A highly polymorphic dinucleotide repeat at the D8S1222 locus

A highly polymorphic dinucleotide (CA) repeat clone was isolated from a CEPH mega-YAC clone (844E2), and was localized to chromosome 8 using a panel of 13 mouse/human somatic cell hybrids.     

Characterisation of a new rare fragile site easily confused with the fragile X.

A new fragile site (FRAXE) in Xq28 is described. It appears to be a typical folate sensitive fragile site. The fragile site is not associated with mental retardation, it does not give abnormal results when subjected to Southern analysis with probe pfxa3 which detects the unstable DNA sequence characteristic of fragile X syndrome. In situ hybridization mapping locates the fragile site between 150 kb and 600 kb distal to FRAXA. The distinction between the two fragile sites is important clinically since cytogenetic detection of FRAXE, without molecular analysis, could result in misdiagnosis of fragile X syndrome.     

Presymptomatic diagnosis in families with adenomatous polyposis using highly polymorphic dinucleotide CA repeat markers flanking the APC gene.

A panel of five multiallelic and highly informative dinucleotide CA repeat markers flanking the APC gene was used for presymptomatic diagnosis of familial adenomatous polyposis coli (FAP). Marker regions were amplified by PCR. DNA fragments were separated by electrophoresis in denaturing polyacrylamide gels and visualised by ethidium bromide staining. Two or more markers were found to be informative in all nine families tested, and all 23 persons at risk could be diagnosed as affected or unaffected by the disease gene, the probability being > 99.9% in 14 cases from six families in which flanking markers were informative. We found no indication for locus heterogeneity of the disease in our sample. The polyposis phenotype and its extracolonic manifestations co-segregated with a distinct haplotype determined by the markers flanking the APC gene. In one family with no remaining living affected members, we could infer the high risk haplotype from genotyping of first degree relatives. The segregation of this haplotype is consistent with the occurrence of CHRPEs in the progeny. In a sporadic case we made use of the typical early extracolonic manifestations of the disease (osteomas, desmoids) to identify the high risk haplotype. We conclude from our experience that indirect genotyping of FAP with this particular panel of closely linked and highly polymorphic microsatellite markers is a rapid, efficient, and highly reliable method for presymptomatic diagnosis of FAP.     

Sequence analysis of the fragile X trinucleotide repeat: implications for the origin of the fragile X mutation

This study addresses mechanism of instability of the FMR-1 (CGG)_n-repeat,and investigates features which may distinguish between normalstable and fragile X unstable repeats. To achieve this, we havesequenced 178 alleles to analyze patterns of AGG interruptionswithin the CGG repeat, and have typed the (CA)_n-repeat at DXS548for 204 chromosomes. Overall, our data is consistent with theidea that the length of uninterrupted CGG repeats determinesInstability. We predict that certain sequence configurations[no AGG, and (CGG)_9–11AGG(CGG)     

Ultrastructure of the fragile X chromosome: new observations on the fragile site

Using a nonair-drying modification of a method for longitudinal sectioning of metaphase spreads on glass slides [Wen et al., 1997], we have studied 14 preidentified X chromosomes (10 from fragile X specimens and 4 controls) with transmission electron microscopy (TEM). Four of 10 X chromosomes from fragile X specimens exhibited lighter chromatin density in the area of and distal to the fragile site, most pronounced under dark-field TEM. A clear line of separation at the fragile site locus was also observed by TEM in an X chromosome with no visible fragile site after Q-banding. We hypothesize that these areas of lighter density, including lines of separation, precede the appearance of the fragile site that is commonly observed using light microscopy.     

Identification and regional localization of a highly polymorphic dinucleotide repeat D11S614 to the interval in 11q23.3 flanked by recurrent translocation breakpoints

A higly informative dinucleotide repeat polymorphism has been identified at the D11S614 locus on chromosome 11q23. Ten differen alleles have been observed at this locus, and the heterozygosity frequency is approximately 85%. Physical localization of this marker in a panel of somatic cell hybrids containing chromosome 11 translocation showed that is maps to 11q23.3, within the interval between the recurrent t(4;11) leukaemia breakpoint and the t(11;22) Ewing's sarcoma breakpoint. This physical mapping data is consistent withs the genetic mapping which indicates tight linkage to other markers in the q23.3 region including PBGD, CD3D and D11S29. Regional localization of highly informative markers such as D11S614 will facilitate integration of the genetic and physical maps.     

