Reduced telomere length in amniocytes: an early biomarker of abnormal fetal development?

Telomeres protect chromosome ends and control cell division and senescence. During organogenesis, telomeres need to be long enough to ensure the cell proliferation necessary at this stage of development. Previous studies have shown that telomere shortening is associated with growth retardation and congenital malformations. However, these studies were performed in newborns or postnatally, and data on telomere length (TL) during the prenatal period are still very limited. We measured TL using quantitative PCR in amniotic fluid (AF) and chorionic villi (CV) samples from 69 control fetuses with normal ultrasound (52 AF and 17 CV) and 213 fetuses (165 AF and 48 CV) with intrauterine growth retardation (IUGR) or congenital malformations diagnosed by ultrasound. The samples were collected by amniocentesis at the gestational age (GA) of 25.0 ± 5.4 weeks and by CV biopsy at 18.1 ± 6.3 weeks. In neither sample type was TL influenced by GA or fetal sex. In AF, a comparison of abnormal versus normal fetuses showed a significant telomere shortening in cases of IUGR (reduction of 34%, P < 10-6), single (29%, P < 10-6) and multiple (44%, P < 10-6) malformations. Similar TL shortening was also observed in CV from abnormal fetuses but to a lesser extent (25%, P = 0.0002; 18%, P = 0.016; 20%, P = 0.004, respectively). Telomere shortening was more pronounced in cases of multiple congenital anomalies than in fetuses with a single malformation, suggesting a correlation between TL and the severity of fetal phenotype. Thus, TL measurement in fetal samples during pregnancy could provide a novel predictive marker of pathological development.

Spatial organization of chromosome territories in the interphase nucleus of trisomy 21 cells.

In the interphase cell nucleus, chromosomes adopt a conserved and non-random arrangement in subnuclear domains called chromosome territories (CTs). Whereas chromosome translocation can affect CT organization in tumor cell nuclei, little is known about how aneuploidies can impact CT organization. Here, we performed 3D-FISH on control and trisomic 21 nuclei to track the patterning of chromosome territories, focusing on the radial distribution of trisomic HSA21 as well as 11 disomic chromosomes. We have established an experimental design based on cultured chorionic villus cells which keep their original mesenchymal features including a characteristic ellipsoid nuclear morphology and a radial CT distribution that correlates with chromosome size. Our study suggests that in trisomy 21 nuclei, the extra HSA21 induces a shift of HSA1 and HSA3 CTs out toward a more peripheral position in nuclear space and a higher compaction of HSA1 and HSA17 CTs. We posit that the presence of a supernumerary chromosome 21 alters chromosome compaction and results in displacement of other chromosome territories from their usual nuclear position.

Sperm meiotic segregation of a balanced interchromosomal reciprocal insertion resulting in recurrent spontaneous miscarriage.

RESEARCH QUESTION: Is sperm fluorescence in-situ hybridization (FISH) useful to evaluate the risk of chromosomally unbalanced gametes in interchromosomal reciprocal insertion (IRI) carriers? How do these imbalances lead to recurrent miscarriages? DESIGN: This study reports a clinical and molecular study of a rare familial balanced IRI resulting in recurrent spontaneous miscarriage. Sperm FISH was performed to estimate the number of unbalanced gametes. RESULTS: A 31-year-old healthy male (proband) and his 28-year-old female partner were referred to the Genetics Department for three spontaneous miscarriages occurring during the first trimester of pregnancy. FISH analysis of the proband with the LSI TRA/D (14q11.2) and DiGeorge N25 (22q11.2) break-apart probes showed the presence of a balanced IRI between 14q11.2 and 22q11.2 chromosomal regions. This IRI was also identified in the proband's father. Sperm FISH with the same probes showed that more than 40% of gametes of the proband were unbalanced for either 14q11.2 or 22q11.2, despite normal sperm parameters. FISH analysis of a product of conception indicated that unbalanced gametes result in a non-viable fetus. CONCLUSIONS: This study shows the value of sperm FISH analysis in improving genetic reproductive advice for IRI carriers. Disruption of critical genes through this rearrangement and their consequent functional impairment could result in recurrent miscarriages. In this case, several genes located in the 14q11.2 region, particularly RNase 3, would be good candidates to explain the lethality of the imbalances.

