The clinical value of optical genome mapping in the rapid characterization of RB1 duplication and 15q23q24.2 triplication, for more appropriate prenatal genetic counselling.

BACKGROUND: Despite recent advances in prenatal genetic diagnosis, medical geneticists still face considerable difficulty in interpreting the clinical outcome of copy-number-variant duplications and defining the mechanisms underlying the formation of certain chromosomal rearrangements. Optical genome mapping (OGM) is an emerging cytogenomic tool with proved ability to identify the full spectrum of cytogenetic aberrations. METHODS: Here, we report on the use of OGM in a prenatal diagnosis setting. Detailed breakpoint mapping was used to determine the relative orientations of triplicated and duplicated segments in two unrelated foetuses harbouring chromosomal aberrations: a de novo 15q23q24.2 triplication and a paternally inherited 13q14.2 duplication that overlapped partially with the RB1 gene. RESULTS: OGM enabled us to suggest a plausible mechanism for the triplication and confirmed that the RB1 duplication was direct oriented and in tandem. This enabled us to predict the pathogenic consequences, refine the prognosis and adapt the follow-up and familial screening appropriately. CONCLUSION: Along with an increase in diagnostic rates, OGM can rapidly highlight genotype-phenotype correlations, improve genetic counselling and significantly influence prenatal management.

Episignatures in practice: independent evaluation of published episignatures for the molecular diagnostics of ten neurodevelopmental disorders.

Variants of uncertain significance (VUS) are a significant issue for the molecular diagnosis of rare diseases. The publication of episignatures as effective biomarkers of certain Mendelian neurodevelopmental disorders has raised hopes to help classify VUS. However, prediction abilities of most published episignatures have not been independently investigated yet, which is a prerequisite for an informed and rigorous use in a diagnostic setting. We generated DNA methylation data from 101 carriers of (likely) pathogenic variants in ten different genes, 57 VUS carriers, and 25 healthy controls. Combining published episignature information and new validation data with a k-nearest-neighbour classifier within a leave-one-out scheme, we provide unbiased specificity and sensitivity estimates for each of the signatures. Our procedure reached 100% specificity, but the sensitivities unexpectedly spanned a very large spectrum. While ATRX, DNMT3A, KMT2D, and NSD1 signatures displayed a 100% sensitivity, CREBBP-RSTS and one of the CHD8 signatures reached <40% sensitivity on our dataset. Remaining Cornelia de Lange syndrome, KMT2A, KDM5C and CHD7 signatures reached 70-100% sensitivity at best with unstable performances, suffering from heterogeneous methylation profiles among cases and rare discordant samples. Our results call for cautiousness and demonstrate that episignatures do not perform equally well. Some signatures are ready for confident use in a diagnostic setting. Yet, it is imperative to characterise the actual validity perimeter and interpretation of each episignature with the help of larger validation sample sizes and in a broader set of episignatures.

Prenatal Diagnosis of Primrose Syndrome.

Primrose syndrome is a very rare congenital malformation. Symptoms of this disorder may appear during childhood, but the diagnosis is identified in adulthood in the majority of cases. The prenatal diagnosis of Primrose syndrome is not developed in the literature. We present herein a case series of 3 cases with characteristic sonographic features. A dysmorphic metopic suture, downslanting palpebral fissures, a wide forehead, and agenesis of corpus callosum are the main signs. A missense mutation in ZBTB20 identified in whole exome sequencing can confirm the prenatal diagnosis of Primrose syndrome.

Spermatogonial depletion and a spermatogenesis defect in the Dp(16)1Yey mouse model of Down syndrome.

Down syndrome (DS), or Trisomy 21, is the most common chromosomal disorder in humans. Men with DS are infertile. The DYRK1A gene on Hsa21 is involved in several features of DS. Overexpression of the homolog dyrk1A disrupts primordial germ cell migration in zebrafish, and overexpression of Dyrk1A impairs gonadotropic axis function and the early stages of spermatogenesis in the mouse. Other genes on Hsa21 might be involved in the pathogenesis of infertility in DS. We investigated the Dp(16)1Yey mouse model of DS, which features segmental duplication of chromosome Mmu16 (orthologous to a large part of Hsa21 and carrying Dyrk1A and 112 other genes). Using an immunohistochemical assay for the spermatogonial marker STRA8, we observed spermatogonial depletion in the Dp(16)1Yey mouse. This was correlated with low mRNA expression of GFR1 (a marker of the self-renewal stem cell pool) in an RT-qPCR assay and low protein expression of PLZF (a marker of differentiating stem cells) in a slot-blot assay. Spermatogenesis was present but impaired, with a low sperm count, low protamine-1 expression, a low testis weight, and a low seminiferous tubule diameter. Low circulating luteinizing hormone and follicle-stimulating hormone levels and an elevated testis anti-Müllerian hormone level (as measured in ELISAs) revealed the presence of hypogonadotropic hypogonadism. The Dp(16)1Yey mouse model of DS recapitulates observations made in zebrafish and mice overexpressing DYRK1A homologs. The presence of an excess of Mmu16 material perturbs spermatogenesis and the gonadotropic axis. More generally, DYRK1A's role in human infertility (outside DS) remains to be characterized.