A deletion of 1.6 kb proximal to the CGG repeat of the FMR1 gene causes the clinical phenotype of the fragile X syndrome

vast majority of individuals with the fragile X syndrome showexpanded stretches of CGG repeats In the 5' non-coding regionof FMR1. This expansion coincides with abnormal methylatlonpatterns in that area resulting in the silencing of the FMR1gene. Evidence is accumulating that this directly causes thefragile X phenotype. Very few other mutations in FMR1, causingthe fragile X phenotype have been reported thus far and allconcerned isolated cases. We, however, report a family, In which11 Individuals have a deletion of 1.6 kb proximal to the CGGrepeat of the FMR1 gene. Although fragile X chromosomes werenot detected, all 4 affected males and 2 of the carrier femalesshow characteristics of the fragile X phenotype. Using RT-PCRwe could demonstrate that FMR1 Is not expressed In the affectedmales, strongly suggesting that the FMR1 promoter sequences5' to the CGG repeat are missing. The deletion patients haveapproximately 45 CGG repeats in their FMR1 gene, though notinterspersed by AGG triplets that are usually present in bothnormal and expanded repeats. It Is hypothesized that prior tothe occurrence of the deletion, an expansion of the repeat occurred,and that the deletion removed the 5' part of the CGG repeatcontaining the AGG triplets. Transmission of the deletion throughthe family could be traced back to the deceased grandfatherof the affected males, which supports the hypothesis that theFMR1 gene product is not required for spermatogenesls. Finally,the data provide additional evidence that the fragile X syndromeis a single gene disorder.     

Standardization of PCR amplification for fragile X trinucleotide repeat measurements

To provide the clinical diagnostics community with accurate protocols and measurements for the detection of genetic disorders, we have established a quantitative measurement program for trinucleotide repeats associated with human disease. In this study, we have focused on the triplet repeat associated with fragile X syndrome. Five cell lines obtained from the Coriell Cell Repository were analyzed after polymerase chain reaction (PCR) amplification and size separation. These cell lines were reported to contain CGG repeat elements (ranging from 29 to 110 repeats). Our initial measurements focused on measurement variability: (a) between slab-PAGE and capillary (CE) separation systems (b) interlane variability (slab-PAGE) (c) intergel variability, and (d) variability associated with amplification. Samples were run in triplicate for all measurements, and the analysis performed using Gene Scan analysis software. The repeat sizes were verified by DNA sequence analyzes. The standard deviations for interlane measurements in slab-gels ranged from 0.05 to 0.35. There was also little variation in size measurements performed on different gels and among PCR amplifications. The CGG repeat measurements performed by capillary electrophoresis were more precise, with standard deviations ranging from 0.02 to 0.29. The slab-PAGE and CE size measurements were in agreement except for the pre-mutation alleles, which yielded significantly smaller sizes by CE.     

Precursor arrays for triplet repeat expansion at the fragile X locus

To determine factors governing triplet repeat expansion at FMR1,we need to understand the basis of normal variation. We havesequenced the FMR1 repeat from 102 normal X chromosomes andshow that most are interrupted with a regularly spaced AGG trinucleotidegiving an ordered structure to the array. Five types of arrayswere identified consisting of varying numbers of a core unitwith consensus [AGG(CGG)_g]. Additional variation in the lengthof the (CGG)_n portion within each unit generates the continuumof lengths seen on normal chromosomes. Ten per cent containlong, uninterrupted tracts of (CGG)_n, and their lengths suggestthey have arisen by the loss of AGG triplets from longer interruptedarrays. Haplotype analysis of arrays carrying long, uninterrupted(CGG)_n tracts suggests that they occur more frequently on geneticbackgrounds which are more highly represented on fragile X chromosomes.These arrays may well be precursors from which the larger fragileX associated arrays have arisen by further expansion.     

