor (RYR1) and for which several mutations have been described, no direct molecular evidence for a mutation in another gene has been reported so far. In this study we show that the CACNL1A3 gene encoding the alpha 1-subunit of the human skeletal muscle dihydropyridine-sensitive L-type voltage-dependent calcium channel (VDCC) represents a new MHS locus and is responsible for the disease in a large French family. Linkage analysis performed with an intragenic polymorphic microsatellite marker of the CACLN1A3 gene generated a two-point LOD score of 4.38 at a recombinant fraction of 0. Sequence analysis of the coding region of the CACLN1A3 gene showed the presence of an Arg-His substitution at residue 1086, resulting from the transition of A for G3333, which segregates perfectly with the MHS phenotype in the family. The mutation is localized in a very different part of the alpha 1-subunit of the human skeletal muscle VDCC, compared with previously reported mutations found in patients with hypokalemic periodic paralysis, and these two diseases might be discussed in terms of allelic diseases. This report is the first direct evidence that the skeletal muscle VDCC is involved in MHS, and it suggests a direct interaction between the skeletal muscle VDCC and the ryanodine receptor in the skeletal muscle sarcoplasmic reticulum.

Molecular screening of families affected by familial adenomatous polyposis (FAP).

To assess the risk of developing familial adenomatous polyposis (FAP) in presymptomatic individuals using APC gene flanking and intragenic polymorphic markers. Twenty families enrolled in the Italian Registry of Polyposis comprising a total of 217 individuals, including 53 (24%) presymptomatic subjects with a 50% a priori risk of FAP, were analysed. Direct analysis techniques had previously failed to identify the FAP mutation in these families. DNA isolated from peripheral mononuclear blood cells and tissue sections was analysed by the polymerase chain reaction and a panel of seven highly polymorphic markers--YN5.64, CB83, CB26, LNS, APC1458.5, MBC, 37AB. Amplification products were separated by a modified denaturing gel electrophoresis method. The haplotype associated with the disease was identified in 18 families (90%). The segregation of the FAP haplotype in these kindreds showed that 10 presymptomatic individuals had inherited the FAP mutation and carried a high risk of developing the disease. The remaining two families were not informative because of the lack of a sufficient number of probands or biological specimens. These data indicate that indirect analysis with linked DNA markers has a high rate of success in defining the risk of FAP of presymptomatic subjects, provided that a sufficient number of probands or samples is available. Uninformative families accounted for 10% of the total, indicating that linkage analysis may still have higher sensitivity than direct mutation analysis techniques. The combined use of both approaches should be implemented, however, to enhance further the application of molecular genetics to the screening of families with FAP.

The 1591C Mutation in Triosephosphate Isomerase (TPI) Deficiency. Tightly Linked Polymorphisms and a Common Haplotype in All Known Families

ABSTRACT In order to investigate the basis of the repeated occurrence of the 1591C mutation (TPI 1591C, 105 Glu–Asp) in multiple unrelated families throughout the world, we studied five microsatellite and short tandem repeat markers that lie within a 1.77 megabase region which includes the TPI gene. We also studied an intragenic polymorphic marker that lies within intron 5 of the TPI gene. This polymorphism, recently described by others, is characterized by either an A or a G at position 2262 (the A in the initiation ATG is designated as +1 for both genomic and cDNA nucleotides). With very minor exceptions, all of the known families in the world with the 1591C mutation were available for study. These included five families from the U.S., three from France, one from Greece, one (of Turkish origin) from Germany, and two from Australia. Although we did not have the opportunity to directly study five families from the U.K., key data concerning the 2262 intragenic polymorphism in these subjects were made available to us. Four of the microsatellite and short tandem repeat markers were linked, but in apparent equilibrium. In contrast, a polymorphic repeat pentamer in the CD4 gene, thought to lie telomeric to TPI, was in apparent complete linkage disequilibrium with the TPI 1591C mutation. The intragenic polymorphism was also in apparent complete linkage disequilibrium with the mutation. In unrelated persons of known phase (1591C homozygotes or normal controls), the comparative allele frequencies for the CD4 pentameric repeat were 1.0 (14/14 alleles) in homozygous TPI 1591C subjects and 0.412 (28/68 alleles) in normal subjects (p < 0.0001). Again, in persons of known phase, the comparative allele frequencies for the A form of the intragenic 2262 A or G polymorphism were 1.0 (14/14 alleles) in 1591C homozygotes and 0.130 (7/54 alleles) in normals (p < 0.0001). Haplotypes were discernible in all of the 1591C homozygotes and in several of the heterozygotes and normals. The CD4 162, TPI 2262A haplotype was found on only two of thirty-eight normal chromosomes, but was universally associated with 1591C. The data indicate that all TPI 1591C subjects are descendants of a common ancestor who probably lived in what is now England or France. The original mutation probably occurred well in excess of 1000 years ago.

