Intragenic Restriction Fragment Length Polymorphisms Research Articles

Screening for Hemophilia A Carriers: Utility of PCR-RFLP—Based Polymorphism Analysis

Health schemes are promoting application of molecular diagnosis and screening in peripheral diagnostics labs. Intragenic restriction fragment length polymorphisms in the intron 18 (BclI), intron 19 (HindIII), and intron 22 (XbaI) of the Factor VIII gene were studied in 100 patients with hemophilia A and their relatives at risk from different regions of north India. For Bcl I, HindIII, and XbaI, the positive allele frequency was 0.57, 0.38, and 0.43, respectively, and heterozygosity was 0.54, 0.49, 0.41, respectively, whereas the heterozygosity in terms of informativity of the above markers was 53% for BclI, 44% for HindIII, and 34% for XbaI. Combined informativity of these markers was 77%. Review of Indian and world literature shows a marked variation in the informativity of polymorphic sites. Screening for carriers forms the baseline for prevention of hemophilia A. Polymerase chain reaction-restriction fragment length polymorphism is a low-cost procedure, efficient in the north Indian population.

Allelic variation investigation of the estrogen receptor within an Australian multiple sclerosis population

Multiple Sclerosis (MS) is a central nervous system (CNS) chronic inflammatory demyelinating disease leading to various neurological disabilities. The disorder is more prevalent for women with a ratio of 3:2 female to male. Objectives: To investigate variation within the estrogen receptor 1 (ESR1) polymorphism gene in an Australian MS case-control population using two intragenic restriction fragment length polymorphisms; the G594A located in exon 8 detected with the BtgI restriction enzyme and T938C located in intron 1, detected with PvuII. One hundred and ten Australian MS patients were studied, with patients classified clinically as Relapsing Remitting MS (RR-MS), Secondary Progressive MS (SP-MS) or Primary Progressive MS (PP-MS). Also, 110 age, sex and ethnicity matched controls were investigated as a comparative group. No significant difference in the allelic distribution frequency was found between the case and control groups for the ESR1 PvuII ( P = 0.50) and Btg1 ( P = 0.45) marker. Our results do not support a role for these two ESR1 markers in multiple sclerosis susceptibility, however other markers within ESR1 should not be excluded for potential involvement in the disorder.

VARIATION IN THE VITAMIN D RECEPTOR GENE IS ASSOCIATED WITH MULTIPLE SCLEROSIS IN AN AUSTRALIAN POPULATION

Multiple Sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS) resulting in accumulating neurological disability. The disorder is more prevalent at higher latitudes. To investigate VDR gene variation using three intragenic restriction fragment length polymorphisms (Apa I, Taq I and Fok I) in an Australian MS case-control population. One hundred and four Australian MS patients were studied with patients classified clinically as Relapsing Remitting MS (RR-MS), Secondary Progressive MS (SP-MS) or Primary Progressive MS (PP-MS). Also, 104 age-, sex-, and ethnicity-matched controls were investigated as a comparative group. Our results show a significant difference of genotype distribution frequency between the case and control groups for the functional exon 9 VDR marker Taq I (pGen = 0.016) and interestingly, a stronger difference for the allelic frequency (pAll = 0.0072). The Apa I alleles were also found to be associated with MS (pAll = 0.04) but genotype frequencies were not significantly different from controls (pGen = 0.1). The Taq and Apa variants are in very strong and significant linkage disequilibrium (D′ = 0.96, P < 0.0001). The genotypic associations are strongest for the progressive forms of MS (SP–MS and PP–MS). Our results support a role for the VDR gene increasing the risk of developing multiple sclerosis, particularly the progressive clinical subtypes of MS.

Clinical description and exclusion of candidate genes in a novel autosomal recessively inherited vitreoretinal dystrophy.

