CTLA4 deficiency is an inborn error of immunity (IEI) due to heterozygosity for germline loss-of-function variants of the CTLA4 gene located on chromosome 2q33.2. CTLA4 deficiency underlies pleiotropic immune and lymphoproliferation-mediated features with incomplete penetrance. It has been identified in hundreds of patients but copy number variants (CNVs) have been reported in only 12 kindreds, including nine which displayed large 2q33.1-2q33.2 deletions encompassing CTLA4. We conducted a nationwide study in France to identify patients with 2q33 deletions encompassing CTLA4. We investigated the clinical and immunological phenotypes and genotypes of these patients. We identified 12 patients across six unrelated kindreds with clinical immunodeficiency. Neurological features were recorded in three patients, including one with syndromic neurodevelopmental disorder. Single-nucleotide polymorphism (SNP) or comparative genomic hybridization (CGH) array analysis, and targeted high-throughput sequencing revealed five different heterozygous 2q33 deletions of 26 kilobases to 7.12 megabases in size and encompassing one to 41 genes. We identified a contiguous gene syndrome (CGS) due to associated KLF7 deficiency in a kindred with a neurodevelopmental phenotype. Deletions within the 2q33 region encompassing CTLA4 are rare and not extensively explored, and are probably underdiagnosed in cytogenetic practice. A literature review identified 14 different CGS loci including at least one gene responsible for an IEI. The deletions involved in IEIs should be systematically delimited, to facilitate screening for CGS.

Objectives. - The screening of fetal aneuploidies and non-invasive prenatal diagnosis of monogenic diseases (NIPD-MD) both rely on the study of free fetal DNA in maternal circulation, but their respective rise was unequal. Development of NIPD-MD has taken longer as it represents a less attractive commercial dynamic for industry, but also because it usually involves the development of tailored tests specific to each pathogenic variant. Methods. - We have carried out a review of the literature on the various indications and technologies involved in the use of NIPD-MM. We present its current implementation and its development in France. Results. - To date, NIPD-MD has been routinely offered in France for several years by the laboratories of the French NIPD-MD network but remains mostly limited to the exclusion of paternal or de novo variants, the exclusion DPNI-MD. Indeed, it is still difficult to study the transmission of maternal variants from circulating free DNA analysis, due to its biological complexity: coexistence and predominance of similar DNA sequences of maternal origin. Different strategies, either direct or indirect, are being evaluated to establish fetal status regardless of the parental origin of the disease or its transmission mode. The emergence of commercial screening solutions for monogenic diseases complements the arsenal of prenatal exploration tools for these diseases. Conclusion. - The multitude of existing technologies and protocols may complicate the information provided during antenatal consultations, but mastery of know-how and knowledge of ethical issues of NIPD-MD will ensure optimal service and better management of pregnancies at risk of transmitting monogenic disease. (c) 2023 Elsevier Masson SAS. All rights reserved.

The screening of fetal aneuploidies and non-invasive prenatal diagnosis of monogenic diseases (NIPD-MD) both rely on the study of free fetal DNA in maternal circulation, but their respective rise was unequal. Development of NIPD-MD has taken longer as it represents a less attractive commercial dynamic for industry, but also because it usually involves the development of tailored tests specific to each pathogenic variant.We have carried out a review of the literature on the various indications and technologies involved in the use of NIPD-MM. We present its current implementation and its development in France.To date, NIPD-MD has been routinely offered in France for several years by the laboratories of the French NIPD-MD network but remains mostly limited to the exclusion of paternal or de novo variants, the exclusion DPNI-MD. Indeed, it is still difficult to study the transmission of maternal variants from circulating free DNA analysis, due to its biological complexity: coexistence and predominance of similar DNA sequences of maternal origin. Different strategies, either direct or indirect, are being evaluated to establish fetal status regardless of the parental origin of the disease or its transmission mode. The emergence of commercial screening solutions for monogenic diseases complements the arsenal of prenatal exploration tools for these diseases.The multitude of existing technologies and protocols may complicate the information provided during antenatal consultations, but mastery of know-how and knowledge of ethical issues of NIPD-MD will ensure optimal service and better management of pregnancies at risk of transmitting monogenic disease.

