Boleslaw Goldman

Genotyping of patients with sporadic and radiation-associated meningiomas.

Ionizing radiation is the most established risk factor for meningioma formation. Our aim was to evaluate the main effect of selected candidate genes on the development of meningioma and their possible interaction with ionizing radiation in the causation of this tumor. The total study population included 440 cases and controls: 150 meningioma patients who were irradiated for tinea capitis in childhood, 129 individuals who were similarly irradiated but did not develop meningioma, 69 meningioma patients with no previous history of irradiation, and 92 asymptomatic population controls. DNA from peripheral blood samples was genotyped for single nucleotide polymorphisms (SNP) in 12 genes: NF2, XRCC1, XRCC3, XRCC5, ERCC2, Ki-ras, p16, cyclin D1, PTEN, E-cadherin, TGFB1, and TGFBR2. SNP analysis was done using the MassArray system (Sequenom, San Diego, CA) and computerized analysis by SpectroTYPER. Logistic regressions were applied to evaluate main effect of each gene on meningioma formation and interaction between gene and radiation. Intragenic SNPs in the Ki-ras and ERCC2 genes were associated with meningioma risk (odds ratio, 1.76; 95% confidence interval, 1.07-2.92 and odds ratio, 1.68; 95% confidence interval, 1.00-2.84, respectively). A significant interaction was found between radiation and cyclin D1 and p16 SNPs (P for interaction = 0.005 and 0.057, respectively). Our findings suggest that Ki-ras and ERCC2 SNPs are possible markers for meningioma formation, whereas cyclin D1 and p16 SNPs may be markers of genes that have an inverse effect on the risk to develop meningioma in irradiated and nonirradiated populations.

Origin and spread of the 1278insTATC mutation causing Tay-Sachs disease in Ashkenazi Jews: genetic drift as a robust and parsimonious hypothesis

The 1278insTATC is the most prevalent β-hexosaminidasexa0A (HEXA) gene mutation causing Tay-Sachs disease (TSD), one of the four lysosomal storage diseases (LSDs) occurring at elevated frequencies among Ashkenazi Jews (AJs). To investigate the genetic history of this mutation in the AJ population, a conserved haplotype (D15S981:175–D15S131:240–D15S1050:284–D15S197:144–D15S188:418) was identified in 1278insTATC chromosomes from 55 unrelated AJ individuals (15 homozygotes and 40 heterozygotes for the TSD mutation), suggesting the occurrence of a common founder. When two methods were used for analysis of linkage disequilibrium (LD) between flanking polymorphic markers and the disease locus and for the study of the decay of LD over time, the estimated age of the insertion was found to be 40±12 generations (95% confidence interval: 30–50 generations), so that the most recent common ancestor of the mutation-bearing chromosomes would date to the 8th–9th century. This corresponds with the demographic expansion of AJs in central Europe, following the founding of the Ashkenaz settlement in the early Middle Ages. The results are consistent with the geographic distribution of the main TSD mutation, 1278insTATC being more common in central Europe, and with the coalescent times of mutations causing two other LSDs, Gaucher disease and mucolipidosis typexa0IV. Evidence for the absence of a determinant positive selection (heterozygote advantage) over the mutation is provided by a comparison between the estimated age of 1278insTATC and the probability of the current AJ frequency of the mutant allele as a function of its age, calculated by use of a branching-process model. Therefore, the founder effect in a rapidly expanding population arising from a bottleneck provides a robust parsimonious hypothesis explaining the spread of 1278insTATC-linked TSD in AJ individuals.

True hermaphroditism with ambiguous genitalia due to a complicated mosaic karyotype: clinical features, cytogenetic findings, and literature review.

Abnormal recombination between the X and Y chromosomes during meiosis, occurring outside the pseudoautosomal region, can result in translocation of the SRY gene from the Y to the X chromosome, and consequently in abnormal sexual differentiation, such as the development of 46,XX males or true hermaphroditism. In this report we present clinical, cytogenetic, and molecular‐cytogenetic data of a patient with ambiguous genitalia and true hermaphroditism, who had a unique mosaic karyotype, comprising three different cell lines: 46,XXSRY+, 45,XSRY+, and 45,X. The mosaic karyotype of our patient probably represents two different events: abnormal recombination between the X and Y chromosomes during paternal meiosis, and postzygotic loss of one of the X chromosomes. Replication studies demonstrated that in 80% of the XX cells, the SRY sequence was located on the active X chromosome. This finding suggests nonrandom X inactivation and, together with the presence of the SRY gene, explains the male phenotype of our patient. On the other hand, the presence of the 45,X cell line may have contributed to genital ambiguity. We conclude that fluorescence in situ hybridization (FISH) analysis with SRY probes is highly recommended and allows accurate diagnosis and optimal management in cases of 46,XX hermaphroditism and ambiguous genitalia.

Detection of Tay-Sachs disease carriers among individuals with thermolabile hexosaminidase B.

