Chromosome I linkage studies in Charcot-Marie-Tooth neuropathy type I.

Charcot-Marie-Tooth neuropathy type 1 (CMT1) is an autosomal dominant disorder of peripheral nerve. The gene for CMT1 was originally localized to chromosome 1 by linkage to the Duffy blood group, but it has since been shown that not all CMT1 pedigrees show this linkage. We report here the results of linkage studies using five chromosome 1 markers--Duffy (Fy), antithrombin III (AT3), renin (REN), beta-nerve growth factor (NGFB), and salivary amylase (AMY1)--in 16 CMT1 pedigrees. The total lod scores exclude close linkage of CMT1 to any of these markers. However, individual families show probable linkage of CMT1 to Duffy, AT3, and/or AMY1. No linkage was indicated with REN or NGFB. These results indicate the possible location of a CMT1 gene between the AMY1 and AT3 loci at p21 and q23, respectively, on chromosome 1 and support the theory that there is at least one other CMT1 gene.     

Multicolor fluorescence in situ hybridization (FISH) applied to FISH-banding

During the last decade not only multicolor fluorescence in situ hybridization (FISH) using whole chromosome paints as probes, but also numerous chromosome banding techniques based on FISH have been developed for the human and for the murine genome. This review focuses on such FISH-banding techniques, which were recently defined as 'any kind of FISH technique, which provide the possibility to characterize simultaneously several chromosomal subregions smaller than a chromosome arm. FISH-banding methods fitting that definition may have quite different characteristics, but share the ability to produce a DNA-specific chromosomal banding'. While the standard chromosome banding techniques like GTG lead to a protein-related black and white banding pattern, FISH-banding techniques are DNA-specific, more colorful and, thus, more informative. For some, even high-resolution FISH-banding techniques the development is complete and they can be used for whole genome hybridizations in one step. Other FISH-banding methods are only available for selected chromosomes and/or are still under development. FISH-banding methods have successfully been applied in research in evolution- and radiation-biology, as well as in studies on the nuclear architecture. Moreover, their suitability for diagnostic purposes has been proven in prenatal, postnatal and tumor cytogenetics, indicating that they are an important tool with the potential to partly replace the conventional banding techniques in the future.     

Genetic linkage and heterogeneity in type I Charcot-Marie-Tooth disease (hereditary motor and sensory neuropathy type I).

The segregation patterns of DNA markers from the pericentromeric regions of chromosomes 1 and 17 were studied in seven pedigrees segregating an autosomal dominant gene for Charcot-Marie-Tooth neuropathy type I (CMT I; hereditary motor and sensory neuropathy I). A multilocus analysis with four markers (pMCR-3, pMUC10, FY, and pMLAJ1) spanning the pericentromeric region of chromosome 1 excluded the CMT I gene from this region in six pedigrees but gave some evidence for linkage to the region of Duffy in one pedigree. Linkage of the CMT I gene to markers in the pericentromeric region of chromosome 17 (markers pA10-41, pEW301, p3.6, and pTH17.19) was established; however, in these seven pedigrees homogeneity analysis with chromosome 17 markers detected significant genetic heterogeneity. This analysis suggested that three of the seven pedigrees are not linked to this same region. Overall, two of the seven CMT I pedigrees were not linked to markers tested from chromosomes 1 or 17. These results confirm genetic heterogeneity in CMT I and implicate the existence of a third autosomal locus, in addition to a locus on chromosome 17, and a probable locus on chromosome 1. This evidence of etiological heterogeneity, supported by statistical tests, will have to be taken into consideration when fine-structure genetic maps of the regions around CMT I are constructed.     

Segregation analysis incorporating linkage markers. I. Single-locus models with an application to type I diabetes.

A method is described for segregation analysis that incorporates linkage markers. The model allows for segregation (penetrance), linkage (recombination fraction), and association (linkage disequilibrium) parameters. A single-locus-multiple-allele model underlying the trait phenotype is assumed. When families have been ascertained in a systematic fashion, a joint (markers, phenotypes) likelihood with ascertainment is advocated. When ascertainment correction is not feasible, a conditional (markers given phenotypes) approach is recommended, which is also valid in the presence of reduced fertility and assortative mating. This approach, oriented toward determining mode of inheritance, differs from conventional linkage analysis, which is oriented toward detection of linkage. Therefore, it is more appropriately considered an extension of the affected sib-pair method to arbitrary pedigrees, including association information and allowing for multiple alleles. Incorporation of coupling parameters allows for discrimination between pleiotropy and linkage disequilibrium. The method is demonstrated through a reanalysis of four recently published family studies on type 1 diabetes and HLA. Recessive inheritance is rejected in all four data sets. For three of them, dominant inheritance is not rejected, while in the fourth, all two-allele models are rejected in favor of three alleles. Although association with the DR3 and DR4 alleles is quite strong, pleiotropy with regard to these alleles is unlikely. The results also suggest an additional familial factor(s) (e.g., locus).     