Non-invasive prenatal diagnosis of single-gene disorders (SGD-NIPD) has been widely accepted, but is mostly limited to the exclusion of either paternal or de novo mutations. Indeed, it is still difficult to infer the inheritance of the maternal allele from cell-free DNA (cfDNA) analysis. Based on the study of maternal haplotype imbalance in cfDNA, relative haplotype dosage (RHDO) was developed to address this challenge. Although RHDO has been shown to be reliable, robust control of statistical error and explicit delineation of critical parameters for assessing the quality of the analysis have not been fully addressed. We present here a universal and adaptable enhanced-RHDO (eRHDO) procedure through an automated bioinformatics pipeline with a didactic visualization of the results, aiming to be applied for any SGD-NIPD in routine care. A training cohort of 43 families carrying CFTR, NF1, DMD, or F8 mutations allowed the characterization and optimal setting of several adjustable data variables, such as minimum sequencing depth, type 1 and type 2 statistical errors, as well as the quality assessment of intermediate steps and final results by block score and concordance score. Validation was successfully performed on a test cohort of 56 pregnancies. Finally, computer simulations were used to estimate the effect of fetal-fraction, sequencing depth and number of informative SNPs on the quality of results. Our workflow proved to be robust, as we obtained conclusive and correctly inferred fetal genotypes in 94.9% of cases, with no false-negative or false-positive results. By standardizing data generation and analysis, we fully describe a turnkey protocol for laboratories wishing to offer eRHDO-based non-invasive prenatal diagnosis for single-gene disorders as an alternative to conventional prenatal diagnosis.

Hereditary erythrocytosis is a rare hematologic disorder characterized by an excess of red blood cell production. Here we describe a European collaborative study involving a collection of 2,160 patients with erythrocytosis sequenced in ten different laboratories. We focused our study on the EGLN1 gene and identified 39 germline missense variants including one gene deletion in 47 probands. EGLN1 encodes the PHD2 prolyl 4-hydroxylase, a major inhibitor of hypoxia-inducible factor. We performed a comprehensive study to evaluate the causal role of the identified PHD2 variants: (i) in silico studies of localization, conservation, and deleterious effects; (ii) analysis of hematologic parameters of carriers identified in the UK Biobank; (iii) functional studies of the protein activity and stability; and (iv) a comprehensive study of PHD2 splicing. Altogether, these studies allowed the classification of 16 pathogenic or likely pathogenic mutants in a total of 48 patients and relatives. The in silico studies extended to the variants described in the literature showed that a minority of PHD2 variants can be classified as pathogenic (36/96), without any differences from the variants of unknown significance regarding the severity of the developed disease (hematologic parameters and complications). Here, we demonstrated the great value of federating laboratories working on such rare disorders in order to implement the criteria required for genetic classification, a strategy that should be extended to all hereditary hematologic diseases.

ABSTRACT Until recently, fetal genetic testing was possible only via invasive sampling (amniocentesis or chorionic villus sampling). Although such testing allows for prenatal diagnosis (PND) of inherited monogenetic disorders, it is also associated with a risk of miscarriage. Identification of cell-free fetal DNA (cfDNA) from maternal circulation allows for noninvasive fetal sex determination and detection of the common aneuploidies. In fact, the entire fetal genome is represented within the circulating cell-free DNA. Clinical services are now available that provide noninvasive PND for monogenetic disorders, although these remain sparse, and most tests of this nature focus on paternally inherited disorders, given that the fetal DNA is diluted by maternal haploidentical sequences. This study reports on an experimental protocol for droplet digital polymerase chain reaction for paternal pathogenic or de novo variants. Since then, this noninvasive PND service has been offered to couples at risk of monogenic disorders in the fetus who have requested PND as a part of standard care. This report summarizes 3 years of clinical experience. Referral indications included the following: those with 50% risk of transmission of an autosomal dominant disorder from the father, those at 25% risk of autosomal recessive disorders and with different parental pathogenic variants such that they could assess for inheritance of the paternal variant, and those at risk for single-gene disorders due to de novo variants based on evident skeletal dysplasia by ultrasound or possible parental germinal mosaicism in a previously affected child. As a matter of practicality and organization, only ongoing pregnancies had new assays designed and ordered. Local French law regarding prenatal genetic testing requires written consent from both parents. Blood from each pregnant woman and blood samples from both parents for extraction of conventional genomic DNA were also collected. Each new variant required creation of a new assay, whose performance was evaluated. All results were reported with summarized interpretations to the clinicians, and invasive diagnostic testing was recommended in cases of autosomal recessive variant identification where inheritance of the paternal variant was detected to identify maternal inheritance. Sampling errors and identity swaps were ruled out by taking a second sample for every negative result. Development of new assays took 7 to 43 days, and overall, these assays were designed to detect 57 individual variants in 16 genes. A result was obtained for all but 2 cases; these were unsuccessful because of a low proportion of fetal DNA. In the autosomal dominant cases, 27% found fetal inheritance of the paternal variant, whereas 73% ruled out such inheritance. In the autosomal recessive cases, the paternal variant was detected in 56% and not detected in the remainder. In cases in which the paternal variant was detected, invasive diagnostic testing was recommended to determine maternal inheritance and whether the fetus was affected. The single inconclusive result was from a pregnancy obtained after egg donation in which ultimately the egg donor and the father both had the same gene variant, making accurate assessment not possible. There were 198 referrals overall, including 22 referrals due to concern for autosomal dominant disorders, 69 due to risk for an autosomal recessive disorder, and 107 due to concern for a de novo variant either because of ultrasound findings or a family history. There were 202 tests performed for 175 different families. All results reported were considered valid, and there were no false-positive results following invasive sampling for maternal inheritance, and no false-negative results reported following delivery. A limitation of this technique is that it addresses only the paternal variant's fetal status. This approach does not allow testing for all types of disease-causing variations such as repetitive elements, copy number variations, or structural variants. Also, maternally transmitted diseases or recessive conditions with both parents as carriers are also not detectable through this method. In the case of multiple pregnancies, there is no method for determining which twin (or whether both twins) will carry the variant. This test allows couples to avoid the risk of miscarriage while being able to make informed decisions about the pregnancy management. In addition, as new treatments are developed for common disorders (CFTR modulators, gene-replacement therapies, uterine genic therapies, and splicing-modifying therapies), the prenatal testing landscape can be expected to drastically evolve to favor a noninvasive approach.