To describe the clinical phenotype of a novel autosomal recessively inherited vitreoretinal dystrophy in one generation of a family originating from eastern Switzerland. A clinical study including electroretinographic investigations followed by laboratory-based genetic and molecular analysis. Four affected and 3 unaffected members of the family were examined. Ten candidate regions were tested by linkage analysis with highly polymorphic molecular markers or with intragenic restriction fragment length polymorphisms. Of 8 siblings,4 were affected, showing high myopia with pronounced vitreous liquefaction, retinitis pigmentosa-like retinal degeneration, diffuse retinal pigment epithelium atrophy, macular staphylomata, and premature cataract formation. Strikingly abnormal results on electroretinograms, affecting both the rod and the cone systems, revealed an extensive defect of retinal function, unlike those usually found in pathologic myopia. No extraocular manifestations were observed. Three types of nonsyndromic high myopia, Stickler syndrome I, II, and III, Wagner syndrome, Knobloch syndrome, Goldmann-Favre dystrophy, and multiple vitreoretinopathies were excluded by linkage analysis. The reported phenotype as well as the results of molecular linkage analysis in the siblings described here suggest an autosomal recessively inherited vitreoretinal dystrophy, which, to our knowledge, has not been described until now.

Glucose-6-phosphate Dehydrogenase Deficiency in Portugal: Biochemical and Mutational Profiles, Heterogeneity, and Haplotype Association

ABSTRACTGlucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzymopathy. This deficiency in erythrocytes has a prevalence of 0.51 ± 0.109 in the Caucasoid male population of Portugal. The frequency for deficiency-conferring genes is 0.39% in the Portuguese population. In the herein study populations males from areas of Portugal presenting with the highest prevalence of G6PD deficiency (Castelo Branco, Setúbal, Faro, and Lisbon) as well as similar subjects located in the border Center/North area of the country (Viseu) have been analyzed for biochemical parameters and screened for mutations and haplotype-associated mutations commensurate with G6PD deficiency. Six intragenic restriction fragment length polymorphisms (RFLPs) were studied: exon 5, nt 376 A → G, FokI; intron 5, nt 611 C → G, PvuII; intron 8, nt 163 C → T, BspHI; exon 10, nt 116 G → A, PstI; exon 11, nt 1311 C → T, BclI; and intron 11, nt 93 T → C, NlaIII. New haplotypes were constructed with the inclusion of intron 11, nt 93 T → C, NlaIII, and only 5 of 64 possible haplotypes were found to show a marked linkage disequilibrium for several RFLPs and also for mutations and specific haplotypes. The control population (n = 168 males) presented the G6PD B variant and corresponded to haplotypes I (− − + + − −), Ia (− − + + − +), and VIIa (− − + + + +), in 91.8, 2.3, and 5.9%, respectively. The PCR and sequencing analysis of extracted DNAs from the deficient G6PD group showed 48.6% (16/33) of individuals with the G6PD A− mutation, corresponding to haplotype VIa (+ + − + − +); 9% (3/33) with the Betica mutation and 18% (6/33) with the Santa Maria mutation, both of them associated with haplotype IVa (+ − − + − +); 6.1% (2/33) with the Mediterranean mutation associated with haplotype VIIa; 12.3% (4/33) with the Seattle mutation, 3.0% (1/33) with Gaohe mutation; and a new mutation, 3.0% (1/33), which we designated by G6PD Flores, all of them associated with haplotype I.

Carrier detection and prenatal diagnosis of hemophilia A.

The aim of this study was to establish a simple, rapid carrier detection and prenatal diagnosis system for hemophilia A. Intron 22 inversion in FVIII gene was directly examined by long-distance polymerase chain reaction. Polymorphisms of factor VIII intragenic restriction fragment length polymorphism of Bcl I, short tandem repeat (STR) within intron 13 and 22, and extragenic DXS 52 (St 14) variable number tandem repeats (VNTR) loci were assessed by hereditary linkage analysis. The diagnostic rates for these loci were 47.6% (intron 22 inversion), 27.8% (Bcl I, 28.6% and 29.4% (STR within intron 13 and 22), and 81.3% (DXS52), respectively. The overall diagnostic rate in 21 families was 94.7%. The diagnosis in hemophilia A patients or carriers can be made if intron 22 inversion is present. The intragenic and extragenic loci hereditary linkage analysis could be used to establish the diagnosis in intron 22 inversion-negative patients.

