Performance of Massive Parallel Sequencing-Based Cell-Free DNA Testing in Compromised Pregnancies.

Background/Objectives: Non-Invasive prenatal test (NIPT) is used as a universal or contingent test after prior risk assessment. Screening is mainly performed for common trisomies (T21, T13, T18), although other chromosomal anomalies may be detected. Our objective was to study the performance of GWNIPT in the detection of chromosomal abnormalities in pregnancies in which an invasive prenatal study was performed and in early pregnancy losses, in comparison with the reference test. Method: VeriSeqTM NIPT Solution v2, a genome-wide NIPT (GWNIPT), was performed prior to invasive testing in fetal diagnostic study cases (FDS, n = 155) and in early pregnancy losses (EPL, n = 68). Results: In the FDS group, the diagnostic test (QFPCR, array and karyotype) detected anomalies in 32 pregnancies (21%), in twenty of them (61%) also detected by GWNIPT. Eleven of the twelve cases undetected by GWNIPT were balanced translocations (n = 4) or deletions/duplications <7 Mb (n = 7). In the EPL group, GWNIPT detected anomalies in 46% of cases (31/68) but comparison with reference test (QFPCR and karyotype) in products of conception (POC) was only possible in 18 cases. Concordant results between POC and GWNIPT test were obtained in 16 of the 18 cases. In EPL, with GWNIPT testing, common trisomies accounted for 25.8% of cases (8/31), rare trisomies 54.8% (17/31) and microdeletions/duplications 16.1% (5/31). Conclusions: The GWNIPT test may be useful in clinical practice in prenatal and in EPL's genetic diagnosis when the appropriate sample is not available.

ZDHHC15 as a candidate gene for autism spectrum disorder.

The phenotypic repercussion of ZDHHC15 haploinsufficiency is not well-known. This gene was initially suggested as a candidate for X-linked mental retardation, but such an association was later questioned. We studied a multiplex family with three members with autism spectrum disorder (ASD) by array CGH, karyotype, exome sequencing and X-chromosome inactivation patterns. Medical history interviews, cognitive and physical examinations, and sensory profiling were also assessed. The three family members with ASD (with normal cognitive abilities and an abnormal sensory profile) were the only carriers of a 1.7 Mb deletion in the long arm of chromosome X, involving: ZDHHC15, MAGEE2, PBDC1, MAGEE1, MIR384 and MIR325. The normal chromosome X was preferentially inactivated in female carriers, and the whole exome sequencing of an affected family member did not reveal any additional genetic variant that could explain the phenotype. Thus, in the present family, ASD segregates with a deletion on chromosome X that includes ZDHHC15. Considering our results together with gene data (regarding function, expression, conservation and animal/cellular models), ZDHHC15 is a candidate gene for ASD. Emerging evidence also suggests that this gene could be associated with other neurodevelopmental disorders, with incomplete penetrance and variable expressivity.

A Novel Intragenic Duplication in the HDAC8 Gene Underlying a Case of Cornelia de Lange Syndrome.

Cornelia de Lange syndrome (CdLS) is a multisystemic genetic disorder characterized by distinctive facial features, growth retardation, and intellectual disability, as well as various systemic conditions. It is caused by genetic variants in genes related to the cohesin complex. Single-nucleotide variations are the best-known genetic cause of CdLS; however, copy number variants (CNVs) clearly underlie a substantial proportion of cases of the syndrome. The NIPBL gene was thought to be the locus within which clinically relevant CNVs contributed to CdLS. However, in the last few years, pathogenic CNVs have been identified in other genes such as HDAC8, RAD21, and SMC1A. Here, we studied an affected girl presenting with a classic CdLS phenotype heterozygous for a de novo ~32 kbp intragenic duplication affecting exon 10 of HDAC8. Molecular analyses revealed an alteration in the physiological splicing that included a 96 bp insertion between exons 9 and 10 of the main transcript of HDAC8. The aberrant transcript was predicted to generate a truncated protein whose accessibility to the active center was restricted, showing reduced ease of substrate entry into the mutated enzyme. Lastly, we conclude that the duplication is responsible for the patient's phenotype, highlighting the contribution of CNVs as a molecular cause underlying CdLS.

Genomic Chaos (Multiple Copy Number Variations and Structural Reorganization) Detected in Two Prenatal Cases.