Strategy for molecular cloning of the fragile X site DNA

Fragile X syndrome is a common form of mental retardation associated with a fragile site on the human X chromosome. We have recently demonstrated that the fragile X chromosome, when isolated within a somatic cell hybrid, often participates in translocations involving rodent chromosome arms. Cytogenetic and molecular evidence strongly suggests that the human breakpoint of these translocations is within the fragile X sequence. Hence, the joining of heterologous DNA (i.e. from two species) may permit the molecular cloning of the fragile X site. We describe here the cloning approach employed to enhance the isolation of interspecific chromosome translocation junctions. The human portion of the translocation junction should be derived from the fragile X site sequence.     

Validation of linkage-based DNA-diagnosis of fragile X gene carriers with the CGG repeat probe.

We have evaluated our carrier testing for the fragile X [fra(X)] syndrome, which was based on linked DNA markers, with the direct analysis of the CGG repeat sequence in the fra(X) gene. PstI and EcoRI blots were hybridized with a probe derived from the region just 3' of the CGG repeat in Xq27.3. We found the mutation analysis to be very sensitive as all 71 obligate gene carriers as well as 135 fra(X) patients tested showed evidence for an increased restriction fragment length encompassing the CGG repeat sequence with or without dispersion of the hybridization signal (mosaicism). Based on linked DNA markers, 6 out of 50 cytogenetic negative and mentally normal males at risk and 15 of 72 females at risk had inherited the allele at risk. All of these diagnoses could be confirmed by analysis of the CGG repeat length.     

Linkage analysis in the fragile X syndrome using multiple distal Xq polymorphic DNA markers.

Linkage data using the polymorphic loci F9, DXS105, DXS98, DXS52, DXS15, and F8 and the DNA probe 1A1 are presented from 14 families segregating for fragile X [fra(X)] syndrome. Recombination fractions corresponding to the maximum LOD scores obtained by two-point linkage analysis suggest that DXS98 (Zmax = 3.23, theta = 0.0) and DXS105 (Zmax = 2.09, theta = 0.0) are the closest markers proximal to FRAXA and that DXS52 is the closest distal marker (Zmax = 3.55, theta = 0.16). FRAXA is located within a 25 cM interval between F9 and DXS52, coincident with DXS98, on multipoint linkage analysis. Phase-known three way crossover information places F8 outside the cluster (DXS52, DXS15, 1A1). Confidence limits for the markers DXS98 and DXS52 are relatively wide (0.0-0.15 and 0.06-0.31, respectively), but when used in combination with cytogenetic examination offer improved carrier detection in comparison with cytogenetic analysis alone.     

A highly polymorphic CA repeat marker at the interleukin-11 locus

The interleukin-11 (IL-11) stimulates T cell-dependent development of immunoglobulin-producing B cells and collaborates with IL-3 in supporting murine megakaryocyte colony formation. The interleukin-11 (IL-11) also stimulates osteoclast formation and inhibits osteoclast function in vitro and has been implicated in estrogen deficiency-induced bone loss. We isolated a polymorphic dinucleotide (CA) repeat sequence from a genomic clone containing the IL-11 gene located at 19q13.3-q13.4. High heterozygosity (0.81) makes this polymorphism a useful marker in genetic study of disorders affecting immune response and bone metabolism.     

The common fragile site in band q27 of the human X chromosome is not coincident with the fragile X

The common fragile site on the end of the long arm of the human X chromosome has been shown to be at a different location from the rare fragile site which produces the fragile X syndrome of intellectual handicap. The different locations can be clearly seen in chromosomes at about the 550 band level of resolution. This finding should help resolve difficulties in fragile X cytogenetics where expression of the common fragile site can lead to false positive diagnoses.     

Tightly linked polymorphic markers for fragile X syndrome and prenatal cytogenetic diagnostic experience.

