K. Holman

Mapping of DNA instability at the fragile X to a trinucleotide repeat sequence p(CCG)n

The sequence of a Pst I restriction fragment was determined that demonstrate instability in fragile X syndrome pedigrees. The region of instability was localized to a trinucleotide repeat p(CCG)n. The sequence flanking this repeat were identical in normal and affected individuals. The breakpoints in two somatic cell hybrids constructed to break at the fragile site also mapped to this repeat sequence. The repeat exhibits instability both when cloned in a nonhomologous host and after amplification by the polymerase chain reaction. These results suggest variation in the trinucleotide repeat copy number as the molecular basis for the instability and possibly the fragile site. This would account for the observed properties of this region in vivo and in vitro.

Fragile X genotype characterized by an unstable region of DNA

DNA sequences have been located at the fragile X site by in situ hybridization and by the mapping of breakpoints in two somatic cell hybrids that were constructed to break at the fragile site. These hybrids were found to have breakpoints in a common 5-kilobase Eco RI restriction fragment. When this fragment was used as a probe on the chromosomal DNA of normal and fragile X genotype individuals, alterations in the mobility of the sequences detected by the probe were found only in fragile X genotype DNA. These sequences were of an increased size in all fragile X individuals and varied within families, indicating that the region was unstable. This probe provides a means with which to analyze fragile X pedigrees and is a diagnostic reagent for the fragile X genotype.

Fragile X syndrome: genetic localisation by linkage mapping of two microsatellite repeats FRAXAC1 and FRAXAC2 which immediately flank the fragile site.

We report the genetic localisation of the fragile site at Xq27.3 associated with fragile X syndrome. The position of the fragile site within the multipoint linkage map was determined using two polymorphic microsatellite AC repeat markers FRAXAC1 and FRAXAC2. These markers were physically located within 10 kilobases and on either side of the p(CCG)n repeat responsible for the fragile site. FRAXAC1 has five alleles with heterozygosity of 44% and is in strong linkage disequilibrium with FRAXAC2 which has eight alleles and a heterozygosity of 71%. No recombination was observed either between these markers in 40 normal CEPH pedigrees or with the fragile X in affected pedigrees. These markers provide the means for accurate diagnosis of the fragile X genotype in families by rapid polymerase chain reaction analysis and were used to position the fragile X within the multipoint map of the X chromosome to a position 3.7 cM distal to DXS297 and 1.2 cM proximal to DXS296.

Isolation and characterisation of (AC)n microsatellite genetic markers from human chromosome 16

A cosmid library of human chromosome 16 has been subcloned, and (AC)n microsatellite positive clones have been identified and sequenced. Oligonucleotide primers flanking the repeat were designed and synthesized for (AC)n microsatellites with n greater than 16. These microsatellite loci were then mapped by PCR using a somatic cell hybrid panel of human chromosome 16, and their heterozygosities and allele frequencies determined. Fourteen (AC)n microsatellites were mapped to discrete physical intervals of human chromosome 16 defined by a mouse/human hybrid panel. Nine of these have expected heterozygosities ranging between 0.60 and 0.79, four have expected heterozygosities between 0.02 and 0.49, and one detected three loci where the alleles could not be resolved.

High-Resolution Cytogenetic-Based Physical Map of Human Chromosome 16

A panel of 54 mouse/human somatic cell hybrids, each possessing various portions of chromosome 16, was constructed; 46 were constructed from naturally occurring rearrangements of this chromosome, which were ascertained in clinical cytogenetics laboratories, and a further 8 from rearrangements spontaneously arising during tissue culture. By mapping 235 DNA markers to this panel of hybrids, and in relation to four fragile sites and the centromere, a cytogenetic-based physical map of chromosome 16 with an average resolution of 1.6 Mb was generated. Included are 66 DNA markers that have been typed in the CEPH pedigrees, and these will allow the construction of a detailed correlation of the cytogenetic-based physical map and the genetic map of this chromosome. Cosmids from chromosome 16 that have been assembled into contigs by use of repetitive sequence fingerprinting have been mapped to the hybrid panel. Approximately 11% of the euchromatin is now both represented in such contigs and located on the cytogenetic-based physical map. This high-resolution cytogenetic-based physical map of chromosome 16 will provide the basis for the cloning of genetically mapped disease genes, genes disrupted in cytogenetic rearrangements that have produced abnormal phenotypes, and cancer breakpoints.

A refined physical map of the long arm of human chromosome 16

Mapping of 33 anonymous DNA probes and 12 genes to the long arm of chromosome 16 was achieved by the use of 14 mouse/human hybrid cell lines and the fragile site FRA16B. Two of the hybrid cell lines contained overlapping interstitial deletions in bands q21 and q22.1. The localization of the 12 genes has been refined. The breakpoints present in the hybrids, in conjunction with the fragile site, can potentially divide the long arm of chromosome 16 into 16 regions. However, this was reduced to 14 regions because in two instances there were no probes or genes that mapped between pairs of breakpoints.