Functional and clinical studies reveal pathophysiological complexity of CLCN4-related neurodevelopmental condition.

Missense and truncating variants in the X-chromosome-linked CLCN4 gene, resulting in reduced or complete loss-of-function (LOF) of the encoded chloride/proton exchanger ClC-4, were recently demonstrated to cause a neurocognitive phenotype in both males and females. Through international clinical matchmaking and interrogation of public variant databases we assembled a database of 90 rare CLCN4 missense variants in 90 families: 41 unique and 18 recurrent variants in 49 families. For 43 families, including 22 males and 33 females, we collated detailed clinical and segregation data. To confirm causality of variants and to obtain insight into disease mechanisms, we investigated the effect on electrophysiological properties of 59 of the variants in Xenopus oocytes using extended voltage and pH ranges. Detailed analyses revealed new pathophysiological mechanisms: 25% (15/59) of variants demonstrated LOF, characterized by a "shift" of the voltage-dependent activation to more positive voltages, and nine variants resulted in a toxic gain-of-function, associated with a disrupted gate allowing inward transport at negative voltages. Functional results were not always in line with in silico pathogenicity scores, highlighting the complexity of pathogenicity assessment for accurate genetic counselling. The complex neurocognitive and psychiatric manifestations of this condition, and hitherto under-recognized impacts on growth, gastrointestinal function, and motor control are discussed. Including published cases, we summarize features in 122 individuals from 67 families with CLCN4-related neurodevelopmental condition and suggest future research directions with the aim of improving the integrated care for individuals with this diagnosis.

Mutations in RNU7-1 Weaken Secondary RNA Structure, Induce MCP-1 and CXCL10 in CSF, and Result in Aicardi-Goutières Syndrome with Severe End-Organ Involvement.

BACKGROUND: Aicardi-Goutières syndrome (AGS) is a type I interferonopathy usually characterized by early-onset neurologic regression. Biallelic mutations in LSM11 and RNU7-1, components of the U7 small nuclear ribonucleoprotein (snRNP) complex, have been identified in a limited number of genetically unexplained AGS cases. Impairment of U7 snRNP function results in misprocessing of replication-dependent histone (RDH) pre-mRNA and disturbance of histone occupancy of nuclear DNA, ultimately driving cGAS-dependent type I interferon (IFN-I) release. OBJECTIVE: We performed a clinical, genetic, and immunological workup of 3 unrelated patients with uncharacterized AGS. METHODS: Whole exome sequencing (WES) and targeted Sanger sequencing of RNU7-1 were performed. Primary fibroblasts were used for mechanistic studies. IFN-I signature and STAT1/2 phosphorylation were assessed in peripheral blood. Cytokines were profiled on serum and cerebrospinal fluid (CSF). Histopathology was examined on brain and kidney tissue. RESULTS: Sequencing revealed compound heterozygous RNU7-1 mutations, resulting in impaired RDH pre-mRNA processing. The 3' stem-loop mutations reduced stability of the secondary U7 snRNA structure. A discrete IFN-I signature in peripheral blood was paralleled by MCP-1 (CCL2) and CXCL10 upregulation in CSF. Histopathological analysis of the kidney showed thrombotic microangiopathy. We observed dysregulated STAT phosphorylation upon cytokine stimulation. Clinical overview of all reported patients with RNU7-1-related disease revealed high mortality and high incidence of organ involvement compared to other AGS genotypes. CONCLUSIONS: Targeted RNU7-1 sequencing is recommended in genetically unexplained AGS cases. CSF cytokine profiling represents an additional diagnostic tool to identify aberrant IFN-I signaling. Clinical follow-up of RNU7-1-mutated patients should include screening for severe end-organ involvement including liver disease and nephropathy.

Outcome of publicly funded nationwide first-tier noninvasive prenatal screening.

