Evaluation of Quantitative Fluorescence in situ Hybridization for Relative Measurement of Telomere Length in Placental Mesenchymal Core Cells.

BACKGROUND: Reduced telomere length in placental mesenchymal core cells has been reported during pregnancies complicated by intrauterine growth restriction. To estimate telomere length, a precise, accurate and reproducible technique must be used. OBJECTIVE: We evaluated the characteristics of a quantitative fluorescence in situ hybridization (Q-FISH) technique for measuring relative telomere length in placental mesenchymal core cells. METHODS: From late chorionic villus samplings, telomere length in placental mesenchymal core cells was estimated by a Q-FISH technique using peptide nucleic acid telomere probes. The main characteristics of the Q-FISH technique, such as precision and reproducibility, were evaluated. RESULTS: The telomere length of the cultured placental mesenchymal cells did not follow a normal distribution. When the Q-FISH technique was performed on interphase nuclei of uncultured mesenchymal core cells, normal telomere length distribution was observed. The precision of the technique when applied to cultured placental mesenchymal core cells was estimated to be <6%, and its reproducibility ranged from to 92.9 to 104.7%. CONCLUSION: Our results showed that cell culture of placental villi produced a non-normal telomere length distribution, probably related to telomere DNA replication during the cell cycle. Despite the influence of cell culture, the Q-FISH technique reported herein showed good precision and reproducibility.

Loss-of-function mutations in FGFR1 cause autosomal dominant Kallmann syndrome.

We took advantage of overlapping interstitial deletions at chromosome 8p11-p12 in two individuals with contiguous gene syndromes and defined an interval of roughly 540 kb associated with a dominant form of Kallmann syndrome, KAL2. We establish here that loss-of-function mutations in FGFR1 underlie KAL2 whereas a gain-of-function mutation in FGFR1 has been shown to cause a form of craniosynostosis. Moreover, we suggest that the KAL1 gene product, the extracellular matrix protein anosmin-1, is involved in FGF signaling and propose that the gender difference in anosmin-1 dosage (because KAL1 partially escapes X inactivation) explains the higher prevalence of the disease in males.

A homozygous balanced reciprocal translocation suggests LINC00237 as a candidate gene for MOMO (macrosomia, obesity, macrocephaly, and ocular abnormalities) syndrome.

Macrosomia, obesity, macrocephaly, and ocular abnormalities syndrome (MOMO syndrome) has been reported in only four patients to date. In these sporadic cases, no chromosomal or molecular abnormality has been identified thus far. Here, we report on the clinical, cytogenetic, and molecular findings in a child of healthy consanguineous parents suffering from MOMO syndrome. Conventional karyotyping revealed an inherited homozygous balanced reciprocal translocation (16;20)(q21;p11.2). Uniparental disomy testing showed bi-parental inheritance for both derivative chromosomes 16 and 20. The patient's oligonucleotide array-comparative genomic hybridization profile revealed no abnormality. From the homozygous balanced reciprocal translocation (16;20)(q21;p11.2), a positional cloning strategy, designed to narrow 16q21 and 20p11.2 breakpoints, revealed the disruption of a novel gene located at 20p11.23. This gene is now named LINC00237, according to the HUGO (Human Genome Organization) nomenclature. The gene apparently leads to the production of a non-coding RNA. We established that LINC00237 was expressed in lymphocytes of control individuals while normal transcripts were absent in lymphocytes of our MOMO patient. LINC00237 was not ubiquitously expressed in control tissues, but it was notably highly expressed in the brain. Our results suggested autosomal recessive inheritance of MOMO syndrome. LINC00237 could play a role in the pathogenesis of this syndrome and could provide new insights into hyperphagia-related obesity and intellectual disability.

Proportion of parents agreeing to delay fetal karyotyping until the third trimester of pregnancy in cases with an indication.

