ITPR1: The missing gene in miosis-ataxia syndrome?

The association of early-onset non-progressive ataxia and miosis is an extremely rare phenotypic entity occasionally reported in the literature. To date, only one family (two siblings and their mother) has benefited from a genetic diagnosis by the identification of a missense heterozygous variant (p.Arg36Cys) in the ITPR1 gene. This gene encodes the inositol 1,4,5-trisphosphate receptor type 1, an intracellular channel that mediates calcium release from the endoplasmic reticulum. Deleterious variants in this gene are known to be associated with two types of spinocerebellar ataxia, SCA15 and SCA29, and with Gillespie syndrome that is associated with ataxia, partial iris hypoplasia, and intellectual disability. In this work, we describe a novel individual carrying a heterozygous missense variant (p.Arg36Pro) at the same position in the N-terminal suppressor domain of ITPR1 as the family previously reported, with the same phenotype associating early-onset non-progressive ataxia and miosis. This second report confirms the implication of ITPR1 in the miosis-ataxia syndrome and therefore broadens the clinical spectrum of the gene. Moreover, the high specificity of the phenotype makes it a recognizable syndrome of genetic origin.

Structural Variant Disrupting the Expression of the Remote FOXC1 Gene in a Patient with Syndromic Complex Microphthalmia.

Ocular malformations (OMs) arise from early defects during embryonic eye development. Despite the identification of over 100 genes linked to this heterogeneous group of disorders, the genetic cause remains unknown for half of the individuals following Whole-Exome Sequencing. Diagnosis procedures are further hampered by the difficulty of studying samples from clinically relevant tissue, which is one of the main obstacles in OMs. Whole-Genome Sequencing (WGS) to screen for non-coding regions and structural variants may unveil new diagnoses for OM individuals. In this study, we report a patient exhibiting a syndromic OM with a de novo 3.15 Mb inversion in the 6p25 region identified by WGS. This balanced structural variant was located 100 kb away from the FOXC1 gene, previously associated with ocular defects in the literature. We hypothesized that the inversion disrupts the topologically associating domain of FOXC1 and impairs the expression of the gene. Using a new type of samples to study transcripts, we were able to show that the patient presented monoallelic expression of FOXC1 in conjunctival cells, consistent with the abolition of the expression of the inverted allele. This report underscores the importance of investigating structural variants, even in non-coding regions, in individuals affected by ocular malformations.

Chapitre 7. Un regard de généticien.

Examined through the eyes of the geneticist, the modifications of the bioethics law seem relatively modest with regard to the supervision of the practices of his discipline. The introduction of rules concerning the use of algorithms in medical practice is the truly new point. It seemed beneficial to take into account “the interference of thinking machines” in medical decision-making and to initiate the outlines of a framework. We will debate the proposals and terms. Precisions made to the obligation to inform relatives of the existence of a genetic anomaly are defined around the concept of solidarity. Without neglecting this latter, we will recall other determinants, the complexity and the issues underlying the delivery of predictive genetic information as well as the risks that informed persons may incur. It seems appropriate to also consider the ethical tensions that can impose themselves on the physicians involved in the mandatory process of information..

Chapitre 8. Annoncer ou pas la découverte d'anomalies non sollicitées lors d'un test génétique à séquençage haut débit ?

OBJECTIVE: New genome sequencing techniques allow new approaches in medical genetics, in particular by facilitating the diagnosis of genetic diseases. However, their use also leads to unsolicited genetic findings being uncovered. This type of discovery raises ethical, legal and psychological considerations. The objective of this psychological research was to study the different positions of patients, health professionals and general public regarding the acceptability of the announcement of unsolicited findings revealed during a high-throughput sequencing genetic test. METHOD: the first exploratory study aimed, through non-directive research interviews conducted with 13 patients of a medical genetics service, to understand the psychological repercussions linked to the announcement of a result of a targeted genetic test and to know the patients’ desires regarding the announcement of unsolicited findings if the test had been a high-throughput genetic test. The second study, using a quantitative methodology, aimed to identify the judgment policies of 144 patients, 94 healthcare professionals and 211 people from the general public concerning the acceptability of this type of disclosure. RESULTS: The cluster analyses highlighted six judgment policies as to whether or not to disclose the discovery of unsolicited anomalies: “Tell everything”, “Tell even in part”, “Tell everything unless desperate”, “Undecided”, “Do not tell” and “Do not tell if no prevention”. The participants positioned themselves differently, in particular according to the patient’s consent. CONCLUSION: This research shows the variability of positioning and the importance of consent in the acceptability of the disclosure of unsolicited findings. However, one of the limitations of the study lies in the fact that in medical clinic, acceptability and acceptance may vary over time. A longitudinal study would undoubtedly afford a better understanding of the psychological progress of patients in this type of care pathway..