PURPOSE: Noninvasive prenatal screening (NIPS) using cell-free DNA has transformed prenatal care. Belgium was the first country to implement and fully reimburse NIPS as a first-tier screening test offered to all pregnant women. A consortium consisting of all Belgian genetic centers report the outcome of two years genome-wide NIPS implementation. METHODS: The performance for the common trisomies and for secondary findings was evaluated based on 153,575 genome-wide NIP tests. Furthermore, the evolution of the number of invasive tests and the incidence of Down syndrome live births was registered. RESULTS: Trisomies 21, 18, and 13 were detected in respectively 0.32%, 0.07%, and 0.06% of cases, with overall positive predictive values (PPVs) of 92.4%, 84.6%, and 43.9%. Rare autosomal trisomies and fetal segmental imbalances were detected in respectively 0.23% and 0.07% of cases with PPVs of 4.1% and 47%. The number of invasive obstetric procedures decreased by 52%. The number of trisomy 21 live births dropped to 0.04%. CONCLUSION: Expanding the scope of NIPS beyond trisomy 21 fetal screening allows the implementation of personalized genomic medicine for the obstetric population. This genome-wide NIPS approach has been embedded successfully in prenatal genetic care in Belgium and might serve as a framework for other countries offering NIPS.

Prenatally detected copy number variants in a national cohort: A postnatal follow-up study.

OBJECTIVE: Belgian genetic centers established a database containing data on all chromosomal microarrays performed in a prenatal context. A study was initiated to evaluate postnatal development in children diagnosed prenatally with a non-benign copy number variant (CNV). METHODS: All children diagnosed with a prenatally detected non-benign CNV in a Belgian genetic center between May 2013 and February 2015 were included in the patient population. The control population consisted of children who had undergone an invasive procedure during pregnancy, with no or only benign CNVs. Child development was evaluated at 36 months using three (3) questionnaires: Ages and Stages Questionnaire Third edition, Ages and Stages Questionnaire Social-Emotional Second Edition and a general questionnaire. RESULTS: A significant difference in communication and personal-social development was detected between children with a reported susceptibility CNV and both children with an unreported susceptibility CNV and the control population. The outcome of children with a particular CNV is discussed in a case-by-case manner. CONCLUSION: Our postnatal follow-up project of children with a prenatally detected non-benign CNV is the first nationwide project of its kind. A higher number of cases for each CNV category is however needed to confirm our findings.

Chromosomal microarray analysis in prenatal diagnosis: ethical considerations of the Belgian approach.

Detection of genetic aberrations in prenatal samples, obtained through amniocentesis or chorion villus biopsy, is increasingly performed using chromosomal microarray (CMA), a technique that can uncover both aneuploidies and copy number variants throughout the genome. Despite the obvious benefits of CMA, the decision on implementing the technology is complicated by ethical issues concerning variant interpretation and reporting. In Belgium, uniform guidelines were composed and a shared database for prenatal CMA findings was established. This Belgian approach sparks discussion: it is evidence-based, prevents inconsistencies and avoids parental anxiety, but can be considered paternalistic. Here, we reflect on the cultural and moral bases of the Belgian reporting system of prenatally detected variants.

The Belgian MicroArray Prenatal (BEMAPRE) database: A systematic nationwide repository of fetal genomic aberrations.

OBJECTIVE: With the replacement of karyotyping by chromosomal microarray (CMA) in invasive prenatal diagnosis, new challenges have arisen. By building a national database, we standardize the classification and reporting of prenatally detected copy number variants (CNVs) across Belgian genetic centers. This database, which will link genetic and ultrasound findings with postnatal development, forms a unique resource to investigate the pathogenicity of variants of uncertain significance and to refine the phenotypic spectrum of pathogenic and susceptibility CNVs. METHODS: The Belgian MicroArray Prenatal (BEMAPRE) consortium is a collaboration of all genetic centers in Belgium. We collected data from all invasive prenatal procedures performed between May 2013 and July 2016. RESULTS: In this three-year period, 13 266 prenatal CMAs were performed. By national agreement, a limited number of susceptibility CNVs and no variants of uncertain significance were reported. Added values for using CMA versus conventional karyotyping were 1.8% in the general invasive population and 2.7% in cases with an ultrasound anomaly. Of the reported CNVs, 31.5% would have remained undetected with non-invasive prenatal test as the first-tier test. CONCLUSION: The establishment of a national database for prenatal CNV data allows for a uniform reporting policy and the investigation of the prenatal and postnatal genotype-phenotype correlation.