OBJECTIVE: To assess the extent to which couples who could benefit from fetal karyotyping during the first or second trimester would agree to delay the examination until the third trimester. METHODS: In this prospective monocentric study, the same physician suggested to some couples to delay fetal karyotyping until the third trimester. RESULTS: 458 couples participated in this study. 230 couples (230/458 = 50.2%) refused to delay the examination until the third trimester of pregnancy (group 1). For these patients, four chromosomal abnormalities led to the termination of pregnancy. Fifty-six couples (56/458 = 12.2%) who initially agreed to delay the fetal karyotyping later changed their minds (group 2). 104 couples (104/458 = 22.7%) agreed to delay the examination (group 3). For these patients, one trisomy 21 was diagnosed and led to the subsequent termination of the pregnancy at 33 weeks of amenorrhea. Sixty-eight couples (68/458 = 14.8%) refused any form of invasive prenatal diagnosis (group 4). There was no difference in the rate of preterm premature rupture of membranes, pregnancy term, premature birth rate and birth weight between the four groups. CONCLUSIONS: Our study reports that about a quarter of couples did indeed agree to delay fetal karyotype assessment until the third trimester of pregnancy.

Confined placental mosaicism and pregnancy outcome: a distinction needs to be made between types 2 and 3.

OBJECTIVE: To study the influence of types 2 and 3 confined placental mosaicism (CPM) on pregnancy outcome. METHOD: From 13 809 chorionic villus samplings (CVSs), karyotype after long-term cultured villi (LTC-villi) was systematically performed. Next, in case of suspicion of CPM, karyotype after short-term cultured villi (STC-villi) was established to define type 2 CPM (chromosomal abnormality limited to the mesenchymal core) or type 3 CPM (chromosomal abnormality found both in the cytotrophoblast and the mesenchymal core). Confirmatory amniocentesis was performed to exclude fetal mosaicism. Uniparental disomy (UPD) testing was carried out when the abnormal cell line involved chromosomes 5, 6, 7, 15 or 16. RESULTS: Fifty-seven CPM cases were observed (57/13 809 = 0.41%) and of these, 37 were type 2 and 20 were type 3 CPM. Incidence of preterm infants, neonatal hypotrophy and adverse pregnancy outcome were comparable between patients in whom type 2 CPM was demonstrated and the control population. In contrast, for the type 3 CPM the incidence of these factors was higher than for the control population. CONCLUSION: When a CPM is suspected, it appears essential to determine type, since type 2 has no effect on fetal development and type 3 is associated with preterm infants, low birth weight and adverse pregnancy outcome.

[Maze-like vascular anomaly in partial mole. Interest for the pathological diagnosis of partial mole on chorionic villous sampling]. / Aspect labyrinthique des vaisseaux villositaires dans les môles partielles. Intérêt pour le diagnostic anatomopathologique de môle partielle sur biopsie de villosités choriales.

A case of maze-like angiomatoid anomaly in villi obtained by chorionic villous sampling (CVS) is described. This feature is pathognomonic of partial mole (triploid syndrome) and it was later confirmed by chromosomal analysis. Maze-like angiomatoid anomaly was previously described on specimen submitted after spontaneous or induced abortions, but it was never reported on CVS. This report emphasized that microscopic investigation of CVS cannot be conclusive for cytogenetic anomaly in almost all cases excepted for partial mole where diagnosis criteria are usually characteristic.

[Advantages and limitations of chorionic villous sampling]. / Intérêt et limites de l'examen histopathologique des biopsies de villosités choriales.

Chorionic villous sampling (CVS) has been available for more than twenty years. Together with amniocentesis, it helps the cytogenetician to determine the fetal karyotype for prenatal diagnosis. The choice between these two methods depends on the team and the indication. CVS can now provide sufficient material for both histopathologic and cytogenetic analyses. We evaluated the accuracy of microscopic examination of CVS for detecting primary ovular, uteroplacental vascular (preeclampsia) and inflammatory disorders. Four hundred CVS were examined in the pathology laboratory of Pellegrin Hospital, Bordeaux, France, from January 1995 to February 2008. The results were analyzed according to the indication, the karyotype, the results of placental examination, pregnancy outcome and, when available (following spontaneous or medical termination), fetoplacental findings. The sample was representative of patients requiring CVS for prenatal diagnosis, with respect to maternal age, the stage of pregnancy, and the indications. When used to screen for preeclamsia (prevalence 29.6% in the sample), the sensitivity and specificity of placental biopsy were respectively 56.8% and 87.2% (76.9% in case of intra-uterine growth retardation). When used to screen for chromosomal aberrations (prevalence 7.4%), the specificity was 14.3% and the sensitivity 93.2%. The prevalence of other disorders, and particularly chronic intervillitis, was too low for meaningful analysis. This study shows that histopathologic analysis of chorionic villous samples is useful for detecting the utero-placental vascular origin of intrauterine growth retardation in the absence of other clinical, biological or ultrasound signs, and that it is complementary to cytogenetic analysis. Being a simple and inexpensive examination, histopathologic analysis of CVS could be performed systematically in this indication. Its value and diagnostic signs in other settings need to be determined in larger series.