2p25.3 microduplications involving MYT1L: further phenotypic characterization through an assessment of 16 new cases and a literature review.

Microduplications involving the MYT1L gene have mostly been described in series of patients with isolated schizophrenia. However, few reports have been published, and the phenotype has still not been well characterized. We sought to further characterize the phenotypic spectrum of this condition by describing the clinical features of patients with a pure 2p25.3 microduplication that includes all or part of MYT1L. We assessed 16 new patients with pure 2p25.3 microduplications recruited through a French national collaboration (n = 15) and the DECIPHER database (n = 1). We also reviewed 27 patients reported in the literature. For each case, we recorded clinical data, the microduplication size, and the inheritance pattern. The clinical features were variable and included developmental and speech delays (33%), autism spectrum disorder (ASD, 23%), mild-to-moderate intellectual disability (ID, 21%), schizophrenia (23%), or behavioral disorders (16%). Eleven patients did not have an obvious neuropsychiatric disorder. The microduplications ranged from 62.4 kb to 3.8 Mb in size and led to duplication of all or part of MYT1L; seven of these duplications were intragenic. The inheritance pattern was available for 18 patients: the microduplication was inherited in 13 cases, and all parents but one had normal phenotype. Our comprehensive review and expansion of the phenotypic spectrum associated with 2p25.3 microduplications involving MYT1L should help clinicians to better assess, counsel and manage affected individuals. MYT1L microduplications are characterized by a spectrum of neuropsychiatric phenotypes with incomplete penetrance and variable expressivity, which are probably due to as-yet unknown genetic and nongenetic modifiers.

Prenatal diagnosis by trio exome sequencing in fetuses with ultrasound anomalies: A powerful diagnostic tool.

Introduction: Prenatal ultrasound (US) anomalies are detected in around 5%-10% of pregnancies. In prenatal diagnosis, exome sequencing (ES) diagnostic yield ranges from 6% to 80% depending on the inclusion criteria. We describe the first French national multicenter pilot study aiming to implement ES in prenatal diagnosis following the detection of anomalies on US. Patients and methods: We prospectively performed prenatal trio-ES in 150 fetuses with at least two US anomalies or one US anomaly known to be frequently linked to a genetic disorder. Trio-ES was only performed if the results could influence pregnancy management. Chromosomal microarray (CMA) was performed before or in parallel. Results: A causal diagnosis was identified in 52/150 fetuses (34%) with a median time to diagnosis of 28 days, which rose to 56/150 fetuses (37%) after additional investigation. Sporadic occurrences were identified in 34/56 (60%) fetuses and unfavorable vital and/or neurodevelopmental prognosis was made in 13/56 (24%) fetuses. The overall diagnostic yield was 41% (37/89) with first-line trio-ES versus 31% (19/61) after normal CMA. Trio-ES and CMA were systematically concordant for identification of pathogenic CNV. Conclusion: Trio-ES provided a substantial prenatal diagnostic yield, similar to postnatal diagnosis with a median turnaround of approximately 1 month, supporting its routine implementation during the detection of prenatal US anomalies.

Antenatal ultrasound features of isolated recurrent copy number variation in 7q11.23 (Williams syndrome and 7q11.23 duplication syndrome).

OBJECTIVE: We aimed to gather fetal cases carrying a 7q11.23 copy number variation (CNV) and collect precise clinical data to broaden knowledge of antenatal features in these syndromes. METHODS: We retrospectively recruited unrelated cases with 7q11.23 deletion, known as Williams-Beuren syndrome (WBS), or 7q11.23 duplication who had prenatal ultrasound findings. We collected laboratory and clinical data, fetal ultrasound, cardiac ultrasound and fetal autopsy reports from 18 prenatal diagnostic centers throughout France. RESULTS: 40 fetuses with WBS were collected and the most common features were intra-uterine growth retardation (IUGR) (70.0%, 28/40), cardiovascular defects (30.0%, 12/40), polyhydramnios (17.5%, 7/40) and protruding tongue (15.0%, 6/40). Fetal autopsy reports were available for 11 cases and were compared with ultrasound prenatal features. Four cases of fetuses with 7q11.23 microduplication were collected and prenatal ultrasound signs were variable and often isolated. CONCLUSION: This work strengthens the fact that 7q11.23 CNVs are associated with a broad spectrum of antenatal presentations. IUGR and cardiovascular defects were the most frequent ultrasound signs. By reporting the biggest series of antenatal WBS, we aim to better delineate distinctive signs in fetuses with 7q11.23 CNVs.

