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C S Dobkin

Publications and source records attributed to C S Dobkin.

At least 19 recordsLinked to original sources
PubMed1999

Prenatal fragile X detection using cytoplasmic and nuclear-specific monoclonal antibodies.

We have been carrying out studies aimed at improving prenatal detection of the fragile X chromosome/mutation. Our current protocol requires a turnaround time (TAT) of several days. In an attempt to reduce the TAT, we have turned to the use of monoclonal antibodies (mAbs). Monoclonal antibody 1A1 (provided by Dr. Mandel of INSERM) immunostaining was performed according to a modified three-step immunocytochemical procedure. We found that cytoplasmic staining intensities, using mAb 1A1/avidin biotinylated complex/diaminobenzidine, varied from light to heavy within each sample, with controls exhibiting a majority of heavily stained cells in both chorionic villus (CV) sample and amniotic fluid cultured cells. Using mAb 1A1 and a new nuclear-specific antibody, mAb 3F11, we found that CV cultured cells harboring the FMR1 full mutation could be distinguished from controls as early as 10 weeks of gestation in both male and female specimens. Western blot analysis showed that the antibodies have similar staining patterns but that mAb 3F11 has fewer background/nonspecific bands. Our results demonstrate that it is feasible to detect fragile X full mutations within one day after obtaining cells from CV specimens taken as early as 10 weeks of gestation.

Amino Acid Sequence↗
PubMed1999

FMR1 CGG-repeat instability in single sperm and lymphocytes of fragile-X premutation males.

To determine the meiotic instability of the CGG-triplet repeat in the fragile-X gene, FMR1, we examined the size of the repeat in single sperm from four premutation males. The males had CGG-repeat sizes of 68, 75, 78, and 100, as determined in peripheral blood samples. All samples showed a broad range of variations, with expansions more common than contractions. Examination of single lymphocytes indicated that somatic cells were relatively more stable than sperm. Surprisingly, the repeats in sperm from the 75- and 78-repeat males had very different size ranges and distribution patterns despite the similarity of the repeat size and AGG interruption in their somatic cells. These results suggest that cis or trans factors may have a role in male germline repeat instability.

Adult↗
PubMed1999

Premutation female carriers of fragile X syndrome: a pilot study on brain anatomy and metabolism.

OBJECTIVE: It was thought that premutation carriers of fragile X syndrome (FraX) have no neurobiological abnormalities, but there have been no quantitative studies of brain morphometry and metabolism. Thus the authors investigated brain structure and metabolism in premutation carriers of FraX. METHOD: Eight normal IQ, healthy female permutation FraX carriers aged 39 +/- 9 years (mean +/- SD) and 32 age-sex-handedness-matched controls (39 +/- 10 years) were studied; in vivo brain morphometry was measured using volumetric magnetic resonances imaging, and regional cerebral metabolic rates for glucose were measured using positron emission tomography and (18F)-2-fluoro-2-deoxy-D-glucose. RESULTS: Compared with controls, FraX premutation carriers had a significant (1) decrease in volume of whole brain, and caudate and thalamic nuclei bilaterally; (2) increase in volume of hippocampus and peripheral CSF bilaterally, and third ventricle; (3) relative hypometabolism of right parietal, temporal, and occipital association areas; (4) bilateral relative hypermetabolism of hippocampus; (5) relative hypermetabolism of left cerebellum; and (6) difference in right-left asymmetry of the Wernicke and Broca language areas. CONCLUSIONS: Premutation carriers of FraX, as defined by analysis of peripheral lymphocytes, have abnormalities in brain anatomy and metabolism. The biological basis for this is unknown, but most likely it includes tissue heterogeneity for mutation status. The findings may be of relevance to people counseling families with FraX and to understanding other neuropsychiatric disorders which are associated with expansion of triplet repeats and genetic anticipation.

Adult↗
PubMed1996

Tissue differences in fragile X mosaics: mosaicism in blood cells may differ greatly from skin.

The fragile X mutation is diagnosed from the structure of the FMR1 gene in blood cell DNA. An estimated 12 to 41% of affected males are mosaics who carry both a "full mutation" allele from which there is no gene expression and a "premutation" allele which has normal gene expression. We compared the DNA in blood cells and skin fibroblasts from four mosaic fragile X males to see if there was a difference in the relative amounts of premutation and full mutation alleles within the tissues of these individuals. Two of these males showed striking differences in the ratio of premutation to full mutation in different tissues while the other two showed only slight differences. These observations conform with the widely accepted hypothesis that the fragile X CGG repeat is unstable in somatic tissue during early embryogenesis. Accordingly, the mosaicism in brain and skin, which are both ectodermal in origin, may be similar to each other but different from blood which is not ectodermal in origin. Thus, the ratio of full mutation to premutation allele in skin fibroblasts might be a better indicator of psychological impairment than the ratio in blood cells.

