Exome sequencing has a diagnostic yield ranging from 25% to 70% in rare diseases and regularly implicates genes in novel disorders. Retrospective data reanalysis has demonstrated strong efficacy in ...improving diagnosis, but poses organizational difficulties for clinical laboratories.
We applied a reanalysis strategy based on intensive prospective bibliographic monitoring along with direct application of the GREP command-line tool (to "globally search for a regular expression and print matching lines") in a large ES database. For 18 months, we submitted the same five keywords of interest (
, (
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, and (
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) to PubMed on a daily basis to identify recently published novel disease-gene associations or new phenotypes in genes already implicated in human pathology. We used the Linux GREP tool and an in-house script to collect all variants of these genes from our 5,459 exome database.
After GREP queries and variant filtration, we identified 128 genes of interest and collected 56 candidate variants from 53 individuals. We confirmed causal diagnosis for 19/128 genes (15%) in 21 individuals and identified variants of unknown significance for 19/128 genes (15%) in 23 individuals. Altogether, GREP queries for only 128 genes over a period of 18 months permitted a causal diagnosis to be established in 21/2875 undiagnosed affected probands (0.7%).
The GREP query strategy is efficient and less tedious than complete periodic reanalysis. It is an interesting reanalysis strategy to improve diagnosis.
We report here two new familial cases of associated del15q11 and del7p22, with the latter underlining the clinical variability of this deletion. Two siblings patients presented a similar familial ...imbalanced translocation, originating from a balanced maternal translocation, with deletions of 7p22 and of 15q11 arrGRCh37 7p22.3-p22.2(42976-3736851)x1, 15q11.1-q11.2(20172544-24979427)x1.
We used aCGH array, FISH, and karyotype for studying the phenotype of the two patients.
The 7p22 deletion (3.5 Mb) contained 58 genes, including several OMIM genes. Patients 1 and 2 exhibited acquisition delays, morphological particularities, and hypogammaglobulinemia, which was more severe in patient 1. Patient 1 presented also with cerebral vasculitis.
We discuss here how the PDGFa, CARD11, LFNG, GPER1, and MAFK genes, included in the deletion 7p22, could be involved in the clinical and biological features of the two patients.
Abstract Microtubule associated proteins (MAPs) are widely expressed in the central nervous system, and have established roles in cell proliferation, myelination, neurite formation, axon ...specification, outgrowth, dendrite, and synapse formation. We report eleven individuals from seven families harboring predicted pathogenic biallelic, de novo, and heterozygous variants in the NAV3 gene, which encodes the microtubule positive tip protein neuron navigator 3 (NAV3). All affected individuals have intellectual disability (ID), microcephaly, skeletal deformities, ocular anomalies, and behavioral issues. In mouse brain, Nav3 is expressed throughout the nervous system, with more prominent signatures in postmitotic, excitatory, inhibiting, and sensory neurons. When overexpressed in HEK293T and COS7 cells, pathogenic variants impaired NAV3 ability to stabilize microtubules. Further, knocking-down nav3 in zebrafish led to severe morphological defects, microcephaly, impaired neuronal growth, and behavioral impairment, which were rescued with co-injection of WT NAV3 mRNA and not by transcripts encoding the pathogenic variants. Our findings establish the role of NAV3 in neurodevelopmental disorders, and reveal its involvement in neuronal morphogenesis, and neuromuscular responses.
Purpose:
Patients with rare or ultra-rare genetic diseases, which affect 350 million people worldwide, may experience a diagnostic odyssey. High-throughput sequencing leads to an etiological ...diagnosis in up to 50% of individuals with heterogeneous neurodevelopmental or malformation disorders. There is a growing interest in additional omics technologies in translational research settings to examine the remaining unsolved cases.
Methods:
We gathered 30 individuals with malformation syndromes and/or severe neurodevelopmental disorders with negative trio exome sequencing and array comparative genomic hybridization results through a multicenter project. We applied short-read genome sequencing, total RNA sequencing, and DNA methylation analysis, in that order, as complementary translational research tools for a molecular diagnosis.