Clinical and functional heterogeneity associated with the disruption of retinoic acid receptor beta.

PURPOSE: Dominant variants in the retinoic acid receptor beta (RARB) gene underlie a syndromic form of microphthalmia, known as MCOPS12, which is associated with other birth anomalies and global developmental delay with spasticity and/or dystonia. Here, we report 25 affected individuals with 17 novel pathogenic or likely pathogenic variants in RARB. This study aims to characterize the functional impact of these variants and describe the clinical spectrum of MCOPS12. METHODS: We used in vitro transcriptional assays and in silico structural analysis to assess the functional relevance of RARB variants in affecting the normal response to retinoids. RESULTS: We found that all RARB variants tested in our assays exhibited either a gain-of-function or a loss-of-function activity. Loss-of-function variants disrupted RARB function through a dominant-negative effect, possibly by disrupting ligand binding and/or coactivators' recruitment. By reviewing clinical data from 52 affected individuals, we found that disruption of RARB is associated with a more variable phenotype than initially suspected, with the absence in some individuals of cardinal features of MCOPS12, such as developmental eye anomaly or motor impairment. CONCLUSION: Our study indicates that pathogenic variants in RARB are functionally heterogeneous and associated with extensive clinical heterogeneity.

Clinical and genetic analysis further delineates the phenotypic spectrum of ALDH1A3-related anophthalmia and microphthalmia.

Biallelic pathogenic variants in ALDH1A3 are responsible for approximately 11% of recessively inherited cases of severe developmental eye anomalies. Some individuals can display variable neurodevelopmental features, but the relationship to the ALDH1A3 variants remains unclear. Here, we describe seven unrelated families with biallelic pathogenic ALDH1A3 variants: four compound heterozygous and three homozygous. All affected individuals had bilateral anophthalmia/microphthalmia (A/M), three with additional intellectual or developmental delay, one with autism and seizures and three with facial dysmorphic features. This study confirms that individuals with biallelic pathogenic ALDH1A3 variants consistently manifest A/M, but additionally display neurodevelopmental features with significant intra- and interfamilial variability. Furthermore, we describe the first case with cataract and highlight the importance of screening ALDH1A3 variants in nonconsanguineous families with A/M.

Minigene Splicing Assays and Long-Read Sequencing to Unravel Pathogenic Deep-Intronic Variants in PAX6 in Congenital Aniridia.

PAX6 haploinsufficiency causes aniridia, a congenital eye disorder that involves the iris, and foveal hypoplasia. Comprehensive screening of the PAX6 locus, including the non-coding regions, by next-generation sequencing revealed four deep-intronic variants with potential effects on pre-RNA splicing. Nevertheless, without a functional analysis, their pathogenicity could not be established. We aimed to decipher their impact on the canonical PAX6 splicing using in vitro minigene splicing assays and nanopore-based long-read sequencing. Two multi-exonic PAX6 constructs were generated, and minigene assays were carried out. An aberrant splicing pattern was observed for two variants in intron 6, c.357+136G>A and c.357+334G>A. In both cases, several exonization events, such as pseudoexon inclusions and partial intronic retention, were observed due to the creation or activation of new/cryptic non-canonical splicing sites, including a shared intronic donor site. In contrast, two variants identified in intron 11, c.1032+170A>T and c.1033-275A>C, seemed not to affect splicing processes. We confirmed the high complexity of alternative splicing of PAX6 exon 6, which also involves unreported cryptic intronic sites. Our study highlights the importance of integrating functional studies into diagnostic algorithms to decipher the potential implication of non-coding variants, usually classified as variants of unknown significance, thus allowing variant reclassification to achieve a conclusive genetic diagnosis.