Hypophosphatasia: diagnostic application of linked DNA markers in the dominantly inherited adult form

Hypophosphatasia is a rare disease characterized by low serum levels of tissue non-specific alkaline phosphatase (TNSALP) and a spectrum of skeletal disease varying from the severest form with death in utero to mild with no clinical abnormality in adults. Currently, the diagnosis of hypophosphatasia is made on the basis of clinical findings, radiography, low serum alkaline phosphatase levels and raised abnormal phosphorylated metabolites; there are elevations in serum pyridoxal 5'-phosphate, urinary phosphoethanolamine and inorganic pyrophosphate. In borderline cases the biochemical diagnosis remains uncertain. Prenatally, diagnosis is made using radiography and ultrasonography together with chorionic villus tissue biopsy, in which TNSALP levels are measured using an antibody-based assay. Since hypophosphatasia results from mutations in the TNSALP gene we have, for the first time in two U.K. families, undertaken restriction fragment length polymorphism (RFLP) analysis using three intragenic RFLPs for BclI and MspI at the ALPL locus. One family was informative, and a mutant-allele-specific haplotype with respect to three RFLPs was defined. In the other family the disease was shown to segregate with one allele of the BclI RFLP, but the MspI RFLPs were not informative. The disease segregated in the two families with different alleles of the BclI RFLP, suggesting that the mutations are likely to be different. We confirm that DNA analysis is likely to be the way ahead for diagnosing hypophosphatasia, and that standardized screening methods need to be developed for detecting mutations in these and other families.

Genetic Analysis of Male Pattern Baldness and the 5α-Reductase Genes

Genetic predisposition and androgen dependence are important characteristics of the common patterned loss of scalp hair known as male pattern baldness. The involvement of the 5alpha-reductase enzyme in male pattern baldness has been postulated due to its role in the metabolism of testosterone to dihydrotestosterone. There are two known isozymes of 5alpha-reductase. Type I has been predominantly localized to the skin and scalp. Type II, also present on the scalp, is the target of finasteride, a promising treatment for male pattern baldness. We conducted genetic association studies of the 5alpha-reductase enzyme genes (SRD5A1 on chromosome 5 and SRD5A2 on chromosome 2) using dimorphic intragenic restriction fragment length polymorphisms. From a population survey of 828 healthy families comprising 3000 individuals, we identified 58 young bald men (aged 18-30 y) and 114 older nonbald men (aged 50-70 y) for a case control comparison. No significant differences were found between cases and controls in allele, genotype, or haplotype frequencies for restriction fragment length polymorphisms of either gene. These findings suggest that the genes encoding the two 5alpha-reductase isoenzymes are not associated with male pattern baldness. Finally, no clear inheritance pattern of male pattern baldness was observed. The relatively strong concordance for baldness between fathers and sons in this study was not consistent with a simple Mendelian autosomal dominant inheritance. A polygenic etiology should be considered.

Frequent alterations of cell cycle regulators in early-stage breast lesions as detected by immunohistochemistry.

Progression through G1 phase of the eukaryotic cell cycle is tightly controlled by cyclin-dependent kinases (CDK). These proteins form part of a regulatory pathway including the cyclin-dependent kinase inhibitor (CKI) p16, D-type cyclins and the product of the retinoblastoma gene pRb. Aberration of any one of these components may lead to uncontrolled proliferation contributing to neoplasia. Three of these proteins, cyclin D1, pRb and p16, were analysed by immunohistochemistry on archival paraffin sections to determine whether expression patterns were different in preinvasive ductal carcinoma in situ (DCIS) and invasive breast tumours relative to normal. Genetic analysis of the gene encoding cyclin D1 (CCND1) was also carried out, using an intragenic restriction fragment-length polymorphism (RFLP) to assess possible allelic imbalance. A majority of the tumours studied (approximately 90%) showed abnormalities in expression of at least one of these proteins. Overexpression of cyclin D1 was found in approximately 49% cases, reduced expression of p16 in approximately 46% and reduced expression of pRb in approximately 37%. Allelic imbalance of cyclin D1 was found in approximately 57% cases.

