Nicole Lemieux

Analysis of DNA replication during S-phase by means of dynamic chromosome banding at high resolution

The characteristic patterns of dynamic banding (replication banding) were analysed. Extremely high resolution (850 to 1,250 bands per genome) G- and R-band patterns were obtained after 5-bromo-2′-deoxyuridine (BrdUrd) incorporation either during the early or the late S-phase. We synchronized human lymphocytes with high concentrations of thymidine or BrdUrd as blocking agents, followed by low concentrations of BrdUrd or thymidine respectively as releasing agents, and obtained R- or G-band patterns respectively. The dynamic R-and G-band patterns were complementary for all chromosomes, even for the late-replicating X chromosome. There was no overlapping and every part of each chromosome was positively stained by one of the two banding procedures. The complementarity of the two patterns shows that both high thymidine and high BrdUrd concentrations blocked S-phase progression near the R-band to G-band replication transition in the middle of S-phase. Some bands of the inactive X chromosome replicate before this transition concurrently with R-band replication. The 48 different telomeric regions could be classified into 5 distinct morphotypes based upon the distribution of early and late-replicating DNA in each telomeric region. The dynamic band patterns are particularly useful for the study of the structural and physiological organization of chromosomes at high resolution and should prove invaluable for assessing the replication behavior of rearranged chromosomes.

Del(14)(q22.1q23.2) in a patient with anophthalmia and pituitary hypoplasia

Only few cases with an interstitial deletion of chromosome 14 have been described so far. We report on a 21-month-old girl with an interstitial deletion of the long arm of chromosome 14, del(14)(q22.1q23.2). She presented with bilateral anophthalmia, absent left external auditory canal, facial asymmetry, microretrognathia, hypotonia, and psychomotor retardation. Skeletal X-rays showed lambdoid craniosynostosis, a very small sella turcica and cervical vertebral anomalies. Brain MRI showed the absence of the optic chiasm, an hypoplastic pituitary gland, and cortical atrophy. No cardiac or abdominal malformations were found. Two other patients with a similar deletion, (del(14)(q22.1q23) and del(14)(q22.1q22.3)), are described. Both presented with bilateral anophthalmia and absent pituitary or hypogonadism. These three cases suggest that the region 14q22 is important for eye and pituitary development. Interestingly, the human BMP-4 gene, a member of the TGF-beta superfamily, maps to 14q22-q23 and may play a role in pituitary and eye development.

Chromosomal localization of the ovine beta-casein gene by non-isotopic in situ hybridization and R-banding

The ovine beta-casein gene (CNS2) has been mapped to a specific chromosome band using nonradioactive in situ hybridization and simultaneous fluorescent R-banding. The probe pTZ-E4 was a fragment of the ovine beta-casein gene inserted in the plasmid pTZ18R and labeled with biotin-11-dUTP. It hybridized to band q32 of ovine chromosome 4. The discrepancy between this result and the previous localization of this gene on cattle chromosome 6 may be explained by the very great similarity of the banding patterns of ovine and bovine chromosomes 4 and 6.

Molecular cloning, tissue distribution, and chromosomal localization of MMEL2, a gene coding for a novel human member of the neutral endopeptidase-24.11 family.

Members of the neutral endopeptidase (NEP, also known as MME for membrane metallo-endopeptidase in the Human Gene Nomenclature database) family play significant roles in pain perception, arterial pressure regulation, phosphate metabolism, and homeostasis. In this paper, we report the cloning of a new human member of the NEP family that we named MMEL2 for membrane metallo-endopeptidase-like 2. The MMEL2 protein has the structural characteristics of type II transmembrane proteins, although the presence of a furin-like cleavage site in the ectodomain suggests that it may be released into the medium following proteolytic cleavage. The MMEL2 protein contains the zinc-binding consensus sequence HEXXH and all the residues known to be essential for the enzymatic activity of other members of the family. The MMEL2 mRNA was detected predominantly in testis, but weak expression also was observed in brain, kidney, and heart. The human MMEL2 gene was mapped to 1p36 by fluorescence in situ hybridization. It will be important to test whether MMEL2 defects are associated with diseases such as hereditary motor sensory neuropathy 2A, Schwartz-Jampel-Aberfeld syndrome, or neuroblastoma, which all map to this locus.

Chromosome condensation from prophase to late metaphase: relationship to chromosome bands and their replication time

As chromosomes condense during early mitosis, their subbands fuse in a highly coordinated fashion. Subband fusion occurs when two large subbands flanking one minor subband come together to form one band, which takes on the cytological characteristics of the original flanking subbands. Using four different banding techniques--GTG (G-bands obtained with trypsin and Giemsa), GBG (G-bands obtained with BrdU and Giemsa), RHG (R-bands obtained by heating and Giemsa), and RBG (R-bands obtained with BrdU and Giemsa)--we studied subband fusion from prophase (1,250 bands per haploid set) to late metaphase (300 bands). To quantify the condensation process, a fusion index was established. We found that chromosomes contain preferential zones of condensation. From prophase to late metaphase, the early replicating subbands (R-subbands) fuse more readily with each other than do the late-replicating subbands (G-subbands). R-bands usually replicate early and condense late independently of the adjacent G-bands, which replicate late but condense early. Therefore, chromosome bands can undergo DNA replication and chromatin condensation relatively autonomously. Our data suggest that (1) chromosome replication and condensation are closely connected in time, (2) the metaphase bands represent independent units of chromatin condensation, and (3) the condensation process is an important feature of chromosome organization.

