Giuseppe Simoni

Efficient direct chromosome analyses and enzyme determinations from chorionic villi samples in the first trimester of pregnancy

SummaryChorionic villi were obtained by an aspiration technique which proved to be the best of four alternative procedures. We report in detail the series of experiments which led to (1) successful, rapidly growing cell cultures practically free of maternal cell contamination (the use of hormone-supplemented Chang medium greatly increased the growth rate);(2) an efficient direct method to obtain high quality metaphases from the Langhans cells of the cytotrophoblast tissue and with which the fetal karyotype is defined within a few hours of chorionic villi sampling; and (3) successful testing for the activity of eight enzymes directly from the villi samples, thus showing that this material is suitable for a rapid, direct diagnosis of the related metabolic diseases.

Frequency of monosomy X in women with primary biliary cirrhosis

The mechanisms that cause the female predominance of primary biliary cirrhosis (PBC) are uncertain, but the X chromosome includes genes involved in immunological tolerance. We assessed the rate of X monosomy in peripheral white blood cells from 100 women with PBC, 50 with chronic hepatitis C, and 50 healthy controls, by fluorescence in-situ hybridisation. Frequency of X monosomy increased with age in all groups, but was significantly higher in women with PBC than in controls (p<0.0001); age-adjusted back-transformed mean frequencies were 0.050 (95% CI 0.046-0.055) in women with PBC, 0.032 (0.028-0.036) in those with chronic hepatitis C, and 0.028 (0.025-0.032) in controls. We suggest that haploinsufficiency for specific X-linked genes leads to female susceptibility to PBC.

X Chromosome Monosomy: A Common Mechanism for Autoimmune Diseases

The majority of human autoimmune diseases are characterized by female predominance. Although sex hormone influences have been suggested to explain this phenomenon, the mechanism remains unclear. In contrast to the role of hormones, it has been suggested, based on pilot data in primary biliary cirrhosis, that there is an elevation of monosomy X in autoimmune disease. Using peripheral white blood cells from women with systemic sclerosis (SSc), autoimmune thyroid disease (AITD), or healthy age-matched control women, we studied the presence of monosomy X rates using fluorescence in situ hybridization. We also performed dual-color fluorescence in situ hybridization analysis with a chromosome Y α-satellite probe to determine the presence of the Y chromosome in the monosomic cells. In subsets of patients and controls, we determined X monosomy rates in white blood cell subpopulations. The rates of monosomy X increased with age in all three populations. However, the rate of monosomy X was significantly higher in patients with SSc and AITD when compared with healthy women (6.2 ± 0.3% and 4.3 ± 0.3%, respectively, vs 2.9 ± 0.2% in healthy women, p < 0.0001 in both comparisons). Importantly, X monosomy rate was more frequent in peripheral T and B lymphocytes than in the other blood cell populations, and there was no evidence for the presence of male fetal microchimerism. These data highlight the thesis that chromosome instability is common to women with SSc and AITD and that haploinsufficiency for X-linked genes may be a critical factor for the female predominance of autoimmune diseases.

Diagnostic application of first trimester trophoblast sampling in 100 pregnancies

SummaryThe results of the diagnostic application of first trimester trophoblast sampling in 100 pregnancies are reported in detail. Further improvement of the method for routine, direct chromosome analysis resulted in a technique which proved to be fast, simple, and efficient. We found that short-term incubation of villi permits the application of many experimental methods, such as visualization of sister chromatid exchanges and bromodeoxyuridine (BrdU) incorporation. Fetal karyotyping was successful in each of the 96 pregnancies in which fetal material was obtained from a total of 98 fetuses. There were 42 males and 56 females, and an abnormal chromosome constitution was found in 12 cases. Two trisomic fetuses were found among the eight pregnancies at risk for Duchenne muscular dystrophy, and this indicates that fetal sexing (which is achieved with our method in two hours) should not be performed without chromosome visualization. The results indicate a risk of 8% of an abnormal fetus for mothers aged 35 years or more, while the risk of failure of sampling and of spontaneous abortion after villi sampling were 4 and 6%, respectively. Enzyme determinations were performed in three pregnancies at risk for gangliosidosis GM1, Niemann-Pick disease, and Hurler syndrome. In this last case inconsistency between the results of the assay of iduronidase on chorionic villi and amniotic fluid cells was found. This unexplained error indicates the need for extensive characterisation in chorionic villi of the series of enzymes involved in metabolic diseases.

The 11q;22q translocation: A collaborative study of 20 new cases and analysis of 110 families

SummaryFollowing a previous collaborative study (Fraccaro et al. 1980), 20 new cases of 11q;22q translocation are described. Twelve families were ascertained through an unbalanced carrier of the translocation and eight cases were ascertained as balanced carriers. A segregation analysis was performed on the 110 families so far published. It was concluded that the 11q;22q translocation is a relatively frequent event, and that all the cases thus far reported might have the same breakpoints at 11q23.3 and 22q11.2. The translocation seems to be independent of environmental factors and it seems to have a low rate of mutation as indicated by the scarcity of de novo cases. The new data confirmed that only one type of unbalanced karyotype (47,XX or XY+der(22)t(11;22)(q23.3;q11.2)) is found among the offspring of the translocation carriers. The minimal overall recurrence risk for an unbalanced translocation was estimated to 2%. There was no difference between the recurrence risks for male and female balanced carriers, while the trend was confirmed of an excess of female balanced carriers among the phenotypically normal offspring of the t(11;22) female carriers.