Blood Cells↗
PubMed1996

Mosaicism for the FMR1 gene influences adaptive skills development in fragile X-affected males.

Fragile X syndrome is one of the most common forms of inherited mental retardation, and the first of a new class of genetic disorders associated with expanded trinucleotide repeats. Previously, we found that about 41% of affected males are mosaic for this mutation in that some of their blood cells have an active fragile X gene and others do not. It has been hypothesized that these mosaic cases should show higher levels of functioning than those who have only the inactive full mutation gene, but previous studies have provided negative or equivocal results. In the present study, the cross-sectional development of communication, self-care, socialization, and motor skills was studied in 46 males with fragile X syndrome under age 20 years as a function of two variables: age and the presence or absence of mosaicism. The rate of adaptive skills development was 2-4 times as great in mosaic cases as in full mutation cases. There was also a trend for cases with autism to be more prevalent in the full-mutation group. These results have implications for prognosis, for the utility of gene or protein replacement therapies for this disorder, and for understanding the association between mental retardation, developmental disorders, and fragile X syndrome.

Adolescent↗
PubMed1995

Cognitive and molecular aspects of fragile X.

The relationships among parental origin of the fragile X gene, gene structure, and specific cognitive deficits were evaluated in nonretarded adult female fragile X carriers to determine whether: (1) origin influences gene structure and cognitive function, (2) mild cognitive impairments are associated with altered gene structure, and (3) specific cognitive domains are affected. Results indicated that 56% of women with maternally inherited, but none with paternally inherited, fragile X showed large genomic structural alterations and selective deficits on measures of visual attention. Thus, molecular status of the fragile X gene appears to be linked to parental origin and may selectively affect specific cognitive skills.

Adult↗
PubMed1994

Fragile X induction systems in CVS cultures: effect on cytogenetic, PCR, and genomic Southern Blot DNA analyses of the FMR-1 gene.

Low fragile X frequencies have been commonly observed in chorionic villus sample (CVS) cultures, compared to subsequent analysis in whole blood or products of conception (POC). To investigate possible mechanisms for this effect, CVS cultures from a previously identified fragile X positive male, were restudied and compared to subsequent POC cultures from lung, muscle, skin, and thymus. Cultures were exposed, for the last 24 hours before harvesting, to FUdR, excess thymidine, and a combination of both. For CVS, only those cultures that were exposed to a combination of FUdR and excess thymidine showed positive cytogenetic findings (1/90 or 1.1%), agreeing with our original positive cytogenetic results (2/86 or 2.3%) for cultures exposed to excess thymidine. Fragile X frequencies in the POC tissues from this fetus increased to an average of 14%. PCR analyses showed full mutations (> 200 CGG repeats) in uninduced CVS cultures but induced cultures exhibited apparently smaller sizes in the range of 120-180 repeats. The results showed variability. In one instance, the banding pattern from one of the uninduced cultures was similar to the results where cultures were exposed to a double induction system. When PCR analyses were conducted on induced POC cultures, full mutations were observed in virtually all samples. Southern blot genomic analysis using probe StB12.3 showed an unmethylated full mutation in CVS cultures. Southern blot patterns from cultures of muscle revealed size variations of DNA bands in the premutation range representing unmethylated DNA as well as methylated full mutations. Finally, variations were also observed in lung and skin cultures, compared to CVS and muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

Blotting, Southern↗
PubMed1994

Mosaicism in fragile X affected males.

Fragile X affected males have an expansion of a CGG repeat and a hypermethylated CpG island 5' to the FMR-1 gene. Mosaic males with both a premutation and full mutation have been noted among the affected individuals. Such mosaic males are most easily identified by the presence of a methylated restriction fragment characteristic of the full mutation and an additional unmethylated fragment in the premutation range in Southern analyses with EcoR I and the methylation-sensitive enzyme Eag I and a probe such as StB12.3. We analyzed a group of affected fragile X males by Southern blotting and found 41% (61/148) to be mosaic. The 148 individuals were divided between 36 pairs of brothers and 76 unrelated males. Little difference in the number of mosaics was seen between the brothers and the unrelated males nor was the expected distribution of mosaicism in brother pairs different from observed. Thus, these data do not suggest a familial basis for mosaicism. Our observation that 41% affected fragile X males were mosaic is significantly higher than previous reports. The difference is likely due to technical modifications which permitted the identification of faint premutation bands in some patients. The high percentage of affected males with mosaicism seen here suggests that the occurrence of such individuals may be a much more frequent event than presently recognized.

