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 In this retrospective cohort study, we presented clinical data from an unpublished DMD/BMD cohort of 332 families with incidence of germline mosaicism in families with de novo transmission of 8.1%.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.

Contribution of whole genome sequencing in the molecular diagnosis of mosaic partial deletion of the NF1 gene in neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disease with complete penetrance but highly variable expressivity. In most patients, Next Generation Sequencing (NGS) technologies allow the identification of a loss-of-function pathogenic variant in the NF1 gene, a negative regulator of the RAS-MAPK pathway. We describe the 5-year diagnosis wandering of a patient with a clear NF1 clinical diagnosis, but no molecular diagnosis using standard molecular technologies. The patient presented with a typical NF1 phenotype but NF1 targeted NGS, NF1 transcript analysis, MLPA, and array comparative genomic hybridization failed to reveal a genetic aberration. After 5 years of unsuccessful investigations, trio WGS finally identified a de novo mosaic (VAF ~ 14%) 24.6 kb germline deletion encompassing the promoter and first exon of NF1. This case report illustrates the relevance of WGS to detect structural variants including copy number variants that would be missed by alternative approaches. The identification of the causal pathogenic variant allowed a tailored genetic counseling with a targeted non-invasive prenatal diagnosis by detecting the deletion in plasmatic cell-free DNA from the proband's pregnant partner. This report clearly highlights the need to make WGS a clinically accessible test, offering a tremendous opportunity to identify a molecular diagnosis for otherwise unsolved cases.

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.

Variants in the Oxidoreductase PYROXD1 Cause Early-Onset Myopathy with Internalized Nuclei and Myofibrillar Disorganization.

This study establishes PYROXD1 variants as a cause of early-onset myopathy and uses biospecimens and cell lines, yeast, and zebrafish models to elucidate the fundamental role of PYROXD1 in skeletal muscle. Exome sequencing identified recessive variants in PYROXD1 in nine probands from five families. Affected individuals presented in infancy or childhood with slowly progressive proximal and distal weakness, facial weakness, nasal speech, swallowing difficulties, and normal to moderately elevated creatine kinase. Distinctive histopathology showed abundant internalized nuclei, myofibrillar disorganization, desmin-positive inclusions, and thickened Z-bands. PYROXD1 is a nuclear-cytoplasmic pyridine nucleotide-disulphide reductase (PNDR). PNDRs are flavoproteins (FAD-binding) and catalyze pyridine-nucleotide-dependent (NAD/NADH) reduction of thiol residues in other proteins. Complementation experiments in yeast lacking glutathione reductase glr1 show that human PYROXD1 has reductase activity that is strongly impaired by the disease-associated missense mutations. Immunolocalization studies in human muscle and zebrafish myofibers demonstrate that PYROXD1 localizes to the nucleus and to striated sarcomeric compartments. Zebrafish with ryroxD1 knock-down recapitulate features of PYROXD1 myopathy with sarcomeric disorganization, myofibrillar aggregates, and marked swimming defect. We characterize variants in the oxidoreductase PYROXD1 as a cause of early-onset myopathy with distinctive histopathology and introduce altered redox regulation as a primary cause of congenital muscle disease.

Altered microtubule dynamics and vesicular transport in mouse and human MeCP2-deficient astrocytes.

Rett syndrome (RTT) is a rare X-linked neurodevelopmental disorder, characterized by normal post-natal development followed by a sudden deceleration in brain growth with progressive loss of acquired motor and language skills, stereotypic hand movements and severe cognitive impairment. Mutations in the methyl-CpG-binding protein 2 (MECP2) cause more than 95% of classic cases. Recently, it has been shown that the loss of Mecp2 from glia negatively influences neurons in a non-cell-autonomous fashion, and that in Mecp2-null mice, re-expression of Mecp2 preferentially in astrocytes significantly improved locomotion and anxiety levels, restored respiratory abnormalities to a normal pattern and greatly prolonged lifespan compared with globally null mice. We now report that microtubule (MT)-dependent vesicle transport is altered in Mecp2-deficient astrocytes from newborn Mecp2-deficient mice compared with control wild-type littermates. Similar observation has been made in human MECP2 p.Arg294* iPSC-derived astrocytes. Importantly, administration of Epothilone D, a brain-penetrant MT-stabilizing natural product, was found to restore MT dynamics in Mecp2-deficient astrocytes and in MECP2 p.Arg294* iPSC-derived astrocytes in vitro. Finally, we report that relatively low weekly doses of Epothilone D also partially reversed the impaired exploratory behavior in Mecp2(308/y) male mice. These findings represent a first step toward the validation of an innovative treatment for RTT.