Accuracy and clinical value of maternal incidental findings during noninvasive prenatal testing for fetal aneuploidies.

PURPOSE: Genome-wide sequencing of cell-free (cf)DNA of pregnant women aims to detect fetal chromosomal imbalances. Because the largest fraction of cfDNA consists of maternal rather than fetal DNA fragments, maternally derived copy-number variants (CNVs) are also measured. Despite their potential clinical relevance, current analyses do not interpret maternal CNVs. Here, we explore the accuracy and clinical value of maternal CNV analysis. METHODS: Noninvasive prenatal testing was performed by whole-genome shotgun sequencing on plasma samples. Following mapping of the sequencing reads, the landscape of maternal CNVs was charted for 9,882 women using SeqCBS analysis. Recurrent CNVs were validated retrospectively by comparing their incidence with published reports. Nonrecurrent CNVs were prospectively confirmed by array comparative genomic hybridization or fluorescent in situ hybridization analysis on maternal lymphocytes. RESULTS: Consistent with population estimates, 10% nonrecurrent and 0.4% susceptibility CNVs for low-penetrant genomic disorders were identified. Five clinically actionable variants were reported to the pregnant women, including haploinsufficiency of RUNX1, a mosaicism for segmental chromosome 13 deletion, an unbalanced translocation, and two interstitial chromosome X deletions. CONCLUSION: Shotgun sequencing of cfDNA not only enables the detection of fetal aneuploidies but also reveals the presence of maternal CNVs. Some of those variants are clinically actionable or could potentially be harmful for the fetus. Interrogating the maternal CNV landscape can improve overall pregnancy management, and we propose reporting those variants if clinically relevant. The identification and reporting of such CNVs pose novel counseling dilemmas that warrant further discussions and development of societal guidelines.Genet Med 19 3, 306-313.

Detection of a case of chronic myeloid leukaemia with deletions at the t(9;22) translocation breakpoints by a genome-wide non-invasive prenatal test.

OBJECTIVE: Non-invasive prenatal tests (NIPTs) interrogating the complete genome are able to detect not only fetal trisomy 13, 18 or 21 but additionally provide information on other (sub)chromosomal aberrations that can be fetal or maternal in origin. We demonstrate that in a subset of cases, this information is clinically relevant and should be reported to ensure adequate follow-up. METHOD: Genome-wide NIPT was carried out and followed by a software analysis pipeline optimized to detect subchromosomal aberrations. RESULTS: The NIPT profile showed deletions on chromosomes 9 and 22: NIPT 9q33.3q34.12(129150001-133750000)x1,22q11.23(23550001-25450000)x1,22q13.1(37850001-39600000)x1. This result was confirmed by single nucleotide polymorphism array on maternal genomic DNA, which also demonstrated that the deletions were somatic in nature. Fluorescence in situ hybridization and quantitative real-time polymerase chain reaction revealed that the deletions were flanking the translocation breakpoint on the derivative chromosome 9 as the result of a t(9;22)(q34;q11.2) translocation with BCR-ABL1 fusion typical for chronic myeloid leukaemia (CML). Multidisciplinary counselling, together with complete blood count, taught that the woman was in an early chronic phase CML. The woman was followed up closely, and treatment could be postponed until after delivery. CONCLUSION: Genome-wide NIPT identified a CML in chronic phase caused by the typical t(9;22)(q34;q11.2) translocation and accompanied by deletions flanking the translocation breakpoints. © 2016 John Wiley & Sons, Ltd.

Human Rab7 mutation mimics features of Charcot-Marie-Tooth neuropathy type 2B in Drosophila.