MPC2 variants disrupt mitochondrial pyruvate metabolism and cause an early-onset mitochondriopathy.

Pyruvate is an essential metabolite produced by glycolysis in the cytosol and must be transported across the inner mitochondrial membrane into the mitochondrial matrix, where it is oxidized to fuel mitochondrial respiration. Pyruvate import is performed by the mitochondrial pyruvate carrier (MPC), a hetero-oligomeric complex composed by interdependent subunits MPC1 and MPC2. Pathogenic variants in the MPC1 gene disrupt mitochondrial pyruvate uptake and oxidation and cause autosomal-recessive early-onset neurological dysfunction in humans. The present work describes the first pathogenic variants in MPC2 associated with human disease in four patients from two unrelated families. In the first family, patients presented with antenatal developmental abnormalities and harboured a homozygous c.148T>C (p.Trp50Arg) variant. In the second family, patients that presented with infantile encephalopathy carried a missense c.2T>G (p.Met1?) variant disrupting the initiation codon. Patient-derived skin fibroblasts exhibit decreased pyruvate-driven oxygen consumption rates with normal activities of the pyruvate dehydrogenase complex and mitochondrial respiratory chain and no defects in mitochondrial content or morphology. Re-expression of wild-type MPC2 restored pyruvate-dependent respiration rates in patient-derived fibroblasts. The discovery of pathogenic variants in MPC2 therefore broadens the clinical and genetic landscape associated with inborn errors in pyruvate metabolism.

Exome sequencing as a first-tier test for copy number variant detection: retrospective evaluation and prospective screening in 2418 cases.

BACKGROUND: Despite the availability of whole exome (WES) and genome sequencing (WGS), chromosomal microarray (CMA) remains the first-line diagnostic test in most rare disorders diagnostic workup, looking for copy number variations (CNVs), with a diagnostic yield of 10%-20%. The question of the equivalence of CMA and WES in CNV calling is an organisational and economic question, especially when ordering a WGS after a negative CMA and/or WES. METHODS: This study measures the equivalence between CMA and GATK4 exome sequencing depth of coverage method in detecting coding CNVs on a retrospective cohort of 615 unrelated individuals. A prospective detection of WES-CNV on a cohort of 2418 unrelated individuals, including the 615 individuals from the validation cohort, was performed. RESULTS: On the retrospective validation cohort, every CNV detectable by the method (ie, a CNV with at least one exon not in a dark zone) was accurately called (64/64 events). In the prospective cohort, 32 diagnoses were performed among the 2418 individuals with CNVs ranging from 704 bp to aneuploidy. An incidental finding was reported. The overall increase in diagnostic yield was of 1.7%, varying from 1.2% in individuals with multiple congenital anomalies to 1.9% in individuals with chronic kidney failure. CONCLUSION: Combining single-nucleotide variant (SNV) and CNV detection increases the suitability of exome sequencing as a first-tier diagnostic test for suspected rare Mendelian disorders. Before considering the prescription of a WGS after a negative WES, a careful reanalysis with updated CNV calling and SNV annotation should be considered.

Toward clinical and molecular dissection of frontonasal dysplasia with facial skin polyps: From Pai syndrome to differential diagnosis through a series of 27 patients.

Unique or multiple congenital facial skin polyps are features of several rare syndromes, from the most well-known Pai syndrome (PS), to the less recognized oculoauriculofrontonasal syndrome (OAFNS), encephalocraniocutaneous lipomatosis (ECCL), or Sakoda complex (SC). We set up a research project aiming to identify the molecular bases of PS. We reviewed 27 individuals presenting with a syndromic frontonasal polyp and initially referred for PS. Based on strict clinical classification criteria, we could confirm only nine (33%) typical and two (7%) atypical PS individuals. The remaining ones were either OAFNS (11/27-41%) or presenting with an overlapping syndrome (5/27-19%). Because of the phenotypic overlap between these entities, OAFNS, ECCL, and SC can be either considered as differential diagnosis of PS or part of the same spectrum. Exome and/or genome sequencing from blood DNA in 12 patients and from affected tissue in one patient failed to identify any replication in candidate genes. Taken together, our data suggest that conventional approaches routinely utilized for the identification of molecular etiologies responsible for Mendelian disorders are inconclusive. Future studies on affected tissues and multiomics studies will thus be required in order to address either the contribution of mosaic or noncoding variation in these diseases.

