Shifting the landscape: Dominant C-terminal rare missense FOXL2 variants in non-syndromic primary ovarian failure etiology.

Pathogenic germline variants in the FOXL2 gene are associated with Blepharophimosis, Ptosis, and Epicanthus Inversus syndrome (BPES) in humans, an autosomal dominant condition. Two forms of BPES have emerged: (i) type I (BPES-I), characterized by ocular signs and primary ovarian failure (POI), and (ii) type II (BPES-II) with no systemic associations. This study aimed to compare the distribution of FOXL2 variants in idiopathic POI/DOR (diminished ovarian reserve) and both types of BPES, and to determine the involvement of FOXL2 in non-syndromic forms of POI/DOR. We studied the whole coding region of the FOXL2 gene using next-generation sequencing in 1282 patients with non-syndromic POI/DOR. Each identified FOXL2 variant was compared to its frequency in the general population, considering ethnicity. Screening of the entire coding region of the FOXL2 gene allowed us to identify 10 different variants, including nine missense variants. Of the patients with POI/DOR, 14 (1%) carried a FOXL2 variant. Significantly, six out of nine missense variants (67%) were overrepresented in our POI/DOR cohort compared to the general or specific ethnic subgroups. Our findings strongly suggest that five rare missense variants, mainly located in the C-terminal region of FOXL2 are high-risk factors for non-syndromic POI/DOR, though FOXL2 gene implication accounts for approximately 0.54% of non-syndromic POI/DOR cases. These results support the implementation of routine genetic screening for patients with POI/DOR in clinical settings.

SpliceAI-visual: a free online tool to improve SpliceAI splicing variant interpretation.

SpliceAI is an open-source deep learning splicing prediction algorithm that has demonstrated in the past few years its high ability to predict splicing defects caused by DNA variations. However, its outputs present several drawbacks: (1) although the numerical values are very convenient for batch filtering, their precise interpretation can be difficult, (2) the outputs are delta scores which can sometimes mask a severe consequence, and (3) complex delins are most often not handled. We present here SpliceAI-visual, a free online tool based on the SpliceAI algorithm, and show how it complements the traditional SpliceAI analysis. First, SpliceAI-visual manipulates raw scores and not delta scores, as the latter can be misleading in certain circumstances. Second, the outcome of SpliceAI-visual is user-friendly thanks to the graphical presentation. Third, SpliceAI-visual is currently one of the only SpliceAI-derived implementations able to annotate complex variants (e.g., complex delins). We report here the benefits of using SpliceAI-visual and demonstrate its relevance in the assessment/modulation of the PVS1 classification criteria. We also show how SpliceAI-visual can elucidate several complex splicing defects taken from the literature but also from unpublished cases. SpliceAI-visual is available as a Google Colab notebook and has also been fully integrated in a free online variant interpretation tool, MobiDetails ( https://mobidetails.iurc.montp.inserm.fr/MD ).

Stratification of the risk of ovarian dysfunction by studying the complexity of intermediate and premutation alleles of the FMR1 gene.

FMR1 premutation female carriers are at risk of developing premature/primary ovarian insufficiency (POI) with an incomplete penetrance. In this study, we determined the CGG repeat size among 1095 women with diminished ovarian reserve (DOR) / POI and characterized the CGG/AGG substructure in 44 women carrying an abnormal FMR1 repeat expansion number, compared to a group of 25 pregnant women carrying an abnormal FMR1 CGG repeat size. Allelic complexity scores of the FMR1 gene were calculated and compared between the two groups. In the DOR/POI cohort, 2.1% of women presented with an intermediate repeat size and 1.9% with a premutation. Our results suggest that the risk of POI is highest in the mid-range of CGG repeats. We observed that the allelic score is significantly higher in POI women compared to the pregnant women group (p-value = 0.02). We suggest that a high allelic score due to more than 2 AGG interspersions in the context of an intermediate number of repetitions could favor POI. Larger studies are still needed to evaluate the relevance of this new tool for the determination of the individual risk of developing POI in women with abnormal number of CGG repeats.