Non-invasive prenatal diagnosis of paternally inherited disorders from maternal plasma: detection of NF1 and CFTR mutations using droplet digital PCR.

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.

Hyperckemia and myalgia are common presentations of anoctamin-5-related myopathy in French patients.

INTRODUCTION: Patients with anoctamin-5 (ANO5) mutations may present not only with limb-girdle muscular dystrophy type 2L or adult-onset Miyoshi-type myopathy but also with asymptomatic hyperCKemia, exercise intolerance, or rhabdomyolysis. MATERIALS AND METHODS: Data from 38 patients in France with ANO5 mutations with and without muscle weakness on first examination were compared. RESULTS: Twenty patients presented without muscle weakness. Median age at symptom onset or discovery of hyperCKemia was 23 years. Creatine kinase levels ranged from 200 to 40,000 U/L. Electromyography showed a myopathic pattern in 5 patients, and muscle imaging showed involvement of posterior calf muscles in 10 patients. Mild cardiac involvement was observed in 2 patients. Sixteen patients remain free of weakness after a median follow-up period of 5 years. DISCUSSION: Asymptomatic, sometimes mild hyperCKemia or exercise intolerance is a presentation of ANO5-related myopathy and may remain isolated or precede muscle weakness by many years. Muscle Nerve 56: 1096-1100, 2017.

Droplet digital PCR combined with minisequencing, a new approach to analyze fetal DNA from maternal blood: application to the non-invasive prenatal diagnosis of achondroplasia.

BACKGROUND: Achondroplasia is generally detected by abnormal prenatal ultrasound findings in the third trimester of pregnancy and then confirmed by molecular genetic testing of fetal genomic DNA obtained by aspiration of amniotic fluid. This invasive procedure presents a small but significant risk for both the fetus and mother. Therefore, non-invasive procedures using cell-free fetal DNA in maternal plasma have been developed for the detection of the fetal achondroplasia mutations. METHODS: To determine whether the fetus carries the de novo mis-sense genetic mutation at nucleotide 1138 in FGFR3 gene involved in >99% of achondroplasia cases, we developed two independent methods: digital-droplet PCR combined with minisequencing, which are very sensitive methods allowing detection of rare alleles. RESULTS: We collected 26 plasmatic samples from women carrying fetus at risk of achondroplasia and diagnosed to date a total of five affected fetuses in maternal blood. The sensitivity and specificity of our test are respectively 100% [95% confidence interval, 56.6-100%] and 100% [95% confidence interval, 84.5-100%]. CONCLUSIONS: This novel, original strategy for non-invasive prenatal diagnosis of achondroplasia is suitable for implementation in routine clinical testing and allows considering extending the applications of these technologies in non-invasive prenatal diagnosis of many other monogenic diseases. © 2016 John Wiley & Sons, Ltd.

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.

Droplet Digital PCR for Fast and Accurate Characterization of NF1 Locus Deletions: Confirmation of the Predominant Maternal Origin of Type-1 Deletions.

Neurofibromatosis type-1 is a genetic disorder caused by loss-of-function variants in the tumor-suppressor NF1. Approximately 4% to 11% of neurofibromatosis type-1 patients have a NF1 locus complete deletion resulting from nonallelic homologous recombination between low copy repeats. Codeleted genes probably account for the more severe phenotype observed in NF1-deleted patients. This genotype-phenotype correlation highlights the need for a detailed molecular description. A droplet digital PCR (ddPCR) set along the NF1 locus was designed to delimitate the three recurrent NF1 deletion breakpoints. The ddPCR was tested in 121 samples from nonrelated NF1-deleted patients. Classification based on ddPCR versus multiplex ligation-dependent probe amplification (MLPA) was compared. In addition, microsatellites were analyzed to identify parental origin of deletions. ddPCR identified 77 type-1 (64%), 20 type-2 (16%), 7 type-3 (6%), and 17 atypical deletions (14%). The results were comparable with MLPA, except for three atypical deletions misclassified as type-2 using MLPA, for which the SUZ12 gene was not deleted. A significant maternal bias (25 of 30) in the origin of deletions was identified. This study proposes a fast and efficient ddPCR quantification to allow fine NF1 deletion classification. It indicates that ddPCR can be implemented easily into routine diagnosis to complement the techniques dedicated to NF1 point variant identification. This new tool may help unravel the genetic basis conditioning phenotypic variability in NF1-deleted patients and offer tailored genetic counseling.