Linkage disequilibrium between four intragenic polymorphic microsatellites of the NF1 gene and its implications for genetic counselling.

Four intragenic polymorphic microsatellite markers, AAAT Alu repeat, IVS27AC28.4, ACI27.2, and IVS38GT53.0, located along a 65 kb DNA region of the NF1 gene, were used to genotype 64 Spanish families with neurofibromatosis type 1 (NF1). Linkage disequilirium between each pair of markers was evaluated. Three of these markers, AAAT Alu repeat, ACI27.2, and IVS38GT53.0, exhibit linkage disequilibrium between each other. Analysis of extended haplotypes provides further evidence of the disequilibrium within this region since only 11 haplotypes account for 52% of the total chromosomes. Because of linkage disequilibrium, the informativeness of marker combinations for genotyping of NF1 families is diminished. There was no difference in the overall distribution of alleles between affected and normal chromosomes. An at risk haplotype was not found, as expected for a disease with at least 50% of cases being sporadic.

Deletion in the peripherin/RDS gene in two unrelated Sardinian families with autosomal dominant butterfly-shaped macular dystrophy.

Autosomal dominant butterfly-shaped macular dystrophy is associated with different mutations of the peripherin/RDS gene. We studied the phenotype of two families with a novel large deletion in the peripherin/RDS gene. Clinical study, fluorescein angiography, color vision testing, automatic perimetry, electrophysiologic studies, and DNA analysis were performed on all the members of the two families. Fundus examination in patients aged 30 to 60 years showed yellow deposits in the macula with a butterfly-shaped pattern. Central choroidal atrophy was present in the older patients only. Macular visual function tests (color vision and central visual field) were abnormal, and electro-oculograms were slightly subnormal in five individuals tested. Electroretinograms and results of dark adaptometry were normal. Linkage analysis with intragenic polymorphic markers and quantitative polymerase chain reaction showed heterozygosity for a large deletion that removed exons 2 and 3 of the peripherin/RDS gene in all affected members of two families. This deletion escaped detection by direct analysis of amplified exons and was identified by intragenic polymorphic markers analysis, resulting in loss of heterozygosity from affected parents to affected children, and by quantitative polymerase chain reaction. The delineation of the molecular defect associated with the disease in these two families allows us to verify the presence or absence of the disease in clinically unaffected members.

Clinical Use of Molecular Genetic Studies in Retinoblastoma

With a population incidence of about 1 in 15.000 retinoblastoma is the most frequent intraocular tumor in infancy and early childhood. It occurs in a hereditary form due to a germline mutation in about 40% of patients (30% de novo mutation and 10% transmission from an affected parent) and in a non-hereditary form due to a somatic mutation. The retinoblastoma gene is located on chromosome 13q14. This large gene of about 180 kb, consists of 27 exons of rather different sizes and encodes a 4.7 kb transcript with important function in cell cycle regulation. Individuals with bilateral, multifocal tumors are assumed to carry a germline mutation, whereas unilateral and unifocal tumors are generally due to the somatic form. Both copies of the RB1 gene must be in inactivated before a tumor develops. In about half of patients with the germline mutation the second event inactivating the second allele can be shown by loss of heterozygosity in tumor tissues compared to surrounding somatic tissues.Knowledge of the RB1 gene locus affords an opportunity to specify the type of mutation in many patients and arrive at a definitive molecular diagnosis. This is the basis for clinical evaluation and genetic counseling. The types of mutation are large scale deletions, small deletions and insertions, and base substitutions. There is no hot-spot for mutations.During the last several years we have studied more than 200 patients in search for large scale and small deletions and insertions, and missense mutations. Using intragenic polymorphic DNA markers we were able to identify the mutant haplotype in all familial cases. Direct DNA analysis identified a mutation in about 25% of patients. The distribution of lesions will be described in relation to the clinical situation.