Phenotypic variability in isodicentric Y patients: study of nine cases.

Isodicentric chromosomes are the most commonly reported aberrations of the human Y chromosome. As they are unstable during cell division and can generate various types of cell lines, most reported patients are chromosomal mosaics, generally including a 45,X cell line. Phenotypes depend on the location of the breakpoints as well as on the proportion of each cell line and vary from male to abnormal female or individual with ambiguous genitalia. Although phenotypic variability is known to also depend on the degree of mosaicism in the various tissues, gonads are rarely studied. We report nine cases of isodicentric Y chromosomes studied by conventional and molecular cytogenetic: three males, five females, and one individual with sexual ambiguity. Two males had a non‐mosaic karyotype, while the third male was a mosaic with a predominant 46,XY cell line. Three of the females had a major 45,X cell line, while the last two females and the patient with ambiguous genitalia had a major 46,X,idic(Y) cell line. Analyses of gonadal tissues from the individual with sexual ambiguity and of three of the five female patients gave results concordant with their phenotype, allowing us to better understand the sexual differentiation of these patients.

Familial deletion 18p syndrome: case report

BackgroundDeletion 18p is a frequent deletion syndrome characterized by dysmorphic features, growth deficiencies, and mental retardation with a poorer verbal performance. Until now, five families have been described with limited clinical description. We report transmission of deletion 18p from a mother to her two daughters and review the previous cases.Case presentationThe proband is 12 years old and has short stature, dysmorphic features and moderate mental retardation. Her sister is 9 years old and also has short stature and similar dysmorphic features. Her cognitive performance is within the borderline to mild mental retardation range. The mother also presents short stature. Psychological evaluation showed moderate mental retardation. Chromosome analysis from the sisters and their mother revealed the same chromosomal deletion: 46, XX, del(18)(p11.2). Previous familial cases were consistent regarding the transmission of mental retardation. Our family differs in this regard with variable cognitive impairment and does not display poorer verbal than non-verbal abilities. An exclusive maternal transmission is observed throughout those families. Women with del(18p) are fertile and seem to have a normal miscarriage rate.ConclusionGenetic counseling for these patients should take into account a greater range of cognitive outcome than previously reported.

Cytogenetic Analysis of a Parachordoma

We report the cytogenetic and histopathological findings in a 7-year-old female child with an intranasal tumor that is most consistent with a parachordoma. Karyotypic analysis of the tumor revealed clonal numerical and structural chromosome abnormalities. Seven cells displayed recurrent changes: der(2)t(2;4), del(3q), and the loss of chromosomes 9, 10, 20, and 22. Four cells showed a loss of chromosome 17. To the best of our knowledge, these are the first clonal chromosome abnormalities described in parachordoma.

High-resolution dynamic and morphological G-bandings (GBG and GTG): a comparative study

SummaryA high-resolution replication banding technique, dynamic GBG banding (G-bands after 5′-bromodeoxyuridine [BrdUrd] and Giemsa), showed that, at a resolution of 850 bands/genome, GBG banding and GTG banding (G-bands after trypsin and Giemsa) produce almost identical patterns. RBG band (R-bands after BrdUrd and Giemsa) and RHG band (R-bands after heat denaturation and Giemsa) patterns were previously shown to be only 75%–85% coincident; thus GTG banding more accurately reflects replication patterns than does RHG banding. BrdUrd synchronization uses high concentrations of BrdUrd both to substitute early replicating DNA and to arrest cells before the late bands replicate. Release from the block is via a low thymidine concentration. The banding is revealed by the fluorochrome-photolysis-Giemsa (FPG) technique and produces the GBG banding that includes concomitant staining of constitutive heterochromatin. As opposed to other replication G-banding procedures, BrdUrd synchronization and GBG banding produces a reproducible replication band pattern. The discordance between homologs after GBG banding is similar to that after GTG banding and no lateral asymmetry of the constitutive heterochromatin has been observed. Also, BrdUrd synchronization neither significantly depresses the mitotic index, nor induces chromosome breaks. Thus, GBG banding seems as clinically useful as GTG banding and provides important information regarding replication time.

Fragile site and interstitial telomere repeat sequences at the fusion point of a de novo (Y;13) translocation

Abstract We describe a novel fragile site in a rearranged chromosome, associated with the presence of telomeric repeat sequences at the fusion point of a translocation between chromosomes 13 and Y. The case reported in this study shows a de novo (Y;13) translocation, which appears to represent fusion of an apparently intact chromosome Y with a chromosome 13 that has lost only part of its short arm. Ten percent of the cells show a normal karyotype without the (Y;13) translocation. Molecular cytogenetic studies of the derived Y;13 chromosome revealed three hybridization sites of the telomeric probes – one at each end and one at the breakpoint junction. A fragile site is also observed in the intrachromosomic telomeric region. This coincidence suggests that the telomere repeat sequences (TTAGGG)n, when present at an interstitial chromosomal location, can promote the formation of a novel fragile site.