A novel 2q14.1q14.3 deletion involving GLI2 and RNU4ATAC genes associated with partial corpus callosum agenesis and severe intrauterine growth retardation.

BACKGROUND: Microdeletions encompassing chromosome bands 2q14.1q14.3 are rare. To date, eight reports of relatively large deletions of this region (∼20 Mb) but only two small deletions (<6 Mb) have been reported. These deletions can cause a variable phenotype depending on the size and location of the deletion. Cognitive disability, facial dysmorphism, and postnatal growth retardation are the most common phenotypic features. CASE: We report on a novel 5.8 Mb deletion of 2q14.1q14.3 identified by array comparative genomic hybridization in a fetus with severe intrauterine growth retardation and partial agenesis of the corpus callosum. The deletion contained 24 coding genes including STEAP3, GLI2, and RNU4ATAC and was inherited from the mild affected mother. A sibling developmental delay and similar dysmorphic facial features was found to have inherited the same deletion. CONCLUSION: This case emphasizes the variable expressivity of the 2q14 microdeletion and reinforces the hypothesis that agenesis of corpus callosum, microcephaly, developmental delay, and distinctive craniofacial features may be part of the phenotypic spectrum characterizing the affected patients. We suggest that GLI2 is a dosage-sensitive gene that may be responsible for the agenesis of corpus callosum observed in the proband. Birth Defects Research (Part A) 106:793-797, 2016. © 2016 Wiley Periodicals, Inc.

Prenatal Screening of 21 Microdeletion/Microduplication Syndromes and Subtelomeric Imbalances by MLPA in Fetuses with Increased Nuchal Translucency and Normal Karyotype.

Fetuses with increased nuchal translucency thickness (NT) are at increased risk for chromosomal abnormalities. In case of a normal karyotype, a minority of them may present with structural abnormalities or genetic syndromes, which may be related to submicroscopic chromosomal imbalances. The objective of this study was to evaluate whether MLPA screening of 21 syndromic and subtelomeric regions could improve the detection rate of small chromosomal aberrations in fetuses with increased NT and a normal karyotype. A total of 106 prenatal samples from fetuses with NT ≥ 99th centile and normal R- and G-banding were analyzed by MLPA for subtelomeric imbalances (SALSA P036 and P070) and 21 syndromic regions (SALSA P245). One sample showed a benign CNV (dup(8)pter, FBXO25 gene), and 1 patient was found to have a loss of 18 qter and a gain of 5 pter as a result of an unbalanced translocation. The incidence of cryptic pathogenic variants was <1% or 2.7% when only fetuses with other ultrasound abnormalities were taken into account. Submicroscopic imbalances in fetuses with increased NT may be individually rare, and genome-wide screening seems more likely to improve the diagnostic yield in these fetuses.

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

OBJECTIVES: 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-Beads(TM) (PNBoBs(TM) ) have thus been applied. The aim of this study was to provide the frequencies of the submicroscopic defects detectable by PNBoBs(TM) under different prenatal indications. METHODS: A total of 9648 prenatal samples were prospectively analyzed by karyotyping plus PNBoBs(TM) and classified by prenatal indication. The frequencies of the genomic defects and their 95%CIs were calculated for each indication. RESULTS: The overall incidence of cryptic imbalances was 0.7%. The majority involved the DiGeorge syndrome critical region (DGS). The additional diagnostic yield of PNBoBs(TM) in the population with a low a priori risk was 1/298. The prevalences of DGS microdeletion and microduplication in the low-risk population were 1/992 and 1/850, respectively. CONCLUSIONS: The constant a priori risk for common pathogenic cryptic imbalances detected by this technology is estimated to be ~0.3%. A prevalence higher than that previously estimated was found for the 22q11.2 microdeletion. Their frequencies were independent of maternal age. These data have implications for cell-free DNA screening tests design and justify prenatal screening for 22q11 deletion, as early recognition of DGS improves its prognosis.