Results:
The cohort was mainly composed of pediatric individuals with a median age of 13.7 years (4 years and 6 months to 35 years and 1 month). Genome sequencing alone identified at least one variant with a high level of evidence of pathogenicity in 8/30 individuals (26.7%) and at least a candidate disease-causing variant in 7/30 other individuals (23.3%). RNA-seq data in 23 individuals allowed two additional individuals (8.7%) to be diagnosed, confirming the implication of two pathogenic variants (8.7%), and excluding one candidate variant (4.3%). Finally, DNA methylation analysis confirmed one diagnosis identified by genome sequencing (Kabuki syndrome) and identified an episignature compatible with a BAFopathy in a patient with a clinical diagnosis of Coffin-Siris with negative genome and RNA-seq results in blood.
Conclusion:
Overall, our integrated genome, transcriptome, and DNA methylation analysis solved 10/30 (33.3%) cases and identified a strong candidate gene in 4/30 (13.3%) of the patients with rare neurodevelopmental disorders and negative exome sequencing results.
The molecular basis of McCune Albright syndrome (MAS) is a recurrent GNAS Postzygotic gain of function sporadic mutation, resulting in a mosaic disease. Most of girls present precocious puberty, ...caused by the development of recurrent ovarian cysts with autonomous Hyperestrogenic stimulation. After menarche, the majority of patients with ovarian GNAS mutation have menstrual disturbances and infertility.
We wanted to focus on the fertility of MAS females and propose an appropriate management, by a detailed case report and an exhaustive review of the literature on fertility and pregnancy in MAS females.
We present the case of a 29-year-old MAS female, who had previously undergone a unilateral ovariectomy and was managed by in vitro fertilization (IVF). Eight oocytes with many morphological abnormalities were retrieved. The GNAS mutation was found at a low frequency in follicular cells. The ovarian histopathological examination showed developing follicles of all stages, strongly expressing AMH by immunohistochemistry. In addition, AMH was high (45.5 pmol/L) and the AMH / AFC ratio (5.69 pmol/L per follicle) was much higher than in PCOS and control groups (2.16, and 1.34 respectively).
Ovarian and endometrial involvement can be responsible for infertility in MAS women. IVF and oophorectomy may be useful in management. The genetic characterization of the different tissues may have a prognostic utility. Moreover, we suggest that the AMH could be a marker of the ovarian activity in MAS. Further studies are needed to clarify the potential oocyte abnormalities and the risk of miscarriages in order to guide genetic counseling.
Multi-omics offer worthwhile and increasingly accessible technologies to diagnostic laboratories seeking potential second-tier strategies to help patients with unresolved rare diseases, especially ...patients clinically diagnosed with a rare OMIM (Online Mendelian Inheritance in Man) disease. However, no consensus exists regarding the optimal diagnostic care pathway to adopt after negative results with standard approaches.
In 15 unsolved individuals clinically diagnosed with recognizable OMIM diseases but with negative or inconclusive first-line genetic results, we explored the utility of a multi-step approach using several novel omics technologies to establish a molecular diagnosis. Inclusion criteria included a clinical autosomal recessive disease diagnosis and single heterozygous pathogenic variant in the gene of interest identified by first-line analysis (60%-9/15) or a clinical diagnosis of an X-linked recessive or autosomal dominant disease with no causative variant identified (40%-6/15). We performed a multi-step analysis involving short-read genome sequencing (srGS) and complementary approaches such as mRNA sequencing (mRNA-seq), long-read genome sequencing (lrG), or optical genome mapping (oGM) selected according to the outcome of the GS analysis.
SrGS alone or in combination with additional genomic and/or transcriptomic technologies allowed us to resolve 87% of individuals by identifying single nucleotide variants/indels missed by first-line targeted tests, identifying variants affecting transcription, or structural variants sometimes requiring lrGS or oGM for their characterization.
Hypothesis-driven implementation of combined omics technologies is particularly effective in identifying molecular etiologies. In this study, we detail our experience of the implementation of genomics and transcriptomics technologies in a pilot cohort of previously investigated patients with a typical clinical diagnosis without molecular etiology.