A retrospective cohort study and review of the literature about germline mosaicism in Duchenne/Becker muscular dystrophy prenatal counseling: How to estimate the recurrence risk in clinical settings?

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are the most common inherited neuromuscular diseases. Following the identification of a pathogenic causative variant in the DMD gene of a proband, potential carriers can be informed of their risk of having offspring with the disease. Germline mosaicism is a variant that is confined to the gonads that can be transmitted to offspring and is usually reported when a non-carrier of a DMD pathogenic variant has two or more offspring carrying the variant in question. On average, one third of cases are the result of a de novo variant, and as DMD and BMD are prone to germline mosaicism, its inclusion in genetic counseling is mandatory. In this retrospective cohort study, we presented clinical data from an unpublished DMD/BMD cohort of 332 families with incidence of germline mosaicism in families with de novo transmission of 8.1%. This is also the first systematic literature review searching PubMed to provide an accurate assessment of the current literature on germline mosaicism in DMD and BMD, including 17 case reports and 20 original studies. The incidence of documented germline mosaicism in de novo event families ranged from 6.0 to 40%, with a mean of 8.3%. The estimated recurrence risk for mothers of a patient with a proven de novo causal variant ranged from 4.3 to 11%, with a mean of 5.8% for a male fetus. By providing an up-to-date and comprehensive overview of the literature, this review aims to improve our understanding of germline mosaicism in DMD and to promote the development of effective strategies and reliable data for occurrence risk assessment in genetic counseling of de novo event families.

NOBOX gene variants in premature ovarian insufficiency: ethnicity-dependent insights.

PURPOSE: Premature ovarian insufficiency (POI) affects approximately 1% of women before the age of 40. Genetic contribution is a significant component of POI. The NOBOX gene was considered one of the major genetic causes of POI. However, the pathogenicity and the penetrance of NOBOX variants remain unclear. METHODS: We studied the whole coding region of the NOBOX gene by next generation sequencing in a cohort of 810 patients with POI, and we compared the frequency of each identified NOBOX variant to the general population taking into account the ethnicity of each individual. RESULTS: Screening of the whole coding region of the NOBOX gene allowed us to identify 35 different variants, including 5 loss-of-function variants. In total, 171 patients with POI (25%) carried out at least one NOBOX variant. Regarding missense variants, we observed a significant overrepresentation of the most frequent ones in our 810 POI patients as compared to the general, except for p.(Arg117Trp). However, taking into account the ethnic origin of the individuals, we observed no significant OR difference for p.(Arg44Leu) and p.(Arg117Trp) in African subgroup and for p.(Asp452Asn) in European subgroup. CONCLUSION: This population study suggests that the p.(Arg44Leu) variant could be considered benign variant and that the p.(Asp452Asn) and p.(Arg117Trp) variants could be considered moderate risk pathogenic variants with probably partial and very low penetrance and/or expressivity. In contrast, p.(Gly91Trp) and p.(Gly152Arg) variants could be considered pathogenic variants with a moderate functional impact.

Caveolinopathy: Clinical, histological, and muscle imaging features and follow-up in a multicenter retrospective cohort.

BACKGROUND AND PURPOSE: CAV3 gene mutations, mostly inherited as an autosomal dominant trait, cause various skeletal muscle diseases. Clinical presentations encompass proximal myopathy, distal myopathy, or isolated persistent high creatine kinase (CK) with a major overlapping phenotype. METHODS: Twenty-three patients with CAV3 symptomatic mutations, from 16 different families, were included in a retrospective cohort. Mean follow-up duration was 24.2 ± 15.0 years. Clinical and functional data were collected during the follow-up. The results of muscle imaging, electroneuromyography, muscle histopathology, immunohistochemistry, and caveolin-3 Western blot analysis were also compiled. RESULTS: Exercise intolerance was the most common phenotype (52%). Eighty percent of patients had calf hypertrophy, and only 65% of patients presented rippling. One patient presented initially with camptocormia. A walking aid was required in only two patients. Electroneuromyography was mostly normal. CK level was elevated in all patients. No patient had cardiac or respiratory impairment. Muscle imaging showed fatty involvement of semimembranosus, semitendinosus, rectus femoris, biceps brachialis, and spinal muscles. Almost all (87%) of the biopsies were abnormal but without any specific pattern. Whereas a quarter of patients had normal caveolin-3 immunohistochemistry results, Western blots disclosed a reduced amount of the protein. We report nine mutations, including four not previously described. No phenotype-genotype correlation was evidenced. CONCLUSIONS: Caveolinopathy has diverse clinical, muscle imaging, and histological presentations but often has limited functional impact. Mild forms of the disease, an atypical phenotype, and normal caveolin-3 immunostaining are pitfalls leading to misdiagnosis.