Molecular analyses of unrelated Charcot-Marie-Tooth (CMT) disease patients suggest a high frequency of the CMTIA duplication.

Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral neuropathy. One form of CMT, CMT type 1A, is characterized by uniformly decreased nerve conduction velocities, usually shows autosomal dominant inheritance, and is associated with a large submicroscopic duplication of the p11.2-p12 region of chromosome 17. A cohort of 75 unrelated patients diagnosed clinically with CMT and evaluated by electrophysiological methods were analyzed molecularly for the presence of the CMT1A DNA duplication. Three methodologies were used to assess the duplication: measurement of dosage differences between RFLP alleles, analysis of polymorphic (GT)n repeats, and detection of a junction fragment by pulsed-field gel electrophoresis. The CMT1A duplication was found in 68% of the 63 unrelated CMT patients with electrophysiological studies consistent with CMT type 1 (CMT1). The CMT1A duplication was detected as a de novo event in two CMT1 families. Twelve CMT patients who did not have decreased nerve conduction velocities consistent with a diagnosis of CMT type 2 (CMT2) were found not to have the CMT1A duplication. The most informative molecular method was the detection of the CMT1A duplication-specific junction fragment. Given the high frequency of the CMT1A duplication in CMT patients and the high frequency of new mutations, we conclude that a molecular test for the CMT1A DNA duplication is very useful in the differential diagnosis of patients with peripheral neuropathies.     

Multicolor fluorescence in situ hybridization (M-FISH) on cells from urine for the detection of bladder cancer

Bladder cancer is the fifth most common cancer in adults. Because of the high recurrence rate (up to 70%) new tumor markers for urine are necessary for monitoring patients. In this study, we investigated the value of M-FISH on cells from urine for the detection of bladder cancer. Urine samples from 141 patients suspicious of bladder cancer were analyzed in this study. Cells were isolated from urine before surgical therapy. For FISH analysis, a commercial kit (UroVysion) containing hybridization probes for chromosomes 3, 7, 9p21 and 17, was used. Twenty-five cells were analyzed in each case by two observers. A FISH result was obtained in 121 cases. Overall, sensitivity was 60% and specificity reached 82.6%. Sensitivity and specificity by cytology were 24.1% and 90.5%, respectively. Analyzing results concerning T-category, sensitivity of FISH and cytology was 36.1% and 15% in pTa, 65.2 and 25.7% in pT1, 100% and 66.7% in pT2-3 tumors, respectively. Concerning tumor grade, similar results were obtained: sensitivity was 37% and 14% in G1, 65.4% and 40% in G2, 91.7% and 50% in G3 tumors, for FISH and cytology, respectively. In conclusion, FISH on cells from urine has been shown in all studies to be highly sensitive and specific for detection of bladder cancer. Sensitivity of FISH is higher than conventional cytology and can be used in routine diagnosis additionally to conventional cytology especially in doubtful or negative cases. FISH can detect recurrence earlier than other methods like cytology, cystoscopy or biopsy histological examination.     

Molecular cytogenetic analysis of wheat-barley hybrids using genomic in situ hybridization and barley microsatellite markers.

In the present investigation, genomic in situ hybridization (GISH) and barley microsatellite markers were used to analyse the genome constitution of wheat-barley hybrids from two backcross generations (BC1 and BC2). Two BC1 plants carried 3 and 6 barley chromosomes, respectively, according to GISH data. Additional chromosomal fragments were detected using microsatellites. Five BC2 plants possessed complete barley chromosomes or chromosome segments and six BC2 plants did not preserve barley genetic material. Molecular markers revealed segments of the barley genome with the size of one marker only, which probably resulted from recombination between wheat and barley chromosomes. The screening of backcrossed populations from intergeneric hybrids could be effectively conducted using both genomic in situ hybridization and molecular microsatellite markers. GISH images presented a general overview of the genome constitution of the hybrid plants, while microsatellite analysis revealed the genetic identity of the alien chromosomes and chromosomal segments introgressed. These methods were complementary and provided comprehensive information about the genomic constitution of the plants produced.     