Linkage analysis using the polymorphic loci DXS369, DXS296, DXS297 and DXS306 was carried out on a cohort of 17 families segregating for fragile X syndrome. The observed recombination fractions at: DXS369 (Zmax = 3.02; theta = 0.06), DXS297 (Zmax = 2.92; theta = 0.0), DXS296 (Zmax = 3.82; theta = 0.0), DXA306 (Zmax = 4.55; theta = 0.05) confirm that these loci are tightly linked to FRAXA. Our experience in the cytogenetic analysis of 58 at risk pregnancies by chorionic villus or fetal blood sample examination documents a false negative rate in obligate carrier male pregnancies for CVS of 11% and for FBS of 3%.     

Limited size of the fragile X site shown by fluorescence in situ hybridization.

Cosmids, isolated from a 475 kb YAC that spans the fragile X region, and the YAC itself, were used for fluorescence in situ hybridization (FISH) on metaphase chromosomes from fragile X patients. Cosmid 22.3, containing most of the hybrid translocation breakpoints, shows in situ hybridization signals distal and proximal from the fragile X site. We propose that the size of the fragile site is limited to 20 kb.     

The fragile X syndrome in a large family. III. Investigations on linkage of flanking DNA markers with the fragile site Xq27.

In a large family with the fragile X syndrome, we performed linkage investigations with six probes, detecting RFLPs at both sides of the fragile site Xq27. The nearest flanking markers were cX55.7 (DXS105) on the centromeric side (theta = 0.04, lod 5.0) and St14 (DXS52) on the telomeric side (theta = 0.08, lod 4.0). Non-penetrance could be shown by the presence of the grandpaternal X chromosome in three mentally retarded fra(X) positive males. A second non-penetrant male in this family had inherited an abnormal grandmaternal X chromosome. His carrier mother had two retarded fra(X) positive brothers. Intermediate between the non-penetrant and fully penetrant males was a non-retarded male, who expressed the fragile site in 6% of his cells. His X chromosome showed the same polymorphisms as were found in his seven severely retarded brothers. In five fra(X) negative females the presence of an abnormal X chromosome could be demonstrated. Despite the existence of non-penetrance in this pedigree, there was no close linkage between a factor IX polymorphism and the fragile site (theta = 0.16, lod 1.9). However, in six descendants of a non-penetrant male, the change to penetrance appeared to be accompanied by a low recombination frequency for flanking markers.     

The human caspase-activated DNase gene (hCAD): genomic structure, exonic single-nucleotide polymorphisms, and a highly polymorphic dinucleotide repeat at the hCAD locus

Caspase-activated DNase (CAD) cleaves chromosomal DNA during apoptosis. We determined its genomic structure and identified single-nucleotide polymorphisms (SNPs) within exons 5 and 7, as well as a highly polymorphic dinucleotide repeat of (CT)m(CA)n within the 5′ region of the human CAD gene (hCAD). The genomic structure of hCAD presented here, together with information concerning SNPs within the gene, as well as a highly polymorphic (CT)m(CA)n repeat fragment at the hCAD locus, may assist in the construction of genetic maps for exploring gene(s) that play pivotal roles in carcinogenesis. Received: July 30, 1999 / Accepted: August 14, 1999     

Use of a polymorphic dinucleotide repeat sequence to detect non-blastomeric contamination of the polymerase chain reaction in biopsy samples for preimplantation diagnosis

Using the polymerase chain reaction (PCR), amplification oftwo different target DNA sequences has been achieved with highfrequency using single human blastomeres as template for theduplex reaction. One sequence is located within the –globingene and contains the sickle cell locus, the other is a polymorphicdinucleotide repeat, which, as well as acting as a positivecontrol for amplification, was used to check the origin of theamplified DNA. A comparison of the sequences amplified fromthe blastomere with sequences amplified from parental samplesconfirmed that amplification of blastomeric sequences, but notextraneous contaminating DNA, had taken place in most cases.The efficacy of this system for detecting extraneous DNA waschecked by deliberately contaminating single blastomeres withforeign cells. The presence of contamination was detected bythe amplification of sequences not present in blastomeric DNAand which therefore must have been amplified from extraneouscontaminating DNA.