X-linked dominant chondrodysplasia punctata (CDPX2 or Conradi-Hünermann-Happle syndrome, MIM #302960) is caused by mutations in the EBP gene. Affected female patients present with Blaschkolinear ichthyosis, coarse hair or alopecia, short stature, and normal psychomotor development. The disease is usually lethal in boys. Nevertheless, few male patients have been reported; they carry a somatic mosaicism in EBP or present with Klinefelter syndrome. Here, we report CDPX2 patients belonging to a three-generation family, carrying the splice variant c.301 + 5 G > C in intron 2 of EBP. The grandfather carries the variant as mosaic state and presents with short stature and mild ichthyosis. The mother also presents with short stature and mild ichthyosis and the female fetus with severe limb and vertebrae abnormalities and no skin lesions, with random X inactivation in both. This further characterizes the phenotypical spectrum of CDPX2, as well as intrafamilial variability, and raises the question of differential EBP mRNA splicing between the different target tissues.

Chuvash polycythemia is an autosomal recessive form of erythrocytosis associated with a homozygous p. Arg200Trp mutation in the von Hippel-Lindau (VHL) gene. Since this discovery, additional VHL mutations have been identified in patients with congenital erythrocytosis, in a homozygous or compound-heterozygous state. VHL is a major tumor suppressor gene, mutations in which were first described in patients presenting with VHL disease, which is characterized by the development of highly vascularized tumors. Here, we identify a new VHL cryptic exon (termed E1') deep in intron 1 that is naturally expressed in many tissues. More importantly, we identifymutations in E1' in 7 families with erythrocytosis (1 homozygous case and 6 compound-heterozygous cases with a mutation in E1' in addition to a mutation in VHL coding sequences) and in 1 large family with typical VHL disease but without any alteration in the other VHL exons. In this study, we show that the mutations induced a dysregulation of VHL splicing with excessive retention of E1' and were associated with a downregulation of VHL protein expression. In addition, we demonstrate a pathogenic role for synonymous mutations in VHL exon 2 that altered splicing through E2-skipping in 5 families with erythrocytosis or VHL disease. In all the studied cases, the mutations differentially affected splicing, correlating with phenotype severity. This study demonstrates that cryptic exon retention and exon skipping are new VHL alterations and reveals a novel complex splicing regulation of the VHL gene. These findings open new avenues for diagnosis and research regarding the VHL-related hypoxia-signaling pathway.

