Luc Druart

A simple method for prenatal diagnosis of trisomy 21 on uncultured amniocytes.

Prenatal diagnosis of trisomy 21 would be easier if fluorescence in situ hybridization (FISH) could be applied to interphase nuclei. Therefore, we prepared a chromosome-21 -specific probe by in vitro enzymatic amplification of inter-Alu sequences from YAC clones previously localized to this chromosome. This probe was used for FISH on 22 uncultured amniocyte samples. An easy, rapid, and safe technique is proposed for the prenatal diagnosis of trisomy 21.

Localization of the human coproporphyrinogen oxidase gene to chromosome band 3q12

The human gene encoding coproporphyrinogen oxidase is the defective gene in hereditary coproporphyria. This gene was mapped to chromosome band 3q12 using fluorescent in situ hybridization. The chromosomal localization was confirmed by cosegregation of the human gene with chromosome 3 in a panel of human/rodent somatic hybrids.

Genotype-phenotype correlation in 13q13.3-q21.3 deletion.

Pure interstitial deletions of the long arm of chromosome 13 are correlated with variable phenotypes according to the size and the location of the deleted region. Deletions involving the 13q13q21 region are rare. In order to establish interstitial 13q genotype-phenotype correlation, we used high resolution 244K oligonucleotide array in addition to conventional karyotype and molecular (fluorescent in situ hybridization, microsatellite markers analysis) techniques in two independent probands carrying a deletion 13q13 to 13q21. First patient was a 3-year-old girl with mental retardation and dysmorphy carrying a 13q13.3q21.31 de novo deletion diagnosed post-natally. The second one was a fetus with de novo del(13)(q14q21.2) associated with first trimester increased nuchal translucency. We showed that specific dysmorphic features (macrocephaly, high forehead, hypertelorism, large nose, large and malformed ears and retrognathia) were correlated to the common 13q14q21 chromosomal segment. Physical examination revealed overgrowth with global measurement up to the 95th percentile in both probands. This is the second description of overgrowth in patients carrying a 13q deletion. Haploinsufficiency of common candidates genes such as CKAP2, SUGT1, LECT1, DCLK1 and SMAD9, involved in cell division and bone development, is a possible mechanism that could explain overgrowth in both patients. This study underlines also that cytogenetic analysis could be performed in patients with overgrowth.

Genomic and clinical characteristics of six patients with partially overlapping interstitial deletions at 10p12p11

With the clinical implementation of genomic microarrays, the detection of cryptic unbalanced rearrangements in patients with syndromic developmental delay has improved considerably. Here we report the molecular karyotyping and phenotypic description of six new unrelated patients with partially overlapping microdeletions at 10p12.31p11.21 ranging from 1.0 to 10.6 Mb. The smallest region of overlap is 306 kb, which includes WAC gene, known to be associated with microtubule function and to have a role in cell division. Another patient has previously been described with a 10 Mb deletion, partially overlapping with our six patients. All seven patients have developmental delay and a majority of the patients have abnormal behaviour and dysmorphic features, including bulbous nasal tip, deep set eyes, synophrys/thick eyebrows and full cheeks, whereas other features varied. All patients also displayed various visual impairments and six out of seven patients had cardiac malformations. Taken together with the previously reported patient, our study suggests that the detected deletions may represent a new contiguous gene syndrome caused by dosage-sensitive genes that predispose to developmental delay.

Tetrasomy 13q31.1qter due to an inverted duplicated neocentric marker chromosome in a fetus with multiple malformations

Small supernumerary marker chromosome (sSMC) lacking alpha satellite DNA or endogenous centromere regions are rare and contain fully functional centromeres, called neocentromeres. We report on a woman with a 14‐week gestation pregnancy with a cystic hygroma and cerebellar hypoplasia at ultrasound examination. Cytogenetic studies showed a karyotype 47,XY,+mar dn. This sSMC was observed in chorionic villi, lung, and muscle tissue. Array Comparative Genomic Hybridization showed a gain from 13q31.1 to 13qter region. Fluorescent in situ hybridization with pan alpha satellite probe and probes specific for chromosome 13 showed a marker corresponding to an inversion duplication of the 13q distal chromosomal region without alpha satellite DNA sequence, suggesting the presence of a neocentromere. Examination of the fetus showed dysmorphic features, cystic cervical hygroma, postaxial polydactyly of the right hand and left foot with short fingers, malrotation of the gut, and a micropenis with hypospadias. Genotype–phenotype correlation in tetrasomy 13q is discussed according to the four 13q chromosomal breakpoints reported (13q32, 13q31, 13q21, 13q14) for chromosome 13 supernumerary markers.

Prenatal diagnosis of a (X;X) translocation by fluorescence in situ hybridization and laser scanning image cytometry

A de novo structural abnormality of one X chromosome was prenatally detected in a female fetus. This chromosomal abnormality has been analyzed by conventional cytogenetic methods, fluorescence in situ hybridization, and laser scanning image cytometry. The association of these techniques has demonstrated that this anomaly corresponds to a (X;X) translocation. Analysis of hybridization signals by laser scanning image cytometry allowed to localize that the breakpoints were at the X-centromeric region and Xp11.3, respectively. These results show the usefulness of image analysis and fluorescence in situ hybridization for a rapid characterization of de novo structural chromosome anomalies in prenatal diagnosis.

HUMAN CHROMOSOME MICRODISSECTION AND FLUORESCENCE ANALYSIS BY LASER SCANNING IMAGE CYTOMETRY

Mesentericin YI05 (mYI05) a bacteriocin secreted by Leuconostoc mesenteroides ssp mesenteroides YI05. kills only Listeria types. The bactericidal effect estimates by microbiological methods using well diffusion tests may be observed after an incubation of at least 16 hours. To reduce the analytical delay, we have routinely used fluorescent potential dyes and flow cytometry to determine the bactericidal effect. Because structural parameters (i.e. Forward Angle Scatter, DNA content...) are slowly altered under mY I05 effect, we have preferentially followed the modification of functional parameters such as membrane potential. Decrease of bacteria fluorescence due to release of potential sensitive dye (Rhodamine 123 or DiOC6(3)) is rapidly and significantly observed in less than I rain after bacteriocin incorporation in the incubation medium. This result agrees well with biochemical studies (Maftah et al., 1993, J. Bacteriol. 175, 3232-3235) which assume that mY l05 induces the permeabilization of Listeria plasmic membrane. Potential alterations vary with Listeria species, and for resistant strains potential is slightly modified. Otherwise, analysis carried out with protoplasts of sensitive and resistant Listeria strains, could suggest that one or some components of bacterial wall are thightly implicated in the recognition of mYl05.