DYRK1A Overexpression in Mice Downregulates the Gonadotropic Axis and Disturbs Early Stages of Spermatogenesis.

Down syndrome (DS) is the most common chromosomal disorder. It is responsible for intellectual disability (ID) and several medical conditions. Although men with DS are thought to be infertile, some spontaneous paternities have been reported. The few studies of the mechanism of infertility in men with DS are now dated. Recent research in zebrafish has indicated that overexpression of DYRK1A (the protein primarily responsible for ID in DS) impairs gonadogenesis at the embryonic stage. To better ascertain DYRK1A's role in infertility in DS, we investigated the effect of DYRK1A overexpression in a transgenic mouse model. We found that overexpression of DYRK1A impairs fertility in transgenic male mice. Interestingly, the mechanism in mice differs slightly from that observed in zebrafish but, with disruption of the early stages of spermatogenesis, is similar to that seen in humans. Unexpectedly, we observed hypogonadotropic hypogonadism in the transgenic mice.

Effects of the implementation of second-line prenatal cell-free DNA testing on termination of pregnancy in a French perinatal network.

OBJECTIVE: To evaluate the impact of implementing cell-free DNA (cfDNA) testing on gestational age (GA) at termination of pregnancy in a French perinatal network. METHODS: We conducted a retrospective study. All women having undergone a termination of pregnancy between 1 January 2012 and 31 December 2017 were included. We compared the periods before and after the introduction of second-line cfDNA testing, which started on 1 January 2015. Throughout the study period, the invasive procedures were foetal karyotyping and chromosomal microarray analysis. The primary study outcome was GA at termination. The secondary outcomes were GA at termination for trisomy 21 and the frequency and GA at the time of invasive procedures. RESULTS: During the 6-year study period, 840 women underwent termination. The median GA at termination before and after the implementation of cfDNA testing was 19.4 and 19.0 weeks, respectively (p = 0.38). Although the frequency of termination for trisomy 21 increased significantly from 23% to 32% (p < 0.01), the median GA at termination did not change significantly (p = 0.80). The implementation of cfDNA testing was associated with a decrease in the frequency of invasive procedures in general and chorionic villus sampling in particular (p = 0.04). CONCLUSION: The introduction of cfDNA testing does not increase the GA at termination for trisomy21.

Metabolic Diseases and Down Syndrome: How Are They Linked Together?

Down syndrome is a genetic disorder caused by the presence of a third copy of chromosome 21, associated with intellectual disabilities. Down syndrome is associated with anomalies of both the nervous and endocrine systems. Over the past decades, dramatic advances in Down syndrome research and treatment have helped to extend the life expectancy of these patients. Improved life expectancy is obviously a positive outcome, but it is accompanied with the need to address previously overlooked complications and comorbidities of Down syndrome, including obesity and diabetes, in order to improve the quality of life of Down syndrome patients. In this focused review, we describe the associations between Down syndrome and comorbidities, obesity and diabetes, and we discuss the understanding of proposed mechanisms for the association of Down syndrome with metabolic disorders. Drawing molecular mechanisms through which Type 1 diabetes and Type 2 diabetes could be linked to Down syndrome could allow identification of novel drug targets and provide therapeutic solutions to limit the development of metabolic and cognitive disorders.

Clinical and neuroimaging findings in 33 patients with MCAP syndrome: A survey to evaluate relevant endpoints for future clinical trials.

Megalencephaly-CApillary malformation-Polymicrogyria (MCAP) syndrome results from somatic mosaic gain-of-function variants in PIK3CA. Main features are macrocephaly, somatic overgrowth, cutaneous vascular malformations, connective tissue dysplasia, neurodevelopmental delay, and brain anomalies. The objectives of this study were to describe the clinical and radiological features of MCAP, to suggest relevant clinical endpoints applicable in future trials of targeted drug therapy. Based on a French collaboration, we collected clinical features of 33 patients (21 females, 12 males, median age of 9.9 years) with MCAP carrying mosaic PIK3CA pathogenic variants. MRI images were reviewed for 21 patients. The main clinical features reported were macrocephaly at birth (20/31), postnatal macrocephaly (31/32), body/facial asymmetry (21/33), cutaneous capillary malformations (naevus flammeus 28/33, cutis marmorata 17/33). Intellectual disability was present in 15 patients. Among the MRI images reviewed, the neuroimaging findings were megalencephaly (20/21), thickening of corpus callosum (16/21), Chiari malformation (12/21), ventriculomegaly/hydrocephaly (10/21), cerebral asymmetry (6/21) and polymicrogyria (2/21). This study confirms the main known clinical features that defines MCAP syndrome. Taking into account the phenotypic heterogeneity in MCAP patients, in the context of emerging clinical trials, we suggest that patients should be evaluated based on the main neurocognitive expression on each patient.