BACKGROUND:To limit risks of miscarriages associated with invasive procedures of current prenatal diagnosis practice, we aim to develop a personalized medicine-based protocol for non-invasive prenatal diagnosis (NIPD) of monogenic disorders relying on the detection of paternally inherited mutations in maternal blood using droplet digital PCR (ddPCR). METHODS:This study included four couples at risk of transmitting paternal neurofibromatosis type 1 (NF1) mutations and four couples at risk of transmitting compound heterozygous CFTR mutations. NIPD was performed between 8 and 15 weeks of gestation, in parallel to conventional invasive diagnosis. We designed specific hydrolysis probes to detect the paternal mutation and to assess the presence of cell-free fetal DNA by ddPCR. Analytical performances of each assay were determined from paternal sample, an then fetal genotype was inferred from maternal plasma sample. RESULTS:Presence or absence of the paternal mutant allele was correctly determined in all the studied plasma DNA samples. CONCLUSIONS:We report an NIPD protocol suitable for implementation in an experienced laboratory of molecular genetics. Our proof-of-principle results point out a high accuracy for early detection of paternal NF1 and CFTR mutations in cell-free DNA, and open new perspectives for extending the technology to NIPD of many other monogenic diseases.

: Chuvash polycythemia is an autosomal recessive form of erythrocytosis associated with a homozygous p.Arg200Trp mutation in the von Hippel-Lindau ( VHL ) gene. Since this discovery, additional VHL mutations have been identified in patients with congenital erythrocytosis, in homozygous or compound-heterozygous state. VHL is a major tumor suppressor gene that was first described mutated in heterozygous patients presenting with von Hippel–Lindau disease, which is characterized by the development of highly vascularized tumors. Here, we identified a new VHL cryptic-exon (termed E1’) hidden in intron 1 that is naturally expressed in many tissues. More importantly, we identified mutations in E1' in seven families with erythrocytosis (one homozygous case and six compound-heterozygous cases with a mutation in E1' associated with mutation in VHL coding sequence) and in one large family, members of which develop a typical VHL disease without any alteration in the other VHL exons. We performed a comprehensive study and show that the mutations induced a dysregulation of the VHL splicing with excessive retention of E1’ associated with a downregulation of VHL protein expression. In addition, we demonstrated a pathogenic role of synonymous mutations in VHL -Exon 2 that alter splicing through E2-skipping in five families with erythrocytosis or VHL disease. In all the studied cases, the mutations differentially impact splicing, correlating with phenotype severity. This study demonstrates that cryptic-exon-retention or exon-skipping are new VHL alterations and reveals a novel complex splicing regulation of the VHL gene. These findings open new avenues for diagnosis and research into the VHL-related-hypoxia-signaling pathway. novel cryptic-exon in the VHL gene and a complex regulation of VHL splicing. The hypoxia pathway plays a central role in erythrocytosis or tumors developed by patients carrying VHL mutations. Nevertheless, the full molecular mechanisms at the origin of these different phenotypes remain to be elucidated. To date, the functional studies of VHL mutants have been performed on missense mutations. We describe here, for the first time, functional studies of VHL mutations that do not impact the coding sequence but that influence the VHL splicing. We discovered a complex regulation of VHL splicing that may help to explain the complexity of genotype/phenotype correlations observed in VHL-related disorders. Notably, we demonstrated that synonymous variants (D143D or P138P) can impact VHL splicing and should be considered as pathogenic mutations. Our study points to a particular region in the E2 that may be considered as a splicing regulatory domain. Therefore, it would be interesting to evaluate the impact on splicing of all the nucleotide changes described in VHL -E2 17 in the same way as we described for two missense mutations (P138L, G144R). We observed that, depending on the mutation in this region, the impact on splicing can be moderate (D143D, G144R, P138L) or severe (P138P), which correlates with the severity of the disease developed by individuals carrying these VHL mutations (erythrocytosis versus cancers). This observation confirms the hypothesis of a continuum-model of tumor suppression by VHL. 21,31 erythrocytosis previously associated to a heterozygous mutation in VHL rather than a homozygous mutation. Our investigations further confirm that polycythemia associated with VHL mutation is definitely an autosomal recessive disease. In addition, we identified an E1' homozygous mutation in a patient with an erythrocytosis of unknown origin. This result demonstrates the causal role of the alteration in this new cryptic-exon to the occurrence of erythrocytosis. Importantly, we also identified E1' mutations in patients with unexplained VHL disease. vertebral hemangiomas, varicose veins, thromboembolic events, and pulmonary hypertension, but never with tumors. This study describes families with typical VHL-associated phenotypes associated with an unexpected VHL status (i.e. either synonymous mutations or no alterations in VHL Identification of novel VHL spliced isoforms containing a cryptic-exon that was found α using the HRE-luciferase reporter plasmid. The ability of wild type and mutated X1 to downregulate Firefly luciferase activity (related to HIF activity) was compared to pVHL and in competition with pVHL. An immunoblot using an antibody specific to the hemagglutinin tag was used to detect HA-VHL and HA-X1. The X1-L128V+L138P corresponds to a potential impact of the c.340+648T>C and c.340+617C>G variations on the hypothetical X1 protein. Three independent experiments were performed. ** p<0.005, based on t test . (D) Characterization of VHL -E1' retention by the Minigene experiment (representative picture of agarose gel from n=3). RT-PCR was performed on mRNA obtained from cell lines transfected with a minigene construct containing VHL -E1’ (wild-type or mutated) flanked by large intronic sequences cloned between the SERPING1 exons (exons A and B, targeted by the RT-PCR primers). The plasmids were transfected and the expression of the spliced chimeric transcripts (containing