Development of a microsatellite-based approach to co-segregation analysis of familial hypercholesterolaemic kindreds.

Co-segregation studies based on a selection of intragenic restriction fragment length polymorphisms of the low density lipoprotein receptor (LDLR) gene have been used extensively both for research and diagnostic studies of familial hypercholesterolaemia (FH) families, because direct mutation screening remains complex. Here we describe the development and application of a more efficient approach to co-segregation studies based on highly informative dinucleotide and tetranucleotide repeats flanking the LDLR gene. A series of microsatellites (D19S391, D19S394, D19S221 and D19S179) were selected for study on the basis of linkage analysis in the CEPH families using intragenic polymorphisms for a TA repeat (exon 18) in the LDLR gene, and earlier data for a Pvu II polymorphism (intron 15). A physical map of the region of chromosome 19 also contributed to this selection. One marker in particular, D19S394, sited 150 kilobases telomeric to the gene, was extremely useful, displaying 90% heterozygosity, robust PCR of tetranucleotide repeats without stutter bands, and no recombination with the LDLR gene (theta = 0, LOD 68). Use of this marker in the families of twenty-three FH probands from Hampshire demonstrated co-segregation of the hyperlipidaemia phenotype with the LDLR gene region, except in one family with defective apolipoprotein B-100, and a family turning out to display familial combined hyperlipidaemia. This approach should facilitate the search for any families where FH does not co-segregate with the LDLR gene, and will enhance the repertoire of molecular diagnostic tools available for FH.

Mapping of the human adenosine A2a receptor gene: relationship to potential schizophrenia loci on chromosome 22q and exclusion from the CATCH 22 region.

Chromosome 22 contains two potential schizophrenia loci on chromosomal regions 22q11.2 and 22q12-13. In the present study we report results from linkage mapping of the gene coding for the human A2a adenosine receptor (AR), which is one of two receptors mediating central nervous system effects of adenosine. From seven CEPH (Centre d'Etude du Polymorphisme Humain) families, 120 individuals were typed utilizing an intragenic restriction fragment length polymorphism. Significant linkage was found with many markers on chromosome 22. A 10-cM 1000:1 support interval between markers D22S301 and D22S300 is defined on the CHLC (Cooperative Human Linkage Center) framework map of chromosome 22. Localization of the A2aAR gene outside the CATCH 22 syndrome region on 22q11.2 is demonstrated by the observation of heterozygous individuals with defined 2-Mb deletions from this region. Thus, the A2aAR gene is not the schizophrenia susceptibility gene suspected in the CATCH 22 syndrome region on 22q11.2, but remains a candidate for a schizophrenia susceptibility gene on 22q12-13.

Analysis of the novel cyclin-dependent kinase 4 and 6 inhibitor gene p18 in lymphoma and leukemia cell lines

The genes for the CDK4/6-inhibitors p16 INK4A/MTS1 and p15 INK4B/MTS2 are frequently deleted in hematological malignancies. A new member of this family of CDK4/6 inhibitors is p18. In order to assess p18 growth-suppressor gene alterations in hematological neoplasms, we investigated 31 lymphoma and leukemia cell lines by PCR for both exons of this gene. No homozygous deletions were observed. Investigation of a new intragenic restriction fragment length polymorphism revealed no differences in allele distribution between the tumor cell lines and healthy volunteers. Our results suggest that homozygous deletion of the p18 gene does not play a major role in leukemogenesis or lymphomagenesis.