Charcot-Marie-Tooth disease type 2B (CMT2B) is an inherited axonal peripheral neuropathy. It is characterised by prominent sensory loss, often complicated by severe ulcero-mutilations of toes or feet, and variable motor involvement. Missense mutations in RAB7A, the gene encoding the small GTPase Rab7, cause CMT2B and increase Rab7 activity. Rab7 is ubiquitously expressed and is involved in degradation through the lysosomal pathway. In the neurons, Rab7 plays a role in the long-range retrograde transport of signalling endosomes in the axons. Here we developed the first animal model of CMT2B, modelling one of the mutations (L129F) in Drosophila melanogaster. Behavioural assays show that this model recapitulates several hallmarks of the human disease. Upon expression of mutant Rab7 in the sensory neurons, larvae present with a reduction of temperature and pain perception. Furthermore, the larvae exhibit a crawling defect when the mutant protein is expressed in the motor neurons. Analysis of axonal transport of Rab7 positive vesicles in sensory neurons of Drosophila larvae and in neurites of mammalian neuroblastoma cells demonstrates that mutant vesicles pause less than their wild-type counterparts. This latter finding indicates that alterations in vesicle transport might contribute to the pathomechanism of CMT2B.

Bladder exstrophy-epispadias complex and triple-X syndrome: incidental finding or causality?

BACKGROUND: Bladder exstrophy is a rare malformation. Prenatal diagnosis is usually an incidental finding on routine ultrasound examination. Triple-X syndrome (karyotype 47,XXX) is the most frequent sex chromosome aneuploidy in live-born females (approximately 1 in 1000). The diagnosis is often not made because women with 47,XXX karyotype have no or hardly any clinical symptoms during life. METHODS: Prenatal diagnosis of triple X karyotype is usually an incidental finding when an invasive prenatal diagnosis is performed for other reasons. RESULTS: Here, we report on two cases with bladder exstrophy and triple-X syndrome, one in a fetus and one in an adult. In view of two previous reports of this association in literature, causality of these two conditions should be considered. CONCLUSION: A gene dosage effect as possible underlying mechanisms will be discussed.

The impact of confined placental mosaicism on prenatal cell-free DNA screening: Insights from a monocentric study of 99 cases.

INTRODUCTION: Confined placental mosaicism (CPM) is thought to be one of the main sources of false-positive prenatal cell-free DNA (cfDNA) screening results, but extensive and systematic studies to prove this statement are limited. We evaluate the contribution of CPM to false-positive prenatal cfDNA screening results in the largest cohort published to date. METHOD: We systematically offered postnatal analysis on placenta and umbilical cord to women who had a negative amniocentesis following a positive prenatal cfDNA screening result. A standardized protocol was used in which (when available) biopsies were taken at five locations in the placenta and umbilical cord. RESULTS: We analyzed a series of 99 placentas. CPM could be confirmed in 32.3 % of cases (32/99). CPM was detected across all subtypes of chromosomal aberrations (common and rare autosomal trisomies, sex chromosome abnormalities, copy number variations and autosomal monosomies). A lower detection rate was present in umbilical cord biopsies in comparison with placental biopsies. When comparing different sections of the placenta, no clear difference could be observed with regard to the probability of CPM being present nor to the grade of mosaicism. DISCUSSION: We confirm an important role for CPM in explaining false-positive prenatal cfDNA screening results. Placental regional differences are common. Given its limited clinical relevance, we do however not advocate placental studies in a diagnostic setting.

Novel presentation of AADC deficiency as a mild phenotype with exercise-induced dystonic crises: A case report.

Aromatic l-amino acid decarboxylase (AADC) deficiency is a rare autosomal recessive neurometabolic disorder caused by biallelic pathogenic variants in the DDC gene; approximately 140 patients have been described worldwide. AADC deficiency is characterised by a combined deficiency of dopamine, serotonin, adrenaline and noradrenaline causing a highly variable phenotype with developmental delay, early-onset hypotonia, movement disorders and autonomic symptoms. We expand the phenotype of this neurometabolic disorder by reporting on a paediatric patient with a mild phenotype with atypical exercise-induced dystonic crises, a feature that has not been described in AADC deficiency up till now. Additionally, we also present a second patient with typical characteristics and a severe phenotype. The diagnosis in both patients was confirmed by the presence of a homozygous pathogenic variant in the DDC gene and reduced AADC enzyme plasma activity. The use of whole exome sequencing-based strategies has played a crucial role in diagnosing these two patients.

