Persistent Müllerian duct syndrome due to anti-Müllerian hormone receptor 2 microdeletions: a diagnostic challenge.

The persistent Müllerian duct syndrome (PMDS) is defined by the persistence of Müllerian derivatives in an otherwise normally virilized 46,XY male. It is usually caused by mutations in either the anti-Müllerian hormone (AMH) or AMH receptor type 2 (AMHR2) genes. We report the first cases of PMDS resulting from a microdeletion of the chromosomal region 12q13.13, the locus of the gene for AMHR2. One case involved a homozygous microdeletion of five exons of the AMHR2 gene. In the second case, the whole AMHR2 gene was deleted from the maternally inherited chromosome. The patient's paternal allele carried a stop mutation, which was initially thought to be homozygous by Sanger sequencing. Diagnostic methods are discussed, with an emphasis on comparative genomic hybridization and targeted massive parallel sequencing.

Array comparative genomic hybridization analysis of small supernumerary marker chromosomes in human infertility.

Small supernumerary marker chromosomes (sSMC) are structurally abnormal chromosomes that cannot be unambiguously identified by conventional banding cytogenetics. This study describes four patients with sSMC in relation with infertility. Patient 1 had primary infertility. His brother, fertile, carried the same sSMC (patient 2). Patient 3 presented polycystic ovary syndrome and patient 4 primary ovarian insufficiency. Cytogenetic studies, array comparative genomic hybridization (CGH) and sperm analyses were compared with cases previously reported. sSMC corresponded to the 15q11.2 region (patients 1 and 2), the centromeric chromosome 15 region (patient 3) and the 21p11.2 region (patient 4). Array CGH showed 3.6-Mb gain for patients 1 and 2 and 0.266-Mb gain for patient 4. Sperm fluorescent in-situ hybridization analyses found ratios of 0.37 and 0.30 of sperm nuclei with sSMC(15) for patients 1 and 2, respectively (P < 0.001). An increase of sperm nuclei with disomy X, Y and 18 was noted for patient 1 compared with control and patient 2 (P < 0.001). Among the genes mapped in the unbalanced chromosomal regions, POTE B and BAGE are related to the testis and ovary, respectively. The implication of sSMC in infertility could be due to duplication, but also to mechanical effects perturbing meiosis.

Chromosomal breakpoints characterization of two supernumerary ring chromosomes 20.

The occurrence of an additional ring chromosome 20 is a rare chromosome abnormality, and no common phenotype has been yet described. We report on two new patients presenting with a supernumerary ring chromosome 20 both prenatally diagnosed. The first presented with intrauterine growth retardation and some craniofacial dysmorphism, and the second case had a normal phenotype except for obesity. Conventional cytogenetic studies showed for each patient a small supernumerary marker chromosome (SMC). Using fluorescence in situ hybridization, these SMCs corresponded to ring chromosomes 20 including a part of short and long arms of chromosome 20. Detailed molecular cytogenetic characterization showed different breakpoints (20p11.23 and 20q11.23 for Patient 1 and 20p11.21 and 20q11.21 for Patient 2) and sizes of the two ring chromosomes 20 (13.6 Mb for case 1 and 4.8 Mb for case 2). Review of the 13 case reports of an extra r(20) ascertained postnatally (8 cases) and prenatally (5 cases) showed varying degrees of phenotypic abnormalities. We document a detailed molecular cytogenetic chromosomal breakpoints characterization of two cases of supernumerary ring chromosomes 20. These results emphasize the need to characterize precisely chromosomal breakpoints of supernumerary ring chromosomes 20 in order to establish genotype-phenotype correlation. This report may be helpful for prediction of natural history and outcome, particularly in prenatal diagnosis.

Molecular cytogenetic characterization of a 4p15.1-pter duplication and a 4q35.1-qter deletion in a recombinant of chromosome 4 pericentric inversion.

To date, 10 cases of recombinant of chromosome 4 pericentric inversion involving sub-bands p14p15 and q35 have been described. We report on the first case analyzed using array-CGH in a female infant presenting psychomotor and growth retardation, facial anomalies, axial hypotonia, short neck, wide spaced nipples and cardiac defects. Conventional karyotype associated to FISH revealed a recombinant chromosome 4 with partial 4p duplication and 4q deletion derived from a paternal pericentric inversion. Array-CGH allowed us to precise rec4 breakpoints: the proposita carried a small 4.82-4.97 Mb 4q35.1 terminal deletion and a large 35.3-36.7 Mb 4p15.1 terminal duplication. Duplications of the distal 2/3 of short arm of chromosome 4 give rise to recognizable craniofacial features but no specific visceral malformation. A contrario small terminal 4q deletions are associated with cardiac defects. This case and review of literature suggest that two genes ArgBP2 and PDLIM3, located at 4q35.1 and both involved in cardiac and muscle development, could be responsible for cardiac defects observed in terminal 4q35.1 deletions.