Multicolor fluorescence in situ hybridization with combinatorial labeling probes enables a detailed karyotype analysis of Larix principis-rupprechtii

The chromosomes (2n = 2x = 24) of Larix principis-rupprechtii are composed of six pairs of large metacentrics and six pairs of medium-sized submetacentrics. The identification of homologous pairs is hampered by their high degree of similarity at the morphological level in each group. As one of the most extensively used methods in molecular cytogenetics producing chromosome landmarks, fluorescence in situ hybridization (FISH) has significantly facilitated karyotype construction, especially in species with morphologically similar chromosomes. This study developed a simple but effective use of combinatorial labeling probes to distinguish chromosomes of Larix principis-rupprechtii by multicolor FISH. Three highly repetitive sequences in Larix were selected: 25S rDNA hybridized at all of the secondary constrictions of two pairs of metacentrics and the largest pair of submetacentrics; 5S rDNA hybridized at subtelomeric sites of one pair of metacentrics that also harboured 25S rDNA on different arms; LPD family sequences are tandem repeats hybridized at proximal regions of 22 chromosomes. The three different probes were labeled with only two different labels, hybridized to metaphase chromosomes of Larix principis-rupprechtii, simultaneously visualized, and unequivocally distinguished in a single FISH experiment. These multicolor FISH marks largely improved the karyotype analysis of Larix principis-rupprechtii.     

Multipoint linkage analysis in neurofibromatosis type I: an international collaboration.

In addition to reporting, in accompanying papers, their individual analyses of mapping the neurofibromatosis type 1 (NF1) gene on chromosome 17, members of the International Consortium for NF1 Linkage contributed their data for our joint analysis to determine the exact sequence of flanking markers and to obtain precise estimates and confidence limits of the recombination fractions for the closest markers, in anticipation of clinical use. With specimens from 142 families and more than 700 affected persons, eight teams used 31 markers in the pericentric region of chromosome 17 to perform 13,838 genotypings. With the combined data, we used the computer program CRI-MAP to build the most likely sequence of loci by sequentially adding single loci to a fixed pair of loci and separately calculating the likelihood of all permutations of four consecutive loci. The best order is pter-pA10-41-EW301-centromere (p17H8)-pHHH202-NF1-EW206-EW207-EW203++ +-CRI-L581-CRI-L946-HOX2-NGFR-qter. The total genetic distance from pA10-41 to NGFR is 26 cM in males and 56 cM in females, and the overall difference in sex-specific maps is statistically significant (P = .006). The upper 99% confidence limits of the recombination fraction of the closest proximal marker, pHHH202, is 4%, and that for the closest distal marker, EW206, is 9%. These limits should decrease with the use of additional probes and the further evaluation of DNA from the six persons showing multiple recombinations within short genetic distances. Clinical application is technically feasible with currently available markers, although its appropriate use for prenatal and presymptomatic diagnosis requires further discussion and evaluation.     

Sequential chromosome banding and in situ hybridization analysis.

Different combinations of chromosome N- or C-banding with in situ hybridization (ISH) or genomic in situ hybridization (GISH) were sequentially performed on metaphase chromosomes of wheat. A modified N-banding-ISH/GISH sequential procedure gave best results. Similarly, a modified C-banding - ISH/GISH procedure also gave satisfactory results. The variation of the hot acid treatment in the standard chromosome N- or C-banding procedures was the major factor affecting the resolution of the subsequent ISH and GISH. By the sequential chromosome banding - ISH/GISH analysis, multicopy DNA sequences and the breakpoints of wheat-alien translocations were directly allocated to specific chromosomes of wheat. The sequential chromosome banding- ISH/GISH technique should be widely applicable in genome mapping, especially in cytogenetic and molecular mapping of heterochromatic and euchromatic regions of plant and animal chromosomes.     

The mouse IFN-alpha (Ifa) locus: correlation of physical and linkage maps by in situ hybridization

The physical location of the mouse IFN-alpha locus (Ifa) on chromosome 4 was defined by in situ hybridization of a cloned mouse IFN-alpha probe to metaphase spreads in which one chromosome 4 was present as part of a single metacentric chromosome, all other chromosomes being acrocentric. (This approach greatly facilitates analysis and can be used even when it is difficult to obtain good banding). Using unbanded chromosomes, the grains were localized over the chromosome 4 part of the metacentric, in a region 0.61 +/- 0.07 (SD) of the distance from the centromere to the telomere. In Giemsa-banded spreads, the majority of the grains were in the region 4C3----C6. Consideration of these results and of the known linkage maps for mouse and man indicates that the Galt - Aco-1 - Ifa syntenic group spans a distance of approximately 14 cM and suggests that the same group on human 9p will also occupy a similarly sized region, with GALT proximal and IFL distal to the centromere.     

Bayesian analysis of linkage between genetic markers and quantitative trait loci. I. Prior knowledge

Summary Prior information on gene effects at individual quantitative trait loci (QTL) and on recombination rates between marker loci and QTL is derived. The prior distribution of QTL gene effects is assumed to be exponential with major effects less likely than minor ones. The prior probability of linkage between a marker and another single locus is a function of the number and length of chromosomes, and of the map function relating recombination rate to genetic distance among loci. The prior probability of linkage between a marker locus and a quantitative trait depends additionally on the number of detectable QTL, which may be determined from total additive genetic variance and minimum detectable QTL effect. The use of this prior information should improve linkage tests and estimates of QTL effects.     