The use of new technologies in the routine diagnosis of constitutional abnormalities, such as high-resolution chromosomal microarray and next-generation sequencing, has unmasked new mechanisms for generating structural variation of the human genome. For example, complex chromosome rearrangements can originate by a chromosome catastrophe phenomenon in which numerous genomic rearrangements are apparently acquired in a single catastrophic event. This phenomenon is named chromoanagenesis (from the Greek "chromo" for chromosome and "anagenesis" for rebirth). Herein, we report 2 cases of genomic chaos detected at prenatal diagnosis. The terms "chromothripsis" and "chromoanasynthesis" and the challenge of genetic counseling are discussed.

Chromosomal microarray analysis in fetuses with central nervous system anomalies: An 8-year long observational study from a tertiary care university hospital.

OBJECTIVES: To evaluate the prevalence of DNA copy number variants (CNVs) detected with array comparative genomic hybridization (CGH) in fetuses with central nervous system (CNS) anomalies. Secondary objectives were to describe the prevalence of CNV in specific CNS abnormalities, in isolated defects or associated with other malformations or fetal growth restriction (FGR). METHODS: Observational cohort study in 238 fetuses with CNS anomalies in which an array-CGH had been performed between January 2009 and December 2017. Pathogenic CNV and variants of unknown significance (VUS) were reported. RESULTS: Pathogenic CNVs were found in 16/238 cases (6.7%), VUS in 18/238 (7.6%), and normal result in 204/238 (85.7%) cases. Pathogenic CNVs were more frequent in posterior fossa anomalies (cerebellar hypoplasia 33%, megacisterna magna 20%), moderate ventriculomegaly (11%) and spina bifida (3.7%). Pathogenic CNVs and VUS were found in 7/182 (3.8%) and 14/182 (7.7%) cases of isolated anomalies, in 9/49 (18.4%) and 4/49 (8.2%) presenting another malformation, and in 0/7 and 0/7 cases with associated FGR (P = .001, P = .741, respectively). CONCLUSION: These results provide strong evidence toward performing array in fetuses with CNS anomalies, particular in cases of posterior fossa anomalies. The prevalence of pathogenic CNVs is higher in association with other malformations.

Molecular characterization of Spanish patients with MECP2 duplication syndrome.

MECP2 duplication syndrome (MDS) is an X-linked neurodevelopmental disorder characterized by a severe to profound intellectual disability, early onset hypotonia and diverse psycho-motor and behavioural features. To date, fewer than 200 cases have been published. We report the clinical and molecular characterization of a Spanish MDS cohort that included 19 boys and 2 girls. Clinical suspicions were confirmed by array comparative genomic hybridization and multiplex ligation-dependent probe amplification (MLPA). Using, a custom in-house MLPA assay, we performed a thorough study of the minimal duplicated region, from which we concluded a complete duplication of both MECP2 and IRAK1 was necessary for a correct MDS diagnosis, as patients with partial MECP2 duplications lacked some typical clinical traits present in other MDS patients. In addition, the duplication location may be related to phenotypic severity. This observation may provide a new approach for genotype-phenotype correlations, and thus more personalized genetic counselling.

LRBA Deficiency in a Patient With a Novel Homozygous Mutation Due to Chromosome 4 Segmental Uniparental Isodisomy.

LRBA deficiency was first described in 2012 as an autosomal recessive disorder caused by biallelic mutations in the LRBA gene (OMIM #614700). It was initially characterized as producing early-onset hypogammaglobulinemia, autoimmune manifestations, susceptibility to inflammatory bowel disease, and recurrent infection. However, further reports expanded this phenotype (including patients without hypogammaglobulinemia) and described LRBA deficiency as a clinically variable syndrome with a wide spectrum of clinical manifestations. We present the case of a female patient who presented with type 1 diabetes, psoriasis, oral thrush, and enlarged liver and spleen at the age of 8 months. She later experienced recurrent bacterial and viral infections, including pneumococcal meningitis and Epstein Barr viremia. She underwent two consecutive stem cell transplants at the age of 8 and 9 years, and ultimately died. Samples from the patient and her parents were subjected to whole exome sequencing, which revealed a homozygous 1-bp insertion in exon 23 of the patient's LRBA gene, resulting in frameshift and premature stop codon. The patient's healthy mother was heterozygous for the mutation and her father tested wild-type. This finding suggested that either one copy of the paternal chromosome 4 bore a deletion including the LRBA locus, or the patient inherited two copies of the mutant maternal LRBA allele. The patient's sequencing data showed a 1-Mb loss of heterozygosity region in chromosome 4, including the LRBA gene. Comparative genomic hybridization array of the patient's and father's genomic DNA yielded normal findings, ruling out genomic copy number abnormalities. Here, we present the first case of LRBA deficiency due to a uniparental disomy (UPD). In contrast to classical Mendelian inheritance, UPD involves inheritance of 2 copies of a chromosomal region from only 1 parent. Specifically, our patient carried a small segmental isodisomy of maternal origin affecting 1 Mb of chromosome 4.