Analysis of the elastin gene in 60 patients with clinical diagnosis of Williams syndrome

Williams syndrome (WS) is caused by deletion of the elastin (ELN) gene. We have analyzed an intragenic restriction fragment length polymorphism (RFLP) and the gene dosage of ELN using a new probe (FP4) in a series of 60 sporadic patients with a clinical diagnosis of WS. Deletion of the ELN gene was shown in 54 cases, while clinical revaluation of the 6 patients without the deletion did not confirm the diagnosis of WS. These results support the genetic homogeneity of WS, and the high accuracy of ELN molecular analysis, which can be confidenty used for providing genetic counselling to WS families.

The human plakoglobin gene localizes on chromosome 17q21 and is subjected to loss of heterozygosity in breast and ovarian cancers.

The gene encoding human plakoglobin was mapped to chromosome 17q12-q22. An intragenic restriction fragment length polymorphism was used to localize the plakoglobin gene distal to locus KRT10 and proximal to the marker D17S858. The plakoglobin gene colocalizes with the polymorphic 17q21 marker UM8 on the same cosmid insert. This subregion of chromosome 17 is known to be particularly subjected to genetic alterations in sporadic breast and ovarian tumors. We show loss of heterozygosity of the plakoglobin gene in breast and ovarian tumors. We have identified a low-frequency polymorphism in the plakoglobin coding sequence which results in an arginine to histidine substitution at amino acid position 142 of the protein, as well as a silent mutation at nucleotide position 332 of the coding sequence. This polymorphism allowed us to demonstrate an allelic association of plakoglobin with predisposition to familial breast and ovarian cancers. Our results, together with the present knowledge about the biological function of plakoglobin, suggest that plakoglobin might represent a putative tumor suppressor gene for breast and ovarian cancers.

Intragenic TaqI restriction fragment length polymorphism (RFLP) in CICN4, between the loci for X linked ocular albinism (OA1) and microphthalmia with linear skin defects syndrome (MLS)

A TaqI RFLP was detected within the ClCN4 gene, which lies between the loci for OA1 and MLS. There were no observed recombinations between this RFLP and the OA1 mutation in three informative families. Thus, the marker will be useful for genetic counseling in OA1.

Genetic heterogeneity of severe von Willebrand disease type III in the German population.

The genetic heterogeneity of severe von Willebrand disease (vWd) type III was estimated by analysing extended haplotypes of eleven intragenic restriction fragment length polymorphisms and one variable number of tandem repeat polymorphism in 32 patients from 28 families from Germany or of German origin. All patients were screened for gross deletions and for mutations at potential "hot spot" regions of the von Willebrand factor (vWf) gene. Disease-associated haplotypes were established in 24 families. Only a few, apparently unrelated families shared common haplotypes suggesting a considerable genetic heterogeneity in the German population of vWd type III patients. Defects causing vWd type III were identified on 14 out of 56 chromosomes (25%). Gross deletions were detected in two families. A complete homozygous deletion of the vWf gene was displayed in one patient. Another patient was compound heterozygous for a large deletion of at least 100 kb of the vWf gene with an additional, as yet unidentified, defect. One homozygous missense mutation was detected in exon 10, and two nonsense mutations were detected in exon 8 and exon 45 of the vWf gene, respectively. A frameshift mutation (delta C) in exon 18 was identified in five families and an additional frameshift mutation (delta G) was found in exon 28 in one family. It appears that delta C is the most common molecular defect in German patients with vWd type III. Its association with a number of different haplotypes suggests repeated de novo mutations at a mutation "hot spot". Evidence is presented that particular molecular defects causing vWd type III are associated with different patterns of inheritance, depending on their location within the vWf gene. Complete deletions of the gene and nonsense mutations in the pro-sequence are correlated with recessive inheritance, whereas frameshift and nonsense mutations in the gene sequence corresponding to the mature vWf subunit tend to be inherited in a dominant fashion.

Hemophilia A or von Willebrand disease?