Nutritional and genetic determinants of vitamin B and homocysteine metabolisms in neural tube defects: a multicenter case-control study.

Neural tube defects (NTDs) are severe congenital malformations due to failure of neural tube formation in early pregnancy. The proof that folic acid prevents NTDs raises the question of whether other parts of homocysteine (Hcy) metabolism may affect rates of NTDs. This French case-control study covered: 77 women aged 17-42 years sampled prior to elective abortion for a severe NTDs (cases) and 61 women aged 20-43 years with a normal pregnancy. Plasma and erythrocyte folate, plasma B6, B12 and Hcy were tested as five polymorphisms MTHFR 677 C --> T, MTHFR 1298 A --> C, MTR 2756 A --> G, MTTR 66 A --> G and TCN2 776 C --> G. Cases had significantly lower erythrocyte folate, plasma folate, B12 and B6 concentrations than the controls, and higher Hcy concentration. The odds ratio was 2.15 (95% CI: 1.00-4.59) for women with the MTRR 66 A --> G allele and it was decreased for mothers carrying the MTHFR 1298 A --> C allele. In multivariate analysis, only the erythrocyte folate concentration (P = 0.005) and plasma B6 concentration (P = 0.020) were predictors. Red cell folate is the main determinant of NTDs in France. Folic acid supplement or flour fortification would prevent most cases. Increased consumption of vitamins B12 and B6 could contribute to the prevention of NTDs. Genetic polymorphisms played only a small role. Until folic acid fortification becomes mandatory, all women of reproductive age should consume folic acid in a multivitamin that also contains B12 and B6.

Confined placental mosaicism revisited: Impact on pregnancy characteristics and outcome.

OBJECTIVES: We wanted to re-evaluate the influence of confined placental mosaicism subtypes (type 2 and type 3) on pregnancy characteristics and outcome. MATERIAL AND METHODS: From July 2009 to December 2015, 5512 chorionic villus samplings were performed in our Fetal Medicine Center. Conventional karyotyping was performed after long-term and short-term cultured villi to define type 2 or type 3 confined placental mosaicisms. Karyotype after amniocentesis was performed to exclude true fetal mosaicism, when appropriate. Pregnancy characteristics and outcomes were collected and compared to a control population. RESULTS: Thirty-six (0.65%) confined placental mosaicisms were observed (13 type 2 and 23 type 3). Nuchal translucency was not increased for type 2 and type 3 confined placental mosaicisms. Pregnancy characteristics and outcomes were comparable between type 2 confined placental mosaicisms and the control population. In type 3 confined placental mosaicisms, median first trimester serum pregnancy-associated plasma protein A was lower than for the control population (p<0.001), preterm births were noticed in 56% (p<0.001), small for gestational age newborns in 74% (p<0.001), and adverse pregnancy outcome was reported in 35% (p<0.01). CONCLUSION: Although type 2 confined placental mosaicisms appeared to have no influence on pregnancy characteristics and outcome, type 3 confined placental mosaicisms were associated with low levels of first trimester serum pregnancy-associated plasma protein A, preterm birth, small for gestational age newborns, and adverse pregnancy outcomes.

[Smith-Lemli-Opitz syndrome]. / Syndrome de Smith-Lemli-Opitz.

SLO syndrome is an autosomal recessive condition with multiple malformations. This syndrome is ascribed to deficiency of 7 dehydrocholesterol reductase, an enzyme in the cholesterol biosynthetic pathway. The characteristics of this syndrome are facial anomalies, syndactyly of the second and third toe, postaxial polydactyly and genital anomalies with sexual ambiguity. We report a fetal case with intrauterine growth retardation, genital anomalies, multiple malformations with cardiac anomalies, renal aplasia and facial anomalies detected by prenatal ultrasound. Medical abortion was induced at 24 weeks gestation. The diagnosis was considered after complete pathologic examination and biochemical analysis.

Prenatal diagnosis using array-CGH: a French experience.

