Chromosomal bar codes produced by multicolor fluorescence in situ hybridization with multiple YAC clones and whole chromosome painting probes

Colored chromosome staining patterns, termed chromosomal ‘bar codes’ (CBCs), were obtained on human chromosomes by fluorescence in situ hybridization (FISH) with pools of Alu-PCR products from YAC dones containing human DNA inserts ranging from 100 kbp to 1 Mbp. In contrast to conventional G- or R-bands, the chromosomal position, extent, Individual color and relative signal intensity of each ‘bar’ could be modified depending on probe selection and labeling procedures. Alu-PCR amplification products were generated from 31 YAC clones which mapped to 37 different chromosome bands. For multiple color FISH, Alu-PCR amplification products from various clones were either biotinylated or labeled with digoxigenin. Probes from up to twenty YAC clones were used simultaneously to produce CBCs on selected human chromosomes. Evaluation using a cooled CCD camera and digital image analysis confirmed the high reproducibility of the bars from one metaphase spread to another. Combinatorial FISH with mixtures of whole chromosome paint probes was applied to paint seven chromosomes simultaneously in different colors along with a set of YAC clones which map to these chromosomes. We discuss the potential to construct analytical chromosomal bar codes adapted to particular needs of cytogenetic investigations and automated image analysis

Isolation of YAC Clones From the Pericentromeric Region of Chromosome 10 and Development of New Genetic Markers Linked to the Multiple Endocrine Neoplasia Type 2A Gene

Genetic linkage mapping and contig assembly using yeast artificial chromosome (YAC) technology form the basis of our strategy to clone and define the genomic structure of the pericentromeric region of chromosome 10 containing the multiple endocrine neoplasia type 2A gene. Thus far YAC walks have been initiated from five chromosome 10 pericentromeric loci including RBP3, D10S94, RET, D10Z1, and FNRB. Long range pulsed-field gel electrophoresis maps are constructed from the YACs isolated to define clone overlaps and to identify putative CpG islands. Bidirectional YAC walks are continued by rescreening the YAC library with sequence-tagged site assays developed from endclones. Several new restriction fragment length polymorphisms and simple sequence repeat polymorphism markers have been identified from the YAC clones. In particular, two highly informative (CA)n dinucleotide repeat markers, sTCL-1 from proximal chromosome 10p (16 alleles, PIC = 0.68) and sJRH-1 from the RBP3 locus (18 alleles. PIC = 0.88), provide useful reagents for a polymerase chain reaction-based predictive genetic test that can be performed rapidly from small amounts of DNA

Linkage studies with chromosome 17 DNA markers in 45 neurofibromatosis 1 families

A locus for von Recklinghausen neurofibromatosis (NF1) has recently been mapped near the chromosome 17 centromere. We have extended these linkage studies by genotyping 45 NF1 families with three DNA probes known to be linked to the chromosome 17 centromeric region. Of 34 families informative for NF1 and at least one of the three probes, 28 families show no recombinants with the disease gene. These data provide additional support for genetic homogeneity of NF1 and for a primary NF1 locus linked to the chromosome 17 centromere. Among the informative families were 7 families with apparent new NF1 mutations. Our data suggest that these mutations are probably at the chromosome 17 NF1 locus.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26487/1/0000023.pd

A strategy for the characterization of minute chromosome rearrangements using multiple color fluorescence in situ hybridization with chromosome-specific DNA libraries and YAC clones

The identification of marker chromosomes in clinical and tumor cytogenetics by chromosome banding analysis can create problems. In this study, we present a strategy to define minute chromosomal rearrangements by multicolor fluorescence in situ hybridization (FISH) with whole chromosome painting probes derived from chromosome-specific DNA libraries and Alu-polymerase chain reaction (PCR) products of various region-specific yeast artificial chromosome (YAC) clones. To demonstrate the usefulness of this strategy for the characterization of chromosome rearrangements unidentifiable by banding techniques, an 8p+ marker chromosome with two extra bands present in the karyotype of a child with multiple anomalies, malformations, and severe mental retardation was investigated. A series of seven-color FISH experiments with sets of fluorochrome-labeled DNA library probes from flow-sorted chromosomes demonstrated that the additional segment on 8p+ was derived from chromosome 6. For a more detailed characterization of the marker chromosome, three-color FISH experiments with library probes specific to chromosomes 6 and 8 were performed in combination with newly established telomeric and subtelomeric YAC clones from 6q25, 6p23, and 8p23. These experiments demonstrated a trisomy 6pter6p22 and a monosomy 8pter8p23 in the patient. The present limitations for a broad application of this strategy and its possible improvements are discusse

Fetus in fetu : molecular analysis of a fetiform mass

Fetus-in-fetu is a rare condition presenting as a calcified intra- abdominal mass in the newborn infant. Over 50 cases of fetus-in-fetu have been reported since 1800. Karyotype analysis in 8 cases and protein polymorphisms in 4 documented identical findings in the host and fetiform mass. We report a case of fetus-in-fetu in a newborn female including cytogenetic and molecular studies of both the host and mass. Genotypic information from 7 polymerase chain reaction (PCR) assays representing 4 chromosomes demonstrates heterozygous and identical alleles in the infant and fetus-in-fetu at all loci studied. A review of the literature is provided including a discussion regarding the impact of molecular data on present hypotheses of fetus-in-fetu pathogenesis.9 page(s