The determination of hexosaminidases A and B in most programmes for Tay-Sachs disease carrier detection is based on their different heat sensitivity (hexosaminidase A is the heat labile isoenzyme). This routine cannot be employed for individuals who also possess a thermolabile hexosaminidase B. In Israel, 0.6% of the screened samples have a labile hexosaminidases B (about 110 each year) and the assessment of their hexosaminidase A activity has hitherto been based on isoenzyme separation by ion exchange chromatography. The latter requires relative large serum samples, and the individuals must usually be reappointed. In order to avoid the thermal treatment we have used the alternative technique, which employs two substrates with different specificities for the two isoenzymes: 1. The fluorogenic substance, 4-methylumbelliferyl-N-acetyl-glucopyranoside, which measures total hexosaminidase activity and 2. the derivative, 4-methylumbelliferyl-N-acetyl glucosamine-6-sulphate, which is considerably more specific toward hexosaminidase A. Hexosaminidase A activity was expressed as a ratio of total activities (the ratio of the assay with 4-methylumbelliferyl-N-acetyl glucosamine-6-sulphate to that with 4-methyllumbelliferyl-N-acetyl-glucopyranoside). Using the results from 65 obligate heterozygotes for Tay-Sachs disease, we established our reference ranges for assigning the genotypes with respect to the Tay-Sachs gene. Comparison of the results from 182 unrelated and randomly chosen sera screened by the ratio method and by heat inactivation, showed a very high correlation (r = 0.996). Sixty eight sera with thermolabile hexosaminidase B were tested by ion exchange chromatography and by the double substrate method, and they yielded identical diagnoses with regard to the Tay-Sachs locus.(ABSTRACT TRUNCATED AT 250 WORDS)

Specific mutations in the HEXA gene among Iraqi Jewish Tay-Sachs disease carriers: dating of founder ancestor

The incidence of Tay-Sachs disease (TSD) carriers, as defined by enzyme assay, is 1:29 among Ashkenazi Jews and 1:110 among Moroccan Jews. An elevated carrier frequency of 1:140 was also observed in the Iraqi Jews (IJ), while in other Israeli populations the world’s pan-ethnic frequency of approximately 1:280 has been found. Recently a novel mutation, G749T, has been reported in 38.7% of the IJ carriers (24/62). Here we report a second novel HEXA mutation specific to the IJ TDS carriers: a substitution of cytosine 1351 by guanosine (C1351G), resulting in the change of leucine to valine in position 451. This mutation was found in 33.9% (21/62) of the carriers and in none of 100 non-carrier IJ. In addition to the two specific mutations, 14.5% (9/62) of the IJ carriers bear a known “Jewish” mutation (Ashkenazi or Moroccan) and 11.3% (7/62) carry a known “non-Jewish” mutation. In 1 DNA sample no mutation has yet been detected. To investigate the genetic history of the IJ-specific mutations (C1351G and G749T), the allelic distribution of four polymorphic markers (D15S131, D15S1025, D15S981, D15S1050) was analyzed in IJ heterozygotes and ethnically matched controls. Based on linkage disequilibrium, recombination factor (θ) between the markers and mutated loci, and the population growth correction, we deduced that G749T occurred in a founder ancestor 44.8±14.2 generations (g) ago [95% confidence interval (CI) 17.0–72.6xa0g] and C1351G arose 80.4±35.9xa0g ago (95% CI 44.5–116.3xa0g). Thus, the estimated dates for introduction of mutations are: 626±426xa0A.D. (200–1052xa0A.D.) for G749T and 442±1077 B.C. (1519xa0B.C. to 635xa0A.D.) for C1351G.

The single cell as a tool for genetic testing: credibility, precision, implication

PurposeTo investigate the influence of amplicons size and cell type on allele dropout and amplification failures in single-cell based molecular diagnosis.Methods730 single lymphocytes and amniotic cells were collected from known heterozygotes individuals to one of the common Ashkenazi Jewish mutations: 1278+TATC and IVS12+1G>C which cause Tay Sachs Disease, IVS20+6T and 854A>C which underlie Familial Dysautonomia and Canavan Disease. DNA was extracted and analyzed by our routine methods.ResultsReduced rates of allele dropout and amplification failure were found when smaller amplification product were designed and in amniotic cultured cells compared to peripheral lymphocytes. Cultured lymphocytes, induced to divide, demonstrated significantly less allele dropout than non induced lymphocytes suggesting the role of division potential on amplification efficiency.ConclusionSingle cell based diagnosis should be designed for each mutation. Minimal sized amplicons and cell having division potential should be preferred, as well as sensitive techniques to detect preferential amplification.

Differences between cystic fibrosis and normal cells in the degree of satellite association

SummaryThe degree of satellite association was found to be significantly higher in phytohemagglutinin (PHA)-stimulated lymphocytes from cystic fibrosis (CF) patients than from those of control individuals. PHA-stimulated lymphocytes from obligatory heterozygotes for the CF mutant allele showed an intermediate degree of satellite association. The degree of satellite association was estimated by the frequency of cells exhibiting associations, by the number of associations per cell, and by the number of chromosomes in an association. The differences in the degree of satellite association were dependent on the concentration of colchicine used for cell arrest. These findings may assist in developing a diagnostic method for the early identification of heterozygotes for the CF allele and for prenatal detection of CF homozygous fetuses.