A fertilin-derived peptide improves in vitro maturation and ploidy of human oocytes.

OBJECTIVE: To analyze the effect of a cyclic fertilin-derived peptide (cFEE) on in vitro maturation of human oocytes. DESIGN: Randomized study. SETTING: Fertility center in an academic hospital. PATIENT(S): Not applicable. INTERVENTION(S): Human immature germinal vesicle-stage oocytes (n = 1,629) donated for research according to French bioethics laws were randomly allocated to groups treated with 1 or 100 µM of cFEE or to a control group. They were incubated at 37 °C in 6% CO2 and 5% O2, and their maturation was assessed using time-lapse microscopy over 24 hours. In vitro maturated metaphase II oocytes were analyzed for chromosomal content using microarray comparative genomic hybridization, and their transcriptomes were analyzed using Affymetrix Clariom D microarrays. MAIN OUTCOME MEASURE(S): The percentage of oocytes undergoing maturation in vitro was observed. Aneuploidy and euploidy were assessed for all chromosomes, and differential gene expression was analyzed in oocytes treated with cFEE compared with the control to obtain insights into its mechanism of action. RESULT(S): cFEE significantly increased the percentage of oocytes that matured in vitro and improved euploidy in meiosis II oocytes by the up-regulation of FMN1 and FLNA genes, both of which encode proteins involved in spindle structure. CONCLUSION(S): cFEE improves human oocyte maturation in vitro and reduces aneuploidy. It may prove useful for treating oocytes before fertilization in assisted reproductive technology and for in vitro maturation in fertility preservation programs to improve oocyte quality and the chances for infertile couples to conceive.

Characterization of sSMC by FISH and molecular techniques.

Small supernumerary marker chromosome (sSMC) is a structurally altered additional chromosome that may not be explicitly clarified by conventional karyotyping alone. About one third of sSMC carriers have abnormal phenotypes and its clinical correlation is difficult, especially in prenatal studies. The present study was aimed at characterizing 19 sSMC identified in 15 patients with dysmorphic features with or without multiple congenital anomalies, conspicuous family history, short stature and/or ambiguous genitalia. All the sSMC were primarily identified by routine cytogenetics studies (performed with banding techniques) from peripheral blood except in one patient, where amniotic fluid was used. All sSMCs were further characterized by array-CGH (using 44 K oligonucleotide probe) and/or fluorescence in situ hybridization (FISH) using multicolor banding (MCB), centromere specific multicolor FISH (cenM-FISH), subcentromere-specific multicolor FISH (subcenM-FISH), micro-dissection and/or reverse FISH. This report demonstrates the worth of advanced molecular (cyto)genetic techniques in characterizing sSMC, their utility in genotype-phenotype correlation and risk of clinical presentation.

Identification of the IRXB gene cluster as candidate genes in severe dysgenesis of the ocular anterior segment.

PURPOSE: Anterior segment ocular dysgenesis (ASOD) is a broad heterogeneous group of diseases detectable at the clinical and molecular level. In a patient with bilateral congenital ASOD including aniridia and aphakia, a complex chromosomal rearrangement, inv(2)(p22.3q12.1)t(2;16)(q12.1;q12.2), was characterized at the molecular level, to identify candidate genes implicated in ASOD. METHODS: After negative sequencing of the PAX6, FOXC1, and PITX2 genes, we used fluorescence in situ hybridization (FISH) and Southern blot analysis to characterize the chromosomal breakpoints. Candidate genes were selected, and in situ tissue expression analysis was performed on human fetuses and embryos. RESULTS: Molecular analyses showed that the 16q12.2 breakpoint in this rearrangement occurs in a 625-bp region centromeric to the IRX3 gene, which belongs to the IRXB cluster. In situ hybridization expression studies showed that during early human embryonic development, the IRX3 gene is expressed in the anterior segment of the eye. Of interest, it has been shown previously that a highly conserved noncoding region (HCNCR) is located 300 kb centromeric to the IRX3 gene and induces, in a murine transgenic assay, an expression pattern fitting that of the IRX3 gene. CONCLUSIONS: The authors propose that the 16q12.2 breakpoint of this complex translocation is causally related to the ocular anterior segment dysgenesis observed in this patient. This translocation is assumed to separate the HCNCR from the IRXB cluster genes, thus deregulating the IRXB cluster and leading to the ASOD observed by a positional effect.

Monosomy 19pter and trisomy 19q13-qter in two siblings arising from a maternal pericentric inversion: clinical data and molecular characterization.