Non-invasive prenatal diagnosis of single gene disorders by paternal mutation exclusion: 3 years of clinical experience.

OBJECTIVES: Cell-free fetal DNA (cffDNA) analysis is performed routinely for aneuploidy screening, RhD genotyping or sex determination. Although applications to single gene disorders (SGD) are being rapidly developed worldwide, only a few laboratories offer cffDNA testing routinely as a diagnosis service for this indication. In a previous report, we described a standardised protocol for non-invasive exclusion of paternal variant in SGD. Three years later, we now report our clinical experience with the protocol. DESIGN: Descriptive study. SETTING: Multi-centre French. POPULATION: Indications for referral included pregnancies at risk of 25% or 50% of paternally inherited SGD, and pregnancies associated with an increased risk of SGD due to a de novo variant, either from strongly suggestive ultrasound findings or from a possible parental germinal mosaicism in the context of a previously affected child. METHODS: Non-invasive prenatal diagnosis was performed using custom assays for droplet digital PCR. Feasibility, diagnostic performance and turn-around time were evaluated. RESULTS: Mean time for a new assay design and validation was evaluated at 14 days, and mean result reporting time was 6 days. All referred pathogenic variants could be targeted except one located in a complex genomic region. A result was obtained for every 198 referrals except two. CONCLUSION: This service was successfully implemented as a routine laboratory practice. It has been widely adopted by French clinicians and patients for paternal variant exclusion in various disorders. TWEETABLE ABSTRACT: A robust approach to non-invasive prenatal exclusion of paternal pathogenic variant in a diagnosis setting.

Molecular Characterization of a Rare Case of Monozygotic Dichorionic Diamniotic Twin Pregnancy after Single Blastocyst Transfer in Preimplantation Genetic Testing (PGT).

Preimplantation genetic testing (PGT) is widely used to select unaffected embryos, increasing the odds of having a healthy baby. During the last few decades, it was accepted that monozygotic dichorionic diamniotic twin pregnancies occurred from the embryo splitting before Day 3 postfertilization according to Corner's dogma. Hence, the occurrence of a dichorionic diamniotic twin pregnancy after a single blastocyst transfer was considered a dizygotic pregnancy resulting from blastocyst transfer and concurrent natural fertilization. In our study, we have provided for the first time molecular proof that a single blastocyst transfer can result in a monozygotic dichorionic diamniotic twin pregnancy, invalidating Corner's dogma. In this case, we recommend systematically assessing the genetic status of dichorionic twins after single blastocyst transfer using prenatal diagnosis to exclude the risk from a potential concurrent spontaneous pregnancy and to ensure that both fetuses are unaffected. To achieve this goal, we have developed here an innovative noninvasive prenatal diagnosis by exclusion of paternal variants with droplet digital PCR, maximizing the reliability of genetic diagnosis. Further multicentric prospective studies using genetic testing are now required to establish the rate of blastocyst splitting leading to dichorionic pregnancy in PGT and to identify the risk factors.

The value of plasma cell-free DNA levels as biomarker in patients with eating disorders: A preliminary study.