Novel Double Factor PGT strategy analyzing blastocyst stage embryos in a single NGS procedure.

In families at risk from monogenic diseases affected offspring, it is fundamental the development of a suitable Double Factor Preimplantation Genetic Testing (DF-PGT) method for both single-gene analysis and chromosome complement screening. Aneuploidy is not only a major issue in advanced-maternal-age patients and balanced translocation carriers, but also the aneuploidy rate is extremely high in patients undergoing in vitro fertilization (IVF), even in young donors. To adequate NGS technology to the DF-PGT strategy four different whole genome amplification systems (Sureplex, MALBAC, and two multiple displacement amplification systems-MDA) were tested using TruSight One panel on cell lines and blastocyst trophectoderm biopsies-TE. Embryo cytogenetic status was analyzed by Nexus software. Sureplex and MALBAC DNA products were considered not suitable for PGT diagnosis due to inconsistent and poor results on Trusight one (TSO) panel. Results obtained with both MDA based methods (GEH-MDA and RG-MDA) were appropriate for direct mutation detection by TSO NGS platform. Nevertheless, RG-MDA amplification products showed better coverage and lower ADO rates than GEH-MDA. The present work also demonstrates that the same TSO sequencing data is suitable not only for the direct mutation detection, but also for the indirect mutation detection by linkage analysis of informative SNPs. The present work also demonstrates that Nexus software is competent for the detection of CNV by using with TSO sequencing data from RG-MDA products, allowing for the whole cytogenetic characterization of the embryos. In conclusion, successfully development of an innovative and promising DF-PGT strategy using TSO-NGS technology in TE biopsies, performed in-house in a single laboratory experience, has been done in the present work. Additional studies should be performed before it could be used as a diagnostic alternative in order to validate this approach for the detection of chromosomal aneuploidies.

TNFAIP3 haploinsufficiency is the cause of autoinflammatory manifestations in a patient with a deletion of 13Mb on chromosome 6.

There is scarce literature about autoinflammation in syndromic patients. We describe a patient who, in addition to psychomotor and growth delay, presented with fevers, neutrophilic dermatosis, and recurrent orogenital ulcers. Comparative Genomic Hybridization (CGH) array permitted to identify a 13.13Mb deletion on chromosome 6, encompassing 53 genes, and including TNFAIP3 gene (A20). A20 is a potent inhibitor of the NF-kB signalling pathway and restricts inflammation via its deubiquitinase activity. Western blotting and immunoprecipitation assays showed decreased A20 expression and increased phosphorylation of p65 and IkBa. Patient's cells displayed increased levels of total K63-linked ubiquitin and increased levels of ubiquitinated RIP and NEMO after stimulation with TNF. We describe the molecular characterization of an autoinflammatory disease due to a large chromosomal deletion and review the phenotypes of patients with A20 haploinsufficiency. CGH arrays should be the first diagnostic method for comprehensive analysis of patients with syndromic features and immune dysregulation.

Further delineation of the SOX18-related Hypotrichosis, Lymphedema, Telangiectasia syndrome (HTLS).

The transcription factor SOX18 has been shown to play a role in the development of hair, blood and lymphatic vessels. Mutations in SOX18 result in hereditary lymphedema, with the unique clinical association of hypotrichosis and telangiectasia (HLTS). Some patients present with additional disease features which may be explained by the location of SOX18 mutation. We report a patient with hypotrichosis-lymphedema-telangiectasia syndrome (HLTS) confirmed by detection of a novel mutation in the SOX18 gene. Few cases of HTLS have been reported in the literature. We reviewed all cases reported to date to delineate the clinical manifestations that allow us to prompt diagnosis of this syndrome for appropriate management and genetic counseling.

[Comparative genomic hybridisation as a first option in genetic diagnosis: 1,000 cases and a cost-benefit analysis]. / Array CGH como primera opción en el diagnóstico genético: 1.000 casos y análisis de coste-beneficio.