Fetoplacental mosaicism: potential implications for false-positive and false-negative noninvasive prenatal screening results

Purpose:Noninvasive prenatal screening for fetal aneuploidy analyzes cell-free fetal DNA circulating in the maternal plasma. Because cell-free fetal DNA is mainly of placental trophoblast origin, false-positive and false-negative findings may result from placental mosaicism. The aim of this study was to calculate the potential contribution of placental mosaicism in discordant results of noninvasive prenatal screening.Methods:We performed a retrospective audit of 52,673 chorionic villus samples in which cytogenetic analysis of the cytotrophoblast (direct) and villus mesenchyme (culture) was performed, which was followed by confirmatory amniocentesis in chorionic villi mosaic cases. Using cases in which cytogenetic discordance between cytotrophoblast and amniotic fluid samples was identified, we calculated the potential contribution of cell line–specific mosaicism to false-positive and false-negative results of noninvasive prenatal screening.Results:The false-positive rate, secondary to the presence of abnormal cell line with common trisomies in cytotrophoblast and normal amniotic fluid, ranged from 1/1,065 to 1/3,931 at 10% and 100% mosaicism, respectively; the false-negative rate was calculated from cases of true fetal mosaicism, in which a mosaic cell line was absent in cytotrophoblast and present in the fetus; this occurred in 1/107 cases.Conclusion:Despite exciting advances, underlying biologic mechanisms will never allow 100% sensitivity or specificity.Genet Med 16 8, 620–624.Genetics in Medicine (2014); 16 8, 620–624. doi:10.1038/gim.2014.3

The role of imprinted genes in fetal growth

Genomic imprinting is the phenomenon by which one of the two alleles of a subset of genes is preferentially expressed according to its parental origin. This pattern of inheritance is different from the more frequent mode of Mendelian inheritance, which is not influenced by the parental origin of the allele. The idea that imprinted genes can affect fetal growth is becoming increasingly intriguing as it has been shown that most imprinted genes are expressed in the placenta and some play a role in regulating the interactions between its fetal and maternal interfaces. This article considers genomic imprinting by reviewing recent findings of alterations in fetal growth related to different types of genetic changes affecting the expression of imprinted genes. Among the genetic anomalies, the uniparental disomy (UPD) defines the inheritance of both homologous chromosomes from only one parent. UPDs of a number of chromosomes have been described in association with effects on the phenotype. We reviewed cases of UPD reported till now with particular reference to those associated to growth alterations.

Prevalence of recurrent pathogenic microdeletions and microduplications in over 9500 pregnancies.

The implementation of chromosomal microarray analysis (CMA) in prenatal testing for all patients has not achieved a consensus. Technical alternatives such as Prenatal BACs‐on‐BeadsTM (PNBoBsTM) have thus been applied. The aim of this study was to provide the frequencies of the submicroscopic defects detectable by PNBoBsTM under different prenatal indications.

Loss of the Inactive X Chromosome and Replication of the Active X in BRCA1-Defective and Wild-type Breast Cancer Cells

In females, X chromosome inactivation (XCI) begins with the expression of the XIST gene from the X chromosome destined to be inactivated (Xi) and the coating of XIST RNA in cis. It has recently been reported that this process is supported by the product of the BRCA1 tumor suppressor gene and that BRCA1-/- cancers show Xi chromatin structure defects, thus suggesting a role of XCI perturbation in BRCA1-mediated tumorigenesis. Using a combined genetic and epigenetic approach, we verified the occurrence of XCI in BRCA1-/- and BRCA1wt breast cancer cell lines. It was ascertained that the Xi was lost in all cancer cell lines, irrespective of the BRCA1 status and that more than one active X (Xa) was present. In addition, no epigenetic silencing of genes normally subjected to XCI was observed. We also evaluated XIST expression and found that XIST may be occasionally transcribed also from Xa. Moreover, in one of the BRCA1wt cell line the restoring of XIST expression using a histone deacetylase inhibitor, did not lead to XCI. To verify these findings in primary tumors, chromosome X behavior was investigated in a few BRCA1-associated and BRCA1-not associated primary noncultured breast carcinomas and the results mirrored those obtained in cancer cell lines. Our findings indicate that the lack of XCI may be a frequent phenomenon in breast tumorigenesis, which occurs independently of BRCA1 status and XIST expression and is due to the loss of Xi and replication of Xa and not to the reactivation of the native Xi.

First trimester fetal karyotyping: one thousand diagnoses

SummaryCytogenetic investigations for diagnostic purposes were performed on 1000 first trimester samples of chorionic villi (CVS) in two laboratories using similar techniques. Fetal karyotyping was the primary indication for CVS in 912 and maternal age was the major indication in 758 of them. The risk category “previous child/fetus with chromosome abnormality” included 74 diagnoses, while the category “chromosome abnormality in one of the parents” included 38 diagnoses. Sex determination was the primary indication for CVS in 53 pregnancies. The overall incidence of chromosomal abnormalities was 70, of which 47 were balanced and 23 unbalanced. The results are detailed for each of the risk categories and the incidence of abnormal karyotypes is given for each year of maternal age. In the maternal age of 35–37 years the incidence of unbalanced karyotypes was 2.9% and in the years 38 onwards it was 6.6%. The incidence of unbalanced karyotypes was about 4% when the sampling was made in the weeks 9 to 12 but six abnormal karyotypes were found among 39 CVS performed at the eight week of gestation. The 11 trisomies of the type not found at birth were clustered between the 8th and the 10th week of pregnancy. The technical problems encountered in this experience and the preliminary estimates of fetal loss are discussed.