NBEA is a candidate gene for autism, and de novo variants have been reported in neurodevelopmental disease (NDD) cohorts. However, NBEA has not been rigorously evaluated as a disease gene, and associated phenotypes have not been delineated. We identified 24 de novo NBEA variants in patients with NDD, establishing NBEA as an NDD gene. Most patients had epilepsy with onset in the first few years of life, often characterized by generalized seizure types, including myoclonic and atonic seizures. Our data show a broader phenotypic spectrum than previously described, including a myoclonic‐astatic epilepsy–like phenotype in a subset of patients. Ann Neurol 2018;84:796–803

Autism spectrum disorders are complex neurodevelopmental syndromes characterized by phenotypic and genetic heterogeneity. Further identification of causal genes may help in better understanding the underlying mechanisms of the disorder, thus improving the patients' management. To date, abnormal synaptogenesis is thought to be one of the major underlying causes of autism spectrum disorders. Here, using oligoarray-based comparative genomic hybridization, we identified a de novo deletion at 2q37.2 locus spanning 1 Mb and encompassing AGAP1 and SH3BP4, in a boy with autism and intellectual disability. Both genes have been described as being involved in endosomal trafficking, and AGAP1 in particular has been shown to be expressed in the developing brain and to play a role in dendritic spine formation and synapse function, making it a potential causative gene to our patient's phenotype.

Degradation of proteins by the ubiquitin-proteasome system (UPS) is an essential biological process in the development of eukaryotic organisms. Dysregulation of this mechanism leads to numerous human neurodegenerative or neurodevelopmental disorders. Through a multi-center collaboration, we identified six de novo genomic deletions and four de novo point mutations involving PSMD12, encoding the non-ATPase subunit PSMD12 (aka RPN5) of the 19S regulator of 26S proteasome complex, in unrelated individuals with intellectual disability, congenital malformations, ophthalmologic anomalies, feeding difficulties, deafness, and subtle dysmorphic facial features. We observed reduced PSMD12 levels and an accumulation of ubiquitinated proteins without any impairment of proteasome catalytic activity. Our PSMD12 loss-of-function zebrafish CRISPR/Cas9 model exhibited microcephaly, decreased convolution of the renal tubules, and abnormal craniofacial morphology. Our data support the biological importance of PSMD12 as a scaffolding subunit in proteasome function during development and neurogenesis in particular; they enable the definition of a neurodevelopmental disorder due to PSMD12 variants, expanding the phenotypic spectrum of UPS-dependent disorders.

Introduction: Erythrocytoses are characterized by an elevated red cell mass. The most widely studied disease is Polycythemia Vera (PV), a myeloproliferative neoplasm due to the acquired JAK2-V617F mutation. However, other types of erythrocytoses exist and are of major importance. They can be either inherited (Congenital Erythrocytosis-CE) or diagnosed in adult patients with no family history (Idiopathic Erythrocytosis-IE). CE/IE are not associated with myeloproliferation but they can be associated with severe thrombo-embolic or haemorrhagic events, pulmonary arterial hypertension and, rarely, tumours. The 8 genes identified so far as causing CE lie at the crossroads of major biological pathways (metabolism, inflammation, oncogenesis) and are implicated in multiple diseases. These genes are involved (i) in the regulation of the hypoxia pathway, PHD2 (also called EGLN1), HIF-2A (EPAS1), VHL, (ii) in proliferation and differentiation of erythroid progenitors (EPOR), or (iii) in mature cell function, haemoglobins (HBB, HBA1, HBA2) or bisphosphoglyceratemutase (BPGM). However, in 80% of cases the cause remains unknown meaning that no proper diagnosis can be made, no prognosis or advice can be provided to CE/IE patients and their families, and no curative treatment exists.