Oocyte in-vitro maturation: BCL2 mRNA content in cumulus cells reflects oocyte competency.

BCL2-associated X protein (BAX) and B-cell leukaemia/lymphoma gene-2 (BCL2), which are, respectively, pro- and anti-apoptotic proteins of the BCL2 gene family, participate in the mitochondria-dependent apoptosis pathway. A correlation between low incidence of apoptosis in cumulus cells and oocyte maturation has previously been suggested in ovarian stimulation. However, little is known in unprimed ovaries. These authors have investigated whether BAX and BCL2 expression in cumulus cells affects the competency of in-vitro matured oocytes. We have studied 100 cumulus-oocyte-complexes (COC) recovered from unprimed ovaries of 13 women diagnosed with polycystic ovary syndrome (PCOS) and undergoing in-vitro maturation (IVM) with their informed consent. COC were matured for 24 h in a specific maturation medium and the cumulus was stripped from the oocyte. BAX and BCL2 mRNA content was measured in each COC using real-time polymerase chain reaction. We found that BCL2 mRNA expression was significantly higher in cumulus cells associated with mature oocytes than those associated with immature oocytes while BAX mRNA concentrations did not vary in cumulus cells. Regarding fertilization, higher BCL2 mRNA content was found in cumulus cells enclosing fertilized oocytes (0.140 versus 0.075; P = 0.03). These results suggest that BCL2 expression is strongly associated with the ability of oocytes to complete nuclear maturation and to be fertilized.

Characterization of human PGD blastocysts with unbalanced chromosomal translocations and human embryonic stem cell line derivation?

Novel embryonic stem cell lines derived from embryos carrying structural chromosomal abnormalities obtained after preimplantation genetic diagnosis (PGD) are of interest to study in terms of the influence of abnormalities on further development. A total of 22 unbalanced blastocysts obtained after PGD were analysed for structural chromosomal defects. Morphological description and chromosomal status of these blastocysts was established and they were used to derive human embryonic stem cell (ESC) lines. An outgrowth of cells was observed for six blastocysts (6/22; 27%). For two blastocysts, the exact morphology was unknown since they were at early stage, and for four blastocysts, the inner cell mass was clearly visible. Fifteen blastocysts carried an unbalanced chromosomal defect linked to a reciprocal translocation, resulting in a positive outgrowth of cells for five blastocysts. One human ESC line was obtained from a blastocyst carrying a partial chromosome-21 monosomy and a partial chromosome-1 trisomy. Six blastocysts carried an unbalanced chromosomal defect linked to a Robertsonian translocation, and one showed a positive outgrowth of cells. One blastocyst carried an unbalanced chromosomal defect linked to an insertion and no outgrowth was observed. The efficiency of deriving human ESC lines with constitutional chromosomal disorders was low and probably depends on the initial morphological aspect of the blastocysts and/or the type of the chromosomal disorders.

[Evaluation of the third trimester amniocentesis for fetal karyotyping in women with fear of pregnancy loss]. / Evaluation de l'amniocentèse au troisième trimestre pour le dépistage des anomalies chromosomiques chez les patientes n'acceptant pas le risque de perte foetale.

OBJECTIVES: The aim of this study is to determine the complications of third trimester amniocentesis for fetal karyotyping among women unwilling to accept the fetal loss risks of second trimester amniocentesis. MATERIALS AND METHODS: A retrospective study was carried out from January 1998 to December 2006 of 182 singleton pregnancies that underwent a late amniocentesis (after 32 weeks) for fetal karyotyping. The indications were integrated risk (maternal age, first trimester nuchal translucency, second trimester maternal serum markers) over 1/250 (n=68), isolated maternal age over 38 years (n=51), isolated abnormal second trimester biochemical markers (n=34), history of personal or familial a chromosomal abnormality (n=21) or maternal choice (n=8). Presence of fetal abnormalities at ultrasound or context of viral or parasitologic seroconversion as well as multiple pregnancies were considered as non-inclusion criteria. RESULTS: Median maternal age and gestational age at sampling were 39 years (range 23-48) and 32.4 weeks (29.5-37.6). Median interval between amniocentesis and definitive results of amniocentesis on the one hand, and delivery on the on the hand were 15 days (7-42) and 47 days (8-69), respectively. There were no chromosomal abnormality and non-termination of pregnancy. Nine patients out of 182(5%) had a spontaneous labour followed by premature delivery before 37 weeks and six women (3.3%) among those nine displayed preterm premature rupture of membranes (PPROM). Four patients out of 182 (2%) gave birth before definitive karyotyping result but all of them had a direct fluorescence in situ hybridisation analysis with a normal karyotyping result known well before delivery. CONCLUSIONS: The risk of preterm premature rupture of membrane is 3.3%, with a 5% risk of premature delivery before 37 weeks. This late procedure provides a safe reassurance to women who are unwilling to accept the risks of earlier amniocentesis. However, it should only be used in particular situation and in countries were legislation allows late termination of pregnancy.