Array-CGH or Chromosomal Microarray Analysis (CMA) is increasingly used in prenatal diagnosis throughout the world. However, routine practices are very different among centers and countries, regarding CMA indications, design and resolution of microarrays, notification and interpretation of Copy Number Alterations (CNA). We present our data and experience from our Fetal Medicine Center on 224 prospective prenatal diagnoses. Our approach is practical, and aims to propose a strategy to offer Chromosomal Microarray Analysis (CMA) to selected fetuses and to help to interpret CNA. We hope that this publication could encourage development of CMA in centers that have not started yet this activity in prenatal routine, and could contribute to edict guidelines in this field.

Reduced placental telomere length during pregnancies complicated by intrauterine growth restriction.

OBJECTIVES: Recent studies have shown that telomere length was significantly reduced in placentas collected at delivery from pregnancies complicated by intrauterine growth restriction secondary to placental insufficiency. Placental telomere length measurement during ongoing pregnancies complicated by intrauterine growth restriction has never been reported. This was the main objective of our study. METHODS: In our center, late chorionic villus samplings were performed between 18 and 37 weeks of amenorrhea in 24 subjects with severe intrauterine growth restriction (cases) and in 28 subjects with other indications for prenatal diagnosis (controls). Placental insufficiency was assessed by histo-pathological examination. Relative measurement of telomere length was carried out prospectively by quantitative Fluorescent In Situ Hybridization using fluorescent Peptide Nucleic Acid probes on interphase nuclei obtained from long-term cultured villi and with an automated epifluorescent microscope. A quantitative Polymerase Chain Reaction technique was performed to confirm the quantitative Fluorescent In Situ Hybridization results. The number of copies of gene loci encoding the RNA template (hTERC) and the catalytic subunit (hTERT) of the enzyme complex telomerase were also estimated in these placentas by Fluorescent In Situ Hybridization. RESULTS: Mean fluorescence intensity of telomere probes estimated by quantitative Fluorescent In Situ Hybridization was significantly less for cases compared to controls (p<0.001). This result indicated that mean telomere length was significantly reduced in placentas during pregnancies complicated by intrauterine growth restriction. Reduced telomere length was confirmed by the quantitative Polymerase Chain Reaction technique. No copy number variation of the hTERC and hTERT loci was noticed for cases, or for controls. CONCLUSION: This study clearly demonstrates a reduction of placental telomere length in ongoing pregnancies (from 18 to 37 weeks of amenorrhea) complicated by severe intrauterine growth restriction secondary to placental insufficiency.

An unusual chromosome 22q11 deletion associated with an apparent complementary ring chromosome in a phenotypically normal woman.

We report an unusual chromosome 22q11 deletion associated with an apparent complementary ring chromosome in a phenotypically normal woman with a family medical history of 22q11 deletion. Using peripheral blood samples, conventional karyotyping, Fluorescence In Situ Hybridization (FISH) analysis on metaphase spreads and oligo array-based comparative genomic hybridization (oligo array-CGH) were performed. After conventional cytogenetic examination, the chromosome formula was as follows: 47,XX,+r(?)[16]/46,XX[6]. The FISH analysis revealed that this patient had a rearranged chromosome 22 with decreased centromeric fluorescence intensity and deletion of the 22q11.2 locus. She also had a supernumerary ring chromosome composed of an alpha-satellite centromere of 22 origin and 22q11.2 locus. The oligo array-CGH profile showed a deletion of approximately 4.18 Mb on chromosome 22 with a log 2 intensity ratio mean deviation of the deleted region of about -0.29. The 22q11 deletion associated with a complementary ring chromosome described in our patient could be consistent with a centromere misdivision mechanism, with one chromosomal break occurring in the alpha-satellite array and a second one in the 22q11 locus, a mechanism which has recently been referred to as the McClintock mechanism.

Characterization of a de novo balanced translocation t(9;18)(p23;q12.2) in a patient with oculoauriculovertebral spectrum.

We report a patient presenting with oculoauriculovertebral spectrum and a de novo balanced reciprocal translocation t(9;18)(p23;q12.2). Physical mapping of the translocation breakpoints by fluorescent in situ hybridization showed that the breakpoints are located in two regions encompassing gene deserts. An additional paternally inherited duplication in 18p11.23p11.31 was identified by array-CGH. We discuss the possible involvement of these chromosomal abnormalities in OAVS.