Seven members of the same family were studied on several occasions due to a history of hemorrhages. The propositus, a 12-year-old boy, his sister, one brother, and their father all had a low plasma factor VIII (FVIII) level. Von Willebrand factor (vWF) activity, vWF multimeric analysis, and vWF factor domain for binding to FVIII were normal in all seven subjects. The sister had a normal 46XX karyotype. The study of two intragenic restriction fragment length polymorphisms (RFLPs) and two closely linked, highly polymorphic extragenic markers showed a phenotypic expression of mild hemophilia A, which suggests that the sister of the propositus is homozygous or compound heterozygous at the hemophilia A locus. She would have inherited two hemophilic genes: one from her carrier mother and the other from her father, a mild hemophiliac.

Carrier detection of ovine hemophilia A using an RFLP marker, and mapping of the factor VIII gene on the ovine X-chromosome.

Ovine hemophilia A is an X-linked recessive bleeding disorder. For diagnostic purposes, restriction fragment length polymorphism (RFLP) analysis in the region of the factor VIII (F-VIII) gene was carried out using human F-VIII gene probes. The probe St14, known to detect a highly polymorphic region that is closely linked to the F-VIII gene in humans, hybridized nonspecifically with DNA from sheep. Searching for intragenic RFLPs, the entire 9.0-kb coding sequence of the human F-VIII was used as a probe. Using the 1.8-kb SstI/KpnI F-VIII cDNA probe for hybridization, an MspI-RFLP with allelic bands of 5.8 kb (A1) and 4.2 kb (A2) was detected. A1 was in linkage phase with the mutated allele responsible for hemophilia A. The F-VIII locus in the sheep genome was assigned to the long arm of the X-chromosome in the region Xq24-q33, using in situ hybridization with a 3-kb human F-VIII cDNA probe to QFQ banded sheep metaphase chromosomes.

Determination of haemophilia A carrier status from hair samples using polymerase chain reaction technique

The usefulness of intragenic restriction fragment length polymorphisms (RFLPs) for BclI, HindIII and XbaI, adapted for polymerase chain reaction (PCR), was tested for the detection of haemophilia A carrier status in the consultant of a family in which only haematological information was available on the inheritance of the trait. Hair follicles were used as the non-invasive source of DNA. The mother was found to be homozygous for BclI and heterozygous for HindIII sites, whereas her status as regards informativeness could not be established for XbaI. On the basis of HindIII RFLP, the daughter was found to be a carrier of the haemophilia trait. This was confirmed by sequencing the amplified intron 19 of the mother and the daughter. The RFLP for XbaI did not appear to be suitable for carrier detection using PCR due to the difficulty of establishing homozygosity or heterozygosity from the results of digestion of the amplified product.

Haemophilia A diagnosis by simultaneous analysis of two variable dinucleotide tandem repeats within the factor VIII gene

Haemophilia A is a bleeding disorder caused by defects in the gene coding for the co-factor, factor VIII (FVIII). The few available intragenic restriction fragment length polymorphisms (RFLPs) currently used in carrier detection and prenatal diagnosis of haemophilia A are informative in only about 65% of cases. We earlier reported a multi-allelic dinucleotide tandem repeat, (CA)n, specific to intron 13, which remains the single most informative marker within the FVIII gene. We here report a second informative dinucleotide repeat of the form (GT)n (AG)n, located to intron 22 of the FVIII gene. The polymerase chain reaction (PCR) method was used to examine the variability of the repeat in 60 individuals (75 X-chromosomes) and revealed four alleles. The calculated heterozygosity rate is 45%, and family studies showed X-linked mendelian inheritance. The intron 22 dinucleotide repeat is tightly linked with established RFLPs and tracks with haemophilia A in family studies. We now show that by simultaneous amplification of the intron 13 and 22 repeats using PCR all alleles for both markers are detectable on a single polyacrylamide gel. The information thus obtained from a single multiplexed analysis is greater than from multiple RFLP analyses. Hence, rapid haplotype determination by simultaneous amplification and detection of two intragenic dinucleotide repeats should supersede less informative RFLP analysis.