Evaluation of somatic and/or germline mosaicism in congenital malformation of the eye.

Microphthalmia, Anophthalmia and Coloboma (MAC) form a spectrum of congenital eye malformations responsible for severe visual impairment. Despite the exploration of hundreds of genes by High-Throughput Sequencing (HTS), most of the patients remain without genetic diagnosis. One explanation could be the not yet demonstrated involvement of somatic mosaicism (undetected by conventional analysis pipelines) in those patients. Furthermore, the proportion of parental germline mosaicism in presumed de novo variations is still unknown in ocular malformations. Thus, using dedicated bioinformatics pipeline designed to detect mosaic variants, we reanalysed the sequencing data obtained from a 119 ocular development genes panel performed on blood samples of 78 probands with sporadic MAC without genetic diagnosis. Using the same HTS strategy, we sequenced 80 asymptomatic parents of 41 probands carrying a disease-causing variant in an ocular development gene considered de novo after Sanger sequencing of both parents. Reanalysis of the previously sequencing data did not find any mosaic variant in probands without genetic diagnosis. However, HTS of parents revealed undetected SOX2 and PAX6 mosaic variants in two parents. Finally, this work, performed on two large cohorts of patients with MAC spectrum, provides for the first time an overview of the interest of looking for mosaicism in ocular development disorders. Somatic mosaicism does not appear to be frequent in MAC spectrum and might explain only few diagnoses. Thus, other approaches such as whole genome sequencing should be considered in those patients. Parental mosaicism is however not that rare (around 5%) and challenging for genetic counselling.

Bi-allelic variants in WNT7B disrupt the development of multiple organs in humans.

BACKGROUND: Pulmonary hypoplasia, Diaphragmatic anomalies, Anophthalmia/microphthalmia and Cardiac defects delineate the PDAC syndrome. We aim to identify the cause of PDAC syndrome in patients who do not carry pathogenic variants in RARB and STRA6, which have been previously associated with this disorder. METHODS: We sequenced the exome of patients with unexplained PDAC syndrome and performed functional validation of candidate variants. RESULTS: We identified bi-allelic variants in WNT7B in fetuses with PDAC syndrome from two unrelated families. In one family, the fetus was homozygous for the c.292C>T (p.(Arg98*)) variant whereas the fetuses from the other family were compound heterozygous for the variants c.225C>G (p.(Tyr75*)) and c.562G>A (p.(Gly188Ser)). Finally, a molecular autopsy by proxy in a consanguineous couple that lost two babies due to lung hypoplasia revealed that both parents carry the p.(Arg98*) variant. Using a WNT signalling canonical luciferase assay, we demonstrated that the identified variants are deleterious. In addition, we found that wnt7bb mutant zebrafish display a defect of the swimbladder, an air-filled organ that is a structural homolog of the mammalian lung, suggesting that the function of WNT7B has been conserved during evolution for the development of these structures. CONCLUSION: Our findings indicate that defective WNT7B function underlies a form of lung hypoplasia that is associated with the PDAC syndrome, and provide evidence for involvement of the WNT-ß-catenin pathway in human lung, tracheal, ocular, cardiac, and renal development.

GM2 gangliosidosis AB variant: first case of late onset and review of the literature.

AB variant is the rarest form of GM2 gangliosidosis, neurodegenerative diseases caused by lysosomal accumulation of GM2 gangliosides. Less than thirty cases are referenced in the literature, and to date, no late-onset form has been described. Our proband is a 22-year-old male with spinocerebellar ataxia and lower limbs motor deficiency. His symptoms started at the age of 10. A genetic analysis revealed two mutations in the GM2A gene encoding the GM2 activator protein (GM2-AP), an essential co-factor of hexosaminidase A. Both mutations, GM2A:c.79A > T:p.Lys27* and GM2A:c.415C > T:p.Pro139Ser, were inherited respectively from his father and his mother. The nonsense mutation was predicted to be likely pathogenic, but the missense mutation was of unknown significance. To establish the pathogenicity of this variant, we studied GM2 accumulation and GM2A gene expression. Electron microscopy and immunofluorescence performed on patient's fibroblasts did not reveal any lysosomal accumulation of GM2. There was also no difference in GM2A gene expression using RT-qPCR, and both mutations were found on cDNA Sanger sequencing. Measurement of plasma gangliosides by liquid-phase chromatography-tandem mass spectrometry showed an accumulation of GM2 in our patient's plasma at 83.5 nmol/L, and a GM2/GM3 ratio at 0.066 (median of negative control at 30.2 nmol/L [19.7-46.8] and 0.019 respectively). Therefore, the association of both p.Lys27* and p.Pro169Ser mutations leads to a GM2-AP functional deficiency. Whereas the first mutation is more likely to be linked with infantile form of GM2 gangliosidosis, the hypomorphic p.Pro169Ser variant may be the first associated with a late-onset form of AB variant.