Blotting, Southern↗
PubMed1989

Gamma delta beta-thalassemia due to a de novo mutation deleting the 5' beta-globin gene activation-region hypersensitive sites.

gamma delta beta-Thalassemia is a rare disorder of hemoglobin biosynthesis, characterized molecularly by partial or complete deletions of the beta-globin gene complex of 100 kilobases (kb) or greater. Common to all mutants described has been the deletion of the most-5' sequences of the beta-globin complex. We have used the techniques of pulsed-field gel electrophoresis and polymerase chain reaction to study a patient with a clinical gamma delta beta-thalassemia phenotype. This subject developed a de novo deletion on a maternally inherited beta-globin gene chromosome involving approximately 30 kb of sequences 5' to the epsilon gene; the deletion extends from -9.5 kb to -39 kb 5' of epsilon and includes three of the four DNase I hypersensitive sites (at -10.9 kb, -14.7 kb, and -18 kb 5' of epsilon). The remaining sequences of the beta-globin complex, including the DNase I hypersensitive sites at -6.1 kb and all structural genes in cis to the deletion are physically intact, but presumably nonfunctional, as evidenced by the presence of a beta S-globin gene that is not expressed as a sickle hemoglobin. Deletion of DNase I hypersensitive sites on a previously functional beta-globin gene complex confirms the significance of these sites in regulating globin gene expression.

Base Sequence↗
PubMed1987

Hypothesis regarding the nature of the fragile X mutation. A reply to Winter and Pembrey.

Pembrey et al. (1985) proposed a hypothesis regarding the nature of the fragile X [fra(X)] mutation. Recently they analyzed DNA linkage data (Winter and Pembrey 1986) that we and others have published on fra(X) pedigrees, found significant linkage heterogeneity, and modified their hypothesis to explain the observations. We would like to point out that their modified hypothesis is not supported by the data available.

Fragile X Syndrome↗
PubMed1982

Five nucleotide changes in the large intervening sequence of a beta globin gene in a beta+ thalassemia patient.

A beta globin gene from a patient with homozygous beta+ thalassemia has been cloned and completely sequenced. No changes from normal are found in the 200 nucleotides 5' to the cap site, in the 3' untranslated region up to the poly A addition site, in the small intervening sequence (IVS 1), or in the coding sequence except for a third base change in codon 2. The only other differences are in the large intervening sequence (IVS 2). One of these, at a position 16 nucleotides from the 5' end of IVS 2, has been reported previously in normal individuals, and is probably a polymorphism. Four other changes, at positions 74, 81, 666, and 705 are also seen in IVS 2. Abnormal beta globin mRNA precursors detected in the bone marrow cells of this patient, and abnormal beta globin RNA splicing observed when this gene is transcribed in a tissue culture system taken together with these IVS 2 changes, suggest that the beta+ thalassemia phenotype is produced by a decrease in normal beta globin mRNA processing.

Base Sequence↗
PubMedDate not supplied

In situ nick translation of the fragile X region.

At the present time, the molecular nature of the fragile site at Xq27.3 is not well understood. To examine the sensitivity of this region to DNAase I, in situ nick translation was performed on metaphase chromosomes from a fragile X (fra(X] positive individual. In this technique DNAase I is used to nick regions of chromosomal DNA that are in "open" conformation. Biotinylated dUTP was incorporated by nick translation at these sites. The incorporation was identified by double antibody labeling and avidin-horseradish peroxidase staining. Spreads, which had been stained with this technique, were photographed and subsequently trypsin-Giemsa G banded (post-GTG banded) for chromosome identification. In 36 of 44 (82%) fra(X) positive male cells, the region distal to fra(X) (q27.3) was prominently stained in contrast to its light staining appearance in GTG preparations. The fragile site itself was outlined more clearly than can be achieved by GTG or homogeneous staining. When autosomal fragile sites were induced by the addition of 1.5 microM aphidicolin 17 hours prior to harvest, 24 of 27 (89%) fragile sites on the ends of autosomes were prominently stained in regions distal to the break. Because the fra(X) and autosomal fragile regions behaved similarly, this suggests that they have a similar conformation. Thus, while autosomal and Xq27.3 fragile sites are strongly induced by different means, the organization of these sites and the regions distal to them appear to be similar.