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.

[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.

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.

Association Between Plasma Rituximab Concentration and the Risk of Major Relapse in Antineutrophil Cytoplasmic Antibody-Associated Vasculitides During Rituximab Maintenance Therapy.

OBJECTIVE: Interindividual variability in response to rituximab remains unexplored in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides. Rituximab pharmacokinetics (PK) and pharmacodynamics (PD) as well as genetic polymorphisms could contribute to variability. This ancillary study of the MAINRITSAN 2 trial aimed to explore the relationship between rituximab plasma concentration, genetic polymorphisms in PK/PD candidate genes, and clinical outcomes. METHODS: Patients included in the MAINRITSAN2 trial (ClinicalTrials.gov identifier: NCT01731561) were randomized to receive a 500-mg fixed-schedule rituximab infusion or an individually tailored regimen. Rituximab plasma concentrations at month 3 (CM3) were assessed. DNA samples (n = 53) were genotyped for single-nucleotide polymorphisms within 88 putative PK/PD candidate genes. The relationship between PK/PD outcomes and genetic variants was investigated using logistic linear regression in additive and recessive genetic models. RESULTS: One hundred and thirty-five patients were included. The frequency of underexposed patients (<4 µg/ml) in the fixed-schedule group was statistically lower compared to that in the tailored-infusion group (2.0% versus 18.0%; P = 0.02, respectively). Low rituximab plasma concentration at 3 months (CM3 <4 µg/ml) was an independent risk factor for major relapse (odds ratio 6.56 [95% confidence interval (95% CI) 1.26-34.09]; P = 0.025) at month 28 (M28). A sensitivity survival analysis also identified CM3 <4 µg/ml as an independent risk factor for major relapse (hazard ratio [HR] 4.81 [95% CI 1.56-14.82]; P = 0.006) and relapse (HR 2.70 [95% CI 1.02-7.15]; P = 0.046). STAT4 rs2278940 and PRKCA rs8076312 were significantly associated with CM3 but not with major relapse onset at M28. CONCLUSION: These results suggest that drug monitoring could be useful to individualize the schedule of rituximab administration within the maintenance phase.

Two novel variations p.(Ser1275Thr) and p.(Ser1275Arg) in FLT4 causing prenatal hereditary lymphedema type 1.

BACKGROUND: Hereditary lymphedema 1 is a rare congenital condition, characterized by the development of chronic swelling in body parts. It is highly variable in expression and age of onset with different presentations: from feet edema to hydrops fetalis. This affection is genetically heterogeneous with autosomal dominant inheritance and incomplete penetrance due to a mutation in the FLT4 gene in most cases. CASES: In our study, we report on two fetuses harboring congenital lymphedema with FLT4 variation and review the prenatal confirmed ones of the literatures. Our cases were selected within fetuses explored by exome sequencing in a diagnosis setting. Prenatal ultrasonography showed hydrops fetalis in one case and an increased nuchal translucency with hydrothorax in the other. Comparative genomic hybridization array on amniocentesis was normal in both cases. Exome sequencing identified a variation p.(Ser1275Thr) and p.(Ser1275Arg) in fetus 1 and fetus 2 in the FLT4 gene, respectively. A de novo mutation at the same codon was reported in prenatal literature suggesting possible genotype phenotype correlation. CONCLUSION: Cystic hygroma/hydrops fetalis are possible manifestations of several disorders. This study illustrates how the integration of exome sequencing in prenatal clinical practice can facilitate the diagnosis and genetic counseling of heterogeneous developmental affections.

Altered microtubule dynamics in Mecp2-deficient astrocytes.