Considering that some Inherited Metabolic Disorders (IMDs) can be diagnosed in patients with no distinctive clinical features of IMDs, we aimed to evaluate the power of exome sequencing (ES) to ...diagnose IMDs within a cohort of 547 patients with unspecific developmental disorders (DD). IMDs were diagnosed in 12% of individuals with causative diagnosis (177/547). There are clear benefits of using ES in DD to diagnose IMD, particularly in cases where biochemical studies are unavailable.
Exome sequencing and diagnostic rate of Inherited Metabolic Disorders in individuals with developmental disorders.
Background
Exome sequencing (ES) has become the most powerful and cost‐effective molecular tool for deciphering rare diseases with a diagnostic yield approaching 30%–40% in solo‐ES and 50% in ...trio‐ES. We applied an innovative parental DNA pooling method to reduce the parental sequencing cost while maintaining the diagnostic yield of trio‐ES.
Methods
We pooled six (Agilent‐CRE‐v2–100X) or five parental DNA (TWIST‐HCE–70X) aiming to detect allelic balance around 8–10% for heterozygous status. The strategies were applied as second‐tier (74 individuals after negative solo‐ES) and first‐tier approaches (324 individuals without previous ES).
Results
The allelic balance of parental‐pool variants was around 8.97%. Sanger sequencing uncovered false positives in 1.5% of sporadic variants. In the second‐tier approach, we evaluated than two thirds of the Sanger validations performed after solo‐ES (41/59–69%) would have been saved if the parental‐pool segregations had been available from the start. The parental‐pool strategy identified a causative diagnosis in 18/74 individuals (24%) in the second‐tier and in 116/324 individuals (36%) in the first‐tier approaches, including 19 genes newly associated with human disorders.
Conclusions
Parental‐pooling is an efficient alternative to trio‐ES. It provides rapid segregation and extension to translational research while reducing the cost of parental and Sanger sequencing.
We applied an innovative parental DNA pooling method to reduce the parental sequencing cost while maintaining the diagnostic yield of trio‐ES. Parental‐pooling is an efficient alternative to trio‐ES. It provides rapid segregation and extension to translational research while reducing the cost of parental and Sanger sequencing.
The relationships between impaired cortical development and consequent malformations in neurodevelopmental disorders, as well as the genes implicated in these processes, are not fully elucidated to ...date. In this study, we report six novel cases of patients affected by BBSOAS (Boonstra‐Bosch‐Schaff optic atrophy syndrome), a newly emerging rare neurodevelopmental disorder, caused by loss‐of‐function mutations of the transcriptional regulator NR2F1. Young patients with NR2F1 haploinsufficiency display mild to moderate intellectual disability and show reproducible polymicrogyria‐like brain malformations in the parietal and occipital cortex. Using a recently established BBSOAS mouse model, we found that Nr2f1 regionally controls long‐term self‐renewal of neural progenitor cells via modulation of cell cycle genes and key cortical development master genes, such as Pax6. In the human fetal cortex, distinct NR2F1 expression levels encompass gyri and sulci and correlate with local degrees of neurogenic activity. In addition, reduced NR2F1 levels in cerebral organoids affect neurogenesis and PAX6 expression. We propose NR2F1 as an area‐specific regulator of mouse and human brain morphology and a novel causative gene of abnormal gyrification.
Synopsis
This study reveals brain cortical folding defects, linked to intellectual disability, in patients with BBSOA syndrome, a rare genetic disorder caused by haploinsufficiency of the transcription factor NR2F1. In mice, Nr2f1 exerts regional control of neural progenitor (NP) self‐renewal and differentiation.
Human NR2F1 is expressed in a gradient along primary neocortical convolutions.
BBSOAS patients display previously unreported neocortical malformations, such as polymicrogyria‐like aberrant folding and expanded occipital convolutions.
Nr2f1 heterozygous and homozygous mutant mice partially recapitulate the human disease and display neocortical malformations.
Nr2f1 orchestrates NP proliferation and differentiation by acting as a pro‐neurogenic factor.
Nr2f1 modulates NP cell cycle dynamics by regulating P21 and Pax6 expression.
Reduced levels of the neuronal differentiation factor NR2F1/COUP‐TFI impairs brain cortex folding in humans, and disrupts regional neural progenitor dynamics in the neocortex of a BBSOAS disease mouse model.
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