OBJECTIVE: Circulating cell-free DNA (cfDNA) holds promise as a rapid and convenient biomarker for identifying individuals with eating disorders. To investigate this hypothesis, we measured plasma cfDNA in patients with different eating disorders. METHODS: In this study, 110 participants (98 patients with eating disorders divided into 30 patients with bulimia nervosa, 33 patients with anorexia nervosa (AN) Restricting subtype, 35 patients with AN Binge-eating/purging subtype and 12 controls) were enrolled. We measured both cell-free nuclear DNA (cf-nDNA) and cell-free mitochondrial DNA (cf-mtDNA) from plasma using two specific droplet digital PCR assays each, referring to two amplicon sizes. RESULTS: Levels of plasma cf-nDNA and cf-mtDNA showed no significant differences between control participants and those with eating disorders. However, we observed a higher proportion of long cf-nDNA fragments in patients with eating disorders, suggesting its potential as a biomarker for eating disorders. CONCLUSION: This is the first study of cfDNA in patients with eating disorders. Our findings highlight the potential for qualitative exploration of cfDNA, although not of quantitative interest. Full characterization of cfDNA may serve as a valuable biomarker for eating disorders and provide some insights into the hidden mechanisms underlying the chronic development of these conditions. Future studies are needed to confirm or refute this hypothesis.

Revisiting GDF9 variants in primary ovarian insufficiency: A shift from dominant to recessive pathogenicity?

BACKGROUND: Primary ovarian insufficiency (POI) affects around 2-4% of women before the age of 40. Genetic factors play an important role in POI. The GDF9 gene has been identified as a significant genetic contributor of POI. However, the pathogenicity and penetrance of GDF9 variants remain uncertain. METHODS: A next-generation sequencing approach was employed to investigate the entire coding region of the GDF9 gene in a cohort of 1281 patients with POI or diminished ovarian reserve (DOR). The frequency of each identified GDF9 variant was then compared with that of the general population, taking into account the ethnicity of each individual. RESULTS: By screening the entire coding region of the GDF9 gene, we identified 19 different variants, including 1 pathogenic frameshift variant. In total, 36 patients with POI/DOR (2.8%) carried at least one GDF9 variant. With regard to missense variants, no significant overrepresentation of the most common variants was observed in our POI/DOR cohort in comparison to the general or specific ethnic subgroups. Only one homozygous subject had a frameshift loss of function variant. CONCLUSION: This epidemiological study suggests that the vast majority of heterozygous missense variants could be considered as variants of uncertain significance and the homozygous loss-of-function variant could be considered as a pathogenic variant. The identification of a novel case of a homozygous POI patient with a heterozygous mother carrying the same variant with normal ovarian function strongly suggests that GDF9 syndrome is an autosomal recessive disorder.

A systematic literature review and meta-analysis of circulating nucleic acids as biomarkers in psychiatry.

Common mental disorders (CMDs) such as depression, anxiety and post-traumatic stress disorders account for 40% of the global burden of disease. In most psychiatric disorders, both diagnosis and monitoring can be challenging, frequently requiring long-term investigation and follow-up. The discovery of better methods to facilitate accurate and fast diagnosis and monitoring of psychiatric disorders is therefore crucial. Circulating nucleic acids (CNAs) are among these new tools. CNAs (DNA or RNA) can be found circulating in body biofluids, and can be isolated from biological samples such as plasma. They can serve as biomarkers for diagnosis and prognoses. They appear to be promising for disorders (such as psychiatric disorders) that involve organs or structures that are difficult to assess. This review presents an accurate assessment of the current literature about the use of plasma and serum cell-free DNA (cfDNA) as biomarkers for several aspects of psychiatric disorders: diagnosis, prognosis, treatment response, and monitor disease progression. For each psychiatric disorder, we examine the effect sizes to give insights on the efficacy of CNAs as biomarkers. The global effect size for plasma nuclear and mitochondrial cfDNA studies was generally moderate for psychiatric disorders. In addition, we discuss future applications of CNAs and particularly cfDNA as non-invasive biomarkers for these diseases.

How a paternal uniparental isodisomy of chromosome 17 leads to autosomal recessive limb-girdle muscular dystrophy R3.