Rett syndrome (RTT) is a severe neurodevelopmental disorder caused by mutations in the gene MECP2 encoding the methyl-CpG binding protein 2. This genetic disease affects predominantly girls and is characterized by a period of normal development that lasts for 8-18 months, followed by neurologic regression affecting both motor and mental abilities. Previous studies performed on brains from RTT subjects and Mecp2-deficient mice showed striking changes in neuronal maturation and dendritic arborization. Recently, we showed that expression of stathmin-like 2 (STMN2) was significantly reduced in fibroblasts from RTT patients, and similar results were obtained in the cerebellum of Mecp2-deficient mice. Because assembly and dynamics of microtubules are known to be modulated by STMN2, we studied microtubule dynamics in brain cells from Mecp2-deficient mice. We observed that Mecp2 deficiency affects microtubule dynamics in astrocytes from Mecp2-deficient mice. Our data reinforce the fact that the loss of Mecp2 in astrocytes may influence the onset and progression of RTT. These results imply that Mecp2 has a stabilizing role in microtubule dynamics and that Mecp2 deficiency, which is associated with STMN2 down-regulation, could lead to impaired microtubule stability, hence explaining the dendritic abnormalities observed in RTT brains.

Long-Reads Sequencing Strategy to Localize Variants in TTN Repeated Domains.

Titin protein is responsible for muscle elasticity. The TTN gene, composed of 364 exons, is subjected to extensive alternative splicing and leads to different isoforms expressed in skeletal and cardiac muscle. Variants in TTN are responsible for myopathies with a wide phenotypic spectrum and autosomal dominant or recessive transmission. The I-band coding domain, highly subject to alternative splicing, contains a three-zone block of repeated sequences with 99% homology. Sequencing and localization of variants in these areas are complex when using short-reads sequencing, a second-generation sequencing technique. We have implemented a protocol based on the third-generation sequencing technology (long-reads sequencing). This new method allows us to localize variants in these repeated areas to improve the diagnosis of TTN-related myopathies and offer the analysis of relatives in postnatal or in prenatal screening.

Noninvasive Prenatal Diagnosis of a Paternally Inherited MEN1 Pathogenic Splicing Variant.

CONTEXT: Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disease caused by mutations in the tumor suppressor gene MEN1. The uncertainty of pathogenicity of MEN1 variants complexifies the selection of the patients likely to benefit from specific care. OBJECTIVE: MEN1-mutated patients should be offered tailored tumor screening and genetic counseling. We present a patient with hyperparathyroidism for whom genetic analysis identified a variant of uncertain significance in the MEN1 gene (NM_130799.2): c.654G > T p.(Arg218=). Additional functional genetic tests were performed to classify the variant as pathogenic and allowed prenatal testing. DESIGN: Targeted next generation sequencing identified a synonymous variant in the MEN1 gene in a 26-year-old male with symptomatic primary hyperparathyroidism. In silico and in vitro genetic tests were performed to assess variant pathogenicity. RESULTS: Genetic testing of the proband's unaffected parents showed the variant occurred de novo. Transcript study showed a splicing defect leading to an in-frame deletion. The classification of the MEN1 variant as pathogenic confirmed the diagnosis of MEN1 and recommended an adapted medical care and follow-up. Pathogenic classification also allowed to propose a genetic counseling to the proband and his wife. Noninvasive prenatal diagnosis was performed with a personalized medicine-based protocol by detection of the paternally inherited variant in maternal plasmatic cell free DNA, using digital PCR. CONCLUSION: We showed that functional genetic analysis can help to assess the pathogenicity of a MEN1 variant with crucial consequences for medical care and genetic counseling decisions.

A missense mutation within the fork-head domain of the forkhead box G1 Gene (FOXG1) affects its nuclear localization.

The forkhead box G1 (FOXG1)gene has recently been associated with the congenital variant of Rett syndrome, and so far 17 mutations have been reported. We screened the coding region in 150 patients affected by postnatal microcephaly, and identified two mutations: the c.326C>T (p.P109L) substitution inherited from the healthy father; and the de novo c.730C>T transition, which induces the p.R244C mutation within the DNA-binding forkhead domain. This latter mutation is carried by an 8-year-old girl, who presented a phenotype reminiscent of the congenital variant of Rett syndrome. Immunofluorescence analysis of the wild-type protein revealed a homogeneous nuclear staining excluding the nucleoli, while the p.R244C mutant showed abnormal nuclear foci in a large proportion of cells, suggesting that its mislocalization may reduce and/or impair target recognition. Interestingly, this missense mutation results in a mislocalization of FoxG1 to specific nuclear foci referred to as nuclear speckles, and affects the cyclin-dependent kinase inhibitor p21 CDKN1A expression. Because CDKL5, which is involved in the early-onset variant of Rett syndrome, is also located in these speckles, we suggest that disregulation of the dynamic behaviour of nuclear speckles may functionally link these two proteins, which are both involved in atypical forms of Rett syndrome.