Pericentric inversions of chromosome 19 are very rare rearrangements. Only one case was shown to have resulted in duplication deficiency in the offspring. We report a familial case of pericentric inversion of chromosome 19 not detectable by standard karyotype and usual subtelomeric FISH probes. This inversion was transmitted in its balanced and in its recombinant form to the offspring. The two children carrying the recombinant chromosome 19 presented with growth and mental retardation, microcephaly, mild facial dysmorphism and clinodactyly. The recombinant chromosome 19 was characterized by FISH and array CGH. It consisted of a 400kb 19pter deletion and a 6.9Mb (19q13.33-qter) duplication. This observation supports the recombination risk of pericentric inversion of chromosome 19 and emphasizes the role of molecular cytogenetics techniques in the characterization of chromosome 19 rearrangements.

TWIST microdeletion identified by array CGH in a patient presenting Saethre-Chotzen phenotype and a complex rearrangement involving chromosomes 2 and 7.

Saethre-Chotzen syndrome (SCS), also known as acrocephalosyndactyly III, is an autosomal dominant hereditary disorder characterized by craniofacial and limb anomalies. SCS is generally caused by mutations in the TWIST gene, but several 7p21.3 microdeletions involving the entire gene have also been described. The patient reported here presented with craniosynostosis, ptosis, brachydactyly and syndactyly of toes. Standard lymphocyte karyotype showed a de novo apparently balanced but complex constitution with a translocation between the short arms of chromosomes 2 and 7 and an insertion of the 7(q21.3q22) band in the short arm of the same chromosome 7. Interestingly, array CGH displayed a unique 690 kb deletion in 7p21.3 involving the TWIST gene, consistent with the phenotype. This case illustrates the important contribution of array CGH to identification of complex chromosomal rearrangements.

Pure proximal deletion of chromosome 21 and kyphosis.

We report on two unrelated patients with a proximal deletion of the long arm of chromosome 21. The deletion encompassed 14.5Mb of DNA. Molecular studies showed that the two telomeric breakpoints were within the same DNA clone (BAC RP11-56D12). The centromeric breakpoints, however, were separated by only 250kb of DNA (BAC RP11-645E14 and RP11-324B9). The phenotype observed in the two patients was very different, as patient 2, who had the largest deletion, had severe kyphosis not observed in patient 1. Previous studies have identified a 6Mb region of chromosome 21 associated with severe kyphosis. Interestingly, this region overlaps the 250kb segment deleted in patient 2. We suggest that one gene (NT011512.4) located in this small overlapping region might be responsible for severe kyphosis.

Molecular karyotyping in human constitutional cytogenetics.

Using array CGH it is possible to detect very small genetic imbalances anywhere in the genome. Its usefulness has been well documented in cancer and more recently in constitutional disorders. In particular it has been used to detect interstitial and subtelomeric submicroscopic imbalances, to characterize their size at the molecular level and to define the breakpoints of chromosomal translocation. Here, we review the various applications of array CGH in constitutional cytogenetics. This technology remains expensive and the existence of numerous sequence polymorphisms makes its interpretation difficult. The challenge today is to transfer this technology in the clinical setting.

Recurrent partial trisomy 1q22-q44 in clonal intraepithelial lymphocytes in refractory celiac sprue.

BACKGROUND & AIMS: Refractory celiac sprue, a low-grade intraepithelial lymphoma characterized by expansion of clonal intraepithelial lymphocytes with intracellular CD3 epsilon but no surface CD3-T-cell receptor complexes, can be an intermediary step between celiac disease and overt T-cell lymphoma. To gain insight into the mechanisms of lymphomagenesis in celiac disease, we have performed the first cytogenetic study in refractory celiac sprue. METHODS: Karyotypes were performed on: (1) 7 cell lines derived from clonal intraepithelial lymphocytes of patients with refractory celiac sprue; (2) 14 control T-cell lines, either from 4 of 7 patients with refractory celiac sprue or from 10 patients with uncomplicated celiac disease; and (3) bone marrow and peripheral blood lymphocytes in 1 of 7 patients with refractory celiac sprue. Rearrangements were confirmed by in situ hybridization using whole-chromosome painting probes and by comparative genomic hybridization in one patient. RESULTS: A recurrent structural chromosomal aberration leading to partial trisomy of the long arm of chromosome 1 was found in 6 of 7 cell lines from patients with refractory celiac sprue but in none of the control T-cell lines. In one patient with circulating abnormal intraepithelial lymphocytes, the partial trisomy 1q was confirmed on cells freshly isolated from bone marrow and blood. CONCLUSIONS: Refractory celiac sprue is strongly associated with partial trisomy of the 1q region. Gain of chromosome 1q, recently found in 16% of enteropathy-type T-cell lymphoma, may be an early event in lymphomagenesis related to celiac disease and provides a key to investigating molecular mechanisms of lymphoid transformation in this disease.