BACKGROUND AND OBJECTIVE: Conventional cytogenetics diagnoses 3-5% of patients with unexplained developmental delay/intellectual disability and/or multiple congenital anomalies. The Multiplex Ligation-dependent Probe Amplification increases diagnostic rates from between 2.4 to 5.8%. Currently the comparative genomic hybridisation array or aCGH is the highest performing diagnostic tool in patients with developmental delay/intellectual disability, congenital anomalies and autism spectrum disorders. Our aim is to evaluate the efficiency of the use of aCGH as first-line test in these and other indications (epilepsy, short stature). PATIENTS AND METHOD: A total of 1000 patients referred due to one or more of the abovementioned disorders were analysed by aCGH. RESULTS: Pathogenic genomic imbalances were detected in 14% of the cases, with a variable distribution of diagnosis according to the phenotypes: 18.9% of patients with developmental delay/intellectual disability; 13.7% of multiple congenital anomalies, 9.76% of psychiatric pathologies, 7.02% of patients with epilepsy, and 13.3% of patients with short stature. Within the multiple congenital anomalies, central nervous system abnormalities and congenital heart diseases accounted for 14.9% and 10.6% of diagnoses, respectively. Among the psychiatric disorders, patients with autism spectrum disorders accounted for 8.9% of the diagnoses. CONCLUSIONS: Our results demonstrate the effectiveness and efficiency of the use of aCGH as the first line test in genetic diagnosis of patients suspected of genomic imbalances, supporting its inclusion within the National Health System.

A Novel Recurrent Breakpoint Responsible for Rearrangements in the Williams-Beuren Region.

Copy number variants (CNVs) of the Williams-Beuren syndrome (WBS) 7q11.23 region are responsible for neurodevelopmental disorders with multisystem involvement and variable expressivity. We found 2 patients with a deletion and 1 patient with a duplication in this region sharing a common breakpoint located between the LIMK1 and EIF4H(WBSCR1) genes. One patient had a WBS phenotype, although testing with a commercially available FISH assay was negative for the deletion. A further test using array CGH showed an atypical WBS region deletion. The second patient showed global developmental delay, speech delay and poor motor skills with a deletion outside the WBS region. The third patient had manifestations compatible with an autism spectrum disorder showing a duplication in the WBS region. Our findings point to the existence of a previously unrecognized recurrent breakpoint responsible for rearrangements in the WBS region. Given that most commercial FISH assays include probes flanking this novel breakpoint, further testing with array CGH should be performed in patients with WBS and negative FISH results.

PIAS4 is associated with macro/microcephaly in the novel interstitial 19p13.3 microdeletion/microduplication syndrome.

Array comparative genomic hybridization (aCGH) is a powerful genetic tool that has enabled the identification of novel imbalances in individuals with intellectual disability (ID), autistic disorders and congenital malformations. Here we report a 'genotype first' approach using aCGH on 13 unrelated patients with 19p13.3 submicroscopic rearrangement (11 deletions and 2 duplications) and review cases in the literature and in public databases. Shared phenotypic features suggest that these patients represent an interstitial microdeletion/microduplication syndrome at 19p13.3. Common features consist of abnormal head circumference in most patients (macrocephaly with the deletions and microcephaly with the duplications), ID with developmental delay (DD), hypotonia, speech delay and common dysmorphic features. The phenotype is associated with at least a ~0.113 Mb critical region harboring three strong candidate genes probably associated with DD, ID, speech delay and other dysmorphic features: MAP2K2, ZBTB7A and PIAS4, an E3 ubiquitin ligase involved in the ubiquitin signaling pathways, which we hypothesize for the first time to be associated with head size in humans.

Three-Year Follow-Up of a Prenatally Ascertained Apparently Non-Mosaic sSMC(10): Delineation of a Non-Critical Region.

Small supernumerary marker chromosomes (sSMC) originating from chromosome 10 are rare and usually found in mosaic form. We present a de novo apparently non-mosaic sSMC(10) prenatally diagnosed in amniotic fluid and postnatally confirmed in peripheral blood. Characterization by array-CGH showed a pericentromeric duplication of 7.1 Mb of chromosome 10. The fetus did not show ultrasound abnormalities, and a normal female phenotype was observed during a 3-year postnatal follow-up. The absence of phenotypic abnormalities in the present case provides evidence of a non-critical pericentromeric region in 10p11.21q11.1 (hg19 35,355,570-42,448,569) associated with a duplication.

Intrachromosomal 3p insertion as a cause of reciprocal pure interstitial deletion and duplication in two siblings: further delineation of the emerging proximal 3p deletion syndrome.