Prenatal exome sequencing in 65 fetuses with abnormality of the corpus callosum: contribution to further diagnostic delineation.

PURPOSE: Abnormality of the corpus callosum (AbnCC) is etiologically a heterogeneous condition and the prognosis in prenatally diagnosed cases is difficult to predict. The purpose of our research was to establish the diagnostic yield using chromosomal microarray (CMA) and exome sequencing (ES) in cases with prenatally diagnosed isolated (iAbnCC) and nonisolated AbnCC (niAbnCC). METHODS: CMA and prenatal trio ES (pES) were done on 65 fetuses with iAbnCC and niAbnCC. Only pathogenic gene variants known to be associated with AbnCC and/or intellectual disability were considered. RESULTS: pES results were available within a median of 21.5 days (9-53 days). A pathogenic single-nucleotide variant (SNV) was identified in 12 cases (18%) and a pathogenic CNV was identified in 3 cases (4.5%). Thus, the genetic etiology was determined in 23% of cases. In all diagnosed cases, the results provided sufficient information regarding the neurodevelopmental prognosis and helped the parents to make an informed decision regarding the outcome of the pregnancy. CONCLUSION: Our results show the significant diagnostic and prognostic contribution of CMA and pES in cases with prenatally diagnosed AbnCC. Further prospective cohort studies with long-term follow-up of the born children will be needed to provide accurate prenatal counseling after a negative pES result.

Prenatal diagnosis of 2q13 duplications: The crucial role of the family survey in genetic counseling on novel copy number variations.

In recent years, the introduction of novel genome analysis technologies (such as array comparative genomic hybridization) has enabled the prenatal diagnosis of various recurrent copy number variations (CNVs). Some of these CNVs have been linked to a greater susceptibility of developmental and neuropsychiatric disorders; for example, recurrent duplication at the 2q13 locus is associated with developmental delay, dysmorphism and intellectual disability. However, this CNV has low penetrance and variable clinical expressivity. It also can be observed in healthy controls and can be transmitted by unaffected parents, making genetic counseling especially challenging. Here, we report on the inheritance of a 2q13 duplication in an asymptomatic family; the case highlights the role of the family survey in genetic counseling with regard to novel CNVs diagnosed before birth.

Down syndrome and infertility: what support should we provide?

Down syndrome (DS) is the most common genetic disease at birth; on average, it affects 1 in 700 newborns. The syndrome features cognitive impairment, susceptibility to certain diseases, and (in some cases) congenital malformations. Improvements in medical care for people with DS have led to an increase in life expectancy. Furthermore, the systematic provision of specific support during childhood improves cognitive function and autonomy in adulthood. Consequently, patients and their families are now seeking the same rights as healthy people. Access to procreation is an emerging debate. The presumption of infertility in DS is based on a few old studies. Down syndrome appears to cause spermatogenesis defects in men and premature menopause in women. When assisted reproductive technology makes it possible to solve these problems, the question of fertility in DS must be addressed. Without entering into highly controversial ethical considerations related to parenthood for people with DS, we reviewed the literature on fertility in DS and tried to specify the associated genetic risk.

Mutations in DDHD1, encoding a phospholipase A1, is a novel cause of retinopathy and neurodegeneration with brain iron accumulation.

Defects of phospholipids remodelling and synthesis are inborn errors of metabolism responsible for various clinical presentations including spastic paraplegia, retinopathy, optic atrophy, myo- and cardiomyopathies, and osteo-cutaneous manifestations. DDHD1 encodes a phospholipase A1, which is involved in the remodelling of phospholipids. We previously described a relatively pure hereditary spastic paraplegia (HSP) phenotype associated with mutations in DDHD1. Here we report a complex form of HSP associated with retinal dystrophy and a pattern of neurodegeneration with brain iron accumulation (NBIA) on brain MRI, due to a novel homozygous mutation in DDHD1. This observation enlarges the clinical spectrum of DDHD1-associated disorders and sheds light on a new aetiology for syndromes associating retinopathy and NBIA. It also emphasizes the role of complex lipids in the retina.