Population screening for 15q11-q13 duplications: corroboration of the difference in impact between maternally and paternally inherited alleles.

Maternally inherited 15q11-q13 duplications are generally found to cause more severe neurodevelopmental anomalies compared to paternally inherited duplications. However, this assessment is mainly inferred from the study of patient populations, causing an ascertainment bias towards patients at the more severe end of the phenotypic spectrum. Here, we analyze the low coverage genome-wide cell-free DNA sequencing data obtained from pregnant women during non-invasive prenatal screening (NIPS). We detect 23 15q11-q13 duplications in 333,187 pregnant women (0.0069%), with an approximately equal distribution between maternal and paternal duplications. Maternally inherited duplications are always associated with a clinical phenotype (ranging from learning difficulties to intellectual impairment, epilepsy and psychiatric disorders), while paternal duplications are normal or associated with milder phenotypes (mild learning difficulties and dyslexia). This data corroborates the difference in impact between paternally and maternally inherited 15q11-q13 duplications, contributing to the improvement of genetic counselling. We recommend reporting 15q11-q13 duplications identified during genome-wide NIPS with appropriate genetic counselling for these pregnant women in the interest of both mothers and future children.

Burden re-analysis of neurodevelopmental disorder cohorts for prioritization of candidate genes.

This study aimed to uncover novel genes associated with neurodevelopmental disorders (NDD) by leveraging recent large-scale de novo burden analysis studies to enhance a virtual gene panel used in a diagnostic setting. We re-analyzed historical trio-exome sequencing data from 745 individuals with NDD according to the most recent diagnostic standards, resulting in a cohort of 567 unsolved individuals. Next, we designed a virtual gene panel containing candidate genes from three large de novo burden analysis studies in NDD and prioritized candidate genes by stringent filtering for ultra-rare de novo variants with high pathogenicity scores. Our analysis revealed an increased burden of de novo variants in our selected candidate genes within the unsolved NDD cohort and identified qualifying de novo variants in seven candidate genes: RIF1, CAMK2D, RAB11FIP4, AGO3, PCBP2, LEO1, and VCP. Clinical data were collected from six new individuals with de novo or inherited LEO1 variants and three new individuals with de novo PCBP2 variants. Our findings add additional evidence for LEO1 as a risk gene for autism and intellectual disability. Furthermore, we prioritize PCBP2 as a candidate gene for NDD associated with motor and language delay. In summary, by leveraging de novo burden analysis studies, employing a stringent variant filtering pipeline, and engaging in targeted patient recruitment, our study contributes to the identification of novel genes implicated in NDDs.

Mutations in the SPTLC2 subunit of serine palmitoyltransferase cause hereditary sensory and autonomic neuropathy type I.

Hereditary sensory and autonomic neuropathy type I (HSAN-I) is an axonal peripheral neuropathy associated with progressive distal sensory loss and severe ulcerations. Mutations in the first subunit of the enzyme serine palmitoyltransferase (SPT) have been associated with HSAN-I. The SPT enzyme catalyzes the first and rate-limiting step in the de novo sphingolipid synthesis pathway. However, different studies suggest the implication of other genes in the pathology of HSAN-I. Therefore, we screened the two other known subunits of SPT, SPTLC2 and SPTLC3, in a cohort of 78 HSAN patients. No mutations were found in SPTLC3, but we identified three heterozygous missense mutations in the SPTLC2 subunit of SPT in four families presenting with a typical HSAN-I phenotype. We demonstrate that these mutations result in a partial to complete loss of SPT activity in vitro and in vivo. Moreover, they cause the accumulation of the atypical and neurotoxic sphingoid metabolite 1-deoxy-sphinganine. Our findings extend the genetic heterogeneity in HSAN-I and enlarge the group of HSAN neuropathies associated with SPT defects. We further show that HSAN-I is consistently associated with an increased formation of the neurotoxic 1-deoxysphinganine, suggesting a common pathomechanism for HSAN-I.