[Risk of missed diagnosis of 22q11.2 deletion in a fetal cardiac conotruncal malformation when another chromosomal abnormality is detected]. / Risque d'absence de diagnostic prénatal d'une délétion 22q11.2 associée à une autre anomalie chromosomique dans une malformation cardiaque conotroncale.

We present a rare case of prenatal diagnosis of two de novo chromosome structural rearrangements including a translocation (1;3) associated with a 22q11.2 deletion. The amniocentesis was performed because the systematic ultrasound examination revealed: right aortic cross with double aortic arch, with normal size of aorta and pulmonary artery. Our report emphasises that 22q11.2 deletion must be looked for when a fetal cardiac conotruncal malformation is diagnosed, even in the presence of another chromosomal abnormality. In prenatal diagnosis, this can have implication for patient management and genetic counselling.

Analysis of mtDNA variant segregation during early human embryonic development: a tool for successful NARP preimplantation diagnosis.

BACKGROUND: Diseases arising from mitochondrial DNA (mtDNA) mutations are usually serious pleiotropic disorders with maternal inheritance. Owing to the high recurrence risk in the progeny of carrier females, "at-risk" couples often ask for prenatal diagnosis. However, reliability of such practices remains under debate. Preimplantation diagnosis (PGD), a theoretical alternative to conventional prenatal diagnosis, requires that the mutant load measured in a single cell from an eight cell embryo accurately reflects the overall heteroplasmy of the whole embryo, but this is not known to be the case. OBJECTIVE: To investigate the segregation of an mtDNA length polymorphism in blastomeres of 15 control embryos from four unrelated couples, the NARP mutation in blastomeres of three embryos from a carrier of this mutation. RESULTS: Variability of the mtDNA polymorphism heteroplasmy among blastomeres from each embryo was limited, ranging from zero to 19%, with a mean of 7%. PGD for the neurogenic ataxia retinitis pigmentosa (NARP) mtDNA mutation (8993T-->G) was therefore carried out in the carrier mother of an affected child. One of three embryos was shown to carry 100% of mutant mtDNA species while the remaining two were mutation-free. These two embryos were transferred, resulting in a singleton pregnancy with delivery of a healthy child. CONCLUSIONS: This PGD, the first reported for a mtDNA mutation, illustrates the skewed meiotic segregation of the NARP mtDNA mutation in early human development. However, discrepancies between the segregation patterns of the NARP mutation and the HV2 polymorphism indicate that a particular mtDNA nucleotide variant might differentially influenced the mtDNA segregation, precluding any assumption on feasibility of PGD for other mtDNA mutations.

Common 4q24 deletion in four cases of hematopoietic malignancy: early stem cell involvement?

We determined bone marrow karyotype at diagnosis in four female acute myeloid leukemia (AML) or myelodysplasia patients, aged between 52 and 56 years. In each case, we observed chromosome rearrangement involving the same 4q24 band. Three patients had a balanced reciprocal translocation as the sole abnormality - t(3;4)(q26;q24), t(4;5)(q24;p16) and t(4;7)(q24;q21) - and the fourth had del(4)(q23q24), +4. We used a set of 4q BAC probes for fluorescent in situ hybridization (FISH) in these four cases. We found a 4q24 submicroscopic deletion in all three translocations, with a common deletion of approximately 0.5 Mb. In three cases, we concluded that rearrangement occurred in an early hematopoietic stem cell, as it was detected, in mosaic with a normal karyotype, in a fraction of remission bone marrow cells, peripheral T and B lymphocytes, malignant lymph node T-lymphoma cells in one case and B-lymphoblastoid cell lines established in two cases. Moreover, one of 10 additional AML patients tested by FISH had a normal karyotype and deletion of one of the commonly deleted probe sequences. A tumor suppressor gene may therefore be involved, especially as two patients developed malignant lymphoma at the same time as myeloid proliferation.