Single-cell PCR performed with neurofibroma Schwann cells reveals the presence of both alleles of the neurofibromatosis type 1 (NF1) gene

It is commonly held that Schwann cells (SC) are the progenitor cells of benign neurofibromas. To test for loss of heterozygosity (LOH) at the neurofibromatosis 1 (NF1) gene locus, three intragenic polymorphic markers were analyzed after polymerase chain reaction amplification, starting from 98 single SC isolated from primary cultures of neurofibromas, of five informative NF1 patients. The patterns obtained did not provide evidence for LOH at the NF1 gene. LOH by nondisjunction, large deletions, or somatic recombination in SC seems not to be the mechanism of generation of neurofibromas.

Applications cliniques du diagnostic moléculaire du rétinoblastome dans 15 families

In 40% retinoblastoma (Rb) results from a hereditary mutation of the Rb susceptibility gene (RB1). In this study, we tested the usefulness of intragenic DNA analysis for ophthalmologic follow-up in affected families. Molecular analysis was performed on 103 DNA samples of 15 Rb families. We used 7 intragenic polymorphic markers and one within the ESD gene for mutation linkage analysis. DNA analysis was informative in 88% of relatives at risk of developing Rb. Among them, the presence of a mutated RB1 allele was excluded in 46%, while 29% were unaffected carriers and 25% had inherited the Rb predisposition. In the majority of familial Rb, the DNA analysis allows the identification of children carrying a RB1 mutation and who will need a close ophthalmologic follow-up under general anesthesia. When the mutated gene is absent, ophthalmological examination under narcosis is unnecessary. Finally, identification of asymptomatic carriers improve the accuracy of genetic counselling.

Molecular genetics aspects of factor XI deficiency and Glanzmann thrombasthenia.

Factor XI deficiency and Glanzmann thrombasthenia are among the hereditary disorders frequently encountered in Israel. Factor XI deficiency is particularly frequent in Ashkenazi (European) Jews with 1:190 individuals affected by the severe deficiency and 8.1% of the population being heterozygotes. So far 4 mutations causing factor XI deficiency have been identified of which the type II (a non-sense mutation) and type III (a missense mutation) are predominant and type I and IV observed only in 5 families. Recently, the type II mutation was observed in Iraqui-Jews as well with 3.7% of 400 unrelated subjects being heterozygotes and with the type III mutation completely absent. Since Iraqui-Jews represent the original gene pool of Jews who lived in Babylon 2500 years ago we hypothesize that the type II mutation is ancient and that the type III mutation occurred more recently, after the divergence of the original Babylonian Jews into Ashkenazi, Sephardic (Spanish) and Middle Eastern Jews. Preliminary data on factor XI intragenic polymorphic markers indeed indicate that type II and type III mutations reside on chromosomes each characterized by a different specific haplotype. Fifty living patients with type I Glanzmann thrombasthenia (28 families) have been observed in Israel. Most of them are Iraqui-Jewish and the rest are Arabs (5 families) and one Iranian Jewish. All Iraqui-Jewish patients have an IIbp deletion within exon 12 of the glycoprotein (GP) IIIa resulting in a shift of the reading frame that leads to premature termination of the GPIIIa synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular biology and pathology of type VII collagen.

Type VII collagen is a genetically distinct member of the collagen family of proteins. Type VII collagen has been shown to be the major component of anchoring fibrils, attachment complexes which secure the cutaneous basement membrane of the skin to the underlying dermis. Understanding of the structure of type VII collagen has been advanced by recent cloning of the corresponding gene. Chromosomal mapping of the gene to the short arm of chromosome 3 and identification of intragenic polymorphic markers have allowed demonstration of strong genetic linkage between the type VII collagen locus and the dystrophic forms of EB (epidermolysis bullosa). This overview summarizes the progress made in the molecular genetics of type VII collagen.