Identification of a Human LMX1 (LMX1.1)-Related Gene, LMX1.2: Tissue-Specific Expression and Linkage Mapping on Chromosome 9

LMX1 is a LIM-homeodomain (LIM-HD)-containing protein expressed selectively in insulin-producing β-cell lines, and it it has been shown to activate insulin gene transcription. The human LMX1 gene was mapped by fluorescencein situhybridization to chromosome region 1q22–q23, yet Churchet al.(1994,Nat. Genet.6: 98–105) identified two exon-trapping products from human chromosome 9 that were highly homologous to hamster LMX1. In the current study, we demonstrate tissue-specific expression of an LMX1 (now known as LMX1.1)-related gene, named LMX1.2. The chicken C-LMX1 gene, recently cloned using the hamster LMX1.1 sequence and shown to specify dorsal cell fate during vertebrate limb development (9), is actually more related to human LMX1.2 than LMX1.1. We have identified a unique simple sequence repeat polymorphic marker (hLMX1.2CA1) in a P1 genomic clone containing the human LMX1.2 gene and genetically mapped the marker on chromosome 9 between markers D9S1825 and D9S290 with odds of at least 1000:1. In addition, we localized the human LMX1.1 gene to three CEPH “B” yeast artificial chromosome clones (907A11, 935B12, and 947B2), along with two nearby polymorphic markers (D1S426 and D1S194)). Identification of this new LIM-HD-related gene may provide the opportunity to elucidate further the function of LIM class homeobox genes. Nearby polymorphic markers will be useful in testing the hypothesis that mutations in these LIM-HD genes result in genetic diseases such as non-insulin-dependent diabetes mellitus

Linkage of preaxial polydactyly type 2 to 7q36

We have characterized a 6-generation North American Caucasian kindred segregating one form of preaxial polydactyly type 2 (PPD-2). We demonstrate linkage to the 7q36 region and describe a submicroscopic telomeric chromosomal deletion in phase with the PPD-2 phenotype. Recently, several kindreds segregating triphalangeal thumb (TPT) with and without associated hand anomalies (syndactyly and/or postaxial polydactyly)have also been linked to the subtelomeric region of chromosome 7q [Heutink et al., 1994: Nat Genet 6:287-291; Tsukurov et al., 1994: Nat Genet 6:282-286]. We demonstrate by haplotype analysis that our North American pedigree represents a PPD allele that is independent of the founder PPD allele present in the previously described kindreds.8 page(s

Identification of trinucleotide repeat-containing genes in human pancreatic islets.

In the search for diabetes genes, the combined approaches of positional cloning with random markers and subsequent evaluation of candidate genes mapping to areas of interest will be increasingly used. For islet candidate genes of unknown function, expressed trinucleotide (triplet) repeats represent a unique subset. It is unlikely that abnormal expansion of expressed islet triplet repeats would be a major cause of diabetes, yet the triplet repeats are frequently polymorphic and can thus be used to map the genes in the human genome. In this study, a human islet cDNA library was screened with (CGG)7 and (CAG)7, and 23 triplet repeats were isolated. Sequencing revealed four known and six novel islet genes containing 4-15 triplet repeats. The four known cDNAs included ferritin, the major iron-binding protein in cells; HSGSA2R, a full-length clone of the alpha-subunit of the G-regulatory protein; HUMSATB1A, a DNA-binding protein expressed predominantly in thymus; and HUMPPA-PRO, a ribosomal protein. The triplet repeats in ferritin and HUMPPAPRO were found to be monomorphic. Characterization of the six unique novel expressed islet triplet cDNAs revealed that they were 0.6-1.5 kb in size, contained 4-15 triplet repeats, and were expressed in islets and all other tissues examined. Four of the novel clones, CGG-isl 10, CGG-isl 11, CAG-isl 6, and CAG-isl 7, were mapped to human chromosomes 19, 16, 12, and 3, respectively, via somatic cell hybrids. One islet cDNA, CAG-isl 7, contained a repeat that was highly polymorphic, with 14 alleles (4-18 triplets) in African-Americans (heterozygosity = 0.86) and 6 alleles (heterozygosity = 0.77) in whites. Northern analysis indicated that the mRNA was abundant in pancreatic islets. A putative full-length clone contained an open reading frame encoding 213 amino acids with a variable number of alanines (4-18) within the COOH-terminal. The gene was uniquely mapped with odds > 1,000:1 on chromosome 3p in Centre d'Etude du Polymorphisme Humain pedigrees. There were no differences in CAG-isl 7 allele frequencies between African-American patients with NIDDM (n = 108) and control subjects (n = 116), nor was expansion above 18 repeats noted. Linkage analysis in 14 nonglucokinase maturity-onset diabetes of the young pedigrees showed a cumulative logarithm of odds score of -33.19 at theta = 0.00. Abnormal expansion was not observed in 20 IDDM patients with one NIDDM parent. While these data suggest no major role for CAG-isl 7 in diabetes, at least four of the six novel islet triplet genes are coexpressed in pancreatic islets and neural tissue, and these genes can now be considered as candidates for diabetes and/or neuropsychiatric diseases