Exclusion analysis of Charcot-Marie-Tooth neuropathy (CMT1) with chromosome 1p markers

We previously described a large five-generation family with autosomal dominant inheritance of hereditary motor and sensory neuropathy type I, or Charcot-Marie-Tooth disease (CMT1). The genetic defect in this family was not linked to the Duffy blood group. We investigated the possibility of a disease locus on the short arm of chromosome 1 using 12 anonymous DNA markers. Two markers, D1S2 and D1S22, showed positive linkage, suggesting the existence of a CMT1 locus on 1p. D1S2 and D1S22 are clustered in the 1p31----p22 region. However, multipoint linkage analysis, including additional DNA markers from this chromosome region, excluded a possible CMT1 locus in this part of chromosome 1.     

Multicolor fluorescence in situ hybridization and pulsed field electrophoresis dissect CMT1B gene region

Summary We have used multicolor fluoresence in situ hybridization of banded chromosomes to orient FcRII and clone 1054 on a single early metaphase chromosome band (1q22) representing about 2% of the physical map of chromosome 1 in the Charcot-Marie-Tooth (CMT1B) gene region. These two cloned fragments are on the same partially digested 900-kb MluI fragment detected by pulsed field gel electrophoresis. When applied to data from an earlier study, multicolor in situ hybridization results further refined the CMT1B genetic location from an 18 cM interval to a 6 cM interval and the physical map from 15% of chromosome 1 to 3% of chromosome 1. Occasionally the three FcRII immunoglobulin receptor genes within the 200-kb region are resolved in individual metaphase chromatids.     

Using fluorescence in situ hybridization (FISH) in genome mapping.

Fluorescence in situ hybridization (FISH) provides one of the most effective and rapid approaches for assigning and ordering DNA fragments within single eukaryotic chromosome bands. These techniques have wide applications not only for the mapping of the human genome and the genomes of other organisms, but also in clinical cytogenetics, somatic cell genetics, cancer diagnosis and gene expression studies.     

Genomic in situ hybridization analysis between Rubus coreanus and its relatives in Rubus (Sect. Idaeobatus)

To further investigate the phylogenetic relationship between Rubus coreanus and its relatives in the section Idaeobatus, we used genomic in situ hybridization (GISH) to ascertain the degree of their genomic homology. Genomic DNA from R. parvifolius and R. inopertus hybridized throughout the centromeric and sub-terminal regions on 14 and 12 chromosomes of R. coreanus, respectively. The probes from R. niveus and R. ellipticus var. obcordatus gave robust signals at the same region of eight chromosomes. R. ellipticus and R. pinfaensis generated strong signals at the centromeric and sub-terminal parts of six chromosomes. The hybridization signals from the R. tsangii and R. corchorifolius probes existed only at the telomeric parts of four chromosomes. The two signals at the sub-terminal region on chromosome 6 of R. coreanus might be 45S rDNA repeats. These results indicated that R. coreanus and R. parvifolius shared many repeat sequences. It could be deduced that the genome of R. parvifolius was most closely related to that of R. coreanus among the species tested, R. inopertus came next, while R. tsangii and R. corchorifolius showed the farthest relationship. The phylogenetic relationships between R. parvifolius and R. coreanus, as well as among the five subsections were mainly discussed.     

Multipoint linkage analysis of steroid sulfatase (X-linked ichthyosis) and distal Xp markers

Six families with steroid sulfatase deficiency (STS; X-linked ichthyosis) have been studied with the Xg blood group (XG) and the DNA markers dic56 (DXS143), 782 (DXS85), pD2 (DXS43), and GMGX9. Carrier status of females was determined by assay of STS in hair roots. GMGX9 detects a frequent restriction fragment length polymorphism and also identifies a deletion in the majority of families with STS deficiency, including five of the six reported here. The linkage relationship of this marker to the others was studied in normal three-generation families yielding 32 phase-known meioses informative for two or more markers. No recombinants were observed between STS and GMGX9, giving a maximum lod score of 8.73 at zero recombination. Multipoint linkage analysis taking STS and GMGX9 as a single locus and incorporating two-point marker data and STS-XG data from published studies gave the map (Sequence: see text). This order was 2.4 times more likely than with (STS,GMGX9) and dic56 reversed and is supported by our findings in a male with steroid sulfatase deficiency due to a deletion of Xp22.3 which encompasses the XG locus. He is deleted for GMGX9 but shows normal hybridization to dic56 and 782.