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.

Deep intronic NIPBL de novo mutations and differential diagnoses revealed by whole genome and RNA sequencing in Cornelia de Lange syndrome patients.

Cornelia de Lange syndrome (CdLS; MIM# 122470) is a rare developmental disorder. Pathogenic variants in 5 genes explain approximately 50% cases, leaving the other 50% unsolved. We performed whole genome sequencing (WGS) ± RNA sequencing (RNA-seq) in 5 unsolved trios fulfilling the following criteria: (i) clinical diagnosis of classic CdLS, (ii) negative gene panel sequencing from blood and saliva-isolated DNA, (iii) unaffected parents' DNA samples available and (iv) proband's blood-isolated RNA available. A pathogenic de novo mutation (DNM) was observed in a CdLS differential diagnosis gene in 3/5 patients, namely POU3F3, SPEN, and TAF1. In the other two, we identified two distinct deep intronic DNM in NIPBL predicted to create a novel splice site. RT-PCRs and RNA-Seq showed aberrant transcripts leading to the creation of a novel frameshift exon. Our findings suggest the relevance of WGS in unsolved suspected CdLS cases and that deep intronic variants may account for a proportion of them.

uORF-introducing variants in the 5'UTR of the NIPBL gene as a cause of Cornelia de Lange syndrome.

Cornelia de Lange syndrome (CdLS) is a clinically-recognizable rare developmental disorder. About 70% of patients carry a missense or loss-of-function pathogenic variant in the NIPBL gene. We hypothesized that some variants in the 5'-untranslated region (UTR) of NIPBL may create an upstream open reading frame (uORF), putatively leading to a loss of function. We searched for NIPBL 5'-UTR variants potentially introducing uORF by (i) reannotating NGS data of 102 unsolved CdLS patients and (ii) literature and variant databases search. We set up a green fluorescent protein (GFP) reporter assay and studied NIPBL expression in a lymphoblastoid cell line (LCL). We identified two variants introducing a novel ATG codon sequence in the 5'-UTR of NIPBL, both predicted to introduce uORF: a novel c.-457_-456delinsAT de novo mutation in a 15-year-old male with classic CdLS, and a c.-94C>T variant in a published family. Our reporter assay showed a significant decrease of GFP levels in both mutant contexts, with similar levels of messenger RNA (mRNA) as compared to wt constructs. Assessment of LCL of one patient showed consistent results with decreased NIPBL protein and unchanged mRNA levels. 5'-UTR uORF-introducing NIPBL variants may represent a rare source of pathogenic variants in unsolved CdLS patients.

Natural history of NF1 c.2970_2972del p.(Met992del): confirmation of a low risk of complications in a longitudinal study.

Individuals with the three base pair deletion NM_000267.3(NF1):c.2970_2972del p.(Met992del) have been recognised to present with a milder neurofibromatosis type 1 (NF1) phenotype characterised by café-au-lait macules (CALs) and intertriginous freckling, as well as a lack of cutaneous, subcutaneous and plexiform neurofibromas and other NF1-associated complications. Examining large cohorts of patients over time with this specific genotype is important to confirm the presentation and associated risks of this variant across the lifespan. Forty-one individuals with the in-frame NF1 deletion p.Met992del were identified from 31 families. Clinicians completed a standardised clinical questionnaire for each patient and the resulting data were collated and compared to published cohorts. Thirteen patients have been previously reported, and updated clinical information has been obtained for these individuals. Both CALs and intertriginous freckling were present in the majority of individuals (26/41, 63%) and the only confirmed features in 11 (27%). 34/41 (83%) of the cohort met NIH diagnostic criteria. There was a notable absence of all NF1-associated tumour types (neurofibroma and glioma). Neurofibroma were observed in only one individual-a subcutaneous lesion (confirmed histologically). Nineteen individuals were described as having a learning disability (46%). This study confirms that individuals with p.Met992del display a mild tumoural phenotype compared to those with 'classical', clinically diagnosed NF1, and this appears to be the case longitudinally through time as well as at presentation. Learning difficulties, however, appear to affect a significant proportion of NF1 subjects with this phenotype. Knowledge of this genotype-phenotype association is fundamental to accurate prognostication for families and caregivers.

De novo coding variants in the AGO1 gene cause a neurodevelopmental disorder with intellectual disability.