EPHA2 biallelic disruption causes syndromic complex microphthalmia with iris hypoplasia.

Disruption of any of the ocular development steps can result in ocular defects such as microphthalmia, coloboma and anterior segment dysgeneses including aniridia and cataract. All of these anomalies can be isolated or seen in association with each other. Except for aniridia (almost exclusively due to PAX6 mutations), most of these congenital ocular malformations are related to a wide genetic heterogeneity, as hundreds of genes are implied in ocular development. Here we describe a patient presenting with bilateral microphthalmia, congenital cataract, corneal dystrophy and iris hypoplasia, associated with extra-ocular features, who underwent an analysis of 119 ocular development related genes. Genetic testing revealed the presence of two truncating variants in the EPHA2 gene. While EPHA2 mutations are mainly known to be responsible for isolated dominant congenital cataract, we report here the first case of complex anterior segment dysgenesis caused by a biallelic EPHA2 mutation. This gene should be screened in case of aniridia with a negative PAX6 testing, as the ocular features of our patient clearly mimic those of PAX6 mutated patients. This observation enlarges the phenotype associated with EPHA2 variations and rise the insight of a possible PAX6-EPHA2 interaction that needs further investigations. Moreover, despite a great variability in ocular and extra-ocular phenotypes, mutations type and inheritance pattern, a possible genotype-phenotype correlation can also be drawn for this gene.

First evidence of SOX2 mutations in Peters' anomaly: Lessons from molecular screening of 95 patients.

Peters' anomaly (PA) is a rare anterior segment dysgenesis characterized by central corneal opacity and irido-lenticulo-corneal adhesions. Several genes are involved in syndromic or isolated PA (B3GLCT, PAX6, PITX3, FOXE3, CYP1B1). Some copy number variations (CNVs) have also been occasionally reported. Despite this genetic heterogeneity, most of patients remain without genetic diagnosis. We retrieved a cohort of 95 individuals with PA and performed genotyping using a combination of comparative genomic hybridization, whole genome, exome and targeted sequencing of 119 genes associated with ocular development anomalies. Causative genetic defects involving 12 genes and CNVs were identified for 1/3 of patients. Unsurprisingly, B3GLCT and PAX6 were the most frequently implicated genes, respectively in syndromic and isolated PA. Unexpectedly, the third gene involved in our cohort was SOX2, the major gene of micro-anophthalmia. Four unrelated patients with PA (isolated or with microphthalmia) were carrying pathogenic variants in this gene that was never associated with PA before. Here we described the largest cohort of PA patients ever reported. The genetic bases of PA are still to be explored as genetic diagnosis was unavailable for 2/3 of patients. Nevertheless, we showed here for the first time the involvement of SOX2 in PA, offering new evidence for its role in corneal transparency and anterior segment development.

Safety and efficacy of riluzole in spinocerebellar ataxia type 2 in France (ATRIL): a multicentre, randomised, double-blind, placebo-controlled trial.