Direct detection of guidance receptor activity during border cell migration.

Guidance receptor signaling is crucial for steering migrating cells. Despite this, we generally lack direct measurements of such signaling. Border cells in Drosophila migrate as a tightly associated group, but dynamically, with front and rear cells exchanging places. They use the receptor tyrosine kinase (RTK) PDGF/VEGF receptor (PVR) as a guidance receptor, perceiving the attractant Pvf1. Here we determine the spatial distribution of PVR signaling by generating an antibody that specifically detects activated PVR in situ. PVR activity is very low in migrating border cells, due to strong activity of cellular phosphatases. Measurements of signal at the cell cortex show variability but a strong bias for both total active PVR and specific activity of PVR to be elevated at the front versus side of the leading cell, often with several-fold difference in signal levels. This polarized active PVR signal requires the E3 ubiquitin ligase Cbl and the recycling regulator Rab11, indicating a dependency on receptor trafficking. The endogenous ligand gradient contributes to shaping of signaling by increasing the specific activity of PVR toward the source in front cells. Surprisingly, signaling is also elevated at the back versus the side of rear cells. This distally polarized distribution of active PVR is ligand independent. Thus the actual guidance signal transmitted in border cells appears to integrate perceived ligand distribution with cell polarity or cell orientation with respect to the cluster. A general implication is that both group configuration and extrinsic cues can directly modulate guidance receptor signaling during collective cell migration.

Loss-of-function mutations in LEMD3 result in osteopoikilosis, Buschke-Ollendorff syndrome and melorheostosis.

Osteopoikilosis, Buschke-Ollendorff syndrome (BOS) and melorheostosis are disorders characterized by increased bone density. The occurrence of one or more of these phenotypes in the same individual or family suggests that these entities might be allelic. We collected data from three families in which affected individuals had osteopoikilosis with or without manifestations of BOS or melorheostosis. A genome-wide linkage analysis in these families, followed by the identification of a microdeletion in an unrelated individual with these diseases, allowed us to map the gene that is mutated in osteopoikilosis. All the affected individuals that we investigated were heterozygous with respect to a loss-of-function mutation in LEMD3 (also called MAN1), which encodes an inner nuclear membrane protein. A somatic mutation in the second allele of LEMD3 could not be identified in fibroblasts from affected skin of an individual with BOS and an individual with melorheostosis. XMAN1, the Xenopus laevis ortholog, antagonizes BMP signaling during embryogenesis. In this study, LEMD3 interacted with BMP and activin-TGFbeta receptor-activated Smads and antagonized both signaling pathways in human cells.

Implementation of Exome Sequencing in Prenatal Diagnostics: Chances and Challenges.

Whole exome sequencing (WES) has become part of the postnatal diagnostic work-up of both pediatric and adult patients with a range of disorders. In the last years, WES is slowly being implemented in the prenatal setting as well, although some hurdles remain, such as quantity and quality of input material, minimizing turn-around times, and ensuring consistent interpretation and reporting of variants. We present the results of 1 year of prenatal WES in a single genetic center. Twenty-eight fetus-parent trios were analyzed, of which seven (25%) showed a pathogenic or likely pathogenic variant that explained the fetal phenotype. Autosomal recessive (4), de novo (2) and dominantly inherited (1) mutations were detected. Prenatal rapid WES allows for a timely decision-making in the current pregnancy, adequate counseling with the possibility of preimplantation or prenatal genetic testing in future pregnancies and screening of the extended family. With a diagnostic yield in selected cases of 25% and a turn-around time under 4 weeks, rapid WES shows promise for becoming part of pregnancy care in fetuses with ultrasound anomalies in whom chromosomal microarray did not uncover the cause.