BACKGROUND: High-impact pathogenic variants in more than a thousand genes are involved in Mendelian forms of neurodevelopmental disorders (NDD). METHODS: This study describes the molecular and clinical characterisation of 28 probands with NDD harbouring heterozygous AGO1 coding variants, occurring de novo for all those whose transmission could have been verified (26/28). RESULTS: A total of 15 unique variants leading to amino acid changes or deletions were identified: 12 missense variants, two in-frame deletions of one codon, and one canonical splice variant leading to a deletion of two amino acid residues. Recurrently identified variants were present in several unrelated individuals: p.(Phe180del), p.(Leu190Pro), p.(Leu190Arg), p.(Gly199Ser), p.(Val254Ile) and p.(Glu376del). AGO1 encodes the Argonaute 1 protein, which functions in gene-silencing pathways mediated by small non-coding RNAs. Three-dimensional protein structure predictions suggest that these variants might alter the flexibility of the AGO1 linker domains, which likely would impair its function in mRNA processing. Affected individuals present with intellectual disability of varying severity, as well as speech and motor delay, autistic behaviour and additional behavioural manifestations. CONCLUSION: Our study establishes that de novo coding variants in AGO1 are involved in a novel monogenic form of NDD, highly similar to the recently reported AGO2-related NDD.

Recurrence of an early postzygotic rescue of an inherited unbalanced translocation resulting in mosaic segmental uniparental isodisomy of chromosome 11q in siblings.

Balanced translocations are associated with a risk of transmission of unbalanced chromosomal rearrangements in the offspring. Such inherited chromosomal abnormalities are typically non-mosaic as they are present in the germline. We report the recurrence in two siblings of a mosaicism for a chromosomal rearrangement inherited from their asymptomatic father who carried a balanced t(2;11)(q35;q25) translocation. Both siblings exhibited a similar phenotype including intellectual disability, dysmorphic features, kyphoscoliosis, and cervical spinal stenosis. Karyotyping, fluorescence in situ hybridization and SNP array analysis of blood lymphocytes of both siblings identified two cell lines: one carrying a 2q35q37.3 duplication and a 11q25qter deletion (~90% cells), and one carrying an 11q uniparental isodisomy of maternal origin (~10% cells). We hypothesize that these mosaics were related to a postzygotic rescue mechanism which unexpectedly recurred in both siblings.

Clinical and molecular description of 19 patients with GATAD2B-Associated Neurodevelopmental Disorder (GAND).

De novo pathogenic variants in the GATAD2B gene have been associated with a syndromic neurodevelopmental disorder (GAND) characterized by severe intellectual disability (ID), impaired speech, childhood hypotonia, and dysmorphic features. Since its first description in 2013, nine patients have been reported in case reports and a series of 50 patients was recently published, which is consistent with the relative frequency of GATAD2B pathogenic variants in public databases. We report the detailed phenotype of 19 patients from various ethnic backgrounds with confirmed pathogenic GATAD2B variants including intragenic deletions. All individuals presented developmental delay with a median age of 2.5 years for independent walking and of 3 years for first spoken words. GATAD2B variant carriers showed very little subsequent speech progress, two patients over 30 years of age remaining non-verbal. ID was mostly moderate to severe, with one profound and one mild case, which shows a wider spectrum of disease severity than previously reported. We confirm macrocephaly as a major feature in GAND (53%). Most common dysmorphic features included broad forehead, deeply set eyes, hypertelorism, wide nasal base, and pointed chin. Conversely, prenatal abnormalities, non-cerebral malformations, epilepsy, and autistic behavior were uncommon. Other features included feeding difficulties, behavioral abnormalities, and unspecific abnormalities on brain MRI. Improving our knowledge of the clinical phenotype is essential for correct interpretation of the molecular results and accurate patient management.

X-linked primary ciliary dyskinesia due to mutations in the cytoplasmic axonemal dynein assembly factor PIH1D3.

By moving essential body fluids and molecules, motile cilia and flagella govern respiratory mucociliary clearance, laterality determination and the transport of gametes and cerebrospinal fluid. Primary ciliary dyskinesia (PCD) is an autosomal recessive disorder frequently caused by non-assembly of dynein arm motors into cilia and flagella axonemes. Before their import into cilia and flagella, multi-subunit axonemal dynein arms are thought to be stabilized and pre-assembled in the cytoplasm through a DNAAF2-DNAAF4-HSP90 complex akin to the HSP90 co-chaperone R2TP complex. Here, we demonstrate that large genomic deletions as well as point mutations involving PIH1D3 are responsible for an X-linked form of PCD causing disruption of early axonemal dynein assembly. We propose that PIH1D3, a protein that emerges as a new player of the cytoplasmic pre-assembly pathway, is part of a complementary conserved R2TP-like HSP90 co-chaperone complex, the loss of which affects assembly of a subset of inner arm dyneins.

A nonsense mutation in the DNA repair factor Hebo causes mild bone marrow failure and microcephaly.