BACKGROUND: Riluzole has been reported to be beneficial in patients with cerebellar ataxia; however, effectiveness in individual subtypes of disease is unclear due to heterogeneity in participants' causes and stages of disease. Our aim was to test riluzole in a single genetic disease, spinocerebellar ataxia type 2. METHODS: We did a randomised, double-blind, placebo-controlled, multicentre trial (the ATRIL study) at eight national reference centres for rare diseases in France that were part of the Neurogene National Reference Centre for Rare Diseases. Participants were patients with spinocerebellar ataxia type 2 with an age at disease onset of up to 50 years and a scale for the assessment and rating of ataxia (SARA) score of at least 5 and up to 26. Patients were randomly assigned centrally (1:1) to receive either riluzole 50 mg orally or placebo twice per day for 12 months. Two visits, at baseline and at 12 months, included clinical measures and 3T brain MRI. The primary endpoint was the proportion of patients whose SARA score improved by at least 1 point. Analyses were done in the intention-to-treat population (all participants who were randomly assigned) and were done with only the observed data (complete case analysis). This trial is registered at ClinicalTrials.gov (NCT03347344) and has been completed. FINDINGS: Between Jan 18, 2018, and June 14, 2019, we enrolled 45 patients. 22 patients were randomly assigned to receive riluzole and 23 to receive placebo. Median age was 42 years (IQR 36-57) in the riluzole group and 49 years (40-56) in the placebo group and 23 (51%) participants were women. All participants presented with moderate-stage disease, characterised by a median SARA score of 13·5 (IQR 9·5-16·5). The primary endpoint, SARA score improvement of at least 1 point after 12 months, was observed in seven patients (32%) in the treated group versus nine patients (39%) in the placebo group, with a mean difference of -10·3% (95% CI -37·4% to 19·2%; p=0·75). SARA score showed a median increase (ie, worsening) of 0·5 points (IQR -1·5 to 1·5) in the riluzole group versus 0·3 points (-1·0 to 2·5) in the placebo group (p=0·70). No serious adverse event was reported in the riluzole-treated group whereas four patients in placebo group had a serious adverse event (hepatic enzyme increase, fracture of external malleolus, rectorrhagia, and depression). The number of patients with adverse events was similar in both groups (riluzole 16 [73%] patients vs placebo 19 [83%] patients; p=0·49). INTERPRETATION: We were able to recruit 45 patients moderately affected by spinocerebellar ataxia type 2 for this trial. Riluzole did not improve clinical or radiological outcomes in these patients. However, our findings provide data on progression of spinocerebellar ataxia type 2 that might prove to be valuable for the design of other clinical trials. FUNDING: French Ministry of Health.

Activation of cryptic donor splice sites by non-coding and coding PAX6 variants contributes to congenital aniridia.

BACKGROUND: The paired-domain transcription factor paired box gene 6 (PAX6) causes a wide spectrum of ocular developmental anomalies, including congenital aniridia, Peters anomaly and microphthalmia. Here, we aimed to functionally assess the involvement of seven potentially non-canonical splicing variants on missplicing of exon 6, which represents the main hotspot region for loss-of-function PAX6 variants. METHODS: By locus-specific analysis of PAX6 using Sanger and/or targeted next-generation sequencing, we screened a Spanish cohort of 106 patients with PAX6-related diseases. Functional splicing assays were performed by in vitro minigene approaches or directly in RNA from patient-derived lymphocytes cell line, when available. RESULTS: Five out seven variants, including three synonymous changes, one small exonic deletion and one non-canonical splice variant, showed anomalous splicing patterns yielding partial exon skipping and/or elongation. CONCLUSION: We describe new spliceogenic mechanisms for PAX6 variants mediated by creating or strengthening five different cryptic donor sites at exon 6. Our work revealed that the activation of cryptic PAX6 splicing sites seems to be a recurrent and underestimated cause of aniridia. Our findings pointed out the importance of functional assessment of apparently silent PAX6 variants to uncover hidden genetic alterations and to improve variant interpretation for genetic counselling in aniridia.

Individuals with heterozygous variants in the Wnt-signalling pathway gene FZD5 delineate a phenotype characterized by isolated coloboma and variable expressivity.