[Preimplantation genetic diagnosis (PGD): results from a Parisian center]. / Indications et résultats du diagnostic pré-implantatoire (DPI).

OBJECTIVE: To report the results of preimplantation genetic diagnosis (PGD) cycles performed in our unit from 2000 to 2004. Materials and methods. One hundred and seventy-one couples were enrolled in the PGD program over this period. The collected oocytes were inseminated by intracytoplasmic sperm injection (ICSI). The resulting embryos were biopsied on the third day of development and the genetic analysis was performed on the same day. Embryo transfers were carried out on the fourth day. RESULTS: The 416 stimulation cycles started yielded 280 oocyte pick-ups, 3506 oocytes retrieved, of which 2966 were suitable for ICSI. Among the 1982 embryos obtained, 1337 embryos were biopsied and genetic diagnosis was performed for 1083 (81%) of them. 381 embryos were transferred during the course of 189 transfer procedures. There were 51 clinical and 46 ongoing (35 single, 11 twin) pregnancies. In addition, 25 frozen embryo replacement cycles were initiated, leading to 6 embryo transfers and 1 ongoing pregnancy. A total of 58 unaffected children were born. CONCLUSION: PGD has gained a place among the choices offered to couples at risk of transmission of a serious and incurable genetic disease. It might be a realistic alternative to prenatal diagnosis for patients carrier of chromosomal rearrangements, single gene defects, X-linked disesases or mitochondrial DNA disorders.

[Extending preimplantation genetic diagnosis to HLA typing: the Paris experience]. / Le diagnostic préimplantatoire couplé au typage HLA: l'expérience parisienne.

Preimplantation genetic diagnosis (PGD) consists in the genetic analysis of one or two cells. These cells (blastomeres) are sampled from embryos, obtained by in vitro fertilization, at the third day of development. Since 1998, the bioethical laws (1994) and their decrees restricted PGD practices in France, strictly to the avoidance of the birth of a child affected with a genetic defect. In parallel, works on blood cord transplantation, taken at the birth of a compatible HLA sibling, showed very encouraging results, particularly for the treatment of Fanconi anemia. In 2001, Verlinsky et al., have reported the first PGD for Fanconi anaemia combined with HLA typing, allowing the birth of a healthy child, HLA-identical with his affected sister. The "designer baby" concept was born. The French law, which allowed PGD under specific conditions, i.e. when the genetic defect has been characterized in one parent at least, recently extended PGD to HLA typing when embryos are at risk of a genetic disorder. Article L.2131-4-1 (August 2004) allows the practice of HLA typing for PGD embryos when an elder sibling is affected with a genetic disorder and need stem cell transplantation. The HLA-matched offspring resulting from PGD can give cord blood at birth to supply the necessary therapy. This double selection give rise to serious ethical problems, but technical difficulties and legal restrictions will probably limit the development of such a procedure.

[Cocaine and trisomy 8 associated with prenatal diagnosis of corpus callosum agenesis]. / Cocaïne et trisomie 8 associées au diagnostic prénatal d'agénésie du corps calleux.

We report the case of a newborn presenting an agenesis of corpus callosum (ACC) discovered in the prenatal period and initially related to cocaine exposure during the first trimester of gestation. The cytogenetic analysis revealed a trisomy 8 mosaicism. The putative role of prenatal cocaine exposure and mosaicism for chromosome 8 in ACC are discussed. This report emphasizes the specific analysis of chromosome 8 by using fluorescence in situ hybridization as a complement to routine cytogenetic analysis for prenatal diagnosis of ACC.

[The Paris experience in preimplantation genetic diagnosis: evaluation after the first births]. / L'expérience parisienne du diagnostic génétique pré-implantatoire (DPI): bilan des premières naissances.

OBJECTIVE: To report the birth of the first thirteen infants conceived after preimplantation genetic diagnosis (PGD) within the medical assistance federation of Paris. PATIENTS AND METHODS: Fifty-nine couples were enrolled between January 2000 and July 2001. They had a total of 71 oocyte pick-up cycles. The collected oocytes were inseminated by intracytoplasmic sperm injection. The resulting embryos were biopsied on the third day of development and the genetic analysis was performed on the same day. Most of the embryo transfers were carried out on the fourth day. RESULTS: The 71 oocyte pick-up cycles yielded 872 oocytes of which 731 were suitable for intracytoplasmic sperm injection. 421 embryos were biopsied and genetic diagnosis was obtained from 312 (74%) of these. 127 embryos were transferred during the course of 58 transfer procedures. There were 18 biologic and 12 clinical (7 singles, 4 twins and 1 triple) pregnancies. Thirteen infants have been born and 4 are expected. CONCLUSIONS: PGD has gained a place among the choices offered to couples at risk of transmission of a serious and incurable genetic disease.