Chromosome Banding↗
PubMedDate not supplied

Multipoint linkage of 9 anonymous probes to HPRT, factor 9, and fragile X.

We have analyzed the segregation of restriction fragment length polymorphisms (RFLPs) associated with 9 anonymous probes detecting loci DXS10, DXS15, DXS19, DXS37, DXS51, DXS52, DXS98, DXS99, and DXS100 and probes for HPRT and F9 in a set of 40 families segregating fragile X (fra(X]. Using two-point and multipoint analysis, we have established their relative genetic locations. The results indicate that DXS99 and DXS10, unlike previous reports, are not tightly linked to F9. A new locus was found to map within the F9 - fra(X) region. DXS98 showed 6% recombination with fra(X) and appeared to be the closest locus to fra(X). These results will be useful for mapping the relative position of newly defined X probes in this region and for future genetic studies of families with fra(X), hemophilia B, or Lesch-Nyhan mutations.

Chromosome Mapping↗
PubMedDate not supplied

Pulsed-field gradient-gel studies around the fragile site.

Using pulsed-field gradient-gel electrophoresis (PFGE) we compared two fragile X (fra(X] chromosomes from individuals in families that exhibit linkage heterogeneity between fra(X) and coagulation factor IX (F9). The analysis of very large restriction fragments indicated that there is a structural difference in the interval between fra(X) and F9 near the locus DXS105. Differences were observed in the Sfi I partial digestion analysis and in the Mlu I pattern of the DXS105 region. Digestion with Nru I and Sst II also showed differences between these alleles. The analyses suggest that the alleles differ in a region of greater than 200 kb. Analysis of other normal and fra(X) chromosomes will be necessary to determine whether the observed difference is a normal population variant or if it may be responsible for the linkage heterogeneity observed between these loci.

Chromosome Fragile Sites↗
PubMedDate not supplied

Fragile X screening program in New York State.

Most fragile X [fra(X)] males in New York State have not been identified. Hence, a large number of female relatives are unaware of their risks for having an affected child. A program was established in New York State in 1987 to screen for the fra(X) syndrome in mentally retarded males with living relatives. The goal of the program is to identify affected males and inform their families about the diagnosis. In this way relatives would be able to assess their risks for having a fra(X) male. In order to identify the males a screening form was developed to assess 10 features which included physical characteristics, behavior, and family history. Males who exhibited at least 5 of these manifestations were selected for cytogenetic analysis. Any male who had macroorchidism or a family history of mental retardation was also included. A total of 995 males have been screened of which 352 (35%) were selected for cytogenetic analyses. Seventeen (10.5%) of the 161 completed studies were positive for fra(X). A large number of possible female carriers were identified in the families of the propositi. This program identifies fra(X) males in a population of the mentally retarded for whom there had been no previous diagnosis. By using a two-step procedure, it is possible to screen a large population of the mentally retarded for fra(X) without testing each male cytogenetically.

Abnormalities, Multiple↗
PubMedDate not supplied

Polymerase chain reaction detection of the Dde I polymorphism in the factor 9 gene for fragile X linkage analysis.

We have used the polymerase chain reaction (PCR) to analyze the 50 base pair (bp) insertion/deletion polymorphism in the coagulation factor 9 gene. This procedure is particularly applicable for DNA marker studies in fragile X families. The polymorphism, which can also be detected in Dde I digestions, was detected by the amplification of fragments of 298 and 348 bp. The alleles were distinguished directly by agarose gel electrophoresis. PCR detection of this polymorphism is much simpler, more accurate, and quicker than conventional analysis.

Alleles↗
PubMedDate not supplied

Cloning the ends of size selected Sfi I fragments.

As an initial step in the physical mapping of the fragile X region a library of Sfi I ends was constructed from the size class of human Sfi I DNA fragments, which includes the fragment with the locus DXS105. Since Sfi I recognizes the sequence GGCCNNNNNGGCC and leaves a 3 base indeterminate "sticky" end, we used a mixture of 64 synthetic deoxynucleotide oligomers to modify these ends for cloning. The oligomers were of the general form AATTNNN. Ligation of these heptamers to the indeterminate Sfi I ends converted them to the EcoR I sticky end. A suppressor tRNA gene was ligated onto this end as a selectable marker and the DNA was cloned in the lambda phage vector Charon 21A. Analysis showed that clones selected for the presence of the tRNA gene contained Sfi I ends. Because this library was constructed from a specific size class of fragments, it was very reduced in complexity. This will simplify the process of identifying the clone which contains the end of the DXS105 fragment. The use of this strategy for chromosome "jumping" is discussed.

Bacteriophage lambda↗