BACKGROUND: Anophthalmia, microphthalmia and coloboma are a genetically heterogenous spectrum of developmental eye disorders. Recently, variants in the Wnt-pathway gene Frizzled Class Receptor 5 (FZD5) have been identified in individuals with coloboma and rarely microphthalmia, sometimes with additional phenotypes and variable penetrance. MATERIALS AND METHODS: We identified variants in FZD5 in individuals with developmental eye disorders from the UK (including the DDD Study [www.ddduk.org/access.html]), France and Spain using whole genome/exome sequencing or customized NGS panels of ocular development genes. RESULTS: We report eight new families with FZD5 variants and ocular coloboma. Three individuals presented with additional syndromic features, two explicable by additional variants in other genes (SLC12A2 and DDX3X). In two families initially showing incomplete penetrance, re-examination of apparently unaffected carrier individuals revealed subtle ocular colobomatous phenotypes. Finally, we report two families with microphthalmia in addition to coloboma, representing the second and third reported cases of this phenotype in conjunction with FZD5 variants. CONCLUSIONS: Our findings indicate FZD5 variants are typically associated with isolated ocular coloboma, occasionally microphthalmia, and that extraocular phenotypes are likely to be explained by other gene alterations.

Confirmation of FZD5 implication in a cohort of 50 patients with ocular coloboma.

Defects in optic fissure closure can lead to congenital ocular coloboma. This ocular malformation, often associated with microphthalmia, is described in various clinical forms with different inheritance patterns and genetic heterogeneity. In recent times, the identification of an increased number of genes involved in numerous cellular functions has led to a better understanding in optic fissure closure mechanisms. Nevertheless, most of these genes are also involved in wider eye growth defects such as micro-anophthalmia, questioning the mechanisms controlling both extension and severity of optic fissure closure defects. However, some genes, such as FZD5, have only been so far identified in isolated coloboma. Thus, to estimate the frequency of implication of different ocular genes, we screened a cohort of 50 patients affected by ocular coloboma by using targeted sequencing of 119 genes involved in ocular development. This analysis revealed seven heterozygous (likely) pathogenic variants in RARB, MAB21L2, RBP4, TFAP2A, and FZD5. Surprisingly, three out of the seven variants detected herein were novel disease-causing variants in FZD5 identified in three unrelated families with dominant inheritance. Although molecular diagnosis rate remains relatively low in patients with ocular coloboma (14% (7/50) in this work), these results, however, highlight the importance of genetic screening, especially of FZD5, in such patients. Indeed, in our series, FZD5 variants represent half of the genetic causes, constituting 6% (3/50) of the patients who benefited from a molecular diagnosis. Our findings support the involvement of FZD5 in ocular coloboma and provide clues for screening this gene during current diagnostic procedures.

Attitudes of French populations towards the disclosure of unsolicited findings in medical genetics.

Next-generation sequencing techniques enable unsolicited findings to be detected. This discovery raises ethical questions concerning the return of these findings. Our study aimed to highlight the views of the general public, patients under supervision and health professionals concerning the acceptability of disclosing unsolicited results to patients. In total, 449 participants assessed scenarios, consisted of all combinations of three factors (patient's information and consent, prevention and treatment of the unsolicited disease and doctor's decision). The response profiles were grouped into six clusters. The participants took ethical aspects into account, but health professionals also considered the medical aspects to a greater extent.

Single Circulating Fetal Trophoblastic Cells Eligible for Non Invasive Prenatal Diagnosis: the Exception Rather than the Rule.

Non-Invasive Prenatal Diagnosis (NIPD), based on the analysis of circulating cell-free fetal DNA (cff-DNA), is successfully implemented for an increasing number of monogenic diseases. However, technical issues related to cff-DNA characteristics remain, and not all mutations can be screened with this method, particularly triplet expansion mutations that frequently concern prenatal diagnosis requests. The objective of this study was to develop an approach to isolate and analyze Circulating Trophoblastic Fetal Cells (CFTCs) for NIPD of monogenic diseases caused by triplet repeat expansion or point mutations. We developed a method for CFTC isolation based on DEPArray sorting and used Huntington's disease as the clinical model for CFTC-based NIPD. Then, we investigated whether CFTC isolation and Whole Genome Amplification (WGA) could be used for NIPD in couples at risk of transmitting different monogenic diseases. Our data show that the allele drop-out rate was 3-fold higher in CFTCs than in maternal cells processed in the same way. Moreover, we give new insights into CFTCs by compiling data obtained by extensive molecular testing by microsatellite multiplex PCR genotyping and by WGA followed by mini-exome sequencing. CFTCs appear to be often characterized by a random state of genomic degradation.