Chromosomal mosaicism: Origins and clinical implications in preimplantation and prenatal diagnosis.

The diagnosis of chromosomal mosaicism in the preimplantation and prenatal stage is fraught with uncertainty and multiple factors need to be considered in order to gauge the likely impact. The clinical effects of chromosomal mosaicism are directly linked to the type of the imbalance (size, gene content, and copy number), the timing of the initial event leading to mosaicism during embryogenesis/fetal development, the distribution of the abnormal cells throughout the various tissues within the body as well as the ratio of normal/abnormal cells within each of those tissues. Additional factors such as assay noise and culture artifacts also have an impact on the significance and management of mosaic cases. Genetic counseling is an important part of educating patients about the likelihood of having a liveborn with a chromosome abnormality and these risks differ according to the time of ascertainment and the tissue where the mosaic cells were initially discovered. Each situation needs to be assessed on a case-by-case basis and counseled accordingly. This review will discuss the clinical impact of finding mosaicism through: embryo biopsy, chorionic villus sampling, amniocentesis, and noninvasive prenatal testing using cell-free DNA.

Frequency of fetal karyotype abnormalities in women undergoing invasive testing in the absence of ultrasound and other high-risk indications.

OBJECTIVES: No previous studies have reported the frequencies of individual chromosomal anomalies in normal-appearing fetuses stratified by maternal age (MA) and gestational age (GA). We therefore sought to (1) characterize the frequency of all fetal karyotype anomalies in sonographically normal appearing fetuses without pretest risk factors, and (2) assess MA and GA impact on the proportion of anomalies targeted by screening and consequent impact on residual risk following a negative result. METHODS: Fetal karyotypes from samples without prior risk assessment or ultrasound anomalies were analyzed. We calculated, per single-year MA and in two GA intervals, the predicted frequency of each cytogenetic defect. RESULTS: A total of 129 263 karyotypes were analyzed. The risk for significant, cytogenetically visible chromosomal anomalies, at 15 to 20 weeks GA, varies between 1/301 at MA of 18 years, and 1/9 at MA of 48 years. The proportion of clinically significant anomalies not addressed by current screening methods is 47% at MA of 18 years and 5% at MA of 48 years. CONCLUSIONS: By determining frequencies for individual karyotype anomalies stratified by MA and GA, in the setting of normal-appearing fetuses, a more personalized risk assessment, including the residual risk after a normal fetal aneuploidy screening result, can be provided. © 2016 John Wiley & Sons, Ltd.

Cryptic 13q34 and 4q35.2 Deletions in an Italian Family.

Variations of DNA sequences in the human genome range from large, microscopically visible chromosome anomalies to single nucleotide changes. Submicroscopic genomic copy number variations, i.e. chromosomal imbalances which are undetectable by conventional cytogenetic analysis, play an intriguing clinical role. In this study, we describe the clinical consequences of the concurrent presence of an interstitial deletion in 13q34 and a terminal deletion in 4q35.2 in an Italian family. The index patient, a 19-year-old male, as well as his 12-year-old sister are carriers of both deletions, one of maternal and the other of paternal origin. The phenotype includes language delay, multiorgan involvement and bleeding diathesis with mild deficiency of factors X and VII. In the sister, the concomitant presence of Noonan syndrome may partly explain the clinical symptoms. The deleted region on chromosome 13 involves several genes (ATP11A, MCF2L, F7, F10, PROZ, PCID2, CUL4A, and LAMP1); some of these seem to play a role in the proband's phenotype. The terminal deletion in 4q35.2 contains other OMIM genes (FRG1, FRG2 and DBET); moreover, the 4q region is reported as a susceptibility locus for Crohn's disease, diagnosed in the proband's father. To our knowledge, this is the first report of a family with these 2 submicroscopic copy number changes. We tried to relate the clinical phenotype of the proband and his family to the molecular function of the involved genes.

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.

Application of a new molecular technique for the genetic evaluation of products of conception.

OBJECTIVES: Karyotyping is a well-established method of investigating the genetic content of product of conceptions (POCs). Because of the high rate of culture failure and maternal cell contamination, failed results or 46,XX findings are often obtained. Different molecular approaches that are not culture dependent have been proposed to circumvent these limits. On the basis of the robust experience previously obtained with bacterial artificial chromosomes (BACs)-on-Beads™ (BoBs™), we evaluated the same technology that we had used for the analysis of prenatal samples on POCs. METHOD: KaryoLite™ BoBs™ includes 91 beads, each of which is conjugated with a composite of multiple neighboring BACs according to the hg19 assembly. It quantifies proximal and terminal regions of each chromosome arm. The study included 376 samples. RESULTS: The failure rate was 2%, and reproducibility >99%; false-positive and false-negative rates were <1% for non-mosaic aneuploidies and imbalances effecting all three BACs in a contig. Detection rate for partial terminal imbalances was 65.5%. The mosaic detection threshold was 50%, and the success rate in macerated samples was 87.8%. The aneuploidy detection rate in samples with cell growth failure was 27.8%, and maternal cell contamination was suspected in 23.1% of 46,XX cultured cells. CONCLUSION: KaryoLite™ BoBs™ as a 'first-tier' test in combination with other approaches showed beneficial, cost-effective and clearly enhanced POC testing.

QF-PCR as a substitute for karyotyping of cytotrophoblast for the analysis of chorionic villi: advantages and limitations from a cytogenetic retrospective audit of 44,727 first-trimester prenatal diagnoses.

OBJECTIVES: Karyotyping on chorionic villous samples (CVS) includes the analysis of both cytotrophoblast (STC) and mesenchyme (LTC). This approach requires complex laboratory organization and trained technicians. The introduction of quantitative fluorescent polymerase chain reaction (QF-PCR) instead of conventional karyotyping in low-risk pregnancies opened its application in CVS analysis. Discordant QF-PCR and CVS cytogenetic results were reported, and strategies for CVS analysis were introduced to minimize this risk. The possibility to substitute the STC with QF-PCR was reported. The aim of this study is to evaluate benefits and limitations of the approach QF-PCR + LTC compared with the traditional method STC + LTC and to quantify the associated risks of false results. METHOD: This study is based on a retrospective cytogenetic audit of CVS results (n = 44 727) generated by the STC + LTC analytic approach. False-negative risks related to true fetal mosaicism type IV, imprinting syndromes and maternal contamination in LTC were calculated. RESULTS: Compared with STC + LTC, QF-PCR + LTC approach is associated with a cumulative false-negative risk of ~1/3100-1/4400. Costs and reporting time of STC in a high-throughput cytogenetic lab are similar to a CE-IVD marked QF-PCR analysis. CONCLUSIONS: These results should be clearly highlighted in the pre-test counseling and extensively discussed with the couple prior to testing for informed consent.

Prenatal BACs-on-Beads™: the prospective experience of five prenatal diagnosis laboratories.

OBJECTIVE: We previously reported on the validation of Prenatal BACs-on-Beads™ on retrospectively selected and prospective prenatal samples. This bead-based multiplex assay detects chromosome 13, 18, 21 and X/Y aneuploidies and the nine most frequent microdeletion syndromes. We demonstrated that Prenatal BACs-on-Beads(TM) is a new-generation, prenatal screening tool. Here, we describe the experience of five European prenatal diagnosis laboratories concerning the ongoing use of Prenatal BACs-on-Beads™ . METHODS: Some 1653 samples were analyzed. All results were confirmed by conventional karyotyping or another appropriate technique. All indications for invasive prenatal diagnosis were included. Amniotic fluid and chorionic villus samples were analyzed in equivalent proportions. RESULTS: The failure rate was 3.3% and the overall abnormality detection rate was ~1/10. Eighty-five percent of the detected abnormalities were common aneuploidies. Eleven microdeletions and duplications were identified, thus giving an overall yield for microdeletion and microduplication detection of 1/145. Compared with QF-PCR, Prenatal BACs-on-Beads™ provides an additional detection rate of ~1/250 for low-risk pregnancies. The false positive and negative rates were both <1%. CONCLUSION: When associated with conventional karyotyping, the Prenatal BACs-on-Beads™ assay combines a short turnaround time (typical of rapid aneuploidy detection tests) with valuable detection of the most frequent microdeletion syndromes that cannot be detected in cytogenetic analyses.

Pure monosomy and pure trisomy of 13q21.2-31.1 consequent to a familial insertional translocation: exclusion of PCDH9 as the responsible gene for autosomal dominant auditory neuropathy (AUNA1).

Insertional translocations (IT) are rare structural rearrangements. Offspring of IT balanced carriers are at high risk to have either pure partial trisomy or monosomy for the inserted segment as manifested by "pure" phenotypes. We describe an IT between chromosomes 3 and 13 segregating in a three-generation pedigree. Short tandem repeat (STR) segregation analysis and array-comparative genomic hybridization were used to define the IT as a 25.1 Mb segment spanning 13q21.2-q31.1. The phenotype of pure monosomy included deafness, duodenal stenosis, developmental and growth delay, vertebral anomalies, and facial dysmorphisms; the trisomy was manifested by only minor dysmorphisms. As the AUNA1 deafness locus on 13q14-21 overlaps the IT in the PCDH9 (protocadherin-9) gene region, PCDH9 was investigated as a candidate gene for deafness in both families. Genotyping of STRs and single nucleotide polymorphisms defined the AUNA1 breakpoint as 35 kb 5' to PCDH9, with a 2.4 Mb area of overlap with the IT. DNA sequencing of coding regions in the AUNA1 family and in the retained homologue chromosome in the monosomic patient revealed no mutations. We conclude that AUNA1 deafness does not share a common etiology with deafness associated with monosomy 13q21.2-q31.3; deafness may result from monosomy of PCHD9 or another gene in the IT, as has been demonstrated in contiguous gene deletion syndromes. Precise characterization of the breakpoints of the translocated region is useful to identify which genes may be contributing to the phenotype, either through haploinsufficiency or extra dosage effects, in order to define genotype-phenotype correlations.

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.

Confirmation of mosaicism and uniparental disomy in amniocytes, after detection of mosaic chromosome abnormalities in chorionic villi.

Chromosome mosaicism is detected in about 1-2% of chorionic villi samples (CVS), and may be due to a postzygotic nondisjunction event generating a trisomic cell line in an initially normal conceptus (mitotic origin) or the postzygotic loss of one chromosome in an initially trisomic conceptus (meiotic origin and trisomy rescue). Depending on the distribution of the abnormal cell line, the mosaic can be confined to the placenta (CPM) or generalised to the fetus (TFM, true fetal mosaicism). Trisomy rescue could theoretically be associated with a 33.3% probability of uniparental disomy (UPD) in the fetus. The aim of this study was to determine the risk of fetal involvement in a cohort of numerical and structural chromosome mosaics revealed in chorionic villi by means of combined direct and long-term culture analyses; we also determined the incidence of UPD associated with mosaic aneuploidies and supernumerary markers involving imprinted chromosomes. A total of 273 of a consecutive series of 15,109 CVS evaluated during a period of 5 years showed a mosaic condition in direct preparations and/or long-term cultures; confirmatory amniocentesis was performed in 203 cases. The abnormal cell line was extended to the fetus in 12.8% cases in terms of structural and numerical abnormalities involving autosomes and sex chromosomes; the risk of TFM varied and depended on the placental tissue distribution of the abnormal cell line. One of the 51 cases in which the mosaic involved an imprinted chromosome showed UPD, thus indicating a risk of 1.96%.

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.

Biparental expression of ESX1L gene in placentas from normal and intrauterine growth-restricted pregnancies.

Equivalent levels of X-linked gene products between males and females are reached by means of X chromosome inactivation (XCI). In the human and murine embryonic tissues, both the paternally and maternally derived X chromosomes (X(P) and X(M)) may be inactivated. In murine extra-embryonic tissues, X(P) is imprinted and always silenced; humans, unlike mice, can inactivate the X(M) in extra-embryonic lineages without an adverse outcome. This difference is probably due to the presence of imprinted placental genes on the murine X chromosome, but not on the human homologue, essential for placental development and function. An example is the paternally imprinted Esx1 gene; mice with a null maternally derived Esx1 allele show intrauterine growth restriction (IUGR) because of placental insufficiency. We investigated the imprinting status of the human orthologous Esx1 gene (ESX1L) in placental samples of four normal full-term and 13 IUGR female fetuses, in which we determined the XCI pattern. Our findings demonstrated that IUGR as well as normal placentas display XCI heterogeneity, thus indicating that the IUGR phenotype is not correlated with a preferential pattern of XCI in placentas. Moreover, ESX1L is equally expressed in IUGR and normal placentas, and shows the same methylation pattern in the presence of both random and skewed XCI. These findings provide evidence that ESX1L is not imprinted in human third-trimester placentas and there is no parent-of-origin effect of chromosome X associated with placental insufficiency.

Prenatal phenotype of Williams-Beuren syndrome and of the reciprocal duplication syndrome.

KEY CLINICAL MESSAGE: Copy losses/gains of the Williams-Beuren syndrome (WBS) region cause neurodevelopmental disorders with variable expressivity. The WBS prenatal diagnosis cannot be easily performed by ultrasound because only few phenotypic features can be assessed. Three WBS and the first reciprocal duplication prenatal cases are described with a review of the literature.

X chromosome inactivation pattern in BRCA gene mutation carriers.

An association of preferential X chromosome inactivation (XCI) with BRCA gene status and breast/ovarian cancer risk has been reported. We evaluated XCI in a large group of BRCA mutation carriers compared to non-carriers and investigated associations between preferential XCI (⩾90:10) and age, mutated gene, cancer development and chemotherapy. XCI was analysed by human androgen receptor (HUMARA) assay and pyrosequencing in 437 BRCA1 or BRCA2 mutation carriers and 445 age-matched controls. The distribution of XCI patterns in the two groups was compared by logistic regression analysis. The association between preferential XCI and selected variables was investigated in both univariate and multivariate fashion. In univariate analyses preferential XCI was not significantly associated with the probability of being a BRCA mutation carrier, nor with cancer status, whereas chemotherapeutic regime and age both showed a significant association. In multivariate analysis only age maintained significance (odds ratio, 1.056; 95% confidence interval, 1.016-1.096). Our findings do not support the usefulness of XCI analysis for the identification of BRCA mutation carriers and cancer risk assessment. The increasing preferential XCI frequency with ageing and the association with chemotherapy justify extending the investigation to other categories of female cancer patients to identify possible X-linked loci implicated in cell survival.

Epigenetic modulation of the IGF2/H19 imprinted domain in human embryonic and extra-embryonic compartments and its possible role in fetal growth restriction.

Genomic imprinting, resulting in parent-of-origin-dependent gene expression, is mainly achieved by DNA methylation. IGF2 and H19, belonging to the same cluster of imprinted genes and regulated by ICR1, DMR2 and H19 promoter elements, play a major role in fetal/placental growth. Using quantitative approaches, we explored the epigenetic modulation of IGF2/H19 during human development in 60 normal and 66 idiopathic IUGR (Intrauterine Growth Restriction) pregnancies, studying embryonic (cord blood) and extraembryonic (placenta and umbilical cord) tissues. We found ICR1 normal methylation levels ( approximately 50%) and H19 promoter/DMR2 hypomethylation in extra-embryonic tissues. In contrast, in embryonic samples the three loci displayed normal methylation values comparable to those in postnatal blood. This feature is stably maintained throughout gestation and does not vary in IUGR cases. We reported asymmetric allelic expression of H19 and IGF2 as a common feature in pre- and post-natal tissues, independent of H19 promoter and DMR2 methylation levels. In addition, we excluded in IUGR post-transcriptional IGF2 interference possibly related to miRNA 483-3p (IGF2, intron 2) expression defects. Through LINE1 methylation analysis, we observed a methylation gradient with increasing methylation from pre- to post-natal life. The involvement of UPD (Uniparental Disomy) in IUGR aetiology was excluded. Our data indicate that: (1) ICR1 methylation status is a necessary and sufficient condition to drive the imprinting of IGF2 and H19 present in embryonic as well as in extra-embryonic tissues; (2) hypomethylation of H19 promoter and DMR2 does not influence the expression pattern of IGF2 and H19; (3) there is a gradient of global methylation, increasing from extra-embryonic to embryonic and adult tissues. Finally, because of placental hypomethylation, cautions should be exercised in diagnosis of imprinting diseases using chorionic villi.

Misbehaviour of XIST RNA in breast cancer cells.

A role of X chromosome inactivation process in the development of breast cancer have been suggested. In particular, the relationship between the breast cancer predisposing gene BRCA1 and XIST, the main mediator of X chromosome inactivation, has been intensely investigated, but still remains controversial. We investigated this topic by assessing XIST behaviour in different groups of breast carcinomas and in a panel of breast cancer cell lines both BRCA1 mutant and wild type. In addition, we evaluated the occurrence of broader defects of heterochromatin in relation to BRCA1 status in breast cancer cells. We provide evidence that in breast cancer cells BRCA1 is involved in XIST regulation on the active X chromosome, but not in its localization as previously suggested, and that XIST can be unusually expressed by an active X and can decorate it. This indicates that the detection of XIST cloud in cancer cell is not synonymous of the presence of an inactive X chromosome. Moreover, we show that global heterochromatin defects observed in breast tumor cells are independent of BRCA1 status. Our observations sheds light on a possible previously uncharacterized mechanism of breast carcinogenesis mediated by XIST misbehaviour, particularly in BRCA1-related cancers. Moreover, the significant higher levels of XIST-RNA detected in BRCA1-associated respect to sporadic basal-like cancers, opens the possibility to use XIST expression as a marker to discriminate between the two groups of tumors.

Prenatal detection by subtelomeric FISH and MLPA of unbalanced meiotic recombinants resulting from parental pericentric inversions.

OBJECTIVE: Pericentric inversion carriers are predisposed to produce unbalanced gametes that result in conceptuses having either a partial trisomy for one distal segment and a partial monosomy for the other or vice versa. Larger inversions result in smaller unbalanced distal segments and a higher likelihood of a viable fetus. In these cases the structure of the recombinant chromosome is similar to the original balanced inverted or normal ones despite the (unbalanced) genetic content. Such cases may not be detected prenatally by conventional cytogenetic analysis. METHODS: In all prenatal samples from the pericentric inversion carriers we applied subtelomeric FISH probes specific for the chromosome involved in order to detect parental meiotic recombinants resulting from a single cross-over event. Confirmatory MLPA was also applied in unbalanced fetuses. RESULTS: The occurrence of a duplication deficiency unbalance from pericentric inversion carriers was successfully detected in all three fetuses by FISH. MLPA assays applied in two cases confirmed these results. CONCLUSIONS: The application of commercial FISH subtelomeric probes is a reliable method that could be routinely applied for the detection of single cross-over meiotic recombinants. MLPA is a sound alternative technique.