De novo 2q36.1q36.3 interstitial deletion involving the PAX3 and EPHA4 genes in a fetus with spina bifida and cleft palate.

BACKGROUND: Interstitial 2q36 deletion is a rare event. Only two previously published cases of 2q36 deletions were characterized using array-CGH. This is the first case diagnosed prenatally. METHODS: We report on the prenatal diagnosis of a 2q36.1q36.3 interstitial deletion in a fetus with facial dysmorphism, spina bifida, and cleft palate. RESULTS: Array-CGH analysis revealed a 5.6 Mb interstitial deletion of the long arm of chromosome 2q36.1q36.3, including the PAX3 and EPHA4 genes. CONCLUSION: The present study reinforces the hypothesis that PAX3 haploinsufficiency may be associated with neural tube defects in humans and suggests that the EPHA4 gene might be implicated during palate development. This report also illustrates the added value of array-CGH to detect cryptic chromosomal imbalances in malformed fetuses and to improve genetic counseling prenatally.

A new case of 8q22.1 microdeletion restricts the critical region for Nablus mask-like facial syndrome.

Microdeletions of 8q21.3-8q22.1 have been identified in all patients with Nablus mask-like facial syndrome (NMLFS). A recent report of a patient without this specific phenotype presented a 1.6 Mb deletion in this region that partially overlapped with previously reported 8q21.3 microdeletions, thus restricting critical region for this syndrome. We report on another case of an 8q21.3 deletion revealed by array comparative genome hybridization (aCGH) in a 4-year-old child with global developmental delay, autism, microcephaly, but without Nablus phenotype. The size of the interstitial deletion was estimated to span 5.2 Mb. By combining the data from previous reports on 8q21.3-8q22.1 deletions and our case, we were able to narrow the critical region of Nablus syndrome to 0.5 Mb. The deleted region includes FAM92A1, which seems to be a potential candidate gene in NMLFS.

Feasibility of Optical Genome Mapping from Placental and Umbilical Cord Sampled after Spontaneous or Therapeutic Pregnancy Termination.

Optical genome mapping (OGM) is an alternative to classical cytogenetic techniques to improve the detection rate of clinically significant genomic abnormalities. The isolation of high-molecular-weight (HMW) DNA is critical for a successful OGM analysis. HMW DNA quality depends on tissue type, sample size, and storage conditions. We assessed the feasibility of OGM analysis of DNA from nine umbilical cord (UC) and six chorionic villus (CV) samples collected after the spontaneous or therapeutic termination of pregnancy. We analyzed quality control metrics provided by the Saphyr system (Bionano Genomics) and assessed the length of extracted DNA molecules using pulsed-field capillary electrophoresis. OMG data were successfully analyzed for all six CV samples. Five of the UC samples did not meet the Saphyr quality criteria, mainly due to poor DNA quality. In this regard, we found that DNA quality assessment with pulsed-field capillary electrophoresis can predict a successful OGM analysis. OGM data were fully concordant with the results of standard cytogenetic methods. Moreover, OGM detected an average of 14 additional structural variants involving OMIM genes per sample. On the basis of our results, we established the optimal conditions for sample storage and preparation required for a successful OGM analysis. We recommend checking DNA quality before analysis with pulsed-field capillary electrophoresis if the storage conditions were not ideal or if the quality of the sample is poor. OGM can therefore be performed on fetal tissue harvested after the termination of pregnancy, which opens up the perspective for improved diagnostic yield.

Optical genome mapping for prenatal diagnosis: A prospective study.