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.

De novo interstitial direct duplication 1(q23.1q31.1) in a fetus with Pierre Robin sequence and camptodactyly.

Interstitial duplications of chromosomal region 1q are rarely seen. We report the first prenatal diagnosis of pure partial trisomy 1q. The fetus was karyotyped for polyhydramnios, micrognathia, and flexion of fingers of both hands. Conventional and molecular cytogenetics showed a de novo direct duplication of the chromosomal region 1q23.1q31.1 leading to a partial trisomy 1q. At autopsy, the fetus showed Pierre Robin sequence (PRS) and camptodactyly. The main histological finding was a decreased number of motoneurons with apoptotic features in the anterior horn of the spinal cord. A literature review and our observations suggest that genetic material mapping to chromosome 1q25 could be responsible for PRS with distal arthrogryposis when this is in triple dose.

De novo inverted duplication 9p21pter involving telomeric repeated sequences.

We report on clinical and cytogenetic findings in a boy with partial 9p duplication, dup(9)(p21pter). Clinical manifestations included facial and hand anomalies and mental retardation. Fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH) were used to characterize further and confirm the conventional banding data. Investigation by FISH using whole chromosome 9 paint probe showed that the additional material was derived from chromosome 9. Using CGH, a region of gain was found in the chromosome segment 9p21pter. YACs and telomeric probes confirmed the duplicated region. Using the all-human telomeric sequences probe, intrachromosomal telomeric signal was noted on the short arm of the abnormal chromosome 9. Mechanism of formation of the duplication, including intrachromosomal telomeric sequences, is discussed.

Altered patterns of DNA methylation on chromosomes from leukemia cell lines: identification of 5-methylcytosines by indirect immunodetection.

An immunodetection technique has been developed to map with high resolution the methylated sites of human chromosomes. We have used this method to define the methylated areas of chromosomes from normal donors and from leukemia cell lines. The chromosomes were exposed for a short time to UV light to induce mild denaturation. The methylated sites were detected in situ by using monoclonal antibodies against 5-methylcytosine (prepared in mouse), and fluorescein-conjugated antimouse immunoglobulins. The chromosomes from normal cells exhibited a fluorescent pattern with RCT banding, although some differences from previously reported patterns could be detected. With this method we have been able to show the presence of two types of R-bands: High fluorescence R-band (HFR) and low fluorescence R-band (LFR). Chromosomes from leukemia cell lines exhibited low global staining with disrupted RCT banding of the chromosomes. The decreased level of the methylation status of the chromosomes from leukemia cells was confirmed by detection of 5-methylcytosines on total immobilized DNA. Thus, we have shown that this method can be used to determine the methylated status of chromosomes and, in turn, to map not only the structural (banding) but also the functional (methylation status) properties of the different chromosome domains in normal and pathologic human cells.

[Contribution of genotyping for fetal sex determination in maternal serum for preimplantation genetic diagnosis of X-linked diseases]. / Intérêt du génotypage du sexe foetal dans le sang maternel dans les indications de diagnostic génétique préimplantatoire de sexe.

OBJECTIVE: Couples with a risk of transmitting X-linked diseases included in a preimplantation genetic diagnosis (PGD) center need early and rapid fetal sex determination during pregnancy in two situations. The first situation corresponds to control of embryo sexing after PGD, the second one being that of couples in PGD program having a spontaneous pregnancy. Determination of fetal sex can be achieved by karyotyping using invasive procedures such as chorionic villus sampling (CVS), amniocentesis or cordocentesis and by non-invasive procedures such as ultrasound (US) examination. CVS is the earliest invasive procedure for fetal sex determination and molecular analysis of X-linked genetic disorders during the first trimester but it is associated with a risk of fetal loss. US allows reliable fetal sex determination only during the second trimester. Recently, reliable non-invasive fetal sex determination was realized by using SRY gene amplification in maternal serum. PATIENTS AND METHODS: We report the prospective use of fetal sex determination in maternal serum in our PGD center. Management of pregnancies was performed using this non-invasive procedure in four cases of embryo sexing control and nine cases of spontaneous pregnancies in couples included in PGD program for X-linked diseases. RESULTS: Fetal sex results using SRY gene amplification on maternal serum were in complete concordance with fetal sex observed by cytogenetic analysis or US examination, as well as at birth. DISCUSSION AND CONCLUSION: This new strategy allowed rapid sex determination during the first trimester and permitted to avoid performing invasive procedures in nine pregnancies.