Uniparental isodisomy is a condition where both chromosomes of a pair are inherited from one parental homologue. If a deleterious variant is present on the duplicated chromosome, its homozygosity can reveal an autosomal recessive disorder in the offspring of a heterozygous carrier. Limb-girdle muscular dystrophy (LGMD) R3 is an autosomal recessive inherited disease that is associated with alpha-sarcoglycan gene (SGCA) variants. We report the first published case of LGMDR3 due to a homozygous variant in SGCA unmasked by uniparental isodisomy. The patient is an 8-year-old who experienced delayed motor milestones but normal cognitive development. He presented with muscle pain and elevated plasma creatine kinase. Sequencing of the SGCA gene showed a homozygous pathogenic variant. Parents were not related and only the father was heterozygous for the pathogenic variant. A chromosomal microarray revealed a complete chromosome 17 copy number neutral loss of heterozygosity encompassing SGCA, indicating paternal uniparental isodisomy.

Non-invasive prenatal diagnosis of single gene disorders with enhanced relative haplotype dosage analysis for diagnostic implementation.

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.

[Towards a generalization of non-invasive prenatal diagnosis of single-gene disorders? Assesment and outlook]. / Vers une généralisation du diagnostic prénatal non-invasif des maladies monogéniques ? État des lieux et perspectives.

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.

An early onset benign myopathy with glycogen storage caused by a de novo 1.4 Mb-deletion of chromosome 14.

Early onset myopathies are a clinically and histologically heterogeneous monogenic diseases linked to approximately 90 genes. Molecular diagnosis is challenging, especially in patients with a mild phenotype. We describe a 26-year-old man with neonatal hypotonia, motor delay and seizures during infancy, and non-progressive, mild muscular weakness in adulthood. Serum Creatine kinase level was normal. Whole-body muscle MRI showed thin muscles, and brain MRI was unremarkable. A deltoid muscle biopsy showed glycogen storage. WGS revealed a de novo 1.4 Mb-deletion of chromosome 14, confirmed by Array-CGH. This microdeletion causes the loss of ten genes including RALGAPA1, encoding for RalA, a regulator of glucose transporter 4 (GLUT4) expression at the membrane of myofibers. GLUT4 was overexpressed in patient's muscle. Here we highlight the importance to search for chromosomal alterations in the diagnostic workup of early onset myopathies.

Muscle MRI as a Diagnostic Challenge in Emery-Dreifuss Muscular Dystrophy.

 Emery-Dreifuss Muscular Dystrophy (EDMD) is an early-onset, slowly-progressive group of myopathies, presenting with joint contractures, muscle weakness and cardiac abnormalities. Variants in the EMD gene cause an X-linked recessive form (EDMD1). The scarce EDMD1 muscle MRI accounts in the literature describe fatty replacement of posterior thigh and leg muscles.We report a 22-year-old patient with early-onset bilateral joint contractures, slowly progressive muscle weakness and minor cardiac rhythm abnormalities. A novel loss-of-function variant of EMD was identified and deemed probably pathogenic in the absence of emerin detection by immunofluorescence and Western Blot. MRI revealed fatty replacement of the lumbar spinal erectors and the posterior compartment of lower limbs. Interestingly, Short Tau Inversion Recovery (STIR) sequences showed a heterogenous hyper signal on the vasti, hamstrings and left lateral gastrocnemius muscles.Oedema-like abnormalities were previously reported in early stages of other muscular dystrophies, preceding fatty replacement and muscle atrophy, but not in EDMD1 patients. We hypothesize that these oedema-like changes may be a marker of early muscle pathology in EDMD1. Further studies focusing on these abnormalities in the early phase of EDMD1 are required to test our hypothesis.

[Usefulness of free light chain measurement in monoclonal gammopathy, other haematological malignancies and autoimmune diseases]. / Intérêt du dosage des chaînes légères libres dans le cadre de gammapathies monoclonales, autres hémopathies malignes et maladies auto-immunes.

Immunoglobulin light chains are called free when they are not linked with heavy chains to form a whole immunoglobulin. Quantification of free light chains is a part of the French national authority for health guidelines for diagnostic and follow-up of light chain, oligo or non-secretory myeloma and AL amyloidosis. Most recently, the World health organisation had included free light chains quantification in prognostic criteria for monoclonal gammopathy of undetermined significance. However the literature bring to light some other potential indications of this analysis in the exploration of monoclonal gammopathy, also in lymphoid malignancies and some autoimmune diseases such as diabetes, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis and Sjögren syndrome.