PURPOSE: Cytogenetic analysis provides important information for prenatal decision-making and genetic counseling. Optical genome mapping (OGM) has demonstrated its performances in retrospective studies. In our prospective study, we assessed the quality of DNA obtained from cultures of amniotic fluid (AF) and chorionic villi (CV) and evaluated the ability of OGM to detect all clinically relevant aberrations identified by standard methods. METHODS: A total of 37 prenatal samples from pregnancies with a fetal anomaly on ultrasound were analyzed prospectively by OGM between January 1, 2021 and June 31, 2022. OGM results were interpreted blindly and compared to the results obtained by standard techniques. RESULTS: OGM results were interpretable in 92% of samples. We observed 100% concordance between OGM and karyotype and/or chromosomal microarray results. In addition, OGM identified a median of 30 small (<100 kb) structural variations per case with the involvement of 12 OMIM genes, of which 3 were OMIM morbid genes. CONCLUSION: This prospective study showed OGM performed well in detecting genomic alterations in cell cultures from prenatal samples. The place of OGM in relation to CMA or exome sequencing remains to be defined in order to optimize the prenatal diagnostic procedure.

Further refining the critical region of 10q26 microdeletion syndrome: A possible involvement of INSYN2 and NPS in the cognitive phenotype.

BACKGROUND: The 10q26 subtelomeric microdeletion syndrome is a rare and clinically heterogeneous disorder. The precise relationships between the causative genes and the phenotype are unclear. CASE PRESENTATION: We report two new cases of 860 kb deletion of 10q26.2 identified by array CGH in a fetus with intrauterine growth retardation and his mother. The deleted region encompassed only four coding genes, DOCK1, INSYN2, NPS and FOX12. The proband had dysmorphic facies characterized by a high forehead, malformed ears, a prominent nose, and retrognathia. He had bilateral club feet, clinodactily and mild psychomotor retardation. His mother had a short stature, microcephaly, a long face with a high forehead and bitemporal narrowing, arched and sparse eyebrows, strabismus, prominent nose and chin, a thin upper lip and large protruding ears, and mild intellectual disability. CONCLUSIONS: This study presents the smallest 10q26.2 deletion so far identified, which further refines the minimal critical region associated with the 10q26 microdeletion syndrome. It focuses on three genes potentially responsible for the phenotype: DOCK1, which is the major candidate gene, and INSYN2 and NPS, which could be involved in cognitive functions.

Prenatal detection of cryptic rearrangements by multiplex ligation probe amplification in fetuses with ultrasound abnormalities.

PURPOSE: Congenital malformations are a major cause of morbidity and mortality in newborn infants, and genomic imbalances are a significant component of their etiology. The aim of this study was to evaluate the ability of prenatal multiplex ligation probe amplification screening to detect cryptic chromosomal imbalances in fetuses with ultrasound abnormalities of unknown etiology. METHODS: Multiplex ligation probe amplification was performed with three separate sets of probes: two for subtelomeric regions and one for mental retardation syndrome loci. Sixty-one fetuses with significant ultrasound anomalies and normal karyotype at a minimum of 400-band resolution were tested between January 2007 and January 2009. RESULTS: We identified four unbalanced rearrangements: one del 18pter/amp 5pter, one del 9pter, one 15q11q13 microdeletion, and one 22q11 microdeletion with atypical presentation. After genetic counseling, two of the pregnancies were terminated. CONCLUSION: Multiplex ligation probe amplification analysis was able to identify clinically significant rearrangements in 6.5% of fetuses with prenatally identified sonographic abnormalities. This prospective study highlights that multiplex ligation probe amplification screening of fetuses with ultrasound abnormalities in the prenatal period is technically feasible and relevant for diagnosis and prognosis.

Fetal skin fibroblasts: a cell model for studying the retinoid pathway in congenital diaphragmatic hernia.