Very few cases of constitutional interstitial deletions of the proximal short arm of chromosome 3 have been reported; however, the proximal 3p deletion is emerging as a clinically recognizable syndrome. We present an intrachromosomal insertion of 3p12.3p14.1 in a phenotypic normal man (46,XY,ins(3)(p25p12.3p14.1)) which is responsible for the unbalanced karyotype in 2 affected offspring, one with a 3p12.3p14.1 interstitial deletion and the other with a reciprocal duplication. The exceptionality of these 2 reciprocal recombinants contributes to a better definition of the proximal 3p deletion syndrome and its duplication counterpart.

Trisomy 18p caused by a supernumerary marker with a chromosome 13/21 centromere: a possible recurrent chromosome aberration.

We present a clinical and molecular cytogenetic characterization of two new patients with a complex supernumerary marker consisting of the entire short arm of chromosome 18 with a chromosome 13/21 centromere. One patient is a girl with a nonsyndromic intellectual disability and the second is a prenatally diagnosed fetus. To our knowledge, these are the fourth and fifth such cases to be described in the literature, suggesting the existence of a possible recurring constitutional structural chromosome abnormality.

De novo MECP2 disomy in a Mexican male carrying a supernumerary marker chromosome and no typical Lubs syndrome features.

Xq28 duplication, including the MECP2 gene, is among the most frequently identified Xq subtelomeric rearrangements. The resulting clinical phenotype is named Lubs syndrome and mainly consists of intellectual disability, congenital hypotonia, absent speech, recurrent infections, and seizures. Here we report a Mexican male patient carrying a supernumerary marker chromosome with de novo Xq28 gain. By MLPA, duplication of MECP2, GDI1, and SLC6A8 was found and a subsequent a-CGH analysis demonstrated that the gain spanned ~2.1Mb. Despite gain of the MECP2 gene, the features of this patient do not evoke Lubs syndrome. Probably the mosaicism of the supernumerary marker chromosome is modifying the phenotype in this patient.

Clinical utility of chromosomal microarray analysis in invasive prenatal diagnosis.

Novel methodologies for detection of chromosomal abnormalities have been made available in the recent years but their clinical utility in prenatal settings is still unknown. We have conducted a comparative study of currently available methodologies for detection of chromosomal abnormalities after invasive prenatal sampling.A multicentric collection of a 1-year series of fetal samples with indication for prenatal invasive sampling was simultaneously evaluated using three screening methodologies: (1) karyotype and quantitative fluorescent polymerase chain reaction (QF-PCR), (2) two panels of multiplex ligation-dependent probe amplification (MLPA), and (3) chromosomal microarray-based analysis (CMA) with a targeted BAC microarray. A total of 900 pregnant women provided informed consent to participate (94% acceptance rate). Technical performance was excellent for karyotype, QF-PCR, and CMA (~1% failure rate), but relatively poor for MLPA (10% failure). Mean turn-around time (TAT) was 7 days for CMA or MLPA, 25 for karyotype, and two for QF-PCR, with similar combined costs for the different approaches. A total of 57 clinically significant chromosomal aberrations were found (6.3%), with CMA yielding the highest detection rate (32% above other methods). The identification of variants of uncertain clinical significance by CMA (17, 1.9%) tripled that of karyotype and MLPA, but most alterations could be classified as likely benign after proving they all were inherited. High acceptability, significantly higher detection rate and lower TAT, could justify the higher cost of CMA and favor targeted CMA as the best method for detection of chromosomal abnormalities in at-risk pregnancies after invasive prenatal sampling.

Assessment of QF-PCR as the first approach in prenatal diagnosis.

Quantitative fluorescent PCR (QF-PCR) has been used by many laboratories for prenatal diagnosis of the most common aneuploidies. QF-PCR is rapid, cost-effective, and suitable for automation and can detect most abnormalities diagnosed by conventional karyotyping. Whether QF-PCR should be used alone in most of the samples and in which karyotyping should also be offered is currently a topic of debate. We evaluated and compared the results obtained from 7679 prenatal samples in which conventional karyotype and QF-PCR had been performed, including 1243 chorionic villi and 6436 amniotic fluid samples. Concordant QF-PCR and karyotype results were obtained in 98.75% of the samples. An abnormal karyotype associated with adverse clinical outcome undetected by QF-PCR was found in 0.05% of samples. Therefore, QF-PCR can be used alone in a large number of samples studied in a prenatal laboratory, thereby reducing both the workload in cytogenetic laboratories and parental anxiety when awaiting results.