Inherited bone marrow failure syndromes are human conditions in which one or several cell lineages of the hemopoietic system are affected. They are present at birth or may develop progressively. They are sometimes accompanied by other developmental anomalies. Three main molecular causes have been recognized to result in bone marrow failure syndromes: (1) defects in the Fanconi anemia (FA)/BRCA DNA repair pathway, (2) defects in telomere maintenance, and (3) abnormal ribosome biogenesis. We analyzed a patient with mild bone marrow failure and microcephaly who did not present with the typical FA phenotype. Cells from this patient showed increased sensitivity to ionizing radiations and phleomycin, attesting to a probable DNA double strand break (dsb) repair defect. Linkage analysis and whole exome sequencing revealed a homozygous nonsense mutation in the ERCC6L2 gene. We identified a new ERCC6L2 alternative transcript encoding the DNA repair factor Hebo, which is critical for complementation of the patient's DNAdsb repair defect. Sequence analysis revealed three structured regions within Hebo: a TUDOR domain, an adenosine triphosphatase domain, and a new domain, HEBO, specifically present in Hebo direct orthologues. Hebo is ubiquitously expressed, localized in the nucleus, and rapidly recruited to DNAdsb's in an NBS1-dependent manner.

RAG2 and XLF/Cernunnos interplay reveals a novel role for the RAG complex in DNA repair.

XRCC4-like factor (XLF) functions in classical non-homologous end-joining (cNHEJ) but is dispensable for the repair of DNA double-strand breaks (DSBs) generated during V(D)J recombination. A long-standing hypothesis proposes that, in addition to its canonical nuclease activity, the RAG1/2 proteins participate in the DNA repair phase of V(D)J recombination. Here we show that in the context of RAG2 lacking the C-terminus domain (Rag2(c/c) mice), XLF deficiency leads to a profound lymphopenia associated with a severe defect in V(D)J recombination and, in the absence of p53, increased genomic instability at V(D)J sites. In addition, Rag2(c/c) XLF(-/-) p53(-/-) mice develop aggressive pro-B cell lymphomas bearing complex chromosomal translocations and gene amplifications involving Igh and c-myc/pvt1 loci. Our results reveal an unanticipated functional interplay between the RAG complex and XLF in repairing RAG-induced DSBs and maintaining genome integrity during antigen receptor gene assembly.

Cernunnos deficiency reduces thymocyte life span and alters the T cell repertoire in mice and humans.

Cernunnos is a DNA repair factor of the nonhomologous end-joining machinery. Its deficiency in humans causes radiosensitive severe combined immune deficiency (SCID) with microcephaly, characterized in part by a profound lymphopenia. In contrast to the human condition, the immune system of Cernunnos knockout (KO) mice is not overwhelmingly affected. In particular, Cernunnos is dispensable during V(D)J recombination in lymphoid cells. Nevertheless, the viability of thymocytes is reduced in Cernunnos KO mice, owing to the chronic activation of a P53-dependent DNA damage response. This translates into a qualitative alteration of the T cell repertoire to one in which the most distal Vα and Jα segments are missing. This results in the contraction of discrete T cell populations, such as invariant natural killer T (iNKT) and mucosa-associated invariant T (MAIT) cells, in both humans and mice.

Stepwise development of MAIT cells in mouse and human.

Mucosal-associated invariant T (MAIT) cells display two evolutionarily conserved features: an invariant T cell receptor (TCR)alpha (iTCRalpha) chain and restriction by the nonpolymorphic class Ib major histocompatibility complex (MHC) molecule, MHC-related molecule 1 (MR1). MR1 expression on thymus epithelial cells is not necessary for MAIT cell development but their accumulation in the gut requires MR1 expressing B cells and commensal flora. MAIT cell development is poorly known, as these cells have not been found in the thymus so far. Herein, complementary human and mouse experiments using an anti-humanValpha7.2 antibody and MAIT cell-specific iTCRalpha and TCRbeta transgenic mice in different genetic backgrounds show that MAIT cell development is a stepwise process, with an intra-thymic selection followed by peripheral expansion. Mouse MAIT cells are selected in an MR1-dependent manner both in fetal thymic organ culture and in double iTCRalpha and TCRbeta transgenic RAG knockout mice. In the latter mice, MAIT cells do not expand in the periphery unless B cells are added back by adoptive transfer, showing that B cells are not required for the initial thymic selection step but for the peripheral accumulation. In humans, contrary to natural killer T (NKT) cells, MAIT cells display a naïve phenotype in the thymus as well as in cord blood where they are in low numbers. After birth, MAIT cells acquire a memory phenotype and expand dramatically, up to 1%-4% of blood T cells. Finally, in contrast with NKT cells, human MAIT cell development is independent of the molecular adaptor SAP. Interestingly, mouse MAIT cells display a naïve phenotype and do not express the ZBTB16 transcription factor, which, in contrast, is expressed by NKT cells and the memory human MAIT cells found in the periphery after birth. In conclusion, MAIT cells are selected by MR1 in the thymus on a non-B non-T hematopoietic cell, and acquire a memory phenotype and expand in the periphery in a process dependent both upon B cells and the bacterial flora. Thus, their development follows a unique pattern at the crossroad of NKT and gammadelta T cells.