BACKGROUND: Although there is strong evidence that genetic factors play a pathogenic role in congenital diaphragmatic hernia (CDH), few causal genes have been identified in humans. A number of studies, essentially in animal models, have suggested that disruption of the retinoid signaling pathway plays a major role in the pathogenesis of CDH. Our hypothesis is that human fetal skin fibroblasts express some metabolic and molecular actors of the retinoid pathway and that they offer convenient cellular material for investigating the molecular retinoid pathway defects associated with CDH. METHODS: We first established the expression of receptors, enzymes and binding proteins involved in the retinoic acid (RA) pathway in non-CDH fetal skin fibroblasts using RT-PCR and immunocytochemistry approaches. We then studied the expression of these genes in skin fibroblasts from seven fetuses with isolated and nonisolated CDH. RESULTS: Fetal skin fibroblasts expressed enzymes involved in RA metabolism as well as nuclear receptors for signal transduction. Basal levels of retinoic acid receptor, retinaldehyde dehydrogenase 2, and CYP26 (cytochrome P450 RAI) expression were altered in two of seven fetuses. Interestingly, these genes were previously described as abnormally expressed in CDH physiopathology. CONCLUSION: Our results suggest that human fetal skin fibroblasts could be useful for studying retinoid signaling pathway disruption in the context of CDH. Our proposal is strengthened by the fact that we identified CDH fetuses for which molecular and metabolic actors of the retinoid pathway were not detected.

Retinoid pathway and congenital diaphragmatic hernia: hypothesis from the analysis of chromosomal abnormalities.

BACKGROUND/OBJECTIVES: Although there is strong evidence implicating genetic factors in congenital diaphragmatic hernia (CDH) pathogenesis, few causal genes have been identified. Many studies suggest that early disruption of the retinoid signaling pathway during gestation may contribute to CDH etiology. Chromosome abnormalities are detected in 10-20% of CDH cases. Chromosomal regions that are involved in balanced translocations or are recurrently deleted or duplicated in patients with CDH are of particular interest to researchers because they are more likely to harbor genes that cause or predispose one to the development of CDH. The aim of this review was to select chromosome loci which have been shown to be associated with CDH and to investigate if these loci contain candidate genes involved in the retinoic signaling pathway. DATA SOURCES: We have re-examined the known CDH-critical chromosomal loci and searched in available databases, such as the UCSC Genome Browser and OMIM, to see whether candidate genes related to the retinoid pathway were present within these loci. RESULTS: Twelve retinoid-related genes have been proposed as potential candidates. Among them, COUP-TFII, FOG2 and GATA4 have already been well studied, especially in animal models. We propose other candidates such as STRA6, LRAT, CRBP1, CRBP2 and CRABP1 are directly implicated in retinoic acid metabolism. CONCLUSION: The identification of CDH-related genes and pathways affecting a normal diaphragm will contribute to the understanding of the pathophysiology of this severe embryopathy and might help to facilitate prenatal management and devise more individual treatment strategies. Further studies are necessary to screen large cohorts of patients with CDH for microimbalances or de novo mutations in these candidate genes. Moreover, functional analyses are needed to establish their exact role in CDH etiology.

Analysis of the cost effectiveness of different strategies for the antenatal diagnosis of chromosomal aberrations in cases of ultrasound-identified fetal abnormalities.

OBJECTIVE: Principal objective of this work was to analyse the cost effectiveness of different sequences of cytogenetic techniques from the hospital's point of view, after prenatal ultrasound has identified fetal malformations. METHODS: Cytogenetic tests were performed for each case in 3 strategies, and their results are reported and compared to one reference strategy. Two new simulated strategies were considered: chromosomal microarrays alone and a direct test + CMA. MAIN OUTCOMES MEASURES: cost-effectiveness ratio. RESULTS: A single test result was positive in 234 of the 835 pregnancies studied (28%). CMA alone would have identified 239 abnormalities. In the simulated direct test + CMA sequence, the direct test alone would have been positive for 66.1% of the abnormalities identified. When testing was indicated for NT, reference strategy (Direct + karyotyping) costs 1 084.8 euros by positive test results. Strategies Direct + CMA and CMA alone cost respectively 992.7 and 550.0 euros by positive test results. For OUM indications, reference strategy costs 2 937.8 euros by positive test results. Strategies Direct + CMA and CMA alone cost respectively, 2 118.4 and 1 304.7 euros by positive test results. CONCLUSIONS: CMA appears to be the most effective test for prenatal cytogenetic diagnosis of fetal abnormalities identified by ultrasound.

