First case of two supernumerary markers derived from chromosome 5 and chromosome 8.

BACKGROUND: Small supernumerary marker chromosomes (sSMC) are additional centric chromosome fragments too small to be identified by banding cytogenetics alone. A sSMC can originate from any chromosome and it is estimated that 70% of sSMC are de novo, while 30% are inherited. Cases of sSMC derived from chromosome 5 (sSMC5) are rare, accounting for1.4% of all reported sSMC cases. In these patients, the most common reported features are macrocephaly, dysmorphic facial features, heart defects, growth retardation, hypotonia, and intellectual disability. Also sSMC derived from chromosome 8 are very rare and the phenotype of patients with sSMC8 is very variable. Common clinical features of the patients include developmental delay, mental retardation, intellectual disability, hypotonia, hypospadias, attention deficit hyperactivity disorders (ADHD), skeletal anomalies, dysmorphic facial features, and renal dysplasia. To the best of our knowledge, in literature there are no cases with coexistence of sSMC5 and sSMC8, so we reviewed the literature to compare cases with SMC5 and those with SMC8 separately. This study is aimed to highlight the unique findings of a patient with the coexistence of sSMC5 and sSMC8. CASE PRESENTATION: We describe a female patient with two supernumerary markers derived from chromosome 5 (SMC5) and chromosome 8 (SMC8). The patient was born prematurely at 30 weeks with respiratory distress and bronchodysplasia. On physical examination she presented dysmorphic features, respiratory issues, congenital heart defect, developmental delay, and intellectual disability. The G-banded chromosome analysis on cultured lymphocytes revealed in all the analyzed cells a female karyotype with the presence of two supernumerary chromosomal markers and the array-CGH highlighted the region and the size of these two duplications. We also used the fluorescent in situ hybridization analysis (FISH) using painting of chromosomes 5 and 8 to confirm the origin of the two sSMC. So, the karyotype of the patient was: 48, XX, +mar1, +mar2. CONCLUSIONS: This is the first case with two markers: one from chromosome 5 and one from chromosome 8. Based on the data reported, we can affirm that the phenotype of our patient is probably caused mainly by the presence of the sSMC.

Isolation and Enrichment of Circulating Fetal Cells for NIPD: An Overview.

Prenatal diagnosis plays a crucial role in clinical genetics. Non-invasive prenatal diagnosis using fetal cells circulating in maternal peripheral blood has become the goal of prenatal diagnosis, to obtain complete fetal genetic information and avoid risks to mother and fetus. The development of high-efficiency separation technologies is necessary to obtain the scarce fetal cells from the maternal circulation. Over the years, multiple approaches have been applied, including choice of the ideal cell targets, different cell recovering technologies, and refined cell isolation yield procedures. In order to provide a useful tool and to give insights about limitations and advantages of the technologies available today, we review the genetic research on the creation and validation of non-invasive prenatal diagnostic testing protocols based on the rare and labile circulating fetal cells during pregnancy.

Mayer-Rokitansky-Küster-Hauser syndrome with 22q11.21 microduplication: a case report.

BACKGROUND: Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome (Online Mendelian Inheritance in Man [OMIM] #277000) is a congenital condition characterized by the total or partial agenesis of vagina and uterus. Agenesis can be isolated (MRKH 1) or associated with other renal, vertebral or cardiac defects (MRKH 2). CASE PRESENTATION: In this paper, we report a case of a Caucasian patient showing the clinical signs associated with MRKH. Array-based comparative genomic hybridization (a-CGH) analysis revealed a microduplication of approximately 3.01 megabases (Mb) located on the long arm of chromosome 22 (22q11.21). Microduplications affecting the 22q11.21 region have been shown to be associated with MRKH syndrome and Müllerian aplasia. The phenotype of patients with 22q11.2 duplication (OMIM #608363) appears extremely variable, ranging from apparently normal to mild learning difficulties or with multiple defects, sharing features with DiGeorge/velocardiofacial (DGS/VCFS) syndrome. CONCLUSIONS: The altered gene expression together with other genetic, nongenetic, epigenetic or environmental factors can cause the extremely variable phenotype in patients carrying such duplication. Therefore, we can consider MRKH syndrome to be one of the clinical features of DGS/VCFS syndrome.

Testing single/combined clinical categories on 5110 Italian patients with developmental phenotypes to improve array-based detection rate.

BACKGROUND: Chromosomal microarray analysis (CMA) is nowadays widely used in the diagnostic path of patients with clinical phenotypes. However, there is no ascertained evidence to date on how to assemble single/combined clinical categories of developmental phenotypic findings to improve the array-based detection rate. METHODS: The Italian Society of Human Genetics coordinated a retrospective study which included CMA results of 5,110 Italian patients referred to 17 genetics laboratories for variable combined clinical phenotypes. RESULTS: Non-polymorphic copy number variants (CNVs) were identified in 1512 patients (30%) and 615 (32%) present in 552 patients (11%) were classified as pathogenic. CNVs were analysed according to type, size, inheritance pattern, distribution among chromosomes, and association to known syndromes. In addition, the evaluation of the detection rate of clinical subgroups of patients allowed to associate dysmorphisms and/or congenital malformations combined with any other single clinical sign to an increased detection rate, whereas non-syndromic neurodevelopmental signs and non-syndromic congenital malformations to a decreased detection rate. CONCLUSIONS: Our retrospective study resulted in confirming the high detection rate of CMA and indicated new clinical markers useful to optimize their inclusion in the diagnostic and rehabilitative path of patients with developmental phenotypes.

Non-invasive prenatal screening: A 20-year experience in Italy.

Over the past two decades, there has been a rapid evolution in prenatal screening for fetal chromosome abnormalities. Initially, testing was focused on the identification of affected pregnancies in either the first, or, the second trimester (e.g. the Combined test or the triple test). This was replaced by sequential modalities (e.g. contingent screening) that have enhanced detection while reducing the need for invasive testing. More recently, the introduction of technologies based on cell-free DNA (cfDNA) in maternal plasma and enrichment of fetal cells in maternal circulation have further refined the concept of sequential screening. In this review, we document our experience with serum and ultrasound-based contingent screening where we were able to achieve a detection rate of 96.8%, a false-positive rate of 2.8% and an odds of being affected given a positive result of 1:11. We also describe our initial experience with a novel sequential protocol that includes the analysis of fetal cells in maternal blood. Methods for enrichment for fetal cells cfDNA and cfDNA technologies offer the possibility of greater sensitivity and specificity as well as expansion in the scope of genetic disorders detectable. As costs decline, these technologies will become increasingly used as primary screening tools. In the meantime, sequential use offers a practical approach to maximizing the benefits of prenatal testing.

Yq Microdeletion in a Patient with VACTERL Association and Shawl Scrotum with Bifid Scrotum: A Real Pathogenetic Association or a Coincidence?

VACTERL association is defined by the occurrence of congenital malformations: vertebral defects, anal atresia, cardiac defects, tracheoesophageal fistula with esophageal atresia, radial and renal dysplasia, and limb defects. No genetic alterations have been discovered except for some sporadic chromosomal rearrangements and gene mutations. We report a boy with VACTERL association and shawl scrotum with bifid scrotum who presented with a de novo Yq11.223q11.23 microdeletion identified by array CGH. The deletion spans 3.1 Mb and encompasses several genes in the AZFc region, frequently deleted in infertile men with severe oligozoospermia or azoospermia. Herein, we discuss the possible explanation for this unusual genotype-phenotype correlation. We suggest that the deletion of the BPY2 (previously VCY2) gene, located in the AZFc region and involved in spermatogenesis, contributed to the genesis of the phenotype. In fact, BPY2 interacts with a ubiquitin-protein ligase, involved in the SHH pathway which is known to be implicated in the genesis of VACTERL association.

Case report of newborn with de novo partial trisomy 2q31.2-37.3 and monosomy 9p24.3.

We describe a newborn female with a de novo duplication of chromosomes 2q31.2 and 2q37.3, and a de novo monosomy 9p24.3. The clinical findings of this patient include congenital heart defects, dysmorphic facial features, hypotonia, feeding difficulties and microcephaly. Ultrasonographic prenatal findings were negative for foetal malformations. Only a mild pyelectasis was reported. This is the first report of molecular cytogenetic characterization of a partial trisomy 2q31.2-37.3 with monosomy 9p24.3.

Sequential combined test, second trimester maternal serum markers, and circulating fetal cells to select women for invasive prenatal diagnosis.

From January 1st 2013 to August 31st 2016, 24408 pregnant women received the first trimester Combined test and contingently offered second trimester maternal serum screening to identify those women who would most benefit from invasive prenatal diagnosis (IPD). The screening was based on first trimester cut-offs of ≥1:30 (IPD indicated), 1:31 to 1:899 (second trimester screening indicated) and ≤1:900 (no further action), and a second trimester cut-off of ≥1:250. From January 2014, analysis of fetal cells from peripheral maternal blood was also offered to women with positive screening results. For fetal Down syndrome, the overall detection rate was 96.8% for a false-positive rate of 2.8% resulting in an odds of being affected given a positive result (OAPR) of 1:11, equivalent to a positive predictive value (PPV) of 8.1%. Additional chromosome abnormalities were also identified resulting in an OAPR for any chromosome abnormality of 1:6.6 (PPV 11.9%). For a sub-set of cases with positive contingent test results, FISH analysis of circulating fetal cells in maternal circulation identified 7 abnormal and 39 as normal cases with 100% specificity and 100% sensitivity. We conclude that contingent screening using conventional Combined and second trimester screening tests is effective but can potentially be considerably enhanced through the addition of fetal cell analysis.

An 11.4-Mb Interstitial Deletion in a Fetus with No Apparent Phenotypic Alterations.

A prenatal case of a de novo interstitial deletion distal to 8q24 was reported. Ultrasound examination and postmortem evaluation demonstrated no apparent phenotypic alterations. Array CGH showed an 11.4-Mb loss in chromosome 8 ranging from 8q24.13 to 8q24.23. This case partially overlaps the 2 cases previously described in the literature.

Aneuploidy screening using circulating fetal cells in maternal blood by dual-probe FISH protocol: a prospective feasibility study on a series of 172 pregnant women.

BACKGROUND: A long sought goal in medical genetics has been the replacement of invasive procedures for the detection of chromosomal aneuploidies by isolating and analyzing fetal cells or free fetal DNA from maternal blood, avoiding risk to the fetus. However, a rapid, simple, consistent, and low-cost procedure suitable for routine clinical practice has not yet been achieved. The purpose of this study was to assess the feasibility of predicting fetal aneuploidy by applying our recently established dual-probe FISH protocol to fetal cells isolated and enriched from maternal blood. METHODS: A total of 172 pregnant women underwent prospective testing for fetal aneuploidy by FISH analysis of fetal cells isolated from maternal blood. Results were compared with the karyotype determined through invasive procedures or at birth. RESULTS: Seven of the samples exhibited fetal aneuploidy, which was confirmed by invasive prenatal diagnosis procedures. After enrichment for fetal cells, the frequency of trisomic cells was at least double in samples from aneuploid pregnancies (range 0.38-0.90%) compared to samples from normal pregnancies (≤0.18%). One false negative result was also obtained. CONCLUSIONS: Noninvasive prenatal aneuploidy screening using fetal cells isolated from maternal blood is feasible and could substantially reduce the need for invasive procedures.

Deletion 18p11.32p11.31 in a Child with Global Developmental Delay and Atypical, Drug-Resistant Absence Seizures.

We report the first case of an 18p11.32 deletion, detected by array CGH, associated with a drug-resistant form of atypical absence epilepsy, global developmental delay and no signs of holoprosencephaly (HPE). In particular, this region encompasses 19 genes, and none of these genes have been strictly associated with epilepsy. Among these, TGIF1 is expressed in the fetal and adult nervous system, and its deletion has been related to central nervous system diseases. TGIF1 deletions have previously been reported in patients with a comparable phenotype as seen in our case and in children whose neurological signs and symptoms were considerable, but not epileptiform. Mutations and deletions involving the TGIF1 gene have been described in patients with HPE in an autosomal dominant model of inheritance. However, TGIF1 mutations have also been reported in normal individuals and in patients with mental retardation or showing a very mild phenotype, suggesting the characteristic of incomplete penetrance and variable expressivity. Therefore, a TGIF1 deletion may not be always related to HPE, and it may have a link to the development of epilepsy.

Human second trimester amniotic fluid cells are able to create embryoid body-like structures in vitro and to show typical expression profiles of embryonic and primordial germ cells.

Human amniotic fluid-derived stem cells (AFSCs) represent a novel class of broadly multipotent stem cells sharing characteristics of both embryonic and adult stem cells. However, both the origin of these cells and their actual properties in terms of pluripotent differentiation potential are still debated. In order to verify the presence of features of pluripotency in human second trimester AFSCs, we have investigated the ability of these cells to form in vitro three-dimensional aggregates, known as embryoid bodies (EBs), and to express specific genes of embryonic stem cells (ESCs) and primordial germ cells (PGCs). EBs were obtained after 5 days of AFSC culture in suspension and showed positivity for alkaline phosphatase (AP) staining and for specific markers of pluripotency (OCT4 and SOX2). Moreover, EB-derived cells showed the expression of specific transcripts of the three germ layers. RT-PCR analysis, carried out at different culture times (second, third, fourth, fifth, and eighth passages), revealed the presence of specific markers of ESCs (such as FGF4 and DAPPA4), as well as of markers typical of PGCs and, in particular, genes involved in early stages of germ cell development (Fragilis, Stella, Vasa, c-Kit, Rnf17). Finally, the expression of genes related to the control of DNA methylation (DNMT3A, DNMT3b1, DNMT1, DNMT3L, MBD1, MBD2, MBD3, MDB4, MeCP2), as well as the lack of inactivation of the X-chromosome in female samples, was also demonstrated. Taken together, these data provide further evidence for the presence of common features among human AFSCs, PGCs, and ESCs.

Prolonged in vitro expansion partially affects phenotypic features and osteogenic potential of ovine amniotic fluid-derived mesenchymal stromal cells.

BACKGROUND AIMS: Ovine amniotic fluid mesenchymal stromal cells (oAFMSCs) are an emerging alternative source of stem cells to develop pre-clinical cell replacement protocols. For tissue engineering purposes, oAFMSCs can be used either immediately after isolation or after in vitro expansion. However, detailed studies are still required to investigate the advantages and drawbacks of their in vitro expansion. METHODS: The phenotype and osteogenic differentiation potential of oAFMSCs were analyzed in relation to in vitro expansion that was carried out for 20 consecutive passages. Expanded oAFMSCs were analyzed for proliferation index, expression profiles of several surface, pluripotency-associated and HLA antigens, global DNA methylation, telomere length and karyotype. The osteogenic differentiation ability of expanded oAFMSCs was assessed by qualitative and quantitative methods. RESULTS: Expanded oAFMSCs reduced their proliferative activity after 10 passages and partially modified the expression of surface antigens and the intracellular distribution of pluripotency-associated markers (NANOG, SOX2 and TERT) after 20 passages. The phenotypic alteration of cultured oAFMSCs was associated with a reduction of in vitro osteogenic plasticity. In detail, after 20 passages of cellular expansion, oAFMSCs lost the ability to increase osteocalcin and decreased collagen type I messenger RNA expression. Also, a lower percentage of cells displayed intracellular calcium release after stimulation with salmon calcitonin. CONCLUSIONS: The results presented here suggest that long-term in vitro expansion may cause significant alterations in phenotypic features and plasticity of oAFMSCs, suggesting a careful re-evaluation of in vitro cultural and temporal conditions before employing expanded oAFMSCs for therapeutic purposes.

Optimal cut-offs for Down syndrome contingent screening in a population of 10,156 pregnant women.

STUDY DESIGN: A population of 10,156 pregnant women with singleton pregnancies were screened by the integrated test. Risks were retrospectively recalculated for contingent test strategies with first step intermediate risk groups defined by first trimester upper cut-offs of 1 : 10, 1 : 30, 1 : 50, and 1 : 70 and lower cut-offs 1 : 1500, 1 : 1200, 1 : 1100, and 1 : 900. The second trimester high risk group was based on a single cut-off of 1 : 250. RESULTS: In the first trimester, the detection rate (DR) ranged from 21% (6/29) to 52% (15/29) as the high risk first trimester cut-off was changed from 1 : 10 to 1 : 70. The corresponding first trimester false positive rate (FPR) increased from 0.2% to 1.4%. In the second trimester, an additional 21/29 (72%) to 12/29 (41%) affected pregnancies could be detected with an additional 1.6% to 2.7% false positives when lower first trimester cut-offs of 1 : 900 to 1 : 1500 were used. The best results were obtained with the upper first trimester cut-off of 1 : 30 and lower first trimester cut-off of 1 : 900, which yielded a rate of women requiring a second trimester test of only 12%, with overall DR and FPR of 93% and 2.8%, respectively. CONCLUSIONS: Although the study population was relatively small, the results confirm the advantage of using contingent screening and suggest optimal first trimester cut-offs of 1 : 30 (lower cut-off) and 1 : 900 (upper cut-off).

Array-CGH characterization of a de novo t(X;Y)(p22;q11) in a female with short stature and mental retardation.

We report the clinical and molecular investigations in a girl with 46,X,-X,+der(X)t(X;Y)(p22;q11) de novo karyotype who presented an intricate phenotype characterized by mental retardation and facial dysmorphisms in combination with short stature. The structure of the derivative X chromosome was studied using BAC array-CGH which disclosed the Xp22 breakpoint between the STS and the VCX3A gene and the presence of the Yq11.1qter chromosome. It is common that females with Xp;Yq translocations present only short stature and are normal in every other aspect. Thus, this would be the first case in which a girl with Xp;Yq translocation presents an unusual phenotype with intermediate male clinical features with Xp;Yq translocations. The risk of developing gonadoblastoma in females with Y chromosome material is also discussed and, to this effect, different explanations related to this apparent variation are also presented.

16q22.1 microdeletion detected by array-CGH in a family with mental retardation and lobular breast cancer.

We describe the case of a boy with psychomotor delay and dysmorphic features, with a germline 16q22.1 microdeletion identified by array-CGH. The deletion spans 0.24Mb and encompasses three genes (ZFP90, CDH3 and CDH1). The deletion has been demonstrated to be inherited from his mother who was affected by lobular breast cancer (LBC) without any other apparently phenotypic features. We suppose that the microdeletion, in particular ZFP90 which is cerebrally expressed, is causative for the boy's phenotype. Mental retardation in the affected boy can recognize several mechanisms such as variable expressivity, non-penetrance, multifactorial/polygenic inheritance, recessive inheritance, a second rearrangement event and epigenetics. Furthermore, we suggest that the deletion of the CDH1, a tumor suppressor gene, involved in hereditary diffuse gastric cancer (HDGC) and LBC predisposed the mother to the carcinoma.

Indirect co-culture with tendons or tenocytes can program amniotic epithelial cells towards stepwise tenogenic differentiation.

BACKGROUND: Amniotic epithelial cells (AEC) have potential applications in cell-based therapy. Thus far their ability to differentiate into tenocytes has not been investigated although a cell source providing a large supply of tenocytes remains a priority target of regenerative medicine in order to respond to the poor self-repair capability of adult tendons. Starting from this premise, the present research has been designed firstly to verify whether the co-culture with adult primary tenocytes could be exploited in order to induce tenogenic differentiation in AEC, as previously demonstrated in mesenchymal stem cells. Since the co-culture systems inducing cell differentiation takes advantage of specific soluble paracrine factors released by tenocytes, the research has been then addressed to study whether the co-culture could be improved by making use of the different cell populations present within tendon explants or of the high regenerative properties of fetal derived cell/tissue. METHODOLOGY/PRINCIPAL FINDINGS: Freshly isolated AEC, obtained from ovine fetuses at mid-gestation, were co-incubated with explanted tendons or primary tenocytes obtained from fetal or adult calcaneal tendons. The morphological and functional analysis indicated that AEC possessed tenogenic differentiation potential. However, only AEC exposed to fetal-derived cell/tissues developed in vitro tendon-like three dimensional structures with an expression profile of matrix (COL1 and THSB4) and mesenchymal/tendon related genes (TNM, OCN and SCXB) similar to that recorded in native ovine tendons. The tendon-like structures displayed high levels of organization as documented by the cell morphology, the newly deposited matrix enriched in COL1 and widespread expression of gap junction proteins (Connexin 32 and 43). CONCLUSIONS/SIGNIFICANCE: The co-culture system improves its efficiency in promoting AEC differentiation by exploiting the inductive tenogenic soluble factors released by fetal tendon cells or explants. The co-cultural system can be proposed as a low cost and easy technique to engineer tendon for biological study and cell therapy approach.

Mosaic 7q31 deletion involving FOXP2 gene associated with language impairment.

We report on a 10-year-old patient with childhood apraxia of speech (CAS) and mild dysmorphic features. Although multiple karyotypes were reported as normal, a bacterial artificial chromosome array comparative genomic hybridization revealed the presence of a de novo 14.8-Mb mosaic deletion of chromosome 7q31. The deleted region involved several genes, including FOXP2, which has been associated with CAS. Interestingly, the deletion reported here was observed in about 50% of cells, which is the first case of mosaicism in a 7q31 deletion. Despite the presence of the deletion in only 50% of cells, the phenotype of the patient was not milder than other published cases. To date, 6 cases with a deletion of 9.1-20 Mb involving the FOXP2 gene have been reported, suggesting a new contiguous gene deletion syndrome characterized mainly by CAS caused by haploinsufficiency of the genes encompassed in the 7q critical region. This report suggests that children found with a deletion involving the FOXP2 region should be evaluated for CAS and that analysis of the FOXP2 gene including array comparative genomic hybridization should be considered in selected patients with CAS. Mosaic deletions in this area may also be considered as causative of CAS.