Primrose syndrome: a phenotypic comparison of patients with a ZBTB20 missense variant versus a 3q13.31 microdeletion including ZBTB20.

Primrose syndrome is characterized by variable intellectual deficiency, behavior disorders, facial features with macrocephaly, and a progressive phenotype with hearing loss and ectopic calcifications, distal muscle wasting, and contractures. In 2014, ZBTB20 variants were identified as responsible for this syndrome. Indeed, ZBTB20 plays an important role in cognition, memory, learning processes, and has a transcription repressive effect on numerous genes. A more severe phenotype was discussed in patients with missense single nucleotide variants than in those with large deletions. Here, we report on the clinical and molecular results of 14 patients: 6 carrying ZBTB20 missense SNVs, 1 carrying an early truncating indel, and 7 carrying 3q13.31 deletions, recruited through the AnDDI-Rares network. We compared their phenotypes and reviewed the data of the literature, in order to establish more powerful phenotype-genotype correlations. All 57 patients presented mild-to-severe ID and/or a psychomotor delay. Facial features were similar with macrocephaly, prominent forehead, downslanting palpebral fissures, ptosis, and large ears. Hearing loss was far more frequent in patients with missense SNVs (p = 0.002), ectopic calcification, progressive muscular wasting, and contractures were observed only in patients with missense SNVs (p nonsignificant). Corpus callosum dysgenesis (p = 0.00004), hypothyroidism (p = 0.047), and diabetes were also more frequent in this group. However, the median age was 9.4 years in patients with deletions and truncating variant compared with 15.1 years in those with missense SNVs. Longer follow-up will be necessary to determine whether the phenotype of patients with deletions is also progressive.

Erratum: Author Correction: A framework to identify contributing genes in patients with Phelan-McDermid syndrome.

[This corrects the article DOI: 10.1038/s41525-017-0035-2.].

Trisomy 20q caused by interstitial duplication 20q13.2: clinical report and literature review.

We report on a 3-year-old boy with moderate developmental delay, abnormal craniofacial features and ventricular septal defect resulting from trisomy of the long arm of chromosome 20. The cytogenetic defect consists of a de novo isolated interstitial duplication in distal 20q [dup(20)(q13.2q13.2)]. The duplication was detected by comparative genomic hybridization (CGH) and confirmed by array CGH. Other cases of comparable trisomies are reviewed. This new case further delineates the recognizable phenotype of trisomy 20q13 --> 20qter and highlights the relevance of CGH for the detection of such rearrangements.

The occurrence of Brugada syndrome and isolated cardiac conductive disease in the same family could be due to a single SCN5A mutation or to the accidental association of both diseases.

AIMS: The distinct cardiac arrhythmia diseases, Brugada syndrome (BS) and isolated cardiac conduction disease (ICCD) are caused by heterozygous mutations in the SCN5A gene. Previous studies have demonstrated an intriguing association between ICCD and BS with the same mutation in the SCN5A gene. METHODS AND RESULTS: The proband of a multigenerational family presented BS and a familial history of sudden death. We performed clinical evaluations in family members including drug testing and screening for SCN5A mutations. Based on electrocardiogram features, we identified four individuals with BS, two with ICCD and one compatible with both. For five individuals, one with BS and ICCD, three with BS and one with ICCD, we characterized a heterozygous C- to T- mutation at position 4313 (P1438L) in the SCN5A gene. Expression studies of the P1438L mutation showed non-functional channels. The proband's father with the BS phenotype was not a carrier of the new SCN5A mutation. CONCLUSION: We report the case of a family with BS and/or ICCD and describe a novel mutation, the P1438L SCN5A mutation. In this family, the occurrence of BS and ICCD could be